Habilitation Thesis
Functions of Plant Proteins associated with
Telomeric Repeats and Telomerase
Mgr. Petra Procházková Schrumpfová, Ph.D.
Brno, 2023
Acknowledgement
I would like to express my deep gratitude to the head of the group, Prof. Jiří Fajkus, who has not only
continuously supported my research, but also motivated me and provided fruitful scientific comments and
advice. Additionally, he has been the person I turned to during challenging moments of my life.
I would also like to acknowledge both current and former colleagues who contributed to the friendly,
tolerant, and supportive atmosphere.
Thanks also go to many of my students for their enthusiasm, patience, and contribution to the successful
work on my scientific project. Their efforts exceeded my expectations, especially when considering the
context of my maternity leave, which made our work more demanding.
The greatest thanks belong to my family, especially my husband Pavel, who has supported me in all aspects
of my life and decisions, and my lovely two children, Verunka and Martínek.
Contents
Commentary….………………………………………………………………………………….….......................................................4
Telomeric repeats and their interactome ……………………………………………………….............................................8
1 Telomeric repeats at the physical ends of linear chromosomes - telomeres............................... 8
1.1 Proteins associated with telomeres in mammals ...............................................................12
1.2 Proteins associated with telomeres in plants .....................................................................15
1.2.1 Telomeric dsDNA associated proteins in plants..........................................................15
Smh/TRB family............................................................................................................16
TRFL family...................................................................................................................22
AID family.....................................................................................................................23
1.2.2 Telomeric ssDNA associated proteins in lants ............................................................23
Proteins with OB-fold...................................................................................................23
Non-OB-fold proteins...................................................................................................26
2 Interstitially located telomeric repeats.......................................................................................27
2.1 Proteins associated with long interstitial telomeric repeats (ITSs).....................................28
2.2 Proteins associated with short internally localized telomeric repeats (telo-boxes)...........29
3 Orchestration of telomere homeostasis.....................................................................................31
3.1 Telomerase..........................................................................................................................32
3.1.1 Telomerase reverse transcriptase (TERT) ...................................................................33
3.1.2 Telomerase RNA (TR) ..................................................................................................34
3.1.3 Telomerase-associated proteins .................................................................................35
3.1.4 Telomerase assembly..................................................................................................38
3.2 Telomere maintenance proteins.........................................................................................44
3.2.1 Mammalian telomere maintenance proteins .............................................................44
3.2.2 Plant telomere maintenance proteins ........................................................................45
3.2.3 HMG proteins..............................................................................................................46
3.3 Telomerase-independent telomere maintenance..............................................................47
Conclusion….………………………………………………………………………………….….........................................................51
Bibliography………………………………………………………………………………………………………………………………………….....52
Supplements…………………………………………………………………………………………………………………………………………….67
4
Commentary
This Habilitation Thesis is a compilation of selected scientific publications in which I have contributed as a primary
author or co-author. The primary goal of my research was to contribute to a fundamental understanding of the nature
and characteristics of proteins and enzymes associated with long or short telomeric repeats.
At the very beginning of my scientific career, I initially focused on characterizing previously unknown plant putative
homologues of proteins linked to telomeric sequences in vitro, known as TRBs. Interestingly, this protein family turned
out to be a fruitful discovery in my scientific journey. Over the course of nearly two decades, we have uncovered that
these proteins not only interact with the physical ends of chromosomes but also serve as the first described plant
interactors of telomerase, the enzyme responsible for elongating telomeres. Furthermore, I found out that TRB
proteins are associated with short telomeric sequences in the promoters of various genes, which resulted in very
fruitful collaborations with laboratories investigating the epigenetic regulation of gene transcription. Moreover, my
exploration of telomeric sequences and associated proteins has led me to the characterization of other diverse
proteins linked to telomeric sequences or telomerase, such as RUVBLs, HMGBs, POTs, the PRC2 complex, and many
others. These investigations have also touched subjects like plant gametogenesis, alternative lengthening of
telomeres, and even the development of novel software for detecting short regulatory motifs within gene promoters.
To investigate and characterize the proteins associated with telomeric repeats and telomerase, we employed a range
of general biochemical and molecular biological techniques. These included cloning, protein expression and
purification, Electrophoretic mobility shift assay (EMSA), yeast two hydrid assay (Y2H), Co-Immunoprecipitation (CoIP),
Bimolecular Fluorescence Complementation (BiFC), Tandem Affinity Purification (TAP-tag), Chromatin
Immunoprecipitation (ChIP) followed by Next-gen sequencing or hybridization and many others. Additionally, I utilized
specialized techniques focused on telomeres or telomerase, such as Telomere Restriction Fragment (TRF) analysis and
the Telomerase Repeated Amplification protocol (TRAP). In order to characterize plant material, I also employed
techniques necessary for the analysis of T-DNA insertion mutant plants or plant cell cultures. Furthermore, as a
supervisor for several bachelor's, master's, and doctoral theses, as well as a principal investigator of grants or a
member of the grant-investigating team, I took on the responsibility of establishing and conducting research on
various topics. This included the characterization of protein localization using microscopic techniques, the
investigation of plant gametogenesis, and even the bioinformatic analysis.
5
Our findings have already been published in total of 20 publications on WoS, including articles and reviews. Among
these, there is 1 correction to a research article (Schrumpfová and Majerská et al., Protoplasma, 2017), where
Schrumpfová P.P. was recognized as the first co-author. Additionally, apart from these 20 publications, there is 1 book
chapter published in Methods in Molecular Biology, The Nucleus, Book Series, Springer protocols (Schořová et al.,
2020) and 1 Meeting abstract (Schrumpfová et al., 2005, FEBS Journal) listed in WoS.
Among these 20 publications, I have served as the primary author in 9 of them and as the corresponding author in 7.
The habitation theses comprise a compilation of 18 of these publications relevant to the thesis title:
*Corresponding Author
[1] Teano G., Concia L., Wolff L., Carron L., Biocanin I., Adamusová K., Fojtová M., Bourge M., Kramdi A., Colot
V., Grossniklaus U., Bowler Ch., Baroux C., Carbone A., Probst A.V., Schrumpfová P.P., Fajkus J., Amiard S., Grob
S., Bourbousse C., and Barneche F. 2023. Histone H1 protects telomeric repeats from H3K27me3 invasion in
Arabidopsis. Cell Reports, 42(8):112894. (IF 8.8, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
0% 5% 5% 10%
[2 ] Kusová A., Steinbachová L., Přerovská T., Drábková Z.L., Paleček J, Khan A., Rigóová G., Gadiou Z., Jourdain
C., Stricker T., Schubert D., Honys D., and Schrumpfová P.P.*. Completing the TRB family: newly characterized
members show ancient evolutionary origins and distinct localization, yet similar interactions. PLANT
MOLECULAR BIOLOGY 112:61–83 2023 (IF 4.076, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
5% 50% 90% 90%
[3] Tomaštíková E.D., Yang F., Mlynárová K., Hafidh S., Schořová Š., Kusová A., Pernisová M., Přerovská T.,
Klodová B., Honys D., Fajkus J., Pecinka A., Schrumpfová P.P.*. RUVBL proteins are involved in plant
gametophyte development. THE PLANT JOURNAL, Volume: 114, Issue: 2 Pages: 325–337, 2023 (IF 7.091, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
5% 90% 90% 80%
[4] Pecinka A., Schrumpfova P.P., Fisher L., Tomastikova E., Mozgova I. The Czech Plant Nucleus Workshop.
Biologia Plantarum, Volume: 66, Pages: 39-45, 2021 (IF 1.122, Q4)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
- - 25% [5]
Schrumpfová P.P.* and Fajkus J. Composition and Function of Telomerase—A Polymerase Associated with
the Origin of Eukaryotes. Biomolecules, 10(10), 2020 (IF 4.879, Q2)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
- - 80% [6]
Schořová Š., Fajkus J., Drábkova L., Honys D., Schrumpfová P.P.* The plant Pontin and Reptin homologues,
RuvBL1 and RuvBL2a, colocalize with TERT and TRB proteins in vivo, and participate in telomerase biogenesis.
THE PLANT JOURNAL, 98(2):195-212, 2019 (IF 5.775, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
- 80% 90% 90%
6
[7] Schrumpfová P.P., Fojtova M., Fajkus J. Telomeres in Plants and Humans: Not So Different, Not So Similar.
CELLS Volume: 8 Issue: 1 Article Number: 58 Published: 2019 (IF 4.366, Q2)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
- - 40% [8]
Schrumpfová P.P., Majerská J., Dokládal L., Schořová Š., Stejskal K., Obořil M., Honys D., Kozáková, L.,
Polanská P.S., Sýkorová E. Tandem affinity purification of AtTERT reveals putative interaction partners of plant
telomerase in vivo. PROTOPLASMA Volume: 254 Issue: 4 Pages: 1547-1562, 2017 (IF 2.457, Q2)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
20% 70% 5% 10%
[9] Schrumpfová P.P.*, Schořová Š., Fajkus J. Telomere- and telomerase-associated proteins and their
functions in the plant cell. FRONTIERS IN PLANT SCIENCE Volume: 7 Article Number: 851, 2016 (IF 4.291, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
- - 80% [10]
Schrumpfová P.P., Vychodilová I., Hapala J., Schořová Š., Dvořáček V., Fajkus J. Telomere binding protein
TRB1 is associated with promoters of translation machinery genes in vivo. PLANT MOLECULAR BIOLOGY
Volume: 90 Issue: 1-2 Pages: 189-206, 2016 (IF 3.359, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
70% 50% 90% 90%
[11] Schrumpfová P.P.*, Vychodilová I., Dvořáčková M., Majerská J., Dokládal L., Schořová S., Fajkus J.
Telomere repeat binding proteins are functional components of Arabidopsis telomeres and interact with
telomerase. PLANT JOURNAL Volume: 77 Issue: 5 Pages: 770-781, 2014 (IF 6.815, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
80% 30% 70% 50%
[12] Schrumpfová P.P.*, Fojtová M., Mokroš P., Grasser K.D., Fajkus J. Role of HMGB proteins in chromatin
dynamics and telomere maintenance in Arabidopsis thaliana. CURRENT PROTEIN & PEPTIDE SCIENCE Volume:
12 Issue: 2 Pages: 105-111, 2011 (IF 3,790, Q2)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
80% - 30% 10%
[13] Peška V., Schrumpfová P.P., Fajkus J. Using the telobox to search for plant telomere binding proteins.
CURRENT PROTEIN & PEPTIDE SCIENCE Volume: 12 Issue: 2 Pages: 75-83, 2011 (IF 3,790, Q2)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
- - 20% -
7
[14] Hofr C., Šultesová P., Zimmermann M., Mozgová I., Schrumpfová P.P., Wimmerová M., Fajkus J. SingleMyb-histone
proteins from Arabidopsis thaliana: a quantitative study of telomere-binding specificity and
kinetics. BIOCHEMICAL JOURNAL Volume: 419 Pages: 221-228, 2009 (IF 3.857, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
10% - - [15]
Mozgová I., Schrumpfová P.P., Hofr C., Fajkus J. Functional characterization of domains in AtTRB1, a
putative telomere-binding protein in Arabidopsis thaliana. PHYTOCHEMISTRY Volume: 69 Issue: 9 Pages:
1814-1819, 2008 (IF 3.186, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
5% 50% 10% 30%
[16] Schrumpfová P.P., Kuchař M., Paleček J., Fajkus J. Mapping of interaction domains of putative telomerebinding
proteins AtTRB1 and AtPOT1b from Arabidopsis thaliana. FEBS LETTERS Volume: 582 Issue: 10 Pages:
1400-1406, 2008 (IF 3.264, Q2)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
70% - 10% [17]
Růčková E., Friml J., Schrumpfová P.P., Fajkus J. Role of alternative telomere lengthening unmasked in
telomerase knock-out mutant plants. PLANT MOLECULAR BIOLOGY Volume: 66 Issue: 6 Pages: 637-646, 2008
(IF 3.543, Q1)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
20% 40% 10% [18]
Schrumpfová P., Kuchař M., Miková G., Skříšovská L., Kubičárová T., Fajkus J. Characterization of two
Arabidopsis thaliana myb-like proteins showing affinity to telomeric DNA sequence. GENOME Volume: 47
Issue: 2 Pages: 316-324, 2004 (IF 2.100, Q2)
Experimental work (%) Supervision (%) Manuscript (%) Research
direction (%)
80% - 20% 10%
8
Telomeric repeats and their interactome
Repetitive G-rich nucleotide sequences have been detected at the ends of the chromosomes of most living
organisms, and hence named telomeric DNA repeats – from the ancient Greek télos 'end' and méros 'part'
(Blackburn & Gall, 1978; reviewed in Jenner et al., 2022). Subsequently, it has become clear that telomeric
motifs are also present within chromosomes. These internally localized telomeric repeats can be
distinguished into two groups: short telomeric DNA repeats called telo-boxes and long telomeric DNA tracts,
called interstitial telomeric sequences (ITSs) (Tremousaygue et al., 1999; Uchida et al., 2002). However, the
defining of these two groups is not entirely precise, it is assumed that telo-boxes are composed of one to
two telomeric DNA units and ITSs contain from several units to hundreds or thousands of telomeric DNA
repeats. Moreover, ITSs are not composed of absolutely pure tracts of telomeric DNA repeats, but they are
generally composed of telomeric repeats interspaced with degenerate repeats. The telomeric repeats,
either located at the ends of or within the chromosomes, act as binding targets for large number of proteins.
Some of these proteins recognize telomeric repeats specifically, while some of the telomeric-sequence
associated proteins show higher sequence variability. Despite the initial assumptions that telomere-binding
proteins are exclusively localized at the terminal telomeric tracts (Palm & de Lange, 2008), nowadays it is
clearer that functions of telomeric-sequence associated proteins, including enzyme elongating telomeres telomerase,
is more complex and these proteins possess a broad spectrum of activities.
1 Telomeric repeats at the physical ends of linear chromosomes -
telomeres
Telomeres are nucleoprotein structures forming and protecting the ends of linear chromosomes. They
serve at least three functions which are essential for cell viability. First, they protect chromosome physical
ends from fusion, endogenous nucleases and erroneous recognition as unrepaired chromosomal breaks.
Secondly, telomeres facilitate the complete replication of the physical ends of the DNA. Finally, telomeres
are implicated in intranuclear chromosome localization and meiotic chromosome pairing (reviewed in
Blackburn et al., 2015; Procházková Schrumpfová et al., 2019; see Suppl. M; Shay & Wright, 2019; Schmidt
& Cech, 2015; Schrumpfová & Fajkus, 2020; see Suppl. O).
For its potential significance to aging, cancer and cell viability serve telomeres, telomerase and telomereassociated
proteins as a subject of intensive research. Barbara McClintock was the first to recognized that
induced chromosome ends were distinctly different from natural ends and Hermann Müller, based in part
on some of the findings of McClintock, called the ends of linear chromosomes "telomeres" (Müller, 1938;
McClintock, 1942). However, the intensive research on the telomeres was started only three decades ago
with a description of telomere DNA component (Blackburn & Gall, 1978), detection of telomerase
9
(ribonucleoprotein with a reverse transcriptase function) (Greider & Blackburn, 1985, 1989) and uncovering
telomere binding proteins (Bianchi et al., 1997; Broccoli et al., 1997).
Figure 1. The replicating DNA in eukaryotes: DNA polymerases involved in replication (adopted from
Schrumpfova et al., 2020; see Suppl. O).
During semiconservative DNA replication, each strand serves as a template for DNA polymerases to
synthesize a new complementary strand. A specialized RNA polymerase (primase), that is a part of DNA
Pol α, synthesizes the RNA primer. A single RNA primer aids DNA replication on the leading strand and
multiple primers initiate Okazaki fragment synthesis on the lagging strand. Further DNA synthesis is carried
out by DNA Pol ε and DNA Pol δ. The newly replicated telomere resulting from the lagging strand synthesis
(Lagging telomere) retains the terminal RNA primer, which is subsequently removed. Attachment of the
last RNA primer more proximally on the DNA strand, together with RNA-primer removal, creates an
overhang on the G-rich strand. The initial product of the leading strand DNA synthesis (Leading telomere)
is a blunt terminus whose C-strand is then resected by an exonuclease to create the mature G-rich
overhang. In cells with an active RNA-dependent DNA polymerase (Telomerase), the G-rich overhangs
originating from Lagging or Leading telomeres, can undergo elongation. Telomerase carries its own RNA
molecule, which is used as a template, and can anneal through the first few nucleotides of its template
region to the distal-most nucleotides of the G-rich overhang of the telomere DNA, add a new telomere
repeat (GGTTAG) sequence, translocate and then repeat the process. The complementary C-strand is then
in-filled by DNA Pol α-primase.
10
Telomeres cannot be fully replicated by enzymes that duplicate DNA. Conventional DNA polymerases
cannot fully replicate telomeres all the way to the end of a chromosome. The synthesis of Okazaki fragments
on the lagging strand requires RNA primers attaching ahead, resulting in shortening of the chromosome's
end with each duplication (Olovnikov, 1973). Moreover, the product of the leading strand DNA synthesis is
a blunt terminus whose C-strand is then resected by an exonuclease to create the mature G-rich overhang
(see Figure 1).
Telomeric DNA in most organisms consists of tandem arrays of a short repetitive sequence. Two strands
are recognized: one strand of the telomeric repeat tract running towards the 3′ end that is rich in guanines,
called G-strand, whereas the complementary strand rich in cytosines is called C-strand (Makarov et al.,
1997). The telomere in most of the species terminates in a 3′ single-stranded G-rich DNA overhang. In
human telomeres a G-overhang is prevalent whose length varies from several tens to 280 base pairs (bp)
(Cimino-Reale et al., 2001; Makarov et al., 1997; Wright et al., 1997). Telomeric sequence is highly
conserved in Unikonta, where telomere motif is composed from (TTAGGG)n (Moyzis et al., 1988). This motif
is the predominant terminal repeat sequence for fungi, animals, and Amoebozoa (Fulnecková et al., 2013)
and sometimes is referred to as the vertebrate telomeric sequence. However even among Unikonts, the
DNA sequence at chromosome ends is not completely uniform, e.g. there was detected presence of
(TTAGGC)n in Nematoda, (TGTGGG)n in Rotifera or (TTAGG)n in insect Coleoptera (Frydrychová & Marec,
2002; Mason et al., 2016; Müller et al., 1991). While most filamentous fungi use (TTAGGG)n at their
chromosome ends, yeasts telomeric sequence is not regular and can be described as T(G1–3) (reviewed in
Kupiec, 2014; Peska et al., 2020; Tomáška et al., 2018). Moreover in addition to 3′ G-overhangs,
Caenorhabditis elegans possess telomeric 5′ C-overhangs (Oganesian & Karlseder, 2011; Raices et al., 2008).
Recently, in Hymenoptera (Insecta) various sequences (e.g. TTAGGTTGGG, TTAGG, TTTAGGTTAGG) were
identified in terminal regions of assembled genomes (Fajkus et al., 2023).
In land plants, the telomere is mostly composed of Arabidopsis-type (TTTAGGG)n repeats (Richards &
Ausubel, 1988; reviewed in Schrumpfová et al., 2016a; see Supp. J; Schrumpfova et al., 2020; see Suppl.
O) (see Figure 2). Several groups of flowering plants are known in which a replacement of the plant
telomere sequence has occurred. Known exceptions are species in the order Asparagales, starting from
divergence of the Iridaceae family. Iridaceae family shares the human-type telomeric repeat (TTAGGG)n,
probably caused by a mutation that altered the telomerase RNA subunit of telomerase ∼80 Mya (Adams et
al., 2001; Sýkorová et al., 2003). The human-type telomere is also shared by species of the Allioideae
subfamily (Sýkorová et al., 2006), except for the Allium genus where unusual telomeric sequence
(CTCGGTTATGGG)n was detected (Fajkus et al., 2016). An unusual telomeric motif (TTTTTTAGGG)n was also
found in the closely related genera Cestrum elegans (family Solanaceae) (Peška et al., 2015). Moreover,
outside of land plants in red and green algae and glaucophytes (Koonin, 2010), telomere types also vary.
11
For example, in algae, not only the Arabidopsis-type of telomeric repeat, but also human-type (TTAGGG)n,
the Chlamydomonas-type (TTTTAGGG)n and a (TTTTAGG)n repeat were described (Fulnecková et al., 2013).
Even within one plant carnivorous genus Genlisea, the telomeric sequence can vary from the Arabidopsistype
telomere repeat present in G. nigrocaulis to two variant sequences (TTCAGG)n and (TTTCAGG)n in G.
hispidula and its close relative G. subglabra (Tran et al., 2015) (reviewed in Schrumpfová et al., 2016a; see
Supp. J; Schrumpfova et al., 2020; see Suppl. O).
In plants, as well as in most of other species, replication of chromosomal ends results in
G-overhangs after degradation of the last RNA primer at the 5’ terminus of a nascent strand. In A. thaliana
or Silene latifolia, relatively short (20–30 nucleotides (nt)) G-overhangs were detected. Moreover, half of
the Arabidopsis and Silene telomeres showed no overhangs or overhangs less than 12 nt in length (Kazda
et al., 2012; Riha et al., 2000). A substantial portion of telomeres in Arabidopsis does not apparently
undergo nucleolytic resection. Říha et al. showed that A. thaliana contain blunt-ended and short (1- to 3nucleotide)
G-overhang-containing telomeres (Riha et al., 2000).
Figure 2. Telomeres in the evolutionary tree (adopted from Schrumpfova et al., 2020; see Suppl. O).
A simplified phylogenetic tree is shown, where telomeres and telomerase evolved upon linearization of
chromosomes by the insertion of Group II self-splicing introns. In the Eukaryote branch, the groupings correspond
to the current ‘supergroups’ according to the recent eukaryotic Tree of Life (eToL). Unresolved branching orders
among lineages are shown as multifurcations. Broken lines reflect lesser uncertainties about the monophyly of
certain groups. Examples of known telomeric repeat variants are listed next to respective supergroups. The major
known telomeric repeat variants in the supergroups are marked with a larger font. Last eukaryote common
ancestor (LECA); last universal common ancestor (LUCA).
12
Human telomere length at birth is highly heterogeneous, ranging from roughly 5-15 kilobase pairs (kb)
(Sanders & Newman, 2013). The length of the germline human telomeres varies from 15–20 kb and
laboratory mice have 25–150 kb telomeres (Sanders & Newman, 2013; Shay & Wright, 2000). It is obvious
that length of the telomeres can vary not only between the species but also in between one genus (Gomes
et al., 2011). Estimates of telomeric bp loss vary between 30–200 bp per division (Lansdorp et al., 1996).
The length of plant telomeric DNA at a single chromosomal arm can be as small as 500 bp in Physcomitrella
patens (Fojtová et al., 2015; Shakirov et al., 2010) as long as 160 kb in Nicotiana tabacum (Fajkus et al.,
1995) or 200 kb in N. sylvestris (Kovařik et al., 1996). Besides the remarkable variation in telomere length
among diverse plant genera or orders, telomere lengths can also vary at the level of the species or ecotypes,
e.g. Arabidopsis telomeres range from 1.5 to 9 kb, depending on the ecotype (Maillet et al., 2006; Shakirov
& Shippen, 2004); telomeres from inbred lines of maize range from 1.8 to 40 kb (Burr et al., 1992).
Additionally, in the long-living organism Betula pendula, telomeres in different genotypes varied from a
minimum length of 5.9 - 9.6 kb to a maximum length of 15.3 - 22.8 kb (Aronen & Ryynänen, 2014) reviewed
in Schrumpfová et al., 2016a; see Supp. J).
1.1 Proteins associated with telomeres in mammals
Telomere-associated proteins can regulate the length of the telomere tract by modulating access of
telomerase or affecting conventional DNA replication machinery. In mammals, telomeric DNA is maintained
primarily by six-protein complex called Shelterin: Telomere Repeat Binding Factors 1 and 2 (TRF1, TRF2),
Protection of telomeres 1 (POT1), TRF1- and TRF2-Interacting Nuclear Protein 2 (hTIN2), telomere
protection protein 1 (TPP1) and Repressor/activator protein 1 (RAP1). Moreover, the physical ends of the
chromosomes are associated with a nucleoprotein complex named CST (Cdc13/CTC1, STN1, TEN1). The
specific telomeric dsDNA binding of Shelterin complex is mediated by TRF1 and TRF2 proteins (Broccoli et
al., 1997) through their Myb-like domain, with an LKDKWRT amino acid motif. The Myb-like domain is
conserved in telomeric sequence binding proteins not only in mammals but also in plants or yeasts (Bilaud
et al., 1996; Feldbrügge et al., 1997). Myb-like domains of the TRF1 and TRF2 proteins are located at their
C-terminus. Another Shelterin subunit - POT1 protein - is linked to the Shelterin complex by TPP1 protein,
which in turn binds to TIN2 and RAP1 proteins and interacts with TRF2 protein (reviewed in Schmidt & Cech,
2015) (see Figure 3A).
13
TRF2 protein has a central protective role in shelterin complex because it specifically inhibits Ataxia
Telangiectasia Mutated (ATM) kinase-dependent DNA damage signalling and the classical Ku70/80- and
Ligase IV-mediated non-homologous enjoining pathway (NHEJ) at telomeres. The POT1 proteins (POT1a
and POT1b in the mouse) associate with single-stranded (ss) telomeric DNA and POT1 protein safeguards
Figure 3. An integrative schematic view of the human and plant terminal telomeric complex (adopted from
Schrumpfová et al. 2019; see Suppl. M).
A) Human active telomerase is associated with chaperones as well as with TR associated conserved scaffold
box H/ACA of small nucleolar RNAs proteins. Mammalian shelterin proteins (TRF1/2, RAP1, TIN2, TPP1 and
POT1) modulate access to the telomerase complex and the ATR/ATM-dependent DNA damage response
pathway. The CST complex (CTC1-STN1-TEN1) affects telomerase and DNA polymerase α recruitment to the
chromosomal termini and, thus, coordinates G-overhang extension by telomerase with fill-in synthesis of
the complementary C-strand (blue dashed line). G-quadruplexes, D-loops and T-loops during telomere
replication are resolved by RTEL helicase. HOT1 directly binds double strand telomere repeats and
associates with the active telomerase. Telomere nucleosomes show a shorter periodicity than that in the
other parts of chromosomes.
B) Arabidopsis telomerase is associated with TRB proteins as well as with POT1a that interacts with the dyskerin
orthologue CBF5. Plants possess all orthologue proteins of conserved scaffold box H/ACA of small nucleolar
RNAs (CBF5, GAR1, NOP10, NHP2). Moreover, TRB proteins interact with the telomeric sequence due to the
same Myb-like binding domain as that in mammalian TRF1/2. TRB proteins interact with TERT and POT1b
and, when localized at chromosomal ends, they are eligible to function as components of the plant shelterin
complex. An evolutionarily conserved CST complex is suggested to coordinate the unique requirements for
efficient replication of telomeric DNA in plants as well as in other organisms. In addition, plant RTEL
contributes to telomere homeostasis. For the sake of clarity, only the situation in telomere with 3' overhang
is depicted. For further information and for human and plant telomere histone modifications see
Schrumpfová et al. (2019; see Suppl. M).
A
B
Telomerase
Telomerase
14
telomeres against Ataxia Telangiectasia and Rad3 related (ATR) kinase pathway (Denchi & de Lange, 2007;
Smogorzewska et al., 2002). A bridge between proteins directly associated with DNA-TRF1, TRF2 and POT1
is mediated by TIN2 and the oligosaccharide/oligonucleotide binding (OB)-fold domain of TPP1 protein
(reviewed in (Schmidt & Cech, 2015). Moreover protein RAP1, interacts with TRF2 (Arat & Griffith, 2012)
and modulates its recruitment to telomeric DNA (Janoušková et al., 2015; Nečasová et al., 2017). The most
of the Shelterin complexes can be purified without dissociation, indicating they form stable complexes at
least in vitro. It was published that TRF2 interacts with TIN2 with an 2:1 stoichiometry in the context of
Shelterin (RAP12:TRF22:TIN21:TPP11:POT11) (Lim et al., 2017).
The maintenance of telomere repeats in most eukaryotic organisms requires enzyme telomerase.
Telomerase consists of a telomerase reverse transcriptase (TERT) and telomerase RNA subunits (TR) that
dictates the synthesis of the G-rich strand of telomere terminal repeats and elongates telomeric tracts at
the chromosomal terminus (Blackburn & Gall, 1978; Greider & Blackburn, 1985, 1989). Most enzymes
encounter their substrates by simple diffusion but both telomerase and its chromosome end substrate have
very low abundance and the telomerase enzyme is recruited to telomeres rather than simply encountering
them by diffusion (Schmidt & Cech, 2015; Xi & Cech, 2014). The Shelterin component TPP1 is the key
telomeric component necessary for telomerase recruitment to telomeres (Xin et al., 2007). In addition,
TPP1 in complex with POT1 stimulates telomerase to synthesize additional telomeric repeats in vitro and
has therefore been proposed to be a processivity factor for telomerase action at telomeres (Wang et al.,
2007). Protein TPP1 is composed of an N-terminal OB-fold domain required for telomerase recruitment, a
central domain that directly binds to POT1 and a C-terminal domain necessary for its association with TIN2.
Loss of any of the members of Shelterin protein complex can result in inappropriate DNA damage response
(DDR), can lead to chromosome fusion, telomere loss or activate replicative senescence or apoptosis (Sfeir,
2012).
Kappei et al. identified by the proteomics of isolated chromatin segments (PICh) approach a telomeric DNA
binding protein named homeobox telomere-binding protein 1 (HOT1). HOT1 directly binds ds telomere
repeats and associates with the active telomerase and is required for telomerase chromatin binding. HOT1
is the telomere-binding protein that acts as a positive regulator of telomere length (Déjardin & Kingston,
2009; Kappei et al., 2013).
15
1.2 Proteins associated with telomeres in plants
1.2.1 Telomeric dsDNA associated proteins in plants
In plants, telomeric dsDNA sequence binding proteins with a Myb-like domain can be classified into three
main groups: (i) with a Myb-like domain at the N-terminus (Smh/TRB family) (ii) with a Myb-like domain at
the C-terminus (TRFL family) (iii) with a Myb-like domain at the C-terminus (AID family) (reviewed in Du et
al., 2013; Peška et al., 2011; Schrumpfová et al., 2016a; see Supp. F and J).
The first group of proteins, with a Myb-like domain at the N-terminus, contain a central histone-like domain
(H1/5 domain) with homology to the H1 globular domain found in the linker histones H1/H5 and is therefore
called the Smh (Single myb histone) family (Marian et al., 2003; Marian & Bass, 2005; Schrumpfová et al.,
2004; see Supp. A). Members belonging to Smh family are frequently named Telomere Repeat Binding
(TRB) proteins so we call this family also Smh/TRB family (see Figure 3B and Figure 5).
Within the second family of the proteins with a Myb-like domain - TRFL family - there were also identified
several plant orthologues. In A. thaliana there were characterised six proteins with the C-terminal Myb-like
domain (AtTBP1, AtTRP1 and AtTRFL1, 2, 4, 9) belonging to the subfamily of proteins named TRFL I with
characteristic features. Proteins from TRFL I family can homo- and heterodimerize and they can efficiently
bind to telomeric DNA in vitro (Karamysheva et al., 2004). A key feature of this subfamily is a ∼30 amino
acid extension of the Myb-like domain that is likely responsible for specific binding to plant telomeric DNA.
Moreover, the TRFL family includes six proteins, that are unable to bind telomeric DNA in vitro and are also
unable to form homo- and heterodimers, despite possessing the C-terminal Myb-like domain. These
proteins are members of subfamily named TRFL II (AtTRFL3, 5, 6, 7, 8, 10) (Karamysheva et al., 2004).
The proteins from the third family contain a single Myb-like domain at the C-terminus and contains only a
few described members.
All three Myb-like protein subfamilies were already detected in the moss P. patens and separation of
Smh/TRB and TRFL and is apparent already in unicellular algae. These data suggest ancient origin of the
three protein subfamilies and their diversification early in evolution of the plant lineage (Fulcher & Riha,
2016).
Especially the first (Smh/TRB) family and the second (TRFL) family contain increased number of family
members. However, this observation is not surprising as whole genome duplication events (WGDs) have
occurred in many plant families (Freeling, 2009; Qiao et al., 2022). These WGDs result in a multitude of
genomic changes, such as deletions of large fragments of chromosomes, silencing of duplicate genes and
recombining of homologous chromosomal segments, as was shown, e.g. in crucifer species (Freeling, 2009;
Mandáková & Lysak, 2008). Increased numbers of genes of the same family may lead to gene sub-
16
functionalization, neo-functionalization and partial or full redundancy, and complicates assignment of an
actual and specific function for individual proteins in vivo.
Overall, the conserved domain composition of the plant proteins with respect to their mammalian
counterparts does not guarantee conservation of their function. It seems that some proteins are involved
in a similar biochemical pathways, but their interaction partners, and consequently potential regulatory
factors, might slightly differ (reviewed in Schrumpfová et al., 2019; see Supp. M).
Smh/TRB family
Screening of Z. mays cDNA led to identification of gene coding ZmSmh1 protein (Marian et al., 2003). The
Smh1 gene is expressed in leaf tissue and the ZmSmh1 protein binds ds oligonucleotide probes with at least
two internal tandem copies of the maize telomere repeat, TTTAGGG.
Simultaneously as Smh protein from Zea was characterized, we searched A. thaliana databases in our
laboratory for putative genes coding for proteins with the Myb-like domain. This search resulted in two
candidate protein sequences at that time, AtTRB2 and AtTRB3, formerly named AtTBP3 and AtTBP2,
respectively (Kuchař & Fajkus, 2004; Schrumpfová et al., 2004; see Supp. A). We characterised these two
candidates and we found out that AtTRB2 and AtTRB3 proteins able to bind the G-rich strand and dsDNA of
plant telomeric sequence with an affinity proportional to a number of telomeric repeats. The binding of
AtTRB proteins to telomeric ds telomeric oligonucleotides is highly specific, because even a 100-fold
abundance of non-telomeric sequence cannot displace their binding to tetramers of the telomeric sequence
(Schrumpfová et al., 2004; see Supp. A). Binding affinity to ds- and ss-oligonucleotides of the plant
telomeric sequence is roughly proportional to the number of telomeric repeat.
Additionally, the later identified member of Smh/TRB family - AtTRB1 protein - is able to bind related
telomeric DNA sequences (plant (TTTAGGG) or human (TTAGGG)) with a certain flexibility, as well as AtTRB2
or AtTRB3 proteins. We analysed DNA-protein interaction of the full-length and truncated variants of
AtTRB1. We showed that preferential interaction of AtTRB1 with ds telomeric DNA is mediated by the Myblike
domain while the H1/5 domain interacts non-specifically with any DNA without preference for either
telomeric or non-telomeric sequence (Ellen & van Holde, 2004; Mozgová et al., 2008; see Supp. D). The
partial non-selective binding of the Myb-like domain to either plant (TTTAGGG) or human (TTAGGG)
telomeric sequence appears to be a general feature of the A. thaliana Smh/TRB family proteins (Mozgová
et al., 2008; Schrumpfová et al., 2004; see Supp. D and A).
Recently, we have completed characterization of the TRB family as we described two novel members of the
TRB family from Arabidopsis (AtTRB4 and AtTRB5) (see Figure 4). The results clearly showed that AtTRB4
and AtTRB5 do preferentially bind long arrays of telomeric sequences. However, the AtTRB minimal
17
recognition motif was newly defined as one telo-box positioned within a non-telomeric DNA sequence
(Kusova et al., 2023; see Suppl. R).
Figure 4. Sequence and structural alignments of TRB family proteins (Kusová et al., 2023; see Supp. R).
A) Schematic representation of the conserved domains of TRBs from A. thaliana. Myb-like, Myb-like domain;
H1/5-like, histone-like domain; coiled-coil, C-terminal domain.
B) Unrooted Maximum likelihood (ML) phylogenetic tree of Brassicaceae TRB proteins. The length of the
branches are proportional, and the black dots indicate the position of TRB1-5 from A. thaliana.
C) Multiple alignments of the Myb-like, H1/5-like and coiled-coil domains. The positions of α-helices or β-sheets
of the uppermost or the lowermost sequence in each alignment are highlighted: bold, experimentally
determined structures (cryo-EM or X-ray crystallography); thin, AlphaFold prediction. Human Telomeric
repeat-binding factor 2 (hTRF2) and Xenopus laevis histone H1.0 (Xl H1.0-B) were used to show the most
conserved amino acid (aa) residues. Amino acid shading indicates the following conserved amino acids: dark
green, hydrophobic and aromatic; light green, polar; blue, basic; magenta, acidic; yellow, without side chain
(glycine and proline). The aa of hTRF2 that mediate intermolecular contacts between telomeric DNA and
hTRF2 are marked with an asterisk.
D) A certain flexibility in binding related telomeric DNA sequences was observed from the A. thaliana telomeric
DNA: A. thaliana (TTTAGGG), Chlamydomonas reinhardtii (TTTTAGGG), human (TTAGGG), Bombyx mori
[TTAGG]5TTAG and Ascaris lumbricoides (TTAGGC) (Niedermaier and Moritz, 2000; Okazaki et al., 1993;
Petracek and Berman, 1992), however, the ability to bind variant telomere sequences decreased with
sequence divergence. In addition to being able to bind ds telomeric sequences, AtTRB proteins can also bind
to the G-rich ss telomeric DNA although with lower affinity compared to ds telomeric sequences. The C-rich
telomeric strand is not preferentially bound (Schrumpfová et al., 2004; see Supp. A). Our observation are
consistent with the findings that also other members of Smh/TRB family proteins in land plants show
telomeric dsDNA binding capability, e.g. Z. mays ZmSMHs or O. sativa OsTRBFs (Byun et al., 2018; Marian et
al., 2003).
18
We performed comprehensive phylogenetic analysis and found out that TRB proteins first evolved in
Streptophyta in Klebsormidiophyceae. In Klebsormidium nites only one TRB homolog was identified.
Following the evolutionary tree, an increasing number of TRB homologues were found in Bryophyta and
Tracheophyta. In seed plants, which have undergone more rounds of WGDs than Bryophyta and Lycophyta
(Clark & Donoghue, 2018), predominantly three TRB proteins were recognized. Within Brassicaceae, which
has undergone an additional recent round of WGD (Walden et al., 2020), five TRB homologs were revealed
(Kusová et al., 2023; see Supp. R).
The ability of AtTRB proteins to bind typical plant and human telomeric motifs with a similar affinity could
be important for an easier adaptation to a change in telomere sequence from a plant to divergent telomeric
motifs, which has occurred during the evolution of plants of several species as was described above. It is
also consistent with the Kováč et al. that argued that there is an upper limit for the specificity of interaction
between binding partners (e.g. enzyme-substrate, ligand-receptor, protein-DNA sequence), since
interactions that are too specific would lack flexibility and a perfect recognition would be too rigid and
possibly non-functional (Kováč, 1987), e.g. Tay1 (telomere-associated in Yarrowia lipolytica 1) protein, the
double strand (ds) sequence telomere-binding protein of the yeast Y. lipolytica, exhibits lower affinity for
its own telomeres (TTAGTCAGGG) than for the mammalian-type telomeric repeats (TTAGGG) (reviewed in
Tomáška et al., 2018).
Another typical character of a telomere dsDNA-binding proteins seems to be capability for multimerization.
Dimerization has been proved to increase the efficiency of the binding of telomere-associated proteins,
TRF1 and TRF2, to telomeric DNA also in mammalian cells. Mammalian TRF1 is a homodimer in vivo and its
accumulation at telomeres depends on homotypic interactions. Similarly, the TRF homology (TRFH) domain
near their N-terminus from TRF2 protein mediates homotypic interactions, but TRF1 and TRF2 do not form
heterodimers (Bianchi et al., 1997; Fairall et al., 2001).
In our studies we demonstrated that AtTRBs show strong mutual and self-interactions using yeast two
hydrid assay (Y2H) assay (Schrumpfová et al., 2004; see Supp. A; Kusova et al., 2023; see Suppl. R).
Additionally, we investigated the ability of the AtTRB1 fragments to form self-dimers or multimers using
Poly Acrylamide Gel Electrophoresis (PAGE) with a weak detergent, perfluoro-octanoic acid (PFO) (Mozgová
et al., 2008; see Supp. D). This method can be used for detection and molecular mass determination of
protein complexes since (in contrast to SDS-PAGE with sodium dodecyl sulfate), PFO-PAGE preserves high
affinity protein–protein interactions (Ramjeesingh et al., 1999). The results confirmed the strong tendency
of the H1/5 domain to multimerize and the same holds true for all the fragments of AtTRB1 which contain
the H1/5 domain. Myb-like domain of the rice RTBP, TRFL family protein, also interacts with plant telomeric
DNA in the form of a homodimer (Yu et al., 2000). In contrast to H1/5 domain, the N-terminal Myb-like
19
domain itself did not form higher molecular weight complexes. According to these results we proposed
model of binding of AtTRB proteins to plant telomeric DNA where the Myb-like domain primarily ensures
direct sequence-specific binding of AtTRB1 to telomeric DNA, while the H1/5 domain may enhance this
binding by protein dimerization and sequence-non-specific binding to DNA (Mozgová et al., 2008; see Supp.
D).
The study of stoichiometry and kinetics of AtTRB1 and AtTRB3 proteins binding to the telomeric DNA
revealed that the affinity of AtTRB1 to telomeric substrate with four telomeric repeats is 4-fold higher than
that of AtTRB3, although AtTRB1 and AtTRB3 are relatively similar in their primary sequences (Hofr et al.,
2009; see Supp. E). Similarly to our results, human hTRF1 binds telomeric DNA with a 4-fold higher affinity
than that of hTRF2 when interacting with human telomeric DNA (Hanaoka et al., 2005). In Mozgova et al.
(2008; see Supp. D) we assumed that the non-specific interaction of H1/5 domain with any DNA without
preference for either telomeric or non-telomeric sequence (Ellen & van Holde, 2004), together with the
high pI of the AtTRB1 fragments, suggests that electrostatic interactions take part in the interaction of the
fragments of AtTRB1 with telomeric dsDNA (Mozgová et al., 2008; see Supp. D). Interestingly, our model
showing model of Myb-like domain revealed that the solution accessible surface of AtTRB4 and AtTRB5
differ to the solution accessible surface of AtTRB2/AtTRB3 and to AtTRB1 (Kusova et al., 2023; see Suppl.
R).
All five AtTRB members preferentially localize to the nucleus and nucleolus during interphase. Both the
central H1/H5‐like domain and the Myb-like domain from AtTRB1 can direct a GFP fusion protein to the
nucleus and nucleolus (Dvořáčková et al., 2010a; Kusova et al., 2023; see Suppl. R). AtTRB1-GFP localization
is cell cycle‐regulated, as the level of nuclear‐associated GFP diminishes during mitotic entry and GFP
progressively re‐associates with chromatin during anaphase/telophase. Although a possible association of
AtTRB1-GFP with the telomere was suggested previously in Dvořáčková et al. (2010a) the small size of
Arabidopsis chromosomes, in combination with short telomere lengths, precluded the authors to visualize
the AtTRB-telomere association. We took advantage of the well-established protocol of Nicotiana
benthamiana leaf infiltration and the fact that N. benthamiana has longer telomeres that are easier to
visualize compared to Arabidopsis. In our study Schrumpfová et al. (2014; see Supp. H) we proved that
AtTRB proteins are not only binding to the telomeric DNA sequence in vitro, as was described above, but
that they also co-localize with telomeres in situ.
Later on, localization of AtTRB1 protein at the plant telomeres in vivo was verified by independent
technique by the teams of Holger Puchta and Andreas Houben. They used imaging technique based on two
orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR
associated protein 9 (Cas9). Dreissig et al. demonstrated not only that CRISPR–dCas9 can be used to
20
visualize specific DNA sequences in combination with fluorescently tagged proteins interacting with those
DNA sequences but they also demonstrated that around 87.6 % of telomeres were simultaneously bound
by AtTRB1 protein and CRISPR–dCas9 signals resembling telomeres (Dreissig et al., 2017).
Telomere shortening was observed in attrb1 mutants in the A. thaliana ecotype Columbia, with otherwisestable
telomere lengths (Shakirov & Shippen, 2004; Schrumpfová et al., 2014; see supp. H). In contrast,
telomere extension was detected in attrb2 knockout mutants of the A. thaliana ecotype Wassilewskija,
which exhibits telomere length polymorphism in wild-type plants (W. K. Lee & Cho, 2016; Maillet et al.,
2006; Shakirov & Shippen, 2004). Triple homozygous mutant plants, containing the alleles from A. thaliana
Columbia (attrb1 and attrb3) and from Wassilewskija (attrb2), exhibit telomere shortening (Zhou et al.,
2016, 2018).
Our suggestion, that AtTRBs are part of telomere-associated interactome was supported by the group of
Simon Amiard and Charles White that used pull-down assays to identify potential telomeric interactors in
the Arabidopsis. They identified several candidate proteins, including TRB1 and TRB3 proteins. The TRB
proteins were enriched in pull-down with telomeric probe even more than the GH1-HMGA1 proteins that
are the main objects their study (Charbonnel et al., 2018).
Involvement of AtTRB proteins in telomere interactome was furthermore boosted by our detection of direct
interaction between AtTERT and AtTRB proteins (Kusova et al., 2023; see Suppl. R). AtTRB proteins interact
in Y2H, Co-Immunoprecipitation (Co-IP) or Bimolecular Fluorescence Complementation (BiFC) systems with
the N-terminal part of AtTERT that contains telomerase-specific motifs. Moreover, AtTRB1 was, among the
others, co-purified with N-terminal constructs of AtTERT from A. thaliana suspension cultures
(Schrumpfová and Majerská et al., 2017, 2018; see Supp. K and L). However, neither of the AtTRB2 and
AtTRB3 proteins purified from Escherichia coli, nor their mixture, had any effect on telomerase activity in
vitro, measured by Telomerase Repeat Amplification Protocol (TRAP) (Schrumpfová et al., 2004; see Supp.
A). Likewise, no changes in telomerase activity or processivity were observed in extracts from attrb1 mutant
plants. Correspondingly, no variations in telomerase activity were detected in transformed plants
(TRB1pro:TRB1-GFP) expressing higher levels of protein (Schrumpfová et al., 2014; see Supp. H).
Kuchař et al. (2004) detected that AtTRB2 and AtTRB3 proteins interact with AtPOT1b protein, one of two
homologues of human telomeric ssDNA binding protein POT1 (Baumann & Cech, 2001; Kuchař & Fajkus,
2004). In our study Schrumpfová et al., (2008; see Supp. C) we found out that also other member of
Smh/TRB family - AtTRB1 protein - interacts with AtPOT1b. Using combination of Y2H and Co-IP we detected
that AtTRB1 protein physically interacts with N-terminus AtPOT1b via its H1/5 domain (Schrumpfová et al.,
2008; see Supp. C). Recently we detected also interaction between AtTRB4 and AtTRB5 and AtPOT1a
(Kusova et al., 2023; see Suppl. R).
21
Moreover, proteins from Smh/TRB family physically interact with AtRUVBLs. RUVBL proteins belong to the
evolutionarily highly conserved AAA+-family (ATPase Associated with various cellular Activities) that are
involved in ATP binding and hydrolysis (Matias et al., 2006). AtTRBs together with AtTERT and AtRUVBLs
form trimeric complex AtTERT-AtTRB-AtRUVBL (see also bellow) (Schořová et al., 2019; see Supp. N). Our
results suggested that AtTRB proteins thus play a role of interaction hubs not only in telomere chromatin
structure but also in telomerase biogenesis.
Figure 5. Telomeric and putative telomeric dsDNA- and ssDNA-binding proteins from A. thaliana (adopted
from Schrumpfová et al., 2016a, see Supp. J).
Myb-like domain (Myb); Myb-extension (-ext); Histone-like domain (H1/5); Coiled Coil Domain (CCD);
Oiigonucleotide/Oligosaccharide-Binding Fold domain (OB); Whirly domain (Whirly); RNA-binding domain (RB);
A. thaliana (At); Telomere Repeat Binding Protein (AtTRB); TRF-like family (TRFL family); Suppressor of cdc
thirteen homolog (AtStn1); Conserved telomere maintenance component 1 (AtCTC1); (CTC1-Stn1-Ten1)
complex (CST); RNA recognition motifs (RRM); Protection of telomeres 1a, b, c (AtPot1 a,b,c); Whirly 1 (Why1);
Single-stranded telomere-binding protein 1 (STEP1).
22
In O. sativa there were three proteins from Smh/TRB family identified (Byun et al., 2008). Proteins OsTRBF1
and OsTRBF2 are constitutively transcribed in rice plants grown under greenhouse conditions. Gel
retardation assays showed that these OsTRBF proteins bind specifically to the plant double-stranded
telomeric sequence, TTTAGGG, with markedly different binding affinities. Y2H and Co-IP assays indicated
that both OsTRBF1 and OsTRBF2 interact with one another to form homo- and hetero-complexes, while
OsTRBF3 appeared to act as a monomer (Byun et al., 2008). In an affinity pull-down technique, 80 proteins
from O. sativa were identified for their ability to bind to a telomeric repeat (He et al., 2013). Among them,
two of three previously reported proteins from the Smh/TRB family - OsTRBF1 and OsTRBF2 were isolated.
TRFL family
The second group of proteins, with a Myb-like domain at the C-terminus, is named TRFL (TRF-like). TRFL
family can be divided into two subfamilies named TRFL I (possess extension of the Myb-like domain (Mybext)
that is likely responsible for specific binding to plant telomeric DNA proteins in vitro) and TRFL II (unable
to bind telomeric DNA in vitro) (Karamysheva et al., 2004; Ko et al., 2008) (see Figure 5).
The first identification of a TRFL family protein from O. sativa - Telomere-binding protein 1 (OsRTBP1) (Yu
et al., 2000) - was soon followed by numerous other TRFL members, e.g. Nicotiana glutinosa (NgTRF1) (Yang
et al., 2003), Solanum lycopersicum (LeTBP1) (Moriguchi et al., 2006), A. thaliana (AtTBP1, AtTRP1, AtTRFL2-
10) (Hwang et al., 2001; Chen et al., 2001; Karamysheva et al., 2004) or Cestrum parqui (CpTBP) (Peška et
al., 2011; see Supp. F). Even though O. sativa or N. glutinosa mutants for TRFL members exhibited markedly
longer telomeres (Hong et al., 2007; Yang et al., 2004), in A. thaliana, a knockout of AtTRP1, member of
TRFLI subfamily with a Myb-ext, did not change telomere length significantly (Chen et al., 2005). In A.
thaliana even multiple knockout plant, deficient for all six proteins from TRFLI subfamily (AtTBP1, AtTRP1,
AtTRFL1, AtTRFL2, AtTRFL4 and AtTRF9) did not exhibit changes in telomere length or phenotypes
associated with telomere dysfunction (Fulcher & Riha, 2016; reviewed in Schrumpfová et al., 2016a; see
Supp. J).
A structurally related member to TRFLI subfamily was found in Cestrum parqui, CpTBP1, a plant species
lacking typical telomeres and telomerase (Peška et al., 2011; see Supp. F). The protein shows nuclear
localisation and association with chromatin while transiently expressed in N. benthamiana after infiltration
Agrobacterium tumefaciens into young leaves.
Although, no functional evidence exists for the role of AtTRFL proteins at telomeres so far, plausible
involvement in telomere maintenance in plants was suggested in Kuchař and Fajkus (Kuchař & Fajkus,
2004). Kuchař and Fajkus observed a specific interaction between AtTRP1 (member of TRFLI subfamily) and
AtKu70. The AtTRP1 domain responsible for AtKu70 interaction occurs between amino acid sequence
positions 80 and 269. It was hypothesized that AtKu, a DNA repair factor with a high affinity for DNA ends,
23
sequesters chromosome termini within its DNA loading channel and protects them from nuclease
processing (Valuchova et al., 2017).
Another member of the TRFL family - ZmIBP2 (Initiator-binding protein) protein – binds not only telomeric
repeats (Moore, 2009), but was originally identified as a promoter binding ligand (Lugert & Werr, 1994).
AID family
The third group with a Myb-like domain at the C-terminus (AID family) contains only a few described
members. The AID family is named according to anther indehiscence 1 (AID) protein from O. sativa - OsAID1
(Zhu et al., 2004). OsAID1 was initially identified as being involved in anther development, however, OsAID1
also isolated in an affinity pull-down technique within 80 proteins from O. sativa showing ability to bind to
a telomeric repeat, while no member with a Myb-like domain at the C-terminus of the TRFL family could be
found (He et al., 2013). Another member of this family - ZmTacs1 (Terminal acidic SANT) from Z. mays - may
function in chromatin remodelling within the meristem. In silico expression analysis revealed that ZmTacs1
is expressed in meristem-enriched tissues and in contrast, the Myb-like domains of known Myb-like domain
such as ZmSMH1, or human TRF1 all have basic isoelectric points (Marian & Bass, 2005; reviewed in
Schrumpfová et al., 2016a; see Supp. J). Marian and Bass proposed that the acidic patches observed on the
surfaces of the plant TACS-type proteins are not compatible with direct DNA binding and may reflect areas
for the binding of basic moieties, such as histone tails or basic regions of other proteins (Marian & Bass,
2005).
1.2.2 Telomeric ssDNA associated proteins in plants
Proteins with OB-fold
The majority of telomeric ssDNA binding proteins bind through OB motifs (oligonucleotide/oligosaccharide
binding, OB-fold) and are required for both chromosomal end protection and regulation of telomere length,
e.g., telomere-binding protein subunit alpha/beta (TEBPαβ) from Oxytricha nova; (C. M. Price & Cech,
1987), Cell division cycle 13 (Cdc13p) from S. cerevisiae (Garvik et al., 1995) and POT1, are present in diverse
organisms including human, mouse, chicken or S. pombe (Baumann & Cech, 2001; Lei et al., 2002; Wei &
Price, 2004; L. Wu et al., 2006).
In A. thaliana, three POT-like proteins were named AtPOT1a (previously named AtPOT1-1, AtPot1),
AtPOT1b (previously named AtPOT1-2, AtPot2) and AtPOT1c (Kuchař & Fajkus, 2004; Lei et al., 2002;
Rossignol et al., 2007; Shakirov et al., 2005; Tani & Murata, 2005). AtPOT1a and AtPOT1b proteins contain
two OB motifs as well as mammalian POT1 proteins, but share only 49 % sequence similarity, while mouse
24
proteins share 72 % similarity. AtPOT1c protein is short version of AtPOT1a and originates by gene
duplication and contain only one OB motif (Rossignol et al., 2007) (see Figure 3B and Figure 5).
However, descriptions of plant POT protein functions and binding properties are not unanimously agreed.
While a very weak, but specific affinity of AtPOT1a and AtPOT1b expressed in E. coli for plant telomeric
ssDNA was originally described (Shakirov et al., 2005), later these authors could not demonstrate AtPOT1a
and AtPOT1b binding to telomeric ssDNA in vitro (Shakirov, McKnight, et al., 2009; Shakirov, Song, et al.,
2009). In our laboratory, AtPOT1a and AtPOT1b proteins were expressed in bacteria or using in vitro WG
transcription/translation extract. Unfortunately, none of these systems or their modifications resulted in
expression of intact AtPOT1 proteins. AtPOT1 proteins were either not expressed or due to their
hydrophobicity localized mainly in the bacterial inclusion bodies or they were co-purified with chaperon
GroEL (Schrumpfova, dissertation thesis). There was no proof that the AtPOT1b, purified from bacterial
extract or expressed in vitro translation extract, had the ability to bind telomeric ss oligonucleotides
(Schrumpfová, 2008; see Supp. C). Subsequently it was demonstrated that functional human and mouse
POT1 should be isolated from baculovirus-infected insect cells (Palm et al., 2009).
Nevertheless, stable telomeric ssDNA binding was observed for two full-length plant POT1 proteins: OlPOT1
from the green alga O. lucimarinus as well as for ZmPOT1b from Z. mays (Shakirov, Song, et al., 2009).
Although POT1 proteins from plant species as diverse as Populus trichocarpa (poplar), Hordeum vulgare
(barley), Gossypium hirsutum (cotton), Helianthus argophyllus (sunflower), S. moellendorffii (spikemoss),
Pinus taeda (pine), Solanum tuberosum (potato), Asparagus officinalis (garden asparagus) and Z. mays
(maize) (ZmPOT1a) failed to bind telomeric DNA when expressed in a RRL expression system in vitro and
subjected to an electrophoretic mobility shift assay (EMSA) (Shakirov, Song, et al., 2009), binding of plant
POT1 proteins to telomeric DNA under native conditions cannot be excluded.
Plants expressing AtPOT1a truncated by an N-terminal OB-fold, showed progressive loss of telomeric DNA.
These findings denote that AtPOT1a plays role in positive regulation of telomere length (Surovtseva et al.,
2007). In contrast, expression of only N-terminal part of AtPOT1b leads to severe defects in plant growth
and development, telomeres are shortened and there is a high formation of anaphase bridges or defective
segregation of chromosome, which means that AtPOT1b plays role in protection of chromosomal ends
(Shakirov et al., 2005).
POT1 proteins from A. thaliana differ not only in their functions, but also have divergent interaction
partners. AtPOT1a binds AtSTN1 and AtCTC1 proteins from CST complex (Renfrew et al., 2014). AtPOT1a,
but not AtPOT1b, is associated with an N-terminal part of AtTERT in nucleoplasm in vitro (Rossignol et al.,
2007). Among other interactors of AtPOT1a belongs CBL-interacting protein kinase (CIPK21). This kinase
belongs to the large family in A. thaliana of which several members were shown to be involved in Ca2+
25
signalling and moreover, CIPK21 is presumed to have a function in DDR signalling (Rossignol et al., 2007).
These data suggest a potential role of AtPOT1a in DDR pathway as was described to many other telomeric
proteins (Gallego & White, 2005). We found AtPOT1a protein among proteins that we co-purified with Nterminal
domains of AtTERT using (TAP-MS) (Schrumpfová and Majerská et al., 2017, 2018; see Supp. K
and L). Using BiFC it was confirmed that AtPOT1a interacts with AtCBF5 protein (Centromere-binding factor
5; a plant homologue of dyskerin) in the cytoplasmic or nucleolus foci (Kannan et al., 2008; Lermontova et
al., 2007; Schořová et al., 2019; see Supp. N). Interestingly, AtPOT1a forms weak interaction with AtRUVBL1
protein. This fact corelates with our recent observation that AtPOT1a, AtTERT, AtTRB, AtCBF5 and
AtRUVBL1 proteins are involved in assembly of the plant telomerase (Schořová et al., 2019; see Supp. N).
Moreover, both AtPOT proteins directly interacts with AtTRB proteins (Kuchař & Fajkus, 2004; Kusova et
al., 2023; see Suppl. R), nevertheless, AtPOT1b does not seem to substantially contribute to telomere
maintenance (Cifuentes-Rojas et al., 2012). Using Y2H and BiFC have also recently detected novel
interaction between AtTRB4-5 and AtPOT1a (Kuchař & Fajkus, 2004; Kusova et al., 2023; see Suppl. R).
CST is an evolutionarily conserved trimeric protein complex that in budding yeast is composed of the
proteins Cdc13, Stn1 and Ten1, whereas in mammals the CST complex consists of the proteins CTC1, STN1
and TEN1. CST complex plays role in DNA replication and telomere maintenance through its ability to
interact with ssDNA. Nevertheless, it was found out that CST is neither a nonspecific nor a telomere ssDNA
specific binder, and rather CST is a tight binder of ssDNA with a preference for G-rich sequences (Hom &
Wuttke, 2017). In yeast, these OB-fold proteins are required for recruitment of telomerase and DNA
polymerase α to the chromosomal termini and thus coordinate G-overhang extension by telomerase with
the fill-in synthesis of the complementary C-strand (Giraud-Panis et al., 2010; Grossi et al., 2004; Qi &
Zakian, 2000; Wellinger & Zakian, 2012). Mammalian CST is ortholog of an archaeal RPA complex and is
involved in the rescue of stalled replication forks either at the telomere or elsewhere in the genome and Cstrand
fill-in. However, CST in mammals is also proposed to limit telomerase action, perhaps by competing
for binding to the telomere protein TPP1 (reviewed in Lue, 2018; Rice & Skordalakes, 2016; Schrumpfova
et al., 2019; see Supp. M) (see Figure 3A).
CST in plants is needed for telomere integrity (Leehy et al., 2013; Surovtseva et al., 2007), however, clear
evidence that would show any direct physical interaction of any component of the CST complex with plant
telomeric DNA is absent. It seems that the CST complex controls access of telomerase, end-joining
recombination and the ATR-dependent (ATM and Rad3-related) DNA damage response pathway at the
chromosomal ends in wild-type plants (see Figure 3B) (Amiard et al., 2011; Boltz et al., 2012; Derboven et
al., 2014; Leehy et al., 2013; reviewed in Schrumpfova et al., 2019; see Supp. M).
26
Non-OB-fold proteins
Aside predominantly characterised proteins with OB-fold domain associated with telomeric ssDNA
sequence in plants, several proteins lacking the OB-fold domain were also identified, such as Whirly proteins
or proteins with RNA recognition (RRM) motifs (see Figure 5).
The transcriptional activator protein Whirly 1 (AtWhy1), from a small protein family found mainly in land
plants (Desveaux et al., 2000, 2002; Krause et al., 2005), was also identified in a fraction of AtTERT binding
proteins in A. thaliana (Schrumpfová and Majerská et al., 2017, 2018; see Supp. K and L). Although a TDNA
insertional mutation of AtWHY1 did not result in detectable abnormal phenotypes, atwhy1 mutant
plants contained longer telomeres, whereas AtWHY1 overexpressing plants showed shortened telomeres
and decreased telomerase activity (Yoo et al., 2007). While proteins from A. thaliana (AtWhy1) and from
Hordeum vulgare (HvWhy1) (Grabowski et al., 2008) were found to bind plant telomeric repeat sequences
in vitro, diverse organelle localization of other Why family members from O. sativa, A. thaliana, S.
tuberosum (Krause et al., 2005; Schwacke et al., 2007) and proposed binding to ssDNA of melted promoter
regions (Desveaux et al., 2002), rather indicate a role in communication between plastid and nuclear genes
encoding photosynthetic proteins (Comadira et al., 2015; Foyer et al., 2014). Overall, it seems that Why
proteins bind to various DNA sequences, including: telomeres; a distal element upstream of a kinesin gene;
the promoter region of the early senescence marker gene AtWRKY53 (in a development-dependent
manner) in Arabidopsis. It was further proposed that WHY1 proteins bind to both ssDNA and RNA in Z. mays
chloroplasts, where it plays a role in intron splicing and WHY1 is associated with intron-containing RNA in
barley chloroplasts (Guan et al., 2018).
Among other proteins lacking OB-fold from A. thaliana, belongs truncated derivative of chloroplast RNAbinding
protein (AtCP31) with RRM motif, named AtSTEP1 (single- stranded telomere-binding protein 1)
(see Figure 5). AtSTEP protein localizes exclusively to the nucleus, specifically binds single-stranded G-rich
plant telomeric DNA sequences and inhibits telomerase-mediated telomere extension (Kwon & Chung,
2004).
A 36-kD protein identified by EMSA that specifically binds the G-strand of telomeric ssDNA from N. tabacum
(NtGTBP1) also contains a tandem pair of RRM motifs (Hirata et al., 2004). NtGTBP1 is not only associated
with telomeric sequences, as well as two additional GTBP paralogs (NtGTBP2 and NtGTBP3), but also
inhibits telomeric strand invasion in vitro and leaves of knockdown tobacco plants contained longer
telomeres with frequent formation of extrachromosomal T-circles (see bellow) (Lee & Kim, 2010). These
observations correspond to a previously detected protein from tobacco nuclei that binds G-rich telomeric
strands and reduces accessibility to telomerase or terminal transferase (Fulnečková & Fajkus, 2000).
Fulnečková and Fajkus detected a 40 kDa polypeptide by SDS-PAGE after cross-linking the complex formed
27
by extracts from tobacco leaf nuclei. In addition to the above described proteins, various telomeric ssDNA
binding proteins have also been reported in nuclear extracts from Glycine max, A. thaliana, O. sativa or
Vigna radiata (Ho Lee et al., 2000; Kim et al., 1998; Kwon & Chung, 2004; Zentgraf, 1995). However, precise
characterization of these proteins, identified by EMSA is mostly missing.
2 Interstitially located telomeric repeats
Telomeric repeats are not exclusively located at the physical ends of chromosomes, known as telomeres.
They are also present in multiple internal sites of chromosomes in many species, where they are referred
to as interstitial telomeric sequences (ITSs) (also named interstitial telomeric repeats (ITRs) or even very
short internally localized telomeric repeats (named telo-boxes). ITSs are relatively abundant in
subtelomeric, pericentromeric, and centromeric regions of most eukaryotic organisms, but can also be
found at various positions throughout chromosomes. Short internally localized telomeric repeats - called
telo-boxes - are composed of one to two telomeric DNA repeats. However, the defining of these groups is
not entirely precise and may vary in various scientific resources (Aksenova & Mirkin, 2019; Tremousaygue
et al., 1999; Uchida et al., 2002).
Most interstitial telomeric sequences studied in the human genome are short ITSs with lengths varying from
2–25 copies. They are present in all human chromosomes in subtelomeric regions as well as far from
chromosomal ends (Aksenova & Mirkin, 2019; Azzalin et al., 2001; Ruiz-Herrera et al., 2009). Recent studies
suggested function of ITSs in the stability of the genome and specifically at the role played by ITSs in
interacting with the nuclear envelope and shaping the genome’s 3D structure. A model of mammalian
chromosomal organization involves interaction of telomeres with ITSs and nuclear Lamins (Lamin A/C)
(Vicari et al., 2022; A. M. Wood et al., 2014). Long ITSs represent fragile parts of chromosomes, which are
prone to rearrangements and recombination’s (reviewed in Aksenova & Mirkin, 2019).
In A. thaliana, 8 regions of long ITSs were described on three chromosomes, ranging from 300 bp to 1.2 kb
(Uchida et al., 2002). Large blocks of telomeric repeats were found in pericentromeric regions of some
chromosomes in representatives of the Solanaceae family (He et al., 2013). Interestingly, the large blocks
of imperfect telomeric repeats were found as well in the proximity of centromeres of all Ballantinia
antipoda (Brassicaceae) chromosomes (Mandáková et al., 2010), however, in N. tabacum, no detectable
ITS regions were observed (Majerová et al., 2014) while telomere lengths ranged from 20 to 160 kb (Fajkus
et al., 1995; Kovařik et al., 1996).
Aside of long telomeric repeats the Arabidopsis genome contains very short interspersed segments (teloboxes)
of the telomeric sequence both mainly in interstitial positions. These short telo-boxes, exhibit a nonrandom
distribution. They were described in the promoters of genes coding for translation elongation
28
factor EF1a (Liboz et al., 1990), promoters of many ribosomal protein coding genes (Tremousaygue et al.,
1999) and promoters of genes involved in the biogenesis of the translation machinery (Gaspin et al., 2010).
We developed our own program, named Gene RegulatOry ELEMents (GOLEM) https://golem.ncbr.muni.cz/
(Nevosad et al., in preparation), to precisely localize the distribution of telo-boxes in the vicinity of the
Transcription Start Site (TSS) and Translation Start Site (ATG). Using this program, we found that most of
the telo-boxes in the Arabidopsis genome are located in very close proximity to the TSS. Additionally, we
discovered that genes with high transcription levels in plant leaves or during certain stages of gametophyte
development tend to have telo-boxes located predominantly 100 bp downstream of the TSS (Klodová et
al., in preparation).
2.1 Proteins associated with long interstitial telomeric repeats (ITSs)
The long extra-telomeric repeats can be recognised by the proteins that were previously characterised as
telomere-binding. In yeast several proteins were found to be associated with an artificial interstitial
telomeric tract or subtelomeric ITSs, e.g. Rap1, KU or Tbf1 proteins (reviewed in Aksenova & Mirkin, 2019).
Also in mammals Shelterin components occupy selective ITSs in the human genome, e.g. long artificial ITSs
showed enrichment in hTRF1 and hTRF2 proteins, as well as in the hTRF2-interacting partner, Apollo
exonuclease (Simonet et al., 2011; Ye et al., 2010). The region 2q14 on human chromosome, containing
stretches of degenerate TTAGGG repeats, binds hTRF1, hTRF2, hRAP1 and hTIN2 proteins (Fan et al., 2002).
These extra-telomere located Shelterin components thus participate in additional roles, e.g. gene activation
and repression, DNA replication, heterochromatin boundary-element formation, creation of hotspots for
meiotic recombination and chromatin opening (reviewed in Aksenova & Mirkin, 2019; Schrumpfová et al.,
2016a; see Supp. J).
Using Chromatin immunoprecipitation assay combined with Next generation sequencing (ChIP-Seq) we
revealed preferential association of AtTRB1 protein with long telomeric repeats, but not centromeric or 18S
rDNA sequences (Schrumpfová et al., 2016b; see Supp. I).
Recently we contributed to the findings that histone H1 selectively prevents accumulation of trimethylation
of lysine 27 of histone H3 (H3K27me3) at telomeres and long-ITSs by restricting DNA accessibility to AtTRB
proteins. It was proposed that H1 safeguards telomeres and long-ITSs against excessive H3K27me3
deposition and preserves their topological organization. Despite low protein sequence similarity of H1/H5
domain of AtTRBs and H1 (14%), AtTRBs display a typical H1/H5 domain, that may antagonize chromatin
incorporation of the H1/H5 of AtTRB and H1 proteins and might modulate PRC2 recruitment at ITSs (Teano
et al, 2023; see Suppl. S).
29
2.2 Proteins associated with short internally localized telomeric repeats
(telo-boxes)
In our study Schrumpfová et al., we were the first group to describe the association of AtTRB1 with teloboxes
in the plant genome (Schrumpfová et al., 2016b; see Supp. I). Moreover, we found out that AtTRB1
is bound to telo-boxes in promoters all over the genome. Almost 28 % of telo-box sequences located in the
5' UTR region of the genes coding proteins are covered by AtTRB1. As telo-box sequences are preferentially
located in the promoters of genes involved in the biogenesis of the translation machinery we proposed role
of AtTRB proteins in regulation of several genes, especially genes involved in biogenesis of the translational
machinery (Schrumpfová et al., 2016b; see Supp. I) (see Figure 6).
Our observation that AtTRB proteins are associated with telo-box sequences located outside the telomeres
was later proven by group of Franziska Turck. Zhou et. al. (2016) showed that AtTRB1 binds to thousands
of genomic sites containing telo-box or related cis-elements with a significant increase of sites and strength
of binding in the mutant plants for Like Heterochromatin Protein 1 (AtLHP1) (Zhou et al., 2016, 2018).
AtLHP1 is a plant Polycomb Repressive Complex 1 (PRC1) component that directly binds to H3K27me3
(Turck et al., 2007).
It was further shown that telo-boxes are part of the cis-regulatory elements that may relate to recruitment
of Polycomb repressive complex 2 (PRC2) which may regulate transcription of target genes through histone
Figure 6. An overview of telomeric and non-telomeric locations of TRB1 protein within A. thaliana nucleus,
where the telomeres are clustered in a rosette-like configuration, including nucleolus-associated telomeres.
Modified from Schrumpfová et al. (2016b, see Supp. I).
30
modifications. Zhou et al., 2018 have show direct interaction between AtTRB1,2,3 and CURLY LEAF (AtCLF)
and SWINGER (AtSWN) subunits of PRC2 complex. Recently we have described novel interaction between
AtTRBs and EMBRYONIC FLOWER 2 (AtEMF2) and VERNALIZATION 2 (AtVRN2) subunits of PRC2 complex
(Kusova et al., 2023; see Suppl. R).
There was also proposed role of AtTRB proteins in PEAT complex (PWWPs-EPCRs-ARIDs-TRBs). PEAT
complex may mediate histone deacetylation and heterochromatin condensation and thereby facilitate
Figure 7. Overview of the main Telomere repeat binding proteins (TRBs) functions (Kusova et al., 2023; see Suppl. R).
A) TRBs are associated with the physical ends of chromosomes (telomeres) via their Myb-like domain (Schrumpfová
et al. 2004; see Suppl. A; Mozgová et al. 2008; see Suppl. D; Dvořáčková et al. 2010; Schrumpfová et al. 2014;
see Suppl. H; Dreissig et al. 2017). TRBs interact with Arabidopsis homologs of the G-overhang binding protein
Protection of telomere 1a, b (POT1a, b) (Schrumpfová et al. 2008; see Suppl. D; Kusova et al., 2023; see Suppl.
R).
B) TRBs mediate interactions of Recombination UV B – like (RUVBL) proteins with the catalytic subunit of telomerase
(TERT) (Oguchi et al. 1999), and participate in telomerase biogenesis (Schrumpfová et al. 2014; see Suppl. H;
Schořová et al. 2019; see Suppl. N). TRBs are associated in the nucleus/nucleolus with POT1a (Schořová et al.
2019; see Suppl. N), and also with a plant orthologue of dyskerin, named CBF5 (Lermontova et al. 2007) that
binds the RNA subunit of telomerase (TR) (Fajkus et al. 2019; Song et al. 2021).
C) TRBs are associated with short telomeric sequences (telo-boxes) in the promoters of various genes in vivo, mainly
with translation machinery genes (Schrumpfová et al. 2016; see Suppl. I) . ORF, Open reading frame.
D) Telo-box motifs recruit Polycomb repressive complexes (PRC2) via interactions of PRC2 subunits with TRB (Zhou
et al. 2016; Zhou et al. 2018, this study) CLF, CURLY LEAF; SWN, SWINGER; EMF2, EMBRYONIC FLOWER 2; VRN2,
VERNALIZATION 2.
E) Histone H1 prevents the invasion of H3K27me3 and TRB1 over telomeres and long interstitial telomeric regions
(Teano et. al, 2023; see Suppl. S).
F) TRB proteins, as subunits of the PEAT (PWO-EPCR-ARID-TRB) complex, are involved in heterochromatin formation
and gene repression, but also have a locus‐specific activating role, possibly through the promotion of histone
acetylation (Tan et al. 2018; Tsuzuki and Wierzbicki 2018; Mikulski et al. 2019).
31
heterochromatin silencing. PEAT complex represses in heterochromatin regions the production of small
interfering RNAs (siRNAs) and DNA methylation in A. thaliana (Tan et al., 2018; Tsuzuki & Wierzbicki, 2018).
On the other hand, PEAT complex may possess a locus‐specific activating role, possibly through promoting
histone acetylation through two MYST-type histone acetyltransferases, AtHAM1 and AtHAM2. The
composition of PEAT indicates that it binds to specific regions of chromatin, probably telo-boxes via AtTRB
protein, and adds or removes acetyl groups from histones (Tan et al., 2018; Tsuzuki & Wierzbicki, 2018).
Additionally, AtTRB2 directly interacts with histone deacetylases, AtHDT4 and AtHDA6, in vitro and in vivo
(Lee & Cho, 2016). Deacetylase activity of AtHDT4 (W. K. Lee & Cho, 2016) and AtHDA6 (To et al., 2011)
against acetylation of lysine 27 of histone H3 (H3K27ac), could be important for subsequent methylations
of H3K27me3, that is among others target also for AtLHP1.
Recently was identified a PWWP Interactor of Polycombs 1 (PWO1) as a novel plant-specific factor
associated with chromatin and PRC (Hohenstatt et al., 2018). PWO1 associates physically with CRWN1, that
is one of the Lamin-like genes in Arabidopsis forming the plant-specific CROWDED NUCLEI (CRWN) family.
The authors speculated that PWO1 links H3K27me3-marked chromatin and the nuclear periphery in plants.
Interestingly, AtTRB1 protein was identified as putative interactors of PWO1 in Co-IP experiments coupled
with MS using the PWO1:PWO1-GFP Arabidopsis transgenic line (Mikulski et al., 2019).
Very recent it was demonstrated AtTRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger
H3K4me3 demethylation at some loci (Wang et al., 2023). JMJ14 is histone H3K4 demethylase regulating
flowering time in Arabidopsis (Lu et al., 2010). The attrb1/2/3 triple mutant and the atjmj14-1 mutant show
an increased level of H3K4me3 over AtTRB and JMJ14 binding sites, resulting in up-regulation of their target
genes (Wang et al., 2023).
Overall, we can hypothesise that although the TRBs were originally characterized as being associated with
long arrays of telomeric repeats (see Figure 7A, E), recent observations indicate broad engagement of TRB
proteins in various cellular pathways via recruiting various complexes to telo-boxes (see Figure 7C, D, F).
3 Orchestration of telomere homeostasis
Regulation of the telomere length homeostasis is very complex problem and is achieved via a balance
between telomere lengthening and erosion over successive cell divisions. Additionally, the processes of
telomere maintenance can be orchestrated by various telomere- and telomerase-associated proteins.
Mammalian telomeres are recognized not only with above mentioned proteins (Shelterin complex, POT
proteins, CST complex etc.) but telomere maintenance mechanisms appear to be affected by hundreds of
proteins, However, activities of these plant telomere, and telomerase-associated proteins, are only partly
understood. Some of these proteins were described in several broad studies, e.g. the hTERT associated
32
proteins (proteins were detected using TAP-MS) (Fu & Collins, 2007), telomeric factors associated with
human telomeric chromatin (Déjardin & Kingston, 2009) or protein network surrounding Shelterin subunits
- TRF1, TRF2, POT1 and TIN2 (Giannone et al., 2010; Grolimund et al., 2013; Nittis et al., 2010). The putative
partners associating with Shelterin proteins fell into functional categories such as DNA damage repair,
ubiquitination, chromosome cohesion, chromatin modification/remodelling, DNA replication, cell cycle and
transcription regulation, nucleotide metabolism, RNA processing and nuclear transport. These putative
protein-protein associations may participate in different biological processes at telomeres or, intriguingly,
outside telomeres.
3.1 Telomerase
As was already described above, telomeres cannot be fully replicated by enzymes that duplicate DNA, so
the telomere shortening occurs with each round of DNA replication. Critically shortened telomeres are no
longer able to protect chromosome ends from DNA repair and degradation activities and these phenomena
can lead to replicative senescence and finally cell death (Lundblad & Szostak, 1989).
Telomerase is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end
of telomeres and elongates the telomeres. In humans, telomerase activity was detected in all early
developmental stages. However, just after birth, telomerase activity in somatic cells is downregulated with
the exception of highly dividing cells (e.g. proliferating cells, T-lymphocytes, hair follicle bulbs) (reviewed in
Schrumpfová et al., 2019; see Supp. M).
However, somatic downregulation of telomerase is not conserved mechanism across species, and the
presence of telomerase activity has to be individually tested in each individual species, tissue or even in
different age-classes (Gomes et al., 2011; Haussmann et al., 2007; Seluanov et al., 2007). For example: most
rodent species show high telomerase activity in multiple somatic tissues (Mus musculus, Rattus norvegicus,
Hetrocephalus glaber etc.) and only beaver and capybara show nearly complete somatic repression of
telomerase activity, similar to humans (Seluanov et al., 2007). There was shown a clear tendency for species
smaller than 1 kg to have long telomeres and active telomerase, but species larger than 1 kg have tendency
to have short telomeres and repress telomerase (Gomes et al., 2011).
Also plant cells possess telomerase which is used for maintenance of their telomeres (Fajkus et al., 1996;
Heller et al., 1996). Active telomerase was detected in organs and tissues containing highly dividing
meristem cells such as seedlings, young and middle-age leaves, root tips, floral buds and flowers (Fajkus et
al., 1998; Fitzgerald et al., 1996). In terminally differentiated tissues (stems, mature leaves), telomerase
activity is suppressed (Jurečková et al., 2017; Ogrocká et al., 2012; Riha et al., 1998; reviewed in
Schrumpfová et al., 2019; see Supp. M ).
33
As was already mentioned above, the two core subunits of telomerase are telomerase reverse transcriptase
(TERT), which possesses catalytic activity, and telomerase RNA subunits (TR), which contain a template
region directing the synthesis of DNA repeats at the ends of chromosomes.
3.1.1 Telomerase reverse transcriptase (TERT)
Telomerase protein catalytic subunit (TERT) contains several conserved motifs and domains. The TERT
protein contains N-terminally located telomerase-specific motifs important for binding the telomerase RNA
subunit (TRBD), centrally located catalytic domains with the RT motifs essential for enzyme activity (RT) and
the C-terminal extension (CTE), which is highly conserved among vertebrates as well as among plants (see
Figure 8B). The motifs localized at the N-terminus are telomerase-specific (T2, CP, QFP and T) and are
A
B
Figure 8. Conservation of functional domains of two core telomerase subunits – TERT and TR (adopted
from Schrumpfová et al., 2020; see Supp. O) .
A) Models of secondary structures of human, Tetrahymena and Arabidopsis TRs suggest conservation
of several structural motives including pseudoknot in the vicinity of the template (t/PK domain) and
stem-loop region. In humans the stem-loop region contains the conserved 4/5 (CR4/5) region, the
H (AnAnnA) and ACA-boxes (H/ACA) domains and the Cajal body box (CAB-box) motif that serve as
binding sites for other protein components of the telomerase holoenzyme complex (dyskerin,
NOP10, NHP2, and GAR1). In Tetrahymena the stem-loop 4 (SL4) is directly bound by p65 protein.
To date, particular interactors and their binding sites have not been demonstrated directly in
Arabidopsis.
B) Domain arrangement of human (Animals), Tetrahymena (Ciliates) and Arabidopsis (Plants) TERTs.
The supergroup for each species is given. N-terminus: telomerase essential N-terminal (TEN)
domain and RNA‐binding domain (TRBD domain) are separated by Linker that contains a nucleus
localization‐like signal (NLS). The central RT domain: catalytical part of the enzyme that contains
seven evolutionary-conserved RT motifs (1, 2, A, B′, C, D and E motifs) and also telomerase specific
3 motif. C-terminus: C‐terminal extension (CTE) domain.
34
important for binding the telomerase RNA subunit. The centrally located RT motifs (1, 2 and A–E) are
essential for enzyme activity (reviewed in Sýkorová & Fajkus, 2009). The human telomerase complex
purified from human cell line overexpressing hTERT and hTR forms a dimeric structure (Sauerwald et al.,
2013). However, the presence of two catalytically active hTERT subunits has been a topic of controversy, as
indicated by other studies. Although, the biological significance of a dimeric telomerase RNP is unclear, it
could perhaps facilitate telomerase recruitment to telomeres by providing multiple binding sites, thus
increasing the affinity for its telomeric receptor (reviewed in Schmidt & Cech, 2015). We performed Y2H
screening of several AtTERT fragments. These fragments of AtTERT were previously designed to variously
cover N-terminal, TRBD, RT or CTE domains (Zachová et al., 2013). According our results, dimerization of
AtTERT in A. thaliana can be mediated by the RNA binding domain (TRBD) that is able to interact separately
with the N-terminal fragments and itself (Schrumpfová and Majerská et al., 2017, 2018; see Supp. K and
L).
In AtTERT, multiple nuclear localization signals (NLS), nuclear export signal (NES) or a mitochondrial
targeting signal were reported (Zachová et al., 2013). Due to the presence of these signals, AtTERT protein
and its domains localize mainly within the nucleus and the nucleolus of A. thaliana (Rossignol et al., 2007;
Zachová et al., 2013). Similarly in our study we localised AtTERT domains in the nucleolus. According to our
observation that AtTERT domains can be colocalized together with the AtRUVBL, AtTRB and AtCBF5 proteins
in the nucleolus, we hypothesised that AtTERT nucleolus localisation may be part of the telomerase
assembly pathway (Schořová et al., 2019; see Supp. N).
Apart from telomeric functions of telomerase in the nucleus, there was reported the presence of
telomerase in other subcellular compartments or telomerase putative involvement in signalling pathways,
transcriptional regulation and stress protection (reviewed in Majerská et al., 2011).
It has been proposed that human telomerase is subjected to posttranslational regulation such as
phosphorylation (Kang et al., 1999). Putative phosphorylation sites were also detected in the TERT
sequences from O. sativa or N. tabacum BY-2 cells but not in AtTERT from A. thaliana (Oguchi et al., 2004;
Yang et al., 2002). Moreover, in tobacco cell culture, phytohormones such as auxin or abscisic acid regulate
phosphorylation of telomerase protein, which is required for the generation of a functional telomerase
complex (Tamura et al., 1999; Yang et al., 2002).
3.1.2 Telomerase RNA (TR)
Compared to the conserved structure of the TERT subunit, TRs show high sequence diversity among more
distant organisms, as exemplified by the length differences of TRs in protozoa (159 nt in ciliate
Tetrahymena, 2200 nt in Plasmodium), zebrafish (317 nt), mouse (397 nt), human (451 nt) and budding
35
yeasts (1160 nt). Even within yeasts, the homology among TRs is rather low and their lengths range from
930 nt to more than 2000 nt (see Figure 8B) (reviewed in Schrumpfová et al., 2019; see Supp. M; Webb &
Zakian, 2016; Zhang et al., 2011).
In A. thaliana, there were earlier reported two AtTR candidates, named AtTER1 and AtTER2 (CifuentesRojas
et al., 2011, 2012). It was shown that AtTER1 is able to provide a templating function in telomerase
reconstitution experiments in vitro but direct evidence of its in vivo function were missing (Fajkus et al.,
2019). However, later it was found out that neither AtTER1 nor AtTER2 serve as RNA subunits of active
telomerase and the article Cifuentes-Rojas et 2011 was retracted.
It seems that the natural templating subunit of telomerase in Arabidopsis, as well in other land plants, are
TRs identified by our group (Fajkus et al., 2019). My colleagues used unusually large length of the Allium
telomere repeat unit (12 nt) and identified the candidate TRs in transcriptomes. Based on the Allium TRs,
they consequently identified TRs orthologs in the other land plants. AtTR has been characterized earlier as
a hypoxic stress-responsive long non-coding RNA (lncRNA) transcribed by RNA polymerase III (Pol III) in A.
thaliana (AtR8) and related Brassicaceae species. All AtTR identified homologs in other plant species
possess the conserved Pol III type 3 promotor with specific localization of USE and TATA boxes and poly-U
terminator elements (Wu et al., 2012, 2019). It seems that land plant TR gene is highly conserved in contrary
to the very divergent TR genes found in animal, yeast or protozoan models (Fajkus et al., 2019).
Models of secondary structures of human, Tetrahymena and Arabidopsis TRs suggest conservation of
several structural motives. The most prominent are pseudoknot in the vicinity of the template (t/PK
domain), stem-loop regions and template boundary element (TBE). In humans the stem-loop region
contains the conserved 4/5 (CR4/5) region, the H (AnAnnA) and ACA-boxes (H/ACA) domains and the Cajal
body box (CAB-box) motif that serve as binding sites for other protein components of the telomerase
holoenzyme complex. The TBE defines the end of the sequence recognized by TERT as a template (reviewed
in Schrumpfová et al., 2020; see Supp. O).
Evolution of both subunits of telomerase, TERT and TR, were discussed at The Czech Plant Nucleus
Workshop 2021. The results, together with other results focused on maintenance of the chromosome ends,
were summarized in the Conference report named The Czech Plant Nucleus Workshop 2021 (Pecinka et
al., 2022; see Supp. P).
3.1.3 Telomerase-associated proteins
Besides these two core subunits, TERT and TR, the telomerase complex comprises several other accessory
proteins with diverse roles in telomerase assembly, trafficking, localization, recruitment to telomeres or the
processivity of telomere synthesis (Chan et al., 2017; Nguyen et al., 2018) (see Figure 9 and Figure 10).
36
In humans the active telomerase is associated with Hsp90 and p23 chaperones as well as with TR associated
with conserved scaffold proteins of box H/ACA small nucleolar RNAs (dyskerin, Non-histone protein 2
(NHP2), Nucleolar protein 10 (NOP10), Glycine arginine rich 1 (GAR1)). The telomerase RNP is probably
retained into the nucleoli through the interaction between TERT and nucleolin. Assembly of TR and TERT
into catalytically active telomerase is aided by Pontin (RUVBL1) and Reptin (RUVBL2) (reviewed in Schořová
et al., 2019; see Supp. N) (see Figure 9A).
In plants, a limited number of proteins that directly interact with TERT were described. Using Tandem
Affinity Purification coupled to Mass Spectrometry (TAP-MS) we co-purified and identified several putative
AtTERT interaction partners (Schrumpfová and Majerská et al., 2017, 2018; see Supp. K and L). To confirm
putative protein-protein interactions between AtTERT and proteins of interest, we used Y2H, Co-IP and BiFC
systems. As some of the proteins of interest showed indirect interaction with AtTERT, to achieve
reproducible results we used many modifications, improvements and mutual combination of Y2H, BiFC and
Co-IP. Our optimized BiFC protocol in A. thaliana protoplasts provided us a robust tool to observe direct or
even indirect interactions of (not only) telomere- and telomerase-associated proteins and to distinguish
nucleus, nucleolus or cytoplasmic localization of these interactions. Our modification of Co-IP technique
(Co-Immunoprecipitation with Three Proteins of Interest) allowed detection not only of two proteins of
interest, as is common, but also detection of trimeric complexes, where two proteins of interest interact
indirectly via a protein sandwiched in between them and mediating the interaction. Our improvements and
modifications of these protein-protein interaction techniques were described in the book chapter named
Figure 9. Comparative model of telomerase in human and Arabidopsis localised in the nucleolus.
A) Human active telomerase is associated with Hsp90 and p23 chaperones as well as with TR associated
with conserved scaffold proteins of box H/ACA small nucleolar RNAs (dyskerin, NHP2, NOP10, GAR1).
The telomerase RNP is retained into the nucleoli through the interaction between TERT and nucleolin.
Assembly of TR and TERT into catalytically active telomerase is aided by Pontin (RUVBL1) and Reptin
(RUVBL2).
B) TERT colocalize with RUVBL proteins, bridged by telomeric TRB proteins, in the nucleolus as well as the
interaction of telomeric protein POT1a with Arabidopsis CBF5 (dyskerin). CBF5 together with GAR1,
NOP10, NHP2, but in contrast with human cells also NAF1, were localized in the plant nucleolus,
however entire association with active telomerase holoenzyme has to be elucidated. Modified from
Schořová et al., 2019; see Supp. N.
A B
37
'Analysis of direct and indirect protein-protein interactions of telomere-associated proteins' (Methods in
Molecular Biology, The Nucleus, Book Series, Springer protocols (Schořová et al., 2020).
In our laboratory, we have demonstrated that AtTRB proteins, physically interact with N-terminal domains
of AtTERT (see Figure 3B and 9B). We also suggested a mediated interaction between Telomeric Repeat
Binding Protein 1 (AtTRP1) protein and AtTERT (Schrumpfová et al., 2014; see Supp. H). Rossignol et al.
observed that the N-terminal part of AtTERT exclusively interacts with AtPOT1a but not AtPOT1b (Rossignol
et al., 2007). As well various other proteins from A. thaliana were shown to be associated with AtTERT:
AtRRM (RNA recognition motif (RRM)), AtARM (armadillo/β-catenin-like repeat-containing protein),
AtCHR19 (chromatin remodeling protein), AtMT2A (Metallothionein-like), AtG2p (RNA-binding), AtPURα1
(Pur-alpha 1), AtNUC-L1 (Nucleolin like 1) or Importin4 (ImpA4) (Dokládal et al., 2015, 2018; Fulnečková et
al., 2022; Pontvianne et al., 2010, 2016; reviewed in Schrumpfová et al., 2019; see Supp. M). Some of these
AtTERT partners that we co-purified with N-terminal fragments of AtTERT are possibly involved also in nontelomeric
functions of telomerase, e.g. the human homologue of the AtPURα1 protein, named PURα, has
been implicated in the control of gene transcription (Safak et al., 1999) and DNA replication (Bergemann &
Johnson, 1992).
Among proteins co-purified with AtTERT fragments using TAP-MS we identified also AtRUVBL1 and
AtRUVBL2a proteins (plant homologues of human Pontin and Reptin) (Schrumpfová and Majerská et al.,
2017, 2018; see Supp. K and L). We closely characterised AtRUVBL1-AtTERT and AtRUVBL2a-AtTERT
interactions in the plant cell and found out that, against mammalian counterparts, interaction between
AtRUVBLs and AtTERT proteins in A. thaliana is not direct and is more likely mediated by one of the AtTRB
proteins. Our data show that AtRUVBLs, together with AtTRBs protein, colocalize with N-terminal part of
AtTERT subunit of plant telomerase in the plant nucleolus. It seems that AtRUVBLs are recruited into the
AtTERT complex through an interaction with AtTRBs protein, which mediate interaction with both proteins:
AtTERT and also with AtRUVBLs. Our data indicate the presence of AtTERT-AtTRB-AtRUVBL complex in the
plant nucleolus (Schořová et al., 2019; see Supp. N).
In humans, proper catalysis, accumulation, 3' end processing, and localization of hTR are necessary for the
creation of functional mature hTR, which provides the template for the synthesis of telomere DNA repeats.
Human TR is associated with dyskerin, NOP10, NHP2 and GAR1, that displaces previously bound Nuclear
assembly factor 1 (hNAF1) in the hTR RNP.
In A. thaliana, expression of putative AtGAR1, AtNOP10, AtNHP2 genes encoding protein components of
the H/ACA box snoRNP complex correlate with that of AtCBF5 - plant homologue of dyskerin (Lermontova
et al., 2007). AtCBF5 has been identified as a component of the enzymatically active A. thaliana telomerase
RNP (Kannan et al., 2008; Lermontova et al., 2007). Scaffold proteins AtCBF5, AtGAR1, AtNOP10, AtNHP2,
38
but in contrast to human cells also AtNAF1, were localized into the plant nucleolus (Lermontova et al., 2007;
Pendle et al., 2005). The association of TRs with dyskerin appears to be conserved between plant and animal
kingdoms as telomerase activity was immunoprecipitated with the anti-plant dyskerin antibody from
protein extract from Allium cepa seedlings (Fajkus et al., 2019). Moreover, despite the absence of a
canonical H/ACA binding motif within AtTR, dyskerin binds AtTR with high affinity and specificity in vitro via
a plant specific three-way junction (Song et al., 2021). However, it has not yet been elucidated whether
plant homologues of human GAR1, NOP10, NHP2, or NAF1 are also part of the active holoenzyme of
telomerase in plants.
Comparative overview of human and plant homologues of proteins associated either with the telomerase
catalytic subunit TERT or with the RNA component of telomerase is reviewed in Schrumpfová et al., 2019
(see Supp. M).
3.1.4 Telomerase assembly
Proper assembly of TERT with TR into active and functional complex is stepwise regulated procedure
governed also by multiple associated proteins (reviewed in Shepelev et al., 2023; reviewed in Schrumpfova
et al., 2020; see Suppl. O). Telomerase and its chromosome end substrate have very low abundance (∼250
telomerases/184 telomeres in a human cell in late S phase) thus, it is perhaps not surprising that the
telomerase enzyme is recruited to telomeres rather than simply encountering them by diffusion (Xi & Cech,
2014).
Transcription of the human TERT gene by RNA polymerase II (RNA Pol II) is regulated by several activators
and repressors acting at the promoter level (e.g., c-MYC, Nuclear Factor κB (NF- κB), Signal Transducer and
Activator of Transcription 3 (hSTAT3), Specificity Protein 1/3 (SP1/3). Histone modification H3K27me3 often
silences hTERT, however the mutated hTERT allele is marked by the active histone marks H3K4me2,
H3K4me3 and H3K9ac. All Pol II transcripts undergo processing events that are essential for their function.
The hTERT pre-mRNA with a 5’ mono-methylguanosine (MMG) cap and poly(A) 3’ tail can be spliced into
full-length (FL) or multiple alternative isoforms (Alternative splicing) that are catalytically inactive or even
inhibit telomerase activity. The binding of heat shock protein 90 (hHsp90) with its co-chaperone (p23) in
the cytoplasm enables hTERT phosphorylation (P). hTERT is further imported back to the nucleus by
Importin α or β1 (hImp) via nuclear pores (n.p.), while the export of hTERT may be mediated by the
chromosome region maintenance 1 protein homolog (hCRM1, also known as exportin-1). The ubiquitin
(Ubq)-proteasomal degradation of hTERT is driven by E3 ubiquitin-protein ligase makorin-1 (MKRN1), heat
shock protein 70 (Hsp70) and carboxyl-terminus of Hsp70 Interacting Protein (CHIP) (see Figure 10A, for
references see Schrumpfova et al., 2020; see Suppl. O).
39
Histone modifications H3K4me2/3 or H3K9Ac help to regulate read-through of the human hTR gene by RNA
Pol II in telomerase-positive cell lines. SHQ1 chaperone and RUVBLs facilitate the assembly of nascent RNA
with RNA scaffold proteins (dyskerin, hNOP10, hNHP2, and hNAF1). Mature hTR is capped with a trimethylguanosine
(TMG) cap at the 5’ end, polyadenylated at the 3’ end and co-transcriptionally associated
with scaffold proteins. The hTR variants with shorter or longer 3’ ends, or those associated with variant
proteins, may lead to the degradation of hTR. hNAF1 is replaced by hGAR1 before the hTR
ribonucleoprotein complex reaches the nucleolus (see Figure 10B).
RUVBLs (Pontin and Reptin) enable telomerase assembly and allow hTERT recruitment to the nucleolus to
form a mature telomerase complex while bound by nucleolin (hNCL). PIN2/TERF1-interacting telomerase
inhibitor 1 (hPINX1), together with nucleophosmin (hNPM) and microspherule protein 2 (hMCRS2), regulate
hTERT availability in a cell cycle-dependent manner. Telomere Cajal body protein 1 (hTCAB1, also known as
hWRAP53) recognizes the Cajal body box (CAB-box) of the hTR in the mature telomerase complex and
recruits it to the Cajal bodies (CBs). In CBs, hTR interacts with local proteins such as coilin while survival
motor neuron protein (hSMN) binds hTERT (see Figure 10C and Figure 11A).
In the S-phase, the CBs colocalize with telomeres and facilitate the recruitment of the mature telomerase
complex to the telomeres via interaction with hTPP1 protein, which is one of the subunits of a protein
complex localized at telomeres, termed as Shelterin. The presence of Shelterin proteins (hTRF1/2, hPOT1,
hTIN2, hRAP1 and TPP1) helps distinguish chromosomal ends (telomeres) from DNA breaks see Figure 10D).
Despite the fact that the entire TERT subunit is highly conserved across the phylogenetic tree and shows
significant sequence homology between humans and plants (as reviewed in Schrumpfova et al., 2020; see
Suppl. O), the assembly pathway of plant telomerase holoenzyme is not fully understood. However, our
research has helped to partially elucidate the proteins associated with plant telomerase and their possible
involvement in telomerase assembly.
In humans, the production of hTERT is highly regulated at the transcriptional levels and also posttranscriptional
levels, whereas the hTR transcript is constitutively produced (Gladych et al., 2011). However,
in plants, the transcription of both telomerase subunits (AtTERT and AtTR) is regulated during the plant
development, as both subunits show high transcription in seedlings and young leaves, but diminished
transcription in fully maturated leaves (Jurečková et al., 2017; Ogrocká et al., 2012; Riha et al., 1998; Fajkus
et al., 2019; reviewed in Schrumpfova et al., 2020; see Suppl. O).
Plant TERT gene has a weak promoter. Fojtova et al. identified region 271 bp upstream of ATG as an putative
’minimal promoter’ able to drive sufficient transcription of the telomerase protein subunit gene, resulting
in normal telomerase function (Fojtová et al., 2011). In Crhak et al. it was proposed that unknown factors
necessary for tissue-specific expression of telomerase activity and restoration of telomerase function in the
40
maintenance of telomere are needed (Crhák et al., 2019). Additionally, AtTERT gene might be regulated by
regulatory element at the 5′ end, e.g. within the intron 1, that has function at the level of transcription,
while it is not involved in tissue-specific regulation (Fojtová et al., 2011).
Figure 10. Regulation of human telomerase biogenesis (for description see text) (Schrumpfova et al., 2020;
see Suppl. O).
A) Transcription of the hTERT is regulated by several activators and repressors acting at the promoter
level.
B) Histone modifications help to regulate read-through of the human telomerase RNA (hTR) gene. Mature
hTR is capped and recognised by several associated proteins.
C) RuvBLs (pontin and reptin) enable telomerase assembly and allow hTERT recruitment to the nucleolus
to form a mature telomerase complex while bound by several other proteins.
D) In the S-phase, the CBs colocalize with telomeres and facilitate the recruitment of the mature
telomerase complex to the telomeres.
A B
C
D
41
In A. thaliana, mutation in the attac1 (Telomerase activator 1) gene led to the induction of telomerase in
fully differentiated leaves without stimulating progression through the cell cycle (Ren et al., 2004).
However, AtTAC1 protein does not directly bind the AtTERT promoter and rather regulates telomerase
activity through regulation of the AtBT2 (protein with BTB,TAZ and calmodulin binding domains) gene
expression (Ren et al., 2007).
Alternatively spliced variants of TERT transcripts were also described in many plant species, e.g. A. thaliana
(AtTERT), Zea mays (ZmTERT), Oryza sativa (OsTERT), Iris tectorum and tobacco (Rossignol et al., 2007;
Sýkorová & Fajkus, 2009).
We have characterised RUVBL homologues in A. thaliana and outlined plausible conservation of the
telomerase trafficking pathway in the land plants. We showed, that RUVBL1 and RUVBL2 proteins from A.
thaliana are able to form either homo- or heteromers as well as their homologues in diverse organisms,
although they preferably form mutual heteromers (Schrumpfová and Majerská et al., 2017; Schořová et
al., 2019; see Supp. K and N). Our experiments with plant RUVBL proteins showed that depletion of
AtRUVBL1 and especially of AtRUVBL2a protein, reduced telomerase activity in plants with T-DNA insertion
in AtRUVBL1 or AtRUVBL2a genes, respectively. We did not observe significant changes in transcripts of
AtTERT gene in AtRUVBL1 heterozygous mutant plants and very slight, though significant, increase, in
transcripts of AtTERT gene in AtRUVBL2 heterozygous mutant plant lines (Schořová et al., 2019; see Supp.
N). Similarly to our results, both human RUVBL proteins, hRUVBL1 and hRUVBL2, regulate hTERT both on
the gene and protein levels, only hRUVBL2 depletion inhibits hTERT promoter activity through the
regulation of c-Myc (Mao & Houry, 2017; Venteicher et al., 2008).
It was already mentioned that our data indicate AtRUVBL1 recruitment into the AtTERT complex through
an interaction with AtTRB3 protein (see above). Formation of AtRUVBL1-AtRUVBL2 heteromer is distributed
in whole nucleus but the localization of protein complex AtRUVBL1-AtTRB3-AtTERT occurs in nucleolus. We
showed, that depletion of AtRUVBL1 and especially of AtRUVBL2 proteins causes reduced telomerase
activity and suggests conserved role of AtRUVBL proteins in maturation of functional telomerase complex
across the mammals and also plant species (see Figure 11B) (Schořová et al., 2019; see Supp. N).
Very recently, we have shown that AtRUVBL1 and AtRUVBL2A play roles in reproductive development. We
showed that mutant plants produce embryo sacs with abnormal structure or with various numbers of nuclei
and pollen grains of heterozygous mutant plants exhibit reduced viability and reduced pollen tube growth
in vitro. The activity of the AtRUVBL1 and AtRUVBL2A promoters was observed in the embryo sac, pollen
grains, and tapetum cells, and for AtRUVBL2A also in developing ovules. It seems that RUVBL proteins are
essential for the proper development of both male and particularly female gametophytes in Arabidopsis
(Tomaštíková et al., 2023, see Supp. Q).
42
Plant homologue of dyskerin, named AtCBF5 (or AtNAP57), is localized within nucleoli and Cajal bodies
(Lermontova et al., 2007) and associates with enzymatically active telomerase RNP particles in an RNAdependent
fashion (Kannan et al., 2008). We observed indirect interaction of AtTRBs with AtCBF5 in plant
nucleus. Moreover, we detected that the AtCBF5 is interacting with AtPOT1a not only in Y2H and Co-IP as
was shown in Kannan et al. (2008) but we also showed nucleolar and partly cytoplasmic localization using
BiFC assay. In addition, we observed weak interaction between AtPOT1a-AtRUVBL1 proteins in Y2H and CoFigure
11. Comparative model of telomerase assembly in human and Arabidopsis (adopted from Schořová
et al., 2019; see Supp. N)
A) Human TR, located in the nucleolus, is bound by dyskerin, NHP2, NOP10 and GAR1 and human TERT
associates with the chaperones Hsp90 and p23. Assembly of TR and TERT into catalytically active
telomerase is aided by RUVBL1 (Pontin) and RUVBL2 (Reptin)) AAA+
ATPases. Telomerase is recruited to
Cajal bodies by its interaction with TCAB1. The CBs will colocalize with telomeres and telomerase is
recruited to telomeres by the interaction with the shelterin component TPP1.
B) Arabidopsis CBF5, GAR1, NOP10, NHP2 and also NAF1, were localized into the plant nucleolus. TERT
interaction with RUVBL proteins is bridged by telomeric TRBs. Arabidopsis telomeres cluster at the
periphery of the nucleolus which is mediated by the presence of nucleolin. Recruitment of the mature
telomerase complex to telomeres with or without commitment of Cajal bodies in Arabidopsis needs
further investigation. Proteins already proven as associated with Cajal bodies are highlighted in Cajal
bodies in color. Proteins that have not yet been experimentally proven as Cajal bodies associated are
marked with black and white.
A
B
43
IP assays (Schořová et al., 2019; see Supp. N). Additionally, Arabidopsis GAR1, NOP10, NHP2 and also NAF1
homologues, were localized into the plant nucleolus (Lermontova et al., 2007; Pendle et al., 2005).
The telomerase trafficking pathway during the telomerase maturation, which comprises movement of
maturating telomerase complex through nucleolus to CBs and finally to the telomeres, may be conserved
also in land plants. Dvořáčkova et al. observed that AtTRBs are located not only in the nucleolus but also in
nuclear bodies of different size, some of which might be CBs adjacent to the nucleolus (visualized by a
marker protein Coilin) (Dvořáčková et al., 2010b). Furthermore, plant dyskerin, AtCBF5, indirectly interacts
with AtTRB proteins not only in the plant nucleolus but also in other nuclear bodies that might be CBs
(Schořová et al., 2019; see Supp. N).
Notably, not all the organisms (e.g., budding yeast and ciliates) rely on the CBs trafficking since telomerase
RNAs from these species do not have H/ACA or CAB box motifs, e.g., in Saccharomyces cerevisiae,
telomerase assembly requires export of the TR out of the nucleus and is regulated in a cell cycle-dependent
manner. RNA component of S. cerevisiae telomerase, named TLC1, is assembled with Sm proteins in
nucleoplasm, 5′ TMG cap is added to the TLC1 in nucleoli, TLC1 is assembled in cytoplasm with holoenzyme
proteins and consequently telomerase holoenzyme is transported again in the nucleoplasm, where
telomerase can be recruited to telomeres (reviewed in Shepelev et al., 2023; R. A. Wu et al., 2017).
As was already mentioned above, we used Y2H, BiFC and Co-IP techniques to detect and characterise
protein-protein interactions of the telomere and telomerase associated proteins. The Co-IP technique is
based on precipitation a of intact protein complexes formed by proteins usually produced in in vitro
transcription/translation systems and using an antibody that specifically binds to the particular protein
antigen. Interestingly, we chose mammalian Reticulocyte lysate (RRL) instead of plant Wheat germ (WG)
system to express plant AtTRB proteins in vitro (Schořová et al., 2019; Schrumpfová et al., 2014; see Supp.
N and H). Wheat germ extract is isolated from embryos of dry wheat seeds while Rabbit reticulocyte lysate
is prepared from anaemic rabbits that are stimulated for production of immature red blood cells
responsible for the synthesis of haemoglobin that have already lost their nuclei (reticulocytes). When we
used for protein expression WG transcription/translation system (TnT Coupled Wheat Germ Extract System,
Promega) instead of RRL (TNT T7 Quick Coupled Transcription/Translation System (Promega)), AtTRB
proteins were successfully expressed but revealed no interactions, including well established positive
controls (Schořová et al., 2020: Methods in Molecular Biology - The Nucleus, Book Series, Springer). This
observation might relate to the HSP90 chaperone. HSP90 chaperone is present in WGE extract but is a
deficient in its function (Antonsson et al., 1995). The addition of purified human or yeast co-chaperone p23
to WGE fully reconstitutes HSP90 chaperone activity (Dittmar et al., 1997; Hutchison et al., 1995). Zhang et
al. showed that p23-like proteins are present in plants, they are capable of binding HSP90, but unlike human
44
p23, the plant p23-like proteins do not reconstitute HSP90 chaperone activity (Zhang et al., 2010). As human
chaperone HSP90 and its co-chaperone p23 participate in the folding of a number of cell regulatory
proteins, stably associate with hTERT and remain associated also with active telomerase (Forsythe et al.,
2001; Holt et al., 1999) it will be interesting to learn whether and how are these chaperones in the plant
cells involved in telomere- and telomerase-associated proteins folding and telomerase assembly.
Generally, assembly of functional AtTR RNP, as well as the assembly of mammalian hTR RNP, is certainly a
multistep process that may include AtTR, AtCBF5, AtTRBs, AtRUVBLs, AtPOT1a and many other factors,
whose presence/participation/mutual interactions will be the subjects of our future research. Dynamics
and complexity of mutual interactions can be demonstrated by the fact that we detect the interacting
complex of AtCBF5-AtPOT1a in the nucleolus or in the cytoplasmic and nuclear foci, while AtCBF5-AtTRBs
interactions are localized entirely to the nucleoli and additional nuclear bodies (Schořová et al., 2019; see
Supp. N). Our first model of plant telomerase holoenzyme assembly was achieved by editors of The Plant
Journal, who wrote a special article named “RESEARCH HIGHLIGHT: The journey to the end of the
chromosome: delivering active telomerase to telomeres in plants.”(Sweetlove & Gutierrez, 2019; see Supp.
N).
As we already mentioned - regulation of telomerase assembly, maturation and trafficking is a very complex
process, involving a wide range of co-factors. Moreover, these co-factors are not involved exclusively into
the telomerase assembly but they also participate in various other biochemical pathways. Although in
mammals the telomerase assembly pathway has been partially described, our understanding of telomerase
assembly in plants is still far to be perfect and is still ongoing process.
3.2 Telomere maintenance proteins
3.2.1 Mammalian telomere maintenance proteins
The mammalian Shelterin complex is involved in the repression of the primary signal transducers of DNA
breakage, two phosphatidylinositol-3-kinase-like (PI3K) protein kinases: ataxia telangiectasia mutated
(ATM) and ATM- and RAD3-related (ATR) kinases. Mice TRF2 acts mainly to protect telomeres against ATM
activation (Celli & de Lange, 2005) and POT1 is principally involved in repression of the ATR pathway (Denchi
& de Lange, 2007; Guo et al., 2007) (see Figure 3A). In mammals as well as in other organisms, DSBs activate
ATM kinase in a manner dependent on the meiotic recombination 11 (MRE11), DNA repair protein 50
(RAD50) and Nijmegen breakage syndrome 1 (NBS1) named MRN complex. The MRN complex has been
found to associate with telomeres and contributes to their maintenance (reviewed in Lamarche et al.,
2010). Other proteins involved in DDR machinery are Ku proteins. Human Ku70 protein directly interacts
45
not only with the Shelterin proteins hTRF1, hTRF2 and hRAP1, but also with telomerase subunits hTERT and
hTR (reviewed in Fell & Schild-Poulter, 2015; Schrumpfová et al., 2019; see Supp. M).
Aside DNA damage factors, the mammalian telomere proteome comprises additional telomere-associated
proteins, e.g. regulator of telomere elongation helicase 1 (RTEL1) (see Figure 3A) and many other proteins
interactors (reviewed in Ghisays et al., 2021; Lazzerini-Denchi & Sfeir, 2016). RTEL1 helicase connects
telomeric loops and circles with DNA recombination and telomere replication. RTEL1 play role in dissolving
higher-order structures referred as the telomeric loops (T-loops). These lariat structures are composed of
each chromosome terminus being folded back upon itself, which enables the G-rich DNA overhang to
invade and base-pair with the complementary strand (Griffith et al., 1999). The 3′ G-strand extension that
invades the duplex telomeric repeats forms a D-loop (displacement loop, ~150 bp) (Greider, 1999). In
addition to its role in T-loop stability, mouse RTEL1 can dissolve G4-DNA structures (quadruplexes), which
are predicted to form in the G-rich telomeric regions and might block replication fork progression and the
extension of telomeres by telomerase.
3.2.2 Plant telomere maintenance proteins
In A. thaliana short telomeres in telomerase-deficient plants activate both the ATM and ATR, whereas
absence of members of the plant CST complex initiates only AtATR-dependent, but not AtATM-dependent
DNA damage response (see Figure 3B) (Amiard et al., 2011; Boltz et al., 2012). In contrast to a massive loss
of telomeric DNA that was observed in human cells (Wang et al., 2009), mutations in Ku70 and Ku80 in the
dicotyledonous A. thaliana, as well as in the monocotyledonous O. sativa, resulted in longer telomeres,
suggesting their conserved role in the negative regulation of plant telomerase (Bundock et al., 2002; Gallego
et al., 2003; Hong et al., 2010; Riha et al., 2002).
A. thaliana RTEL1 homolog suppresses HR and is involved in processing DNA replication intermediates and
interstrand and intrastrand DNA cross-links. Deficiency of the AtRTEL1 triggers a SOG1-dependent
replication checkpoint in response to DNA crosslinks. AtSOG1 targets numerous genes required for repair
by HR, including AtRAD51 (Ogita et al., 2018). Similarly to the situation in mammals, the Arabidopsis RTEL1
contributes to telomere homeostasis (Recker et al., 2014).
In contrast with the effects of the loss of function of HR factors, the loss of key factors of NHEJ (MRE11,
RAD50, NBS1, KU70 and LIG4) has little or no impact on growth phenotype, overall DSB repair and telomere
maintenance in P. patens, while a clear telomere phenotype can be seen in the corresponding A. thaliana
mutants. Therefore, it is not possible to simply generalize the results obtained in only one of these model
plants as applying to DNA repair and telomere biology in all plants (Fojtová et al., 2015; Goffová et al., 2019;
Holá et al., 2013).
46
3.2.3 HMG proteins
Proteins classified within the High Mobility Group (HMG) family have been observed to exhibit the capacity
to impact the maintenance of telomeres. These HMG proteins constitute a diverse cohort of non-histone
proteins that are comparatively small in size, and are relatively abundant within the chromatin of eukaryotic
organisms. There are three structurally distinct classes of HMG proteins: the HMG-nucleosome binding
subfamily (HMGN), the HMG-AT-hook subfamily (HMGA) and the HMG-box subfamily (HMGB) (reviewed in
Reeves, 2015).
In mammals, the HMGA subfamily is composed of two proteins: HMGA1 and HMGA2. Both proteins are
expressed in embryonic tissues and embryonic stem cells, are absent in most somatic adult cells and,
interestingly, are highly abundant in tumorigenic cells. HMGA proteins are believed to play a role in
transcription by promoting the joining of regulatory elements and were shown to have a clear role in
development (Ozturk et al., 2014). There was indicated a role for HMGA1 in TERRA (TElomeric Repeatcontaining
RNA) localization to the telomeres (Scheibe et al., 2013). HMGA2 positively regulates the
transcription of the catalytic subunit of the telomerase in human HeLa cells (Li et al., 2011) and increases
telomere stability in cancer cells (Natarajan et al., 2016).
In Arabidopsis, several uncharacterized HMGA proteins are present, including AtGH1-HMGA1 (reviewed in
Kotliński et al., 2017). Chabonnel et al. performed a label-free quantitative proteomics analysis of a
telomere pull-down with either the Arabidopsis TTTAGGG repeat sequence or a shuffled DNA control. They
identified several candidate proteins, including AtTRB1, AtTRB3 and AtGH1-HMGA1 enriched with the
telomeric bait. AtGH1-HMGA1 can be present at some DNA extremities but is not associated exclusively
with the telomeres. AtGH1-HMGA1 is required for efficient DNA damage repair and telomere integrity in
Arabidopsis. AtGH1-HMGA1 mutants exhibit developmental and growth defects, accompanied by ploidy
defects, increased telomere dysfunction-induced foci, mitotic anaphase bridges and degraded telomeres.
It seems that GH1-HMGA1 in A. thaliana is involved directly in the repair process by allowing the completion
of homologous recombination (Charbonnel et al., 2018). Interestingly, AtTRB proteins, associated with
telomeres, possess centrally located H1/H5 domain (Schrumpfová et al., 2004; see Supp. A) (see Figure 5)
that are evolutionary related to the H1/H5 domain located at the N-terminus of the AtGH1-HMGBA proteins
in A. thaliana (Kotliński et al., 2017).
In mammals, it has been observed that additional members of the HMG family originating from the HMGB
subfamily, particularly HMGB1, are capable of regulating the activity of telomerase. However, this effect
was not due to changes in expression of either of the telomerase subunits, but rather through the
involvement of the HMGB1 in assembly of telomerase nucleoprotein complex. Accordingly, HMGB1
47
physically interacts with both mouse TERT and TR, as well as with active telomerase complex in vitro
(Polanská et al., 2012).
In contrast to mammalian HMGB proteins, which contain two HMG-box domains, the typical plant HMGBtype
proteins have a single HMG-box domain, which is flanked by a basic N-terminal domain and an acidic
C-terminal domain. The HMG-box domains of the various plant HMGB proteins are relatively conserved,
but compared to the mammalian homologues the basic and acidic flanking regions vary considerably in
length and sequence (Pedersen & Grasser, 2010). According to results of in vitro studies, plant HMGB
proteins bind linear DNA in non-sequence-specifical manner with moderate affinity. They also recognise
specifically certain DNA structures such as minicircles and four-way junctions and they severely bend linear
DNA upon binding. In Arabidopsis is complicated by the existence of seven proteins that contain HMG-box
domain flanked by a basic and acidic domain and thus can be classified as HMGB-type proteins (Lildballe et
al., 2008). Most of the AtHMGB proteins were shown to be involved in various stress-response pathways
(Roy et al., 2016).
In our study Schrumpfová et al., 2011, T-DNA insertion lines with athmgb1 gene knockout were
characterised. AtHMGB1 protein appears as a typical member of the plant HMGB-type proteins in A.
thaliana and could be regarded as the ortholog of mammalian HMGB1, but not necessarily performing the
equivalent functions. Similarly to mammals, general telomere lengths were significantly shortened in
mutant athmgb1 plants compared to wild-type plants. In accordance with these results, in the plant lines
overexpressing AtHMGB1, elongated telomeres are not dispersed continuously but they rather migrate on
agarose gel as discrete bands, which is typical for telomeres generated by alternative lengthening of
telomeres (ALT) (see below). These observations were proven by fluorescence in situ hybridisation on
metaphase chromosomes where moderate but significant increase of telomeric signal in the AtHMGB1
overexpressing line samples as compared to the wild type (Schrumpfová et al., 2011; see Supp. G).
However, the pathway mediating this effect seems to be in different between plant and mammals. While
the telomere shortening in mouse cells lacking mHMGB1 can be attributed to the insufficient telomerase
activity, no changes in telomerase activity and telomerase processivity could be observed in either athmgb1
or AtHMGB1 overexpressing plants. From our results we can conclude that AtHMGB1 protein does not exert
its effect on telomere length via direct regulation of telomerase, however, AtHMGB1 is involved in the
stress- or stimulus-responsive pathways affecting telomere length (Schrumpfová et al., 2011; see Supp. G).
3.3 Telomerase-independent telomere maintenance
Besides the telomerase-based mechanism of telomere elongation, various organisms as well as plants,
utilize a telomerase-independent telomere maintenance mechanism: alternative lengthening of telomeres
(ALT). The exact mechanism behind telomere maintenance in the ALT pathway is unclear, but likely is based
48
on homologous recombination (HR) and may become active upon the loss of telomerase (Dunham et al.,
2000; Min et al., 2017; Zhang et al., 2019).
ALT relies on the formation of terminal T-loops, which parallels the first steps of HR (see Figure 12). The
eventual resolution of these T-loops and aberrant HR at telomeres generates not only telomeres of highly
heterogeneous lengths but also extrachromosomal T-circles, which are the known hallmarks of ALT. These
ALT hallmarks include not only already mentioned heterogenous distribution of the telomere lengths and
several classes of extrachromosomal telomeric repeats in the nucleus. ALT-positive cells show also
increased telomere sister chromatid exchanges (T-SCE) and presence of promyelocytic leukemia nuclear
bodies (APBs) associated with some telomeres. APBs are special variety of PML (promyelocytic leukemia)
nuclear bodies found in the normal interphase nucleus. PML bodies are donut-shaped nuclear domains
composed of PML and SP100 proteins, which are stabilized by non-covalent interactions of the
posttranslational modification SUMO but they do not contain nucleic acids in normal cells (reviewed in
Corpet et al., 2020). However, in ALT-positive cells, a subset of PML nuclear bodies, APBs, co-localizes with
telomeric DNA. APBs contain PML nuclear bodies components such as PML, SP100 and SUMO and,
moreover, telomeric DNA and telomere associated proteins including the Shelterin components TRF1,
TRF2, POT1, TIN2, TPP1 and RAP1. Additionally, APBs contain factors that are involved in DNA damage
response (DDR) and repair reviewed in Corpet et al., 2020). ALT mechanism is predominantly activated in a
Figure 12. T-loops and Alternative lengthening of telomeres (ALT).
A) Specific telomeric structure T-loop, where the 3′ G-strand extension invades the duplex telomeric
repeats and forms a D-loop (displacement loop), prevent telomerase access to the telomeres. Figure
modified from de Lange, 2004.
B) Telomeres progressively shorten in normal cells with each division in the absence of a telomere
maintenance mechanism. In in ~10–15% of tumours, a DNA homologous recombination mechanism,
instead of telomerase activation, can be engaged. ALT cells use a telomeric DNA template that is copied
to a telomere of a non-homologous chromosome, This telomeric DNA could add telomeric repetitive
sequences to another region of the same telomere via loop formation or to the telomere of a sister
chromatid (Shay and Wright, 2019).
A
B
49
number of human tumours and in human cells immortalized in culture but also was observed in normal
somatic tissues (Neumann et al., 2013). ALT is active in about 10-15 % of cancers (Heaphy et al., 2011).
Telomerase-mediated synthesis of telomeres is also essential for sustained growth and propagation in
plants. Inactivation of a gene coding for catalytical subunit of telomerase, AtTERT, leads to a gradual
shortening of telomeres by 200-500 nt per generation (Fitzgerald et al., 1999; Riha et al., 2001). After 6-8
generations, some telomeres in attert mutants shorten to ∼300-400 nt and start fusing with other
chromosome ends (Heacock et al., 2007; Riha et al., 2001). Plants with such dysfunctional telomeres exhibit
developmental defects and reduced fertility. The severity of these phenotypes worsens with progressive
telomere shortening and mutant populations cannot be propagated beyond 8-10 generations (Riha et al.,
2001).
However, while yeast and human telomerase-deficient cell lines appear to readily adopt ALT for telomere
maintenance, extensive selection of cells derived from Arabidopsis attert mutants failed to recover cultures
featuring hallmarks of ALT (Watson et al., 2005; Zellinger et al., 2007; reviewed in Schrumpfová et al., 2019;
see Supp. M).
In our study Růčková et al. (2008) we described that the ALT mechanism is activated not only in mutant
plants with telomerase dysfunction but possibly also during the earliest stages of normal plant development
(Růčková et al., 2008; see Supp. B). In this study we hypothesised that extremely low rates of telomere
shortening per plant generation (250-500 nt) in telomerase-deficient A. thaliana mutants (attert) does not
correspond to the expected outcome of replicative telomere shortening. The meristem cells in A. thaliana,
which give rise to all tissues including germ-line cells, undergo many divisions, calculated by Andrew Leitch
(Queen Mary, University of London) and Jiří Friml (Institute of Science and Technology Austria, Austria) at
approximately 1000 divisions from seed to seed. When considering only 5-10 nt lost per cell division (the
average length of RNA primer for synthesis of Okazaki fragments) as the minimum plausible loss of
telomeric DNA at each round of replication (under the very improbable scenario that the primer sits exactly
at the 3’ end of the parental DNA strand), then the number of cell divisions accounting for the observed
telomere erosion per generation in attert mutants would be only 25-50 cell divisions. Moreover, in
mammalian cells the primer does not sit exactly at the 3’ end of the parental DNA strand and the loss at
telomere is between 50-100 nt per cell division. Then it would be only 5-10 cell divisions per plant
generation but not already 1000 division as was stated for A. thaliana (Fajkus et al., 2005).
We propagated attert mutant plants from seeds coming either from the Lower-most or the Upper-most
siliques and we followed the length of their telomeres over several generations. We proved that in the
absence of telomerase, the number of cell divisions within one generation influences the control of
telomere lengths. Our data showed a fast and efficient activation of a telomerase-independent mechanism
50
in response to the loss of telomerase activity and imply that ALT is probably involved also in normal plant
development (Fajkus et al., 2005; Růčková et al., 2008; see Supp. B).
The group of Karel Říha speculated that the meristem cells, however, do not undergo so many cell divisions
as was proposed above, as they observed that the number of DNA replications is only slightly increased in
plant growing under long-lived conditions in comparison to the plant growing in short-day conditions. They
showed that the cell depth of gametes is not linearly proportional to the vegetative growth period and
suggested that older plants may not be passing on more mutations to their offspring relative to younger
plants (Watson et al., 2016).
The involvement of ALT in the earliest stages of normal plant development is still questionable and needs
further investigation.
51
Conclusion
The ends of the linear chromosomes, called telomeres, are shields that protect the exposed chromosome
ends from DNA damage machinery. Due to their significance in cell viability, cancer, and ageing, there has
been intensive research on telomeric DNA, telomere-associated proteins, and telomere-related proteins
for over four decades. However, the protein interactome associated with plant telomeres and telomerase
is not as well-studied as the mammalian telomeric proteome. It is interesting to note that telomeric repeats
can also be found dispersed throughout the genome as interstitial telomeric tracts or short telo-boxes.
In plants, telomeres are primarily composed of short tandem repeats that are associated with various
proteins involved in regulating telomere maintenance and the telomerase holoenzyme complex's access.
Telomere Repeat Binding proteins (TRBs) play a crucial role in telomere maintenance, and they are
associated not only with terminally located telomeric repeats but also with telo-boxes, which are mainly
found in gene promoters. These TRBs can recruit and regulate various protein complexes and significantly
influence the chromatin's epigenetic status.
In my habilitation thesis, I presented data that expand our understanding of plant telomere biology,
telomeric sequence- or telomerase-associated proteins, and their roles in telomere homeostasis
maintenance. I discussed the involvement of these proteins in telomerase assembly, recruitment, and
activity, as well as their role in regulating and protecting the chromosomes' physical ends' genomic
integrity. Additionally, I comment on the involvement of these proteins in non-telomeric functions in
epigenetic regulations.
52
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Supplements
Relevant publications of the applicant arranged in chronological order.
All publications referred in Supplements have been included in the thesis (references in bold and marked
with letters of alphabet in the text).
*Corresponding Author
Supplement A
Schrumpfová, P., Kuchar, M., Miková, G., Skrísovská, L., Kubicárová, T., Fajkus, J., 2004. Characterization of
two Arabidopsis thaliana myb-like proteins showing affinity to telomeric DNA sequence. Genome 47, 316–
324
Supplement B
Růčková, E., Friml, J., Schrumpfová, P.P., Fajkus, J., 2008. Role of alternative telomere lengthening
unmasked in telomerase knock-out mutant plants. Plant Mol. Biol. 66, 637–646
Supplement C
Schrumpfová, P.P., Kuchar, M., Palecek, J., Fajkus, J., 2008. Mapping of interaction domains of putative
telomere-binding proteins AtTRB1 and AtPOT1b from Arabidopsis thaliana. FEBS Lett. 582, 1400–1406.
Supplement D
Mozgová, I., Schrumpfová, P.P., Hofr, C., Fajkus, J., 2008. Functional characterization of domains in AtTRB1,
a putative telomere-binding protein in Arabidopsis thaliana. Phytochemistry 69, 1814–1819
Supplement E
Hofr, C., Sultesová, P., Zimmermann, M., Mozgová, I., Schrumpfová, P.P., Wimmerová, M., Fajkus, J., 2009.
Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study of telomere-binding specificity
and kinetics. Biochem. J. 419, 221–228
Supplement F
Peška, V., Schrumpfová, P.P., Fajkus, J., 2011. Using the telobox to search for plant telomere binding
proteins. Curr. Protein Pept. Sci. 12, 75–83
Supplement G
Schrumpfová, P.P.*, Fojtová, M., Mokroš, P., Grasser, K.D., Fajkus, J., 2011. Role of HMGB proteins in
chromatin dynamics and telomere maintenance in Arabidopsis thaliana. Curr. Protein Pept. Sci. 12, 105–
111
Supplement H
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Schrumpfová, P.P., Vychodilová, I., Hapala, J., Schořová, Š., Dvořáček, V., Fajkus, J., 2016. Telomere binding
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Schrumpfová, P.P., Majerská, J., Dokládal, L., Schořová, Š., Stejskal, K., Obořil, M., Honys, D., Kozáková, L.,
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Schrumpfová, P.P., Majerská, J., Dokládal, L., Schořová, Š., Stejskal, K., Obořil, M., Honys, D., Kozáková, L.,
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Schrumpfová, P.P.* and Fajkus, J. 2020. Composition and Function of Telomerase - a polymerase associated
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plant gametophyte development, The Plant Journal 114, 325–337
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Kusova A., Steinbachova L., Přerovská T., Záveská Drábková L., Paleček J., Khan A., Rigóová G., Gadoiu Z.,
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Supplement A
Schrumpfová, P., Kuchar, M., Miková, G., Skrísovská, L., Kubicárová, T., Fajkus, J.,
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Supplement B
Růčková, E., Friml, J., Schrumpfová, P.P., Fajkus, J., 2008. Role of alternative telomere
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length measurement) and participated in the ms writing and editing
Role of alternative telomere lengthening unmasked in telomerase
knock-out mutant plants
Eva Ru˚cˇkova´ Æ Jirˇı´ Friml Æ Petra Procha´zkova´ Schrumpfova´ Æ
Jirˇı´ Fajkus
Received: 23 November 2007 / Accepted: 14 January 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Telomeres in many eukaryotes are maintained
by telomerase in whose absence telomere shortening
occurs. However, telomerase-deficient Arabidopsis thaliana
mutants (Attert-/)
show extremely low rates of
telomere shortening per plant generation (250–500 bp),
which does not correspond to the expected outcome of
replicative telomere shortening resulting from ca. 1,000
meristem cell divisions per seed-to-seed generation. To
investigate the influence of the number of cell divisions per
seed-to-seed generation, Attert-/mutant
plants were
propagated from seeds coming either from the lower-most
or the upper-most siliques (L- and U-plants) and the length
of their telomeres were followed over several generations.
The rate of telomere shortening was faster in U-plants, than
in L-plants, as would be expected from their higher number
of cell divisions per generation. However, this trend was
observed only in telomeres whose initial length is relatively
high and the differences decreased with progressive general
telomere shortening over generations. But in generation 4,
the L-plants frequently show a net telomere elongation,
while the U-plants fail to do so. We propose that this is due
to the activation of alternative telomere lengthening (ALT),
a process which is activated in early embryonic development
in both U- and L-plants, but is overridden in U-plants
due to their higher number of cell divisions per generation.
These data demonstrate what so far has only been speculated,
that in the absence of telomerase, the number of cell
divisions within one generation influences the control of
telomere lengths. These results also reveal a fast and efficient
activation of ALT mechanism(s) in response to the
loss of telomerase activity and imply that ALT is probably
involved also in normal plant development.
Keywords Alternative telomere lengthening Á ALT Á
Replicative telomere shortening Á
Telomerase-deficient plants
Introduction
Incomplete replication of chromosome ends results in progressive
telomere shortening unless a mechanism to
elongate telomeres takes effect. It has been known for more
than a decade that the common system of telomere maintenance
in plants is provided by telomerase (Fajkus et al.
1996; Heller et al. 1996), although exemptions, in which a
different type of telomeres and mechanism of their maintenance
are in use have been described since about the same
time in Allium (Pich et al. 1996; Sykorova et al. 2006) and
later on in three Solanaceae genera (Sykorova et al. 2003).
We hypothesized recently that alternative telomere lengthening
(ALT) mechanisms are probably not restricted to
species possessing ‘‘unusual’’ telomeres, but may be a
normal part of plant development (Fajkus et al. 2005), an
idea explored here. Arabidopsis thaliana knockout mutants
in Telomerase Reverse Transcriptase (AtTERT) exhibit
telomere shortening of 250–500 bp per generation
E. Ru˚cˇkova´ Á J. Friml Á P. Procha´zkova´ Schrumpfova´ Á
J. Fajkus (&)
Department of Functional Genomics and Proteomics, Faculty
of Science, Masaryk University, Building A2 – ILBIT,
Kamenice 5, 625 00 Brno, Czech Republic
e-mail: fajkus@sci.muni.cz
E. Ru˚cˇkova´ Á J. Fajkus
Institute of Biophysics ASCR, v.v.i., Kra´lovopolska´ 135,
61265 Brno, Czech Republic
J. Friml
VIB Department of Plant Systems Biology, Ghent University,
Technologiepark 927, 9052 Gent, Belgium
123
Plant Mol Biol
DOI 10.1007/s11103-008-9295-7
(Fitzgerald et al. 1999) and survive up to 10 generations
with severe cytological and chromosomal abnormalities
occurring after about eight generations (Riha et al. 2001;
Siroky et al. 2003). The rate of the observed inter-generation
shortening of telomeres is surprisingly low, given the
high number of cell divisions per seed-to-seed generation.
The high number results from the mode of plant development,
which does not involve stem cell mobility and cell
lines are not sequestrated for later use (as in the germ line of
mammals). In plants an apical meristem consists of a small
group of stem cells that generate a linear series of cells,
which differentiate into an array of cell types that make a
shoot and root. Flowers initiate from the shoot apical meristem
in mature plants, which is organized in cell layers L1,
L2 and L3, and divisions of those are roughly synchronized.
L2 cell layer derivatives provide the mesodermal cells and
the germ cells of pollen grains and ovules (Fletcher 2002,
Grandjean et al. 2004). Consequently, meristem cells,
which give rise to all tissues, including germ-line cells,
undergo many divisions, calculated in A. thaliana to be
approximately 1,000 divisions from seed to seed (Fajkus
et al. 2005). When considering only 5–10 nucleotides lost
per cell division (the average length of RNA primer for
synthesis of Okazaki fragments) as the minimum plausible
loss of telomeric DNA at each round of replication (under
the very improbable scenario that the primer sits exactly at
the 3’end of the parental DNA strand), then the number of
cell divisions accounting for the observed telomere erosion
per generation in Attert-/mutants
would be only 25–50
cell divisions (as in mammals). But as already stated for A.
thaliana the actual number of divisions is closer to 1,000.
Thus we proposed that ALT system operates in telomerasedeficient
plants and possibly also in normal plant development
to partially compensate for replicative shortening of
telomeres (Fajkus et al. 2005).
To test this hypothesis, we compared telomere shortening
in telomerase knock-out plants differing in the number
of cell divisions per generation. We generated Attert-/-
and
Attert+/+
plants from heterozygous A. thaliana (Attert+/-
)
line (SALK_061434.56.00.X) bearing T-insertion in Attert
gene. In the obtained homozygous Attert-/plants,
seeds
were collected individually from the lowermost or uppermost
siliques and seeds propagated. In subsequent
generations, plants coming from the lowermost siliques
were propagated again through seeds from the lowermost
siliques, while plants coming from the uppermost siliques
were again propagated through seeds from uppermost siliques.
If our hypothesis is correct, the plant propagation
scheme (Fig. 1) should result in additional cell divisions
occurring in ‘‘upper silique’’ lines (U) compared with lower
silique’’ lines (L). The number of additional cell divisions in
U-plants compared with L-plants can be estimated as follows:
the cell division rate after floral transition is 1–2
divisions per 24 h. That corresponds to 1–2 flower initiations
for the same time, so the difference is 1 cell division
between 2 consecutive flower initiations (Grandjean et al.
2004). Since ca. 30 siliques occur between the lowermost
and the uppermost silique, we can expect about 30 additional
cell divisions. Therefore in Attert-/mutants
we
might expect about 150–300 bp-shorter telomere lengths in
the U-lines compared with L-lines.
We show here that differential telomere shortening does
occur between U- and L-lines but that the results are
complicated through the activation of ALT. We reveal a
differential rate of telomere shortening in different generations
of mutants and telomere length oscillations, which,
at least in Attert-/mutants,
cannot be attributed to telomerase
activity.
Material and methods
Plant material
Homozygous mutant Attert-/plants
(M) and corresponding
Attert+/+
controls (Wt) were prepared from
heterozygous A. thaliana line (Attert+/,
H) bearing
T-insertion in Attert gene (SALK_061434.56.00.X). Plants
were initially cultivated on short day (8 h of light) and after
6 weeks at long day (16 h of light) in a greenhouse. Lines
were derived from three heterozygous plant lineages (Ha,
Hb1 and Hb2). Mutant (M) and control plants (Wt) were
selected from their progeny and designated accordingly
(Ma and Wta from Ha; Mb1, Mb2 and Wtb1 and Wtb2
from Hb). First generation plants (G1) were divided into
lines propagated either from the lowermost or the uppermost
siliques (L- and U-plants, respectively, see the
schematic Fig. 1). These lines were cultured until the
fourth generation (G4).
Fig. 1 Schematic diagram of plant propagation system for the
generation of plants derived from the heterozygote Attert+/- mutant
Ha. The same scheme applies to the heterozygous mutants Hb1 and
Hb2 and the nomenclature of the derived plants follow accordingly.
For further details of the source materials see Materials and methods
Plant Mol Biol
123
Genotyping
DNA was extracted according to Edwards et al. (1991). A set
of three primers was used for genotyping. Two primers were
complementary to genomic DNA upstream and downstream
of the T-insertion (tel+ and tel-, respectively) and produced
a 876 bp product in wild-type. In mutants, a 702 bp product
was synthesized using primer LBb1 (complementary to
T-insertion) and tel-. Primer sequences were: tel+: 50
-CTg
CTACTTTCAgCTTCAgC-30
, tel-: 50
-gCAAgAggATgCA
TTgAAgTCCgg-30
, LBb1: 50
-gCgTggACCgCTTgCTgCA
ACT-30
. The reaction mix (15 ll) contained 19 buffer
(DyNAzyme II, Finnzymes), 0.25 mM dNTPs, 0.3 lM forward
and 0.3 lM reverse primer, 0.3 U DyNAzyme II DNA
polymerase (Finnzymes) and 5 ng of DNA. Initial denaturation
(94°C/3 min) was followed by 35 thermal cycles
(94°C/30 s, 56.5°C/30 s and 72°C/1 min), and a final
extension (72°C/10 min).
Induction of callus cultures
Seeds were sterilized by shaking for 15 min in 50 mg ml-1
Ca(OCl)2 solution, then rinsed three times with sterile
water with 0.01% Triton X-100 and placed on solid MS
medium supplemented with 20 g l-1
sucrose. Plants were
genotyped and leaves from mutant, wild-type and heterozygous
plants were harvested, cut and cultivated on solid
MS medium (Duchefa M0231) supplemented with 1 mg/l
2,4-D (2,4-dichlorophenoxyacetic acid), 0.2 mg/l kinetin
and 20 g/l sucrose. Calli were maintained on this medium
and transferred to fresh medium every 4 weeks.
Detection of telomerase activity
Protein extracts from calli were prepared as described in
Fitzgerald et al. (1996). These extracts were then tested for
telomerase activity by plant telomere repeat amplification
protocol (TRAP) as described by Fajkus et al. (1998).
Determination of telomere length
DNA was extracted from three rosette leaves according to
Dellaporta et al. (1983). Primer extension telomere repeat
amplification (PETRA) analysis was performed with an
equivalent amount of DNA as described in Heacock et al.
(2004) and Watson and Shippen (2007). Individual telomeres
were designated according to Heacock et al. (2004)
with a number identifying a chromosome, and R or L letter
indicating a chromosome arm, where R corresponds to
South and L to North (Arabidopsis Genome Initiative
2000). To measure telomere length, signals of PETRA
products were analyzed by TotalLab using a 1-kb DNA
ladder (Fermentas) as standard. The distance of the PETRA
primer to the telomere was subtracted from the total length
of PETRA product to give the actual length of the telomere
tract. The average telomere lengths were visualized using
Southern hybridization of terminal restriction fragments
(TRF) (Fajkus et al. 1995) produced by digestion with MseI
restriction endonuclease. Both PETRA and TRF products
were detected using telomeric oligonucleotide (50
-GGTT
TAGGGTTTAGGGTTTAGGGTTTAG-30
) end-labelled
with [c-32
P]ATP using polynucleotide kinase (NEB).
Results
Arabidopsis tert mutants do not possess residual
telomerase activity
To make sure that A. thaliana Attert-/mutants
do not
possess any residual telomerase activity, tissue cultures
derived from original Attert+/plants
and their mutant
(Attert-/)
and Wt (Attert+/+
) progeny were assayed for
telomerase activity by TRAP assay. The results (Fig. 2)
show the absence of telomerase activity in extracts
obtained from Attert-/mutant,
while both Attert+/-
and
Attert+/+
cultures are telomerase-positive.
Telomere lengthening upon transition from Attert+/(H)
to Attert+/+
(Wt) state
To evaluate telomere length changes between mutant
plants propagated via upper and lower silique seeds
(undergoing a different number of cell divisions per seedto-seed
generation) and to distinguish them from natural
variations in telomere lengths in telomerase-positive plants,
control Wt plants were generated from the same original
heterozygous plants as Attert-/mutants.
Wt plants were
propagated according to the same schematic protocol as
U- and L-lines of Attert-/mutants
(Fig. 1) to reveal any
potential stochastic changes in telomere lengths. Examples
of primary PETRA and TRF results are shown in Fig. 3.
The results were repeated in three independent lineages
(Ha, Hb1 and Hb2). Measurement of PETRA fragment
sizes revealed increases in both 2R and 3L telomeres
in most first generation Attert+/+
plants within a range of
120–380 bp (Figs. 4, 5). The exception is the 3L telomere
in Wta plant where telomere lengths show a mild (90 bp)
shortening (Fig. 5). Telomere dynamics in the following
generations of Wt plants displayed changes in both directions,
but overall most plants showed a net increase in
telomere lengths in G4 compared to their length in the
Plant Mol Biol
123
original heterozygotes. This observation suggests that the
mutation of a single Attert allele acts via haploinsufficiency,
as in human (Hauguel and Bunz 2003; Zhang et al.
2003) and mouse tert genes (Erdmann et al. 2004). No
substantial differences in telomere lengths were observed
between U- and L-lines of corresponding Attert+/+
plants,
revealing that telomerase in meristem cells maintains
telomere stability during plant development (Fajkus et al.
1998; Riha et al. 1998) regardless of number of cell
divisions.
Telomere dynamics in Attert-/-
plants
The key question of this study was to analyze if there is a
relationship between telomere shortening per plant generation
and the number of meristem cell divisions between
generations. To address this question, telomere lengths
were measured during propagation of Attert-/mutants
as
U- and L-lines. Only the first few generations of Attert-/mutants
were analyzed (G1–G4) to avoid accumulated
cytogenetic abnormalities expected from the 6th generation
(Riha et al. 2001). Results of telomere analysis (Figs. 4, 5)
revealed:
(i) Telomere length differences between consecutive
plant generations can be highly variable ranging from
tens of bp to 800 bp. There is some evidence that large
changes in telomere length ([500 bp) in one generation
is followed by smaller changes in the subsequent
generation. For example, in 2R telomeres in mutant
Ma-derived U-plant lines, large changes occur
between G1 and G2 (Figs. 4, 5) and subsequently
are less dramatic. In 3L telomeres of all the Maderived
U-lines, substantial telomere shortening
occurs between G3 and G4, while in earlier generations
there are only moderate changes (Fig. 5).
(ii) Telomeres 2R and 3L in the same plants behave
relatively independently. Compare e.g., a 150 bp
shortening between G1 and G2 generations in the 2R
telomere in L-plants derived from the Mb1 mutant
with ca. 800 bp shortening of 3L telomeres in the
same plants (Figs. 4–6).
(iii) Two patterns were observed in the dynamics of
telomere shortening in Attert-/mutants.
The first is
represented by 2R telomeres of plants derived from
Ma and Mb1 mutants. These results reveal, as
predicted, a faster rate of telomere shortening per
generation in plants propagated through seeds from
the upper siliques (U-lines) than in those from the
lower siliques (L-lines, Figs. 4, 7A). The second is
observed in 3L telomeres of all lines and of 2R
telomeres of Mb2-derived lines. Here the rate of
Fig. 2 Results of TRAP assay in calli derived from the original
heterozygous Attert+/plant
(H), and its progeny—mutant Attert-/(M)
and wild type Attert+/+
(Wt). A 50 bp marker (GeneRuler,
Fermentas) is used as a marker (m). Telomerase extract from standard
A. thaliana wt seedlings was used as positive control (+), and the
extraction buffer served as a negative control (-)
Fig. 3 Example of PETRA and TRF results. One of the Attert+/plants
(Ha) and two generations of Attert-/(Ma)
and Attert+/+
(Wta)
plants derived from the Ha plant, were propagated as L- and U-lines
(as indicated) and assayed by PETRA with a primer specific for the
subtelomere 2R. Apart from stronger bands that correspond to main
products of PETRA there are also weaker bands of a higher mobility.
These weaker bands in a given lane correspond to the stronger bands
in both their number and mutual position. The result labelled as TRF
shows the hybridization pattern of terminal restriction fragments
generated with restriction enzyme MseI in Ha plant. A 1 kb
GeneRuler (Fermentas) has been used as marker (m)
Plant Mol Biol
123
telomere shortening is not substantially different
between U- and L-lines (Figs. 4, 5, 7B). If it is
assumed that ALT becomes activated in response to
telomere shortening, then the mechanism is more
active in 3L telomeres in earlier generations compared
to 2R telomeres, and acts to override any
losses incurred through an increased number of cell
divisions between U- and L-plant generations.
(iv) A difference between U- and L-lines is apparent
from the evaluation of relative telomere length
changes between generations (Fig. 6). While the
overall telomere lengthening is occasionally
observed between G2 and G3 generations in both
U- and L-lines, it is much more frequent between G3
and G4 generations—but only in L-lines, whereas it
is entirely absent in any of the U-line plants between
the same generations. This remarkable difference
suggests that ALT processes may be more frequent in
gametogenesis or early developmental stages. In
L-plants, such elongation can sometimes be even
higher than replicative shortening corresponding to
formation of lower-silique seeds, while in U-plants
the greater number of cell divisions overrides the
efficiency of ALT.
Fig. 4 Telomere lengths in 2R chromosome arms in wild-type (Wta,
Wtb1, Wtb2) and mutant (Ma, Mb1, Mb2) plants. Each graph consists
of consecutive results from the original heterozygous Attert+/-
plant
(Ha or Hb), and four subsequent generations (G1–G4) of the
individual U- or L-lines coming from the given first-generation plant
(Wta, Wtb1, Wtb2 and Ma, Mb1, Mb2). Overlapping points cannot be
seen separately
Fig. 5 Telomere lengths in 3L chromosome arms in wild-type (Wta,
Wtb1, Wtb2) and mutant (Ma, Mb1, Mb2) plants. Each graph consists
of consecutive results from the original heterozygous Attert+/-
plant
(Ha or Hb), and four subsequent generations (G1–G4) of the
individual U- or L-lines coming from the given first-generation plant
(Wta, Wtb1, Wtb2 and Ma, Mb1, Mb2)
Plant Mol Biol
123
Early onset of phenotype changes in Attert mutants
The first phenotypic abnormalities appeared in some
plants from the U- and L-lines in the 4th generation of
Attert-/mutants,
primarily from Mb1 and Mb2 plants.
Some plants showed mild abnormalities, leaves were
asymmetric and lobed. Other plants had more serious
abnormalities both in leaf morphology and shoot structure—stems
were split and in some cases appeared to
have lost the apical dominance; leaves were smaller than
wild-type, had an irregular shape, a rough surface and
were more plentiful than in wild-type plants (Fig. 8);
siliques were smaller with fewer seeds, or were completely
sterile. The most severe phenotype arose in a G4
plant from U-line derived from the Mb1 mutant. In that
plant there were many abnormalities including leaves
with a distinct trichomes, a small stem and sterile
siliques (Fig. 8A–C). 2R and 3L telomeres in this G4
plant were 1340 and 770 bp long, respectively, i.e.
similar to the other G4 mutant plants included in
Figs. 4–7. For other plants, the occurrence of abnormal
phenotypes did not differ substantially between
mutants of the U- and L-lines. The phenotypic abnormalities
observed correspond well to those described by
Riha et al. (2001), but in our experiments some
plants had severe phenotypes in the 4th generation
rather than the 8th generation described in that earlier
work.
Fig. 6 Summary of relative
telomere shortening in each
generation of all three mutant
lines (Ma, Mb1 and Mb2).
Telomere lengths in each
generation were subtracted from
lengths in the previous
generation, therefore values are
positive in case of shortening
and negative in elongation
events
Fig. 7 Graphs showing two different trends observed in rates of
telomere shortening between plants from U- and L-lines. Telomere
lengths of all plants in either U- or L-line were averaged in a given
generation. (A) (2R telomeres in Ma lines) shows example of the case
in which the rate of telomere shortening is faster in the U-line then in
L-line. (B) (2R telomeres in Mb2 lines) exemplifies the similar rate of
telomere shortening in U- and L-lines. The former course was
observed in 2R telomeres in Ma and Mb1 lines, while the latter
occurred in 2R telomeres in Mb2 line and 3L telomeres of all lines
Plant Mol Biol
123
Discussion
Involvement of telomerase-independent processes
in plant telomere dynamics
Our analysis of telomere dynamics in Arabidopsis
Attert-/mutants
challenges current perceptions of the
relative contributions of telomerase activity, ALT and
telomere rapid deletion (TRD) in plant telomere dynamics.
Watson and Shippen (2007) showed that elongated
telomeres in A. thaliana Ku70 mutants shorten to the
length typical for wt plants after three generations when
restored with wild-type Ku70. This corresponds to an
average loss of 2.3 ± 0.8 kb of telomeric DNA per generation,
which is interpreted as the result of TRD, as it
exceeds the previously reported rate of telomere shortening
per generation in tert mutants. The rate of telomere
shortening per generation observed in this work, as well
as in our study was not constant (Fig. 7), but decreased as
telomeres approached the length of about 2 kb. In addition
to the reported TRD, the opposing process—ALT—
was detected in ku70 tert double mutants with elongated
telomeres (Watson and Shippen 2007). Although it is
possible that both TRD and ALT are actively involved in
telomere length regulation in A. thaliana, according to our
previous estimates and calculations (Fajkus et al. 2005),
the observed rate of telomere shortening in Attert-/mutants
is about ten times less than the expected
replicative loss resulting from number of cell divisions
that occurs between generations. As telomeres approach a
critical length, the frequency of ALT events may increase,
which results in more substantial telomere length changes
in both directions. This hypothesis might also explain
why 3L telomeres respond similarly between U- and
L-lines. The initial length of 3L telomeres is about 500 bp
shorter than 2R telomeres. Perhaps because the telomeres
are shorter, stochastic ALT processes are active in both
lines in earlier generations in 3L telomeres. Therefore we
conclude that the apparent slower rates of telomere
attrition in G3–G4 generations are presumably due to the
up-regulation of ALT by shortened telomeres themselves,
or by their changed nucleoprotein structure.
Is ALT restricted to a specific developmental stage?
Frequently telomere elongation was observed between G3
and G4 in L-plants, while they are entirely absent in
U-plants. This remarkable difference suggests that ALT
processes may be more frequent in gametogenesis or early
developmental stages. In L-plants, such elongation can
sometimes be even greater than subsequent replicative
shortening corresponding to formation of lower-silique
seeds, while in U-plants the additional 30 divisions counteract
length gains produced by ALT at a particular time in
development. The activity of ALT in early embryonic
Fig. 8 Examples of mutant
phenotypes. Different life stages
of the G4 plant from U-line,
which had the most serious
phenotype defects (A–C).
Leaves were asymmetric and
rough, had conspicuous
trichomes, stem was short and
thin and the plant was sterile.
A detailed picture of a split
stem is given in panel D
Plant Mol Biol
123
development is supported also by recent observations of
Liu et al. (2007) in mammalian oocyte cells. They show
that oocytes have shorter telomeres than somatic cells and
lack telomerase activity, but their telomeres lengthen
remarkably during early cleavage cycles following fertilization
through a recombination-based mechanism. From
the blastocyst stage onwards, telomerase becomes activated
and maintains the telomere length established by this
alternative mechanism. The involvement of ALT in normal
development thus gains support in such divergent organisms
as mammalians and plants, and this conservation
suggests its key importance.
The observation of the overall telomere elongation
events in L-plants also poses an interesting question: why
is the ALT process not able to compensate completely for
the lack of telomerase in Attert-/mutants?
A possible
explanation is provided by ALT itself. Studies on different
model systems show that ALT involves homologous
recombination (HR) and HR-dependent DNA replication
(reviewed in Cesare and Reddel 2008). Experimental evidence
supports a ‘‘roll and spread’’ model of ALT, in which
a 30
telomeric overhang invades either the duplex region of
its own telomere (forming the t-loop), or an extrachromosomal
telomeric circle (a product of t-loop junction
resolution), and is extended with DNA polymerase using
either of the above templates. The extended telomere can
then spread to other chromosome termini via HR. This
scenario is supported by the presence of both types of
candidate templates (t-loops and t-circles) for the initial
phase of ALT in plants (Cesare et al. 2003; Zellinger et al.
2007). Moreover, it was shown recently that Ku protein
suppresses formation of t-circles and ALT lengthening in
A. thaliana (Zellinger et al. 2007). If initial telomere
elongation events are limited to the early embryogenesis or
gametogenesis, as suggested above, and the ‘‘reservoir’’ of
elongated telomere sequence available for the individual
plant generation is finite, then the extension potential will
depend on the initial length of telomere. In early generations,
when the initial telomere length is relatively long, the
ALT is able to compensate for the replicative telomere
shortening almost completely (compare the expected telomere
loss of several kb per generation in Attert-/-
mutants
with the observed average rate of telomere shortening of
ca. 250–500 bp per generation (Fitzgerald et al. 1999)).
The slow but progressive telomere shortening, nevertheless,
gradually decreases the efficiency of ALT, and the
observed massive increase in genome instability observed
since the 6th generation of Attert mutants (Riha et al. 2001)
may reflect this. Moreover, the increase of recombination
frequency due to the excessive activation of ALT itself can
contribute to an increase in genome instability (Jeyapalan
et al. 2005).
Immortal strand hypothesis resurrected in animal
cells—does it live in plant cells too?
The remarkably low telomere-shortening rate of Attert-/mutants
could be also explained without invoking ALTmechanisms.
The ‘‘immortal strand hypothesis’’ proposed
decades ago (Cairns 1975) suggests stem cells might limit
acquired mutations that give rise to cancer through the
directed inheritance of parental DNA strands. Though
largely disregarded, this hypothesis of the template DNA
strand co-segregation in dividing stem cells and their
progeny has implications in telomere biology. Recent
results (Conboy et al. 2007) provide experimental support
to this hypothesis. These authors used sequential pulses of
three different halogenated thymidine analogs and analyzed
stem cell progeny during induced regeneration
in vivo. They observed extraordinarily high frequencies of
segregation of older and younger template strands during a
period of proliferative expansion of muscle stem cells.
Template strand co-segregation was strongly associated
with asymmetric cell divisions yielding daughters with
divergent fates. Daughter cells inheriting the older templates
retained the more immature phenotype, whereas
daughters inheriting the newer templates acquired a more
differentiated phenotype (Conboy et al. 2007). It has yet to
be shown if this behaviour is also present in plant meristem
cells, but the idea is certainly worth considering and testing
experimentally. While the mechanism could contribute to
the low telomere shortening rate, it does not provide
explanation for the overall telomere lengthening observed
namely between G3 and G4 generations of L-line plants.
The onset of abnormal phenotype preceeds critical
telomere shortening
The onset ofabnormalphenotype effects was observed atleast
two generations earlier in this work than in the previous study
(Riha et al. 2001). The severity of phenotype changes, however,
did not show any direct relationship to telomere lengths
ortheoccurrenceofplantsineitherU-orL-lines.Inparticular,
the severely affected G4 mutant plant did not show markedly
shorter 2R or 3L telomeres than the other mutants of the same
generation. Moreover, we also measured other telomeres
in this particular plant (1R = 840 bp, 1L = 820 bp,
4R = 1,170 bp, 5R = 850 bp, 5L = 970 bp) and all of these
ranged well above the previously published minimum functional
telomere length in Arabidopsis (300–400 bp), which
was based on measurement of telomere fusion sites (Heacock
et al. 2004). Potentially therefore the phenotype characteristics
may not be due to reduced telomere lengths directly, but
perhaps a resultof ALT-associated abundant levelof telomere
recombination, leading to genome instability.
Plant Mol Biol
123
Possible origins of multiple products in PETRA assays
The presence of two or multiple signals coming from a
given chromosome arm using the PETRA technique is
usually attributed to different telomere lengths at homologous
chromosome arms, or to the hypothetical TRD
events in a subpopulation of telomeres (Watson and
Shippen 2007). Besides these bands of comparable intensity,
we observed also some weaker bands of higher
mobility, which corresponded to the stronger bands in the
same lane in their number and mutual positions (see
Fig. 3). Interestingly, the length differences between
weaker and stronger products decreased proportionally
with the size of the stronger band (see Fig. 9). The weaker
band occasionally disappears in association with the
shortest telomeres. We propose that these bands arise
through secondary annealing sites of the telomeric PETRA
primer to the region of the displacement loop (D-loop) at
the site of G-overhang invasion into the double-stranded
telomere region (see Fig. 10). Since the annealing occurs
under native conditions, at least a fraction of t-loops may
be preserved at the initial phase of PETRA. Subsequent
convergence of the sizes of the two products could then be
explained by tightening of the t-loop which is, however
critically limited by bendability of the chromatin fibre
(Fajkus and Trifonov 2001). Continued telomere shortening
below the critical lengths impedes further t-loop
formation (Forsyth et al. 2002), thus leading to a single
telomere product in PETRA.
In conclusion, our results suggest that telomere
dynamics in Attert-/knock-out
mutants can be explained
solely by involvement of ALT, without a necessity to presume TRD events. The ALT events appear to be timelimited,
probably to gametogenesis or early embryonic
development. The fast and efficient activation of ALT in
response to the loss of telomerase activity, and its probable
developmental regulation imply that ALT may be a normal
part of plant development.
Acknowledgements We thank Professor Andrew R. Leitch for
insightful comments and the manuscript revision, Eva Sy´korova´ and
Dagmar Zachova´ for helpful advice for cultivation of plant tissue
cultures. This work was supported by the Czech Ministry of Education
(MSM0021622415, LC06004), Czech Academy of Sciences
(AVOZ50040507) and grants from GACR (521/05/0055) and GA
ASCR (IAA600040505 and IAA601630703).
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Supplement C
Schrumpfová, P.P., Kuchar, M., Palecek, J., Fajkus, J., 2008. Mapping of interaction domains
of putative telomere-binding proteins AtTRB1 and AtPOT1b from Arabidopsis thaliana. FEBS
Lett. 582, 1400–1406
P.P.S. was significantly involved in the experimental part (more then 50 % of
experiments) and participated in the ms writing and editing
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Author's personal copy
Mapping of interaction domains of putative telomere-binding proteins
AtTRB1 and AtPOT1b from Arabidopsis thaliana
Petra Procha´zkova´ Schrumpfova´a,1
, Milan Kucharˇa,1
, Jan Palecˇeka
, Jirˇı´ Fajkusa,b,*
a
Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Faculty of Science, Masaryk University,
Kamenice 5, CZ-62500 Brno, Czech Republic
b
Laboratory of DNA-Molecular Complexes, Institute of Biophysics, Czech Academy of Sciences, Kra´lovopolska´ 135, CZ-61265 Brno, Czech Republic
Received 12 December 2007; revised 19 February 2008; accepted 2 March 2008
Available online 1 April 2008
Edited by Gianni Cesareni
Abstract We previously searched for interactions between plant
telomere-binding proteins and found that AtTRB1, from the single-myb-histone
(Smh) family, interacts with the Arabidopsis
POT1-like-protein, AtPOT1b, involved in telomere capping.
Here we identify domains responsible for that interaction. We
also map domains in AtTRB1 responsible for interactions with
other Smh-family-members. Our results show that the N-terminal
OB-fold-domain of AtPOT1b mediates the interaction with
AtTRB1. This domain is characteristic for POT1- proteins
and is involved with binding the G-rich-strand of telomeric
DNA. AtPOT1b also interacts with AtTRB2 and AtTRB3.
The central histone-globular-domain of AtTRB1 is involved with
binding to AtTRB2 and 3, as well as to AtPOT1b. AtTRB1-heterodimers
with other Smh-family-members are more stable than
AtTRB1-homodimers. Our results reveal interaction networks of
plant telomeres.
Structured summary:
MINT-6440051:
AtTRB1 (uniprotkb:Q8VWK4) physically interacts (MI:0218)
with AtTRB1 (uniprotkb:Q8VWK4) by two-hybrid (MI:0018)
MINT-6440068:
AtTRB2 (uniprotkb:Q8VX38) physically interacts (MI:0218)
with AtTRB1 (uniprotkb:Q8VWK4) by two-hybrid (MI:0018)
MINT-6440083:
AtTRB3 (uniprotkb:Q9M2X3) physically interacts (MI:0218)
with AtTRB1 (uniprotkb:Q8VWK4) by two-hybrid (MI:0018)
MINT-6440099:
AtPOT1b (uniprotkb:Q6Q835) physically interacts (MI:0218)
with AtTRB1 (uniprotkb:Q8VWK4) by two-hybrid (MI:0018)
MINT-6440119:
AtPOT1b (uniprotkb:Q6Q835) physically interacts (MI:0218)
with AtTRB2 (uniprotkb:Q8VX38) by two-hybrid (MI:0018)
MINT-6440138:
AtPOT1b (uniprotkb:Q6Q835) physically interacts (MI:0218)
with AtTRB3 (uniprotkb:Q9M2X3) by two-hybrid (MI:0018)
MINT-6440216:
AtPOT1b (uniprotkb:Q6Q835) physically interacts (MI:0218)
with AtTRB1 (uniprotkb:Q8VWK4) by coimmunoprecipitation
(MI:0019)
MINT-6440157:
AtTRB2 (uniprotkb:Q8VX38) physically interacts (MI:0218)
with AtTRB1 (uniprotkb:Q8VWK4) by coimmunoprecipitation
(MI:0019)
MINT-6440177:
AtTRB3 (uniprotkb:Q9M2X3) physically interacts (MI:0218)
with AtTRB1 (uniprotkb:Q8VWK4) by coimmunoprecipitation
(MI:0019)
Ó 2008 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Keywords: Plant; Telomere; Protein–protein interaction;
AtTRB1; AtTRB2; AtTRB3; AtPOT1b
1. Introduction
Telomere proteins play a role in the protection and maintenance
of chromosome ends. In human cells, the minimal functional
set of proteins participating in telomere protection is
collectively called ‘‘shelterin’’ [1]. Shelterin consists of three
proteins (TRF1, TRF2 and POT1) that directly recognize telomeric
DNA and are interconnected by at least three other proteins
(TIN2, TPP1 and Rap1), forming a telomere-specific
protective cap. Similar complexes are also likely to exist in
plants and these are particularly attractive to study due to
the telomerase-competent status (i.e., reversible telomerase
activity regulation) of plant somatic cells [2,3]. A number of
putative plant telomeric proteins have been found by homology
searches of DNA and protein sequence databases and
tested for their affinity to telomeric DNA sequences in vitro
(reviewed in [4]). There is however very little data relevant to
their telomeric function. Of the putative ‘‘plant shelterin’’ components,
functional data relevant to telomere homeostasis is
available for two Arabidopsis thaliana POT1-like proteins, AtPOT1a
and AtPOT1b. These proteins contain the oligonucleotide-binding
(OB) fold domain which binds to the G-rich
strand of telomeric DNA but their overall sequence similarity
is low (49%). The functions of AtPOT1a and AtPOT1b proteins
are different: AtPOT1a functions mainly in telomerase
regulation, while AtPOT1b contributes to chromosome endprotection
and genome stability [5–9].
Recently, another Arabidopsis protein, AtTBP1, has been
shown to be involved in telomere length regulation [10]. This
*
Corresponding author. Address: Department of Functional Genomics
and Proteomics, Institute of Experimental Biology, Faculty of
Science, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech
Republic. Fax: +420 549492654.
E-mail address: fajkus@sci.muni.cz (J. Fajkus).
1
First two authors contributed equally to this work.
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2008.03.034
FEBS Letters 582 (2008) 1400–1406
Author's personal copy
protein binds double-stranded telomeric DNA in vitro via a
characteristic Myb-like domain, referred to as a telobox, located
at its C-terminus [11,12]. To identify other components
of ‘‘plant shelterin’’, we analyzed a number of putative A. thaliana
telomere proteins for their mutual interactions. We previously
found that AtTRP1, the Arabidopsis myb-like protein
bearing a C-terminal telobox, interacts with AtKu70 [5], which
itself plays a role in plant telomere homeostasis [13,14]. Furthermore
AtTRP1 may be a functional homolog of mammalian
TRF2 [5]. In addition, an Arabidopsis POT1-like
protein, AtPOT1b, interacts with AtTRB1, a protein from
the single myb histone (Smh) family [5]. The Smh family is
characterised by a unique triple motif structure containing a
N-terminal myb-like domain, a central GH1/GH5 histone
globular domain and a C-terminal coiled-coil domain [15].
Proteins of this family in Arabidopsis show specific binding
to telomeric DNA and can form homo- and heteromeric protein–protein
complexes [16].
The abundance of candidate telomere proteins in plants,
arising from numerous paralogs of telomere-binding protein
and plant-specific proteins, coupled with an apparent absence
of some constitutive animals and fungi shelterin components,
makes imperative analyses of interactions between the candidate
plant telomere proteins. Using a combination of the yeast
two-hybrid system (Y2H) and co-immunoprecipitation
(CoIP), we characterise here the protein domains involved in
interactions between AtTRB1 and AtPOT1b, as well as domains
engaged in the formation of homomeric and heteromeric
complexes of AtTRB proteins.
2. Materials and methods
2.1. Cloning of full length proteins and their deletion variants for twohybrid
assay
An overview of cloned candidate telomeric DNA-binding proteins is
given in Table 1. cDNA sequences of AtTRB1, AtTRB2, AtTRB3 and
AtPOT1b have been cloned as described previously [16]. To localize
the interaction domains the deletion forms of AtTRB1 and AtPOT1b
were generated by PCR and cloned into the vector pGBKT7 or
pGADT7, respectively. Sequence-specific primers with restriction sites
were used for cloning individual cDNAs as shown in Table 2.
To localize the interaction domain(s) in AtTRB1, cDNA fragments
were cloned in pGADT7 and denominated according to primers used
(for example, the fragment F1R1 was generated using TRB1 F1 as forward
and TRB1 R1 as reverse primers – see Figs. 1A and 2B). Similarly,
AtPOT1b fragments were generated to localize the region of
AtPOT1b responsible for interaction with AtTRB1 (see Fig. 2C).
Prior to two-hybrid screening, cloned constructs were checked for
the correct reading frame and absence of mutations by DNA sequencing
on an ABI PRISM 310 sequencer (Perkin–Elmer).
2.2. Yeast two-hybrid (Y2H) system
Two strains of Saccharomyces cerevisiae, PJ69-4a and PJ69-4a were
used [17]. Protein AtPOT1b, its deletion variants and AtTRB2, AtTRB3
were expressed from the yeast vector pGBKT7 in strain PJ69-
4a, and AtTRB1 and its fragments from vector pGADT7 in strain
PJ69-4a. This division enabled proper combining of the proteins and
their deletion variants in interaction assays. Both strains, identical except
for the mating type, were mixed on Petri-dishes with YPD medium
(1.1% yeast extract, 2.2% bacteriological peptone, 2% glucose
and 2% agar) to fuse yeast haploid cells of different strains, and incubated
at 30 °C for 8–10 h. The diploid cells were printed by velvet
stamp onto control -Leu,-Trp selective plates (0.67% yeast nitrogen
base, 2% glucose, 0.12% amino acid mixture without Leu and Trp,
2% agar, pH adjusted by NaOH to 6.8) and then onto -Ade selective
plates to test the interaction (0.67% yeast nitrogen base, 2% glucose,
0.12% amino acid mixture without Ade, 2% agar, pH adjusted by
NaOH to 6.8) and were incubated at 30 °C for a few days until colonies
had grown. Alternatively, PJ69-4a cells were cotransformed with
both pGBKT7 and pGADT7 plasmids and grown on -Leu,-Trp plates.
Colonies were inoculated into YPD liquid medium and incubated at
30 °C overnight. Ten-times diluted aliquots were dropped onto both
-Ade and -His plates. For a semi-quantitative test, 5 ll aliquots were
dropped onto selective -His plates containing increasing concentrations
of 3-aminotriazol (3-AT). As the 3-AT inhibits His3 activity,
the ability of yeast cells to grow on higher concentrations correlates
with the higher binding affinity of the hybrid proteins.
To verify our results we also used the yeast strain MaV203, where
the His3-reporter gene is under a less tightly controlled promoter
(Invitrogen). The drop test was executed in the similar way as with
the PJ69 strain.
2.3. In vitro translation and co-immunoprecipitation
Proteins were co-expressed from the same constructs as were used in
Y2H system with an hemagglutinin tag (pGADT) or a myc-tag
(pGBKT) by use of the TNT Quick Coupled Transcription/Translation
System (Promega) in 15–25 ll of each reaction according to the
manufacturerÕs instruction. For Myc pull-down experiments, 15–
25 ll of in vitro-expressed proteins in total volume of 100 ll of HEPES
buffer (25 mM HEPES, 150 mM KCl, 50 mM NaCl, 3 mM MgCl2,
10% glycerol, 0.1% NP-40, 1 mM DTT, 2 mM PMSF, 2 lg/ll leupeptine,
1 lg/ll pepstatine) were mixed with 1 lg anti-Myc-tag polyclonal
antibody (Abcam) and incubated overnight at 4 °C (Input fraction).
10 ll of Protein G magnetic particles (Dynabeads, Invitrogen-Dynal)
were then added, and the mixture was incubated for 1 h/4 °C (UnTable
1
Overview of cloned proteins
Protein Protein group Characteristic domain GenBank accession number Reference
AtTRB1 dsDNA binding N-terminal Myb domain AAL73123 [16]
AtTRB2 Proteins AAL73441 [17]
AtTRB3 NP_190554
AtPOT1b ssDNA binding proteins Pot1 domain NP_196249 [5,9,23]
Table 2
Complete list of primers used for cloning
Primer Restriction
site
Sequence of primer (50
fi 30
)
POT1b F BamHI ATGGATCCTAATGGAGGAGGAGAGAAGAG
POT1b F1 BamHI ATGGATCCTAAAGATTGTGCTGATTAACC
POT1b F2 BamHI TAGGATCCACTTCTTATCGAATCTGAGAG
POT1b F3 BamHI TTGGATCCTTAAGTCAGAAAGGCTTC
POT1b R XhoI ATTCTCGAGTCATGAAGCATTGATCCAAG
POT1b R1 XhoI TTACTCGAGCCCTTCATCAGCATATAGAG
POT1b R2 XhoI TTACTCGAGCCTGTGATTTCAGAATGTG
POT1b R3 XhoI TTACTCGAGGGTTGAAGACAGTGAATG
POT1b R4 XhoI TTACTCGAGATCTTCAAACTTGTACGTG
POT1b R5 XhoI CTTCTCGAGGGTTAATCAGCACAATCTTTA
TRB1 F BamHI ATGGATCCGAATGGGTGCTCCTAAGCAG
TRB1 F1 BamHI CGGGATCCAAGATGCGACCTCTGGACTCC
TRB1 F2 BamHI GAGGATCCAAGGTCTGGGGGTGTTTGGA
TRB1 F01 BamHI CGGGATCCTAGTCATGGCAAATGGCTGG
TRB1 R XhoI TGGCTCGAGAGGCACGGATCATCATTTTG
TRB1 R1 BamHI TCGGATCCTCCAAACACCCCCAGACC
TRB1 R2 BamHI GAGGATCCGGAGTCCAGAGGTCGCATC
TRB1 R12 BamHI CAGGATCCGCGTTTGAAGTCTGGTGGAG
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bound fraction). The beads were washed five times with HEPES buffer
and then incubated with 10 ll of SDS-loading buffer for 10 min/85 °C
to elute bound proteins (Bound fraction). Input, unbound, and bound
fractions were separated by 12% SDS–PAGE and analyzed by
STORM860 (GE Healthcare).
3. Results
3.1. Interactions between AtTRB proteins
We have shown previously that the telomere-binding proteins
AtTRB1, AtTRB2, and AtTRB3 form homodimeric
and heterodimeric complexes in Y2H assays [5,16]. These proteins
are similar to each other at the level of amino acid sequence
and belong to the same family of Smh proteins [15].
In contrast to other myb-like telomere-binding proteins, in
Smh proteins the myb-like domain is N-terminal. The myb-like
domain is followed by GH1/GH5 histone globular domain and
a C-terminal coiled-coil domain (Fig. 1A). Interactions of
AtTRB proteins were expected to be mediated by the C-terminal
coiled-coil domain because this domain supports protein
oligomerization [18]. Therefore, we designed two deletion mutants
AtTRB1 F1R (aa position 101–300) and AtTRB1 F2R
(196–300) that comprised the C-terminus. Each construct
was transformed into PJ69-4a two-hybrid strain and crossed
with PJ69-4a containing full-length (FL) AtTRB constructs.
Only AtTRB1 F1R construct containing both coiled-coil and
Fig. 1. Histone GH1/GH5 globular domain of AtTRB1 binds to AtTRB1, 2 and 3 protein. (A) The AtTRB1 protein contains a myb-like domain
(myb-like), followed by GH1/GH5 histone globular domain and C-terminal coiled-coil domain (coiled-coil). Full-length (FL) AtTRB1, F1R (aa 101–
300), FR1 (aa 1–201), F01R1 (aa 58–201), FR12 (aa 1–159), F01R12 (aa 58–159), F2R (aa 196–300), FR2 (1–106), F1R1 (aa 101–201) fragments (in
PJ69-4a yeast strain) are combined with AtTRB1 FL, AtTRB2 FL, AtTRB3 FL two-hybrid constructs (in PJ69-4a yeast strain) and the diploid cells
are tested on -Ade plates (-Ade) for protein–protein interactions (top and middle panel). Only fragments containing the GH1/GH5 histone globular
domain interact with all three AtTRB proteins. The PJ69-4 cells containing the F1R1 construct are also tested on -His plates with 2 mM
concentration of 3-AT (-His/2 mM). Only the interactions of F1R1 with AtTRB2 and AtTRB3 are detectable (bottom panel). Empty pGBKT7 (right
panel) and pGADT7 vectors are used as negative controls. (B) AtTRB2 and AtTRB3 proteins are able to pull-down F1R1 fragment. The TNT
expressed full-length AtTRB1 FL (lanes 1–3), AtTRB2 FL (lanes 4–6) and/or AtTRB3 FL (lanes 7–9) proteins were mixed with AtTRB1 GH1/GH5
fragment (F1R1, lanes 1–12) and incubated with anti-myc antibody overnight. Then protein G magnetic beads were added and proteins were
immunoprecipitated for 1 h. In the control experiment, the F1R1 fragment was incubated with antibody and beads in the absence of partner protein
(lanes 10–12). Input (I), unbound (U), and bound (B) fractions were collected and run in SDS–12% PAGE gels.
1402 P.P. Schrumpfova´ et al. / FEBS Letters 582 (2008) 1400–1406
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GH1/GH5 histone globular domain supported the growth of
PJ69-4 diploid strain on -Ade plates (Fig. 1A, top panel), suggesting
that the interactions are not mediated by the coiled-coil
region. Instead, interactions between all three AtTRB proteins
and fragments containing the GH1/GH5 histone globular domain
were observed, in particular AtTRB1 FR1 (1–201), AtTRB1
F01R1 (58–201) and AtTRB1 F1R1 (101–201).
Fragments containing truncated or completely deleted GH1/
GH5, as in the AtTRB1 FR2 (1–106), AtTRB1 F01R12 (58–
159) and AtTRB1 FR12 (1–159) constructs, lost the interaction
with AtTRB1, as well as with the other AtTRB proteins.
Thus, the shortest AtTRB1 fragment displaying the interaction
with AtTRB proteins is F1R1 (101–201), which contains a
GH1/GH5 domain and short flanking regions (Fig. 1A, middle
panel).
The PJ69-4 cells containing the F1R1 construct were also
grown on -His plates with increasing concentrations of
3-amino-1,2,3-triazol (3-AT) to compare binding affinities to
Fig. 2. Histone GH1/GH5 globular domain of AtTRB1 binds to the N-terminus of the AtPOT1b protein. (A) Full-length AtPOT1b FL interacts
with AtTRB1 FL, AtTRB2 FL and AtTRB3 FL in two-hybrid assay when scored for growth on -His plates (for Y2H details see Fig. 1A). (B) Yeast
two-hybrid cells containing FR1 (aa 1–201), F01R1 (aa 58–201), FR12 (aa 1–159) fragments are combined with AtPOT1b FL and tested on -Ade
plates for protein–protein interactions. Only fragments containing the GH1/GH5 histone globular domain interact with the AtPOT1b protein. (C) In
the co-immunoprecipitation assay, the TNT expressed AtTRB1 FR1 fragment (lanes 1–6) was mixed with full-length AtPOT1b FL protein (lanes 4–
6) and incubated with anti-myc antibody (same conditions as in Fig. 1B). In the control experiment, the FR1 fragment was incubated with antibody
and beads in the absence of the partner protein (lanes 1–3). (D) The PJ69-4a cells containing AtTRB1 fragment F1R1 (aa 101–201) were
cotransformed with full-length (FL) and/or following fragments F2R (aa 135–454), FR4 (aa 1–150), FR5 (aa 1–90) of AtPOT1b. Transformants
containing FL, FR4 and FR5 fragments grow on -Ade plate (first column), however, a weak self-activation can be seen with FR4 fragment on a
control plate (second column). When these cells were grown on -His plates with increasing concentrations of 3-AT the addition of 3-AT to 5 mM
concentration abolishes the self-activation of FR4 (fourth column) while keeping its specific interaction with the F1R1 fragment of AtTRB1 (third
column). These results suggest that the AtTRB1-AtPOT1b interaction is mediated by the binding of the GH1/GH5 domain of the AtTRB protein to
the N-terminus (bearing the OB-fold domain) of the AtPOT1b protein.
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different AtTRB proteins (data not shown). At 2 mM 3-AT
only the interactions of F1R1 with AtTRB2 and AtTRB3 were
detected (Fig. 1A, bottom panel). To verify these results we
used another two-hybrid strain (MaV203 strain has His3-reporter
gene under a less tightly controlled promoter). When
the full-length AtTRB1 or its FR1 fragment (covering both
myb-like and GH1/GH5 domains) was used in the His-reporter
assay, interactions with all three AtTRB proteins were positive
up to 5 mM 3-AT. A further increase of 3-AT to 20 mM
resulted in a loss of interaction with AtTRB1, while keeping
interactions with AtTRB2 and 3 (Supplementary Fig. S1).
Altogether, these data suggest that heterotypic complexes of
AtTRB1 are more stable than homotypic ones.
To test the above Y2H results by an independent approach,
co-immunoprecipitation (CoIP) assays were performed with
the above AtTRB1 fragments. In particular, interactions were
assayed between F1R1 and the three AtTRB proteins. Fig. 1B
shows that the myc-tagged AtTRB2 and AtTRB3 proteins are
able to pull-down the F1R1 fragment while the full-length
AtTRB1 is not. These results confirm a low affinity of the
GH1/GH5 domain of AtTRB1 to full-length AtTRB1 (insufficient
to provide a positive result in CoIP) and a higher affinity
to both AtTRB2 and AtTRB3.
3.2. AtTRB1 interaction with AtPOT1b
The protein AtPOT1b is thought important for ‘‘chromosome
capping’’ in A. thaliana and interactions previously detected
with AtTRB1 may be of functional significance [5,6].
Fig. 2A shows that the interaction of AtPOT1b is not limited
to AtTRB1, but also occurs with AtTRB2 and AtTRB3 proteins
(Fig. 2A).
For mapping the interaction between the AtTRB1 and AtPOT1b
the same AtTRB1 fragments in PJ69-4a cells (see
Fig. 1A) were crossed with PJ69-4a cells containing full-length
AtPOT1b. Only the diploid cells with AtTRB1 fragments containing
the GH1/GH5 domain grow on -Ade plates (Fig. 2B
and D; data not shown). These results suggest a role of the
GH1/GH5 domain in binding to AtPOT1b.
CoIP assays were performed using the FR1 fragment of
AtTRB1 and the full-length AtPOT1b. The FR1 fragment
co-precipitated with the myc-tagged AtPOT1b protein
(Fig. 2C). This positive result confirms the above findings obtained
by Y2H.
In the case of AtPOT1b the following fragments were generated
and cloned into pGBKT7: POT1b F2R (135–454), POT1b
FR4 (1–150) and POT1b FR5 (1–90). The PJ69-4a cells containing
AtTRB1 fragment F1R1 were cotransformed with
Fig. 3. Schematic depiction of the principal telomere components and their interactions in humans and plants. In humans (A), the complex of
ubiquitously present telomere-associated proteins, termed as shelterin [1], consists of two components that can bind telomeric dsDNA (TRF1,
TRF2), and recruit the shelterin components TIN2, TPP1 and Rap1. The sixth partner in shelterin is the single-stranded TTAGGG repeat-binding
protein, POT1. In addition to its binding to the G-strand of telomeric DNA, it can associate with telomeres also through its interaction with TPP1.
Transitions between these states are illustrated with arrows. Examples of functionally important interactions (with Ku-proteins and telomerase) are
also shown. In plants (B), a number of TRF-like proteins, exemplified here by AtTRP1, have been identified which are able to form homodimers and
bind telomeric dsDNA with their C-terminal telobox Myb-like domain [11,12]. Analogous to TRF2, AtTRP1 is able to interact with AtKu70 [5],
providing thus (in similarity to other organisms) association of Ku-heterodimer with telomeres via protein–protein interaction, in addition to its
possible direct DNA-binding. Besides the TRF-like proteins, plant possess the SMH family of proteins which are characterised by the N-terminal
myb-like domain, a central GH1/GH5 histone globular domain and a C-terminal coiled-coil domain [15]. These proteins form both homomeric and
heteromeric complexes among each other using their central GH1/GH5 domain. This domain can also bind DNA in a sequence-non-specific manner
(not shown), the interaction possibly important to avoid protein aggregation or telomere chromatin folding [23]. In addition, SMH proteins can
interact (using the same GH1/GH5 domain) with AtPOT1b [5], one of the POT1-like proteins in Arabidopsis thaliana, which participates in telomere
end-protection [6]. It is noteworthy that AtPOT1b uses the same domain (N-terminal OB-fold) for interaction with both telomeric ssDNA, and
AtTRB proteins. Transitions of AtPOT1b between its DNA- and SMH-associated state (arrows) may be important for AtPOT1b recruitment to
telomeres and for its protective function. The other AtPOT1 paralog, AtPOT1a, possibly functions in telomerase regulation and recruitment [7,8,22].
The presence of two functionally divergent POT1-like proteins in Arabidopsis is similar to the situation in mice [24]. Only linear telomere
conformation is shown and nucleosomes are not depicted for simplicity.
1404 P.P. Schrumpfova´ et al. / FEBS Letters 582 (2008) 1400–1406
Author's personal copy
full-length (FL) and/or fragments of AtPOT1b. Only transformants
containing FL, FR4 and FR5 fragments grew on -Ade
plates (Fig. 2D), however, a weak self-activation can be seen
with fragment FR4 on the control plate. When these cells were
grown on -His plates with increasing concentrations of 3-AT
the addition of 3-AT to 5 mM concentration abolished the
self-activation of FR4 whilst keeping its specific interaction
with the F1R1 fragment of AtTRB1 (Fig. 2D, right panel).
These results suggest that the AtTRB1-AtPOT1b interaction
is mediated by the binding of GH1/GH5 domain of the AtTRB
protein to the N-terminus (bearing the OB-fold domain) of the
AtPOT1b protein.
4. Discussion
Our results confirm previously published interactions between
AtTRB proteins [16] and interactions between AtTRB1
and AtPOT1b [5]. They also provide a detailed map of those
interactions. Interactions between AtPOT1b and other members
of the Smh family (AtTRB2 and 3) are newly reported
here. The function of AtPOT1b in chromosome protection
and genome stability [6], highlights the importance of its
interactions. AtTRB proteins, the only interaction partners of
AtPOT1b identified so far, are representatives of the plant-specific
Smh family of telomere-binding proteins. They have an
N-terminal myb-like domain (instead of a usual C-terminal position
in TRF-like proteins), and a central GH1/GH5 domain.
The results of the assays show that AtTRB1 uses the central
GH1/GH5 histone globular domain for interaction with AtTRB
proteins and AtPOT1b. The GH1 and GH5 sub-domains
are members of the Ôwinged helixÕ class of DNA-binding domains,
although in contrast to other members of the family,
they contain a distinct, additional cluster of positively charged
amino acids. These residues form a second DNA-binding surface
on the opposite side of the protein to the primary DNAbinding
site [19]. Besides the ability to bind DNA, the GH5 domain
is able to self-associate [20], which mediates AtTRB1 selfinteraction
and interactions with AtTRB2, AtTRB3 and AtPOT1b.
Possibly, the weak interactions of the GH1/GH5 histone
globular domain in AtTRB1 are of a similar nature to
GH5 hydrophobic protein–protein interactions described previously
[20]. The GH1/GH5 histone globular domain can also
bind DNA in a sequence-non-specific manner, while their Nterminal
Myb-like domain [16] provides the sequence-specific
binding of AtTRB to telomeres (Mozgova et al., submitted
for publication).
The use of the same domain for interacting with different
proteins may be of functional importance. It is not only
AtTRB1 which uses the same region to interact with all AtTRB
proteins and AtPOT1b, but this is also observed for the
N-terminus of AtPOT1b, which bears the OB-fold domain.
This domain is thought to be involved in binding to telomere
DNA and interacting with AtTRB proteins. These overlapping
functions may be part of a regulatory mechanism, similar to
that provided by binding properties of the components of
the mammalian shelterin complex (Fig. 3). In the latter complex,
there is a dynamic balance between POT1 bound directly
to the telomere (to telomeric DNA) and via protein–protein
interactions [21]. In plant shelterin, differences in expression
levels of the proteins (see Supplementary figure S2) may also
participate in modulation of telomere metabolism in a tissueand
developmental stage-specific manner. The observed differential
tendency of AtTRB1 to form homomeric and heteromeric
complexes with AtTRB proteins, plus the ability of
AtPOT1b to form complexes with all three tested AtTRB proteins
and the presence of two functionally divergent AtPOT1
proteins in A. thaliana [6–8,22] suggest that plant shelterins
are highly complex and carry features not found in animal
and fungal shelterin.
Acknowledgements: We thank Lumı´r Krejcˇı´ for suggestions regarding
the two-hybrid techniques and Andrew Leitch (Queen Mary University,
London) for critical reading of the manuscript. This work was
supported by the Grant Agency of the Czech Republic (projects 521/
05/0055 and 521/08P452), Czech Ministry of Education (LC06004)
and the institutional support (MSM0021622415 and AVOZ50040507).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.febslet.
2008.03.034.
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Supporting Information
Supplementary Figure S1. AtTRB1 forms preferentially heterodimers with AtTRB2 and 3. Yeast two‐
hybrid strain MaV203 (with His3‐reporter gene under less tightly controlled promoter) was
cotransformed with either full‐length AtTRB1 or FR1 fragment and with either of AtTRB FL constructs
(for further details see Fig. 1). Drop test was performed with ‐His plates containing 5 mM and/or 20
mM 3‐AT. At the 20 mM 3‐AT concentration only AtTRB2 and AtTRB3 transformants grew suggesting
that heterotypic complexes of AtTRB1 are more stable than homotypic ones.
Supplementary Figure S2. Gene expression in various organs of A. thaliana. A. Gene expression of
AtTRB1, 2 and 3 proteins (=SMH). Original data from chip expression database AtGenExpress were
graphically transformed in Arabidopsis Gene Family Profiler (http://agfp.ueb.cas.cz) and modified for
this article. B. Gene expression data for AtPOT1b protein are not available in any of general chip
database. A schematic table of AtPOT1b expression is based on RT‐PCR data published in [6].
105
Supplement D
Mozgová, I., Schrumpfová, P.P., Hofr, C., Fajkus, J., 2008. Functional characterization of
domains in AtTRB1, a putative telomere-binding protein in Arabidopsis thaliana.
Phytochemistry 69, 1814–1819
P.P.S. participated in the experiments design, evaluation of data and the ms editing
This journal did not provide open access, hence the article is not freely available.
Supplement E
Hofr, C., Sultesová, P., Zimmermann, M., Mozgová, I., Schrumpfová, P.P., Wimmerová, M.,
Fajkus, J., 2009. Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study
of telomere-binding specificity and kinetics. Biochem. J. 419, 221–228
P.P.S. was involved in the experimental part (protein cloning, expression and
purification)
Biochem. J. (2009) 419, 221–228 (Printed in Great Britain) doi:10.1042/BJ20082195 221
Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study
of telomere-binding specificity and kinetics
Ctirad HOFR*1
, Pavla ˇSULTESOV´A*, Michal ZIMMERMANN*, Iva MOZGOV´A*, Petra PROCH´AZKOV´A SCHRUMPFOV´A*,
Michaela WIMMEROV´A† and Jiˇr´ı FAJKUS*‡1
*Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Faculty of Science, Masaryk University, CZ-62500 Brno, Czech Republic, †National Centre for
Biomolecular Research and Department of Biochemistry, Faculty of Science, Masaryk University, CZ-61137 Brno, Czech Republic, and ‡Laboratory of DNA–Molecular Complexes,
Institute of Biophysics, Czech Academy of Sciences, CZ-61265 Brno, Czech Republic
Proteins that bind telomeric DNA modulate the structure of chromosome
ends and control telomere function and maintenance.
It has been shown that AtTRB (Arabidopsis thaliana telomererepeat-binding
factor) proteins from the SMH (single-Mybhistone)
family selectively bind double-stranded telomeric DNA
and interact with the telomeric protein AtPOT1b (A. thaliana
protection of telomeres 1b), which is involved in telomere
capping. In the present study, we performed the first quantitative
DNA-binding study of this plant-specific family of proteins.
Interactions of full-length proteins AtTRB1 and AtTRB3 with
telomeric DNA were analysed by electrophoretic mobility-shift
assay, fluorescence anisotropy and surface plasmon resonance to
reveal their binding stoichiometry and kinetics. Kinetic analyses
at different salt conditions enabled us to estimate the electrostatic
component of binding and explain different affinities of the two
proteins to telomeric DNA. On the basis of available data, a
putative model explaining the binding stoichiometry and the
protein arrangement on telomeric DNA is presented.
Key words: Arabidopsis thaliana, fluorescence anisotropy,
kinetics, single-Myb-histone protein (SMH protein), surface
plasmon resonance, telomere protein–DNA interaction.
INTRODUCTION
Telomeres are nucleoprotein complexes consisting of repetitive
DNA sequences, general chromatin proteins and telomere-specific
proteins. Tandem repeats of telomeric DNA are short T- and G-rich
sequences, such as d(GGGTTA) in humans and d(GGGTTTA) in
the majority of plants.
Telomeres form protective capping structures at the ends of
chromosomes [1]. These structures are essential for cell viability
as they prevent chromosomes from unwanted end-to-end joining
and recognition of chromosome tips as unrepaired double-strand
breaks by the repair system of the cell. Changes in telomere
structure and function induce chromosomal abnormalities and are
directly connected with human aging and cancer [2].
Telomeres are usually maintained by telomerase, a ribonucleoprotein
enzyme that adds telomeric repeats to the 3 -overhang of
the G-rich DNA strand. The action of telomerase is regulated by
its expression and by numerous proteins that control telomerase
access to telomeres and organize telomeres into specific capping
structures, such as telomeric loops that were observed in a number
of organisms, including humans and plants [3,4].
Three DNA-binding proteins have been found to be responsible
for specific recognition and direct interactions with the telomeric
repeat sequence in humans. Two of them, TRF1 and TRF2 (where
TRF is telomeric repeat-binding factor), described as negative
regulators of telomere length [5], show substantial structural
similarity and bind double-stranded telomeric DNA. The third
protein, POT1 (protection of telomeres 1), binds the G-rich strand
of telomeric DNA, participates in chromosome capping and is
able to control telomere extension by telomerase, both positively
and negatively [6,7]. The human TRFs and their homologues in
other organisms possess a well conserved DNA-binding structural
motif similar to the c-Myb-family of transcriptional activators [8].
The Myb domain of TRFs is C-terminally positioned and consists
of three helices connected in a helix–turn–helix manner. The third
helix contains a conserved amino acid sequence called a ‘telobox’,
which has been shown to be important for recognition of telomeric
double-stranded DNA [8].
Numerous TRF-like proteins have been identified in plants
(reviewed in [9]), and, in a few cases, the influence of these
proteins on telomere length homoeostasis has been demonstrated
[10,11]. Interestingly, besides the TRF-like proteins, a plantspecific
family of other telobox proteins has been described [12].
This group of proteins, termed the SMH (single-Myb-histone)
family, is characterized by a triple-domain structure consisting of
an N-terminal Myb domain, central globular histone H1/5 domain,
and a C-terminal coiled-coil domain. In Arabidopsis thaliana (At),
five SMH proteins were identified (AtTRB1–AtTRB5, where
TRB is telomere-repeat-binding factor) [12], and three of them
have been characterized [13,14]. These proteins show not only
specific interactions with telomeric DNA, but also a number of
protein–protein interactions functionally related to telomeres. In
addition to their ability to form homodimers (similarly to TRFs),
they can also form heterodimers and both homo- and heterotypic
multimers [13–15] via their H1/5 histone domain. They also
interact (using the same H1/5 domain) with one of the POT1
proteins in A. thaliana, AtPOT1b [15,16], which participates in
telomere capping [17].
The emerging complexity of interactions of AtTRBs urges
more detailed and quantitative studies of their DNA–protein and
protein–protein interactions to reveal principles of their regulatory
role. So far, only structural data for the Myb DNA-binding
Abbreviations used: At, Arabidopsis thaliana; EMSA, electrophoretic mobility-shift assay; FA, fluorescence anisotropy; LB, Luria–Bertani; POT1,
protection of telomeres 1; RedX, Rhodamine Red-X; RT, reverse transcription; SMH, single-Myb-histone; SPR, surface plasmon resonance; TRB, telomererepeat-binding
factor; TRF, telomeric repeat-binding factor.
1
Correspondence may be addressed to either of these authors (email hofr@sci.muni.cz or fajkus@sci.muni.cz).
c The Authors Journal compilation c 2009 Biochemical Society
www.biochemj.org
BiochemicalJournal
222 C. Hofr and others
domain are available [18]. Similarly, kinetic studies are limited
to the interaction of a Myb-domain-bearing fragment with a
short telomeric DNA oligonucleotide (13 bp) [18], and a nonequilibrium
technique was used to describe binding kinetics of
TRFs in rice [19]. In order to describe binding interactions more
thoroughly, associations of the full-length proteins with telomeric
DNA need to be evaluated.
The equilibrium binding kinetics of the full-length proteins can
be studied by quantitative biophysical approaches. The binding
of proteins to fluorescently labelled DNA may be monitored by
FA (fluorescence anisotropy). This method gives well-resolved
binding isotherms at different buffer conditions and therefore
reliable kinetic and energetic parameters of binding. If the solution
contains only free DNA molecules, FA is relatively low, owing
to the fast rotational rearrangement of DNA molecules. After
the binding of protein to DNA, a bulky slower-rotating protein–
DNA complex is formed and the anisotropy is increased. Thus
the anisotropy change of fluorescently labelled DNA duplexes,
after each addition of protein into solution, describes the extent
of protein–DNA binding [20,21].
In the present paper, we report a detailed study to reveal
stoichiometry and kinetics of AtTRB1 and AtTRB3 binding
to telomeric DNA. Proteins AtTRB1 and AtTRB3 have been
chosen for these functional assays because they showed the
highest structural stability within the AtTRB family of proteins.
Interactions of full-length proteins with telomeric DNA are
analysed by a combination of EMSA (electrophoretic mobilityshift
assay) and quantitative biophysical methods employing
FA and SPR (surface plasmon resonance). Kinetic analyses at
different salt conditions enable us to estimate the electrostatic
component of binding and explain different affinities of the
two AtTRBs to telomeric and non-telomeric DNA. The kinetic
measurements also contribute to the estimation of the length of
double-stranded DNA for proper protein binding. On the basis
of these data, a speculative model for binding stoichiometry and
protein arrangement on telomeric DNA is presented.
EXPERIMENTAL
Cloning, expression and purification of AtTRB1 and AtTRB3
The cDNA sequence of AtTRB1 (locus At1g49950) was obtained
by RT (reverse transcription)–PCR from total RNA as described
previously [16]. AtTRB1 has been cloned into pET15b vector
(Novagen) and expressed as a His-tagged fusion protein in
Escherichia coli C41(DE3) cells [14]. The cells were grown on
LB (Luria–Bertani) medium with ampicillin (100 μg/ml) at 37◦
C
overnight. The next day, cells were diluted 20-fold into ZYM
5052 complex autoinducing medium containing ampicilin [22].
The cells were incubated at 37◦
C for 5 h. Then the temperature
was set to 20◦
C, and the incubation continued overnight.
The cDNA sequence of AtTRB3 (locus At3g49850) was
obtained by RT–PCR from total RNA as described previously
[13]. AtTRB3 has been cloned into pET30a(+) vector (Novagen)
and expressed as a His-tagged fusion protein in E. coli
BL21(DE3)pLysS cells. The cells were grown on LB medium
with kanamycin (50 μg/ml) at 37◦
C for 4 h. At a D600 of 0.6,
the overexpression of AtTRB3 was induced by the addition of
IPTG (isopropyl β-D-thiogalactoside) to a concentration of 1 mM.
After lowering the incubation temperature to 25◦
C, the growth
continued for an additional 3 h.
The following extraction and purification steps were the same
for both recombinant proteins. After harvesting by centrifugation
at 8000 g for 8 min, the pellet was dissolved in buffer containing
50 mM sodium phosphate (pH 8.0) with 300 mM NaCl and
Figure 1 Proteins and oligonucleotide duplexes used for binding studies
(a) Organization of the AtTRB1 and AtTRB3 polypeptide chains. The localization of the Myb
domain, histone-like H1/5 domain and coiled-coil domain is shown together with numbers
denoting their positions in the sequence. (b) Base sequence of telomeric oligonucleotide duplex
R4 and R2 along with non-telomeric duplex N. RedX denotes fluorescent label Rhodamine RedX.
The nucleotides of putative Myb-domain-binding sites are shaded grey [18].
10 mM imidazole and was sonicated for 5 min. The sonicated
cell extract was cleared by centrifugation at 14000 rev./min for
1 h at 4◦
C using a Beckman JA 14 rotor and subsequent filtration
(0.45 μm filter). Affinity purification was performed on a column
filled with a TALON® metal-affinity resin (BD Biosciences).
Protein was eluted at 80 mM imidazole. The eluent was loaded on
to a heparin HiTrapTM
column (GE Healthcare). A concentration
gradient of NaCl from 0.4 to 1 M NaCl was used for protein
elution. The fractions containing pure protein were concentrated,
and buffer-exchanged usually into 50 mM sodium phosphate
(pH 7.5) with 100 mM NaCl by ultrafitration (Amicon 10K,
Millipore) or by extensive dialysis. A typical yield was 1 mg of
purified protein per litre of bacterial culture. The concentration
of purified protein was determined using the Bradford assay [23].
DNA substrates
Oligodeoxynucleotides were synthesized and HPLC-purified by
Core Laboratory at Masaryk University. One of the strands in the
duplexes was synthesized with the 3 -end C6 aminoalkyl linker
and labelled with RedX (Rhodamine Red-X) (Molecular Probes)
using the protocol provided by the manufacturer. The duplexes
comprising four and two telomeric repeats were denoted as R4 and
R2 respectively. The DNA duplex with non-telomeric sequence
was denoted as N. The molar absorption coefficients of the single
strands were estimated with the employment of phosphate assay
[24]. Molar absorption coefficients were 281000 (RedX-labelled
strand in R4), 278000 (complementary strand in R4), 148000
(RedX-labelled strand in R2), 140000 (complementary strand in
R2), 284000 (RedX-labelled strand in N) and 265000 M−1
· cm−1
(complementary strand in R2) for DNA oligonucleotides shown
in Figure 1.
EMSA
Protein–DNA-binding reactions were performed in 10 μl volumes
containing the same amount of labelled DNA duplex
(30 pmol) and various concentrations of protein (0–180 pmol)
in 50 mM sodium phosphate (pH 7.0) with 200 mM NaCl.
c The Authors Journal compilation c 2009 Biochemical Society
DNA-binding kinetics of SMH proteins 223
Reaction mixtures were incubated for 10 min at 25◦
C. Protein–
DNA complexes were resolved on horizontal 7.5% (w/v)
acrylamide/0.3% bisacrylamide gels, as described in [25]. The
electrophoresis proceeded at 1.5 V/cm for 30 min and for an
additional 90 min at 3 V/cm. Gels were analysed with a LAS
3000 imaging system (Fujifilm). After the fluorescence imaging,
Coomassie Blue staining of the gel was performed to reveal
protein-containing bands in the gel.
Fluorescence anisotropy
Florescence anisotropy was measured on a FluoroMax-4
spectrofluorimeter (Horiba) with an L-format set up under
control of an Origin-based FluorEssence software (version 2.1.6).
Excitation and emission wavelengths were 572 and 591 nm
respectively, with the same excitation and emission bandpath,
8 nm. The integration time was 3s. For each anisotropy value,
five measurements were averaged. The titration experiments
were carried out in a 10 mm × 4 mm quartz-glass cuvette with
a magnetic bar stirrer. All measurements were conducted at 25◦
C
in 50 mM sodium phosphate buffer (pH 7.5) containing 100 mM
NaCl if not stated otherwise. To 1500 μl of DNA solution (20 nM)
in the buffer, protein solution was added stepwise. The decrease in
DNA concentration during the titration was taken into account
in the analysis of the data. A control titration of protein to RedX
solution (without DNA) has been performed to confirm that there
was no interaction between RedX and protein.
Dissociation constants of protein binding were evaluated by
fitting of dilution-corrected binding isotherms using programs
SigmaPlot 8 (Systat Software) and DynaFit3 (version 3.28)
[26]. Analysis of the binding of protein to DNA duplexes was
performed with the assumption of a non-co-operative binding
mode. The association constants were calculated as reciprocal
values of dissociation constants (Ka = 1/Kd). The association
constants provided the free energies of association.
Electrostatic component of binding
In order to determine the contribution of electrostatic interactions
upon binding of DNA with protein, the equilibrium binding
constant was measured at different concentrations of NaCl (see
Figure 4 and Table 2). The electrostatic component of binding
originates from the formation of ion pairs between the cationic
amino acid residues of the protein and the negatively charged
DNA. The number of ion pairs formed upon protein–DNA binding
and corresponding electrostatic contribution to overall binding
affinity (Ka) could be derived from the dependence of the binding
constant on salt concentration according to the eqn (1):
log Ka = log Ka
nel
− Zϕ · log [NaCl] (1)
where Z is the number of DNA phosphates that interact with the
protein, ϕ is the number of Na+
cations per phosphate released
upon protein binding. For B–DNA duplexes of 24 bp and shorter,
the value for ϕ is approx. 0.64 [27]. The right-hand side of the
equation divides overall binding affinity into the non-electrostatic
part described by Ka
nel
and a salt-dependent electrostatic part
[28,29]. When the linear dependence of log Ka is extrapolated to
the salt concentration of 1 M, the electrostatic term in eqn (1) can
be removed: log Ka = log Ka
nel
, i.e. the binding affinity is given
only by non-electrostatic interactions. Similarly to the binding
affinity, the overall binding energy defined as Ga = −2.3RT · log
Ka could be divided into electrostatic and non-electrostatic terms
Ga = Ga
nel
+ Ga
el
. The electrostatic term Ga
el
disappears
when the salt concentration approaches 1 M and the overall
energy of binding is given only by the non-electrostatic term,
Ga = Ga
nel
= −2.3RT · log Ka
nel
.
Surface plasmon resonance
Sensorgrams were recorded on a Biacore 3000 instrument (GE
Healthcare) using CM5 chips. More details are available in the
Supplementary Online Data at http://www.BiochemJ.org/bj/419/
bj4190221add.htm.
RESULTS
Stoichiometry of protein–DNA complexes
In order to estimate the binding ratio of AtTRBs and DNA,
oligonucleotide substrates containing two or one putative binding
sites were designed. The telomeric duplex R4 covers the length
of four plant telomeric repeats and comprises two putative Mybdomain-binding
sites. The shorter duplex, double-stranded DNA
fragment R2, consists of two telomeric repeats and contains
one Myb-domain-binding site. For comparative purposes,
oligonucleotide duplex N, as a representative of non-telomeric
DNA, was used in the present study (Figure 1).
Both AtTRB1 and AtTRB3 bind telomeric DNA with the
stoichiometry of one protein monomer per one telomeric repeat
The binding stoichiometry was analysed by EMSA with samples
containing a variable protein/DNA ratio. Figure 2 shows fluorescently
visualized bands indicating the mobility of free and
protein-bound DNA duplexes in non-denaturating acrylamide
gels.
Increasing the concentration of protein shifted the free labelled
DNA duplex to a new position corresponding to a protein–DNA
complex. The band corresponding to the free duplex R4
disappeared when the AtTRB1/R4 ratio was 4:1 (Figure 2a).
Similarly, the complete binding of AtTRB3 to substrate R4
was observed at the same protein/DNA ratio (Figure 2b). Both
AtTRB1 and AtTRB3 bind telomeric DNA with the stoichiometry
of one protein monomer per one telomeric repeat.
In order to characterize interaction stoichiometry of AtTRBs
with telomeric DNA further, proteins were allowed to interact with
the shorter substrate R2 bearing two telomeric repetitions
(Figure 2c). The results of EMSA with R2 demonstrate that a
2-fold decrease in the length of DNA reduces the protein/DNA
binding ratio proportionally. These results confirmed that the
stoichiometry of binding is one monomer of AtTRB1 or AtTRB3
per one telomeric repeat. If we consider binding of protein in
dimeric form, as was shown in our recent study [14], then two
protein dimers bind one R4 substrate (four telomeric repeats) or,
in other words, one dimer of AtTRB binds the fragment R2 (two
telomeric repeats). On the basis of these data, we could rephrase
our initial statement regarding stoichiometry to the following
form: one dimer of AtTRB binds the region of two telomeric
repeats.
AtTRB1 shows the same binding stoichiometry for telomeric and
non-telomeric DNA sequences, whereas AtTRB3 exhibits different
binding capacities for telomeric and non-telomeric DNA sequences
The effect of DNA sequence on binding ability of AtTRB1
and AtTRB3 was analysed by comparing the protein/DNA ratio
needed for complete saturation of telomeric R4 and non-telomeric
N substrate. In this respect, AtTRB1 behaves similarly in both
cases; the binding stoichiometry of AtTRB1 remained the same,
as demonstrated in Figure 2(a).
c The Authors Journal compilation c 2009 Biochemical Society
224 C. Hofr and others
Figure 2 Non-denaturing EMSA
(a) AtTRB1 binding to fluorescently labelled oligonucleotide R4 with telomeric sequence and
oligonucleotide N with non-telomeric sequence. (b) AtTRB3 binding to DNA oligonucleotides R4
and N. (c) AtTRB1 or AtTRB3 binding to oligonucleotide R2 with the sequence of two telomeric
repetitions. The DNA oligonucleotides and AtTRBs were incubated with increasing amounts of
protein. The numbers under electrophoretic lanes denote the stoichiometric protein/DNA ratio.
The protein/DNA ratio corresponding to binding saturation is indicated with a grey line.
In contrast, AtTRB3 exhibits a markedly stronger dependence
of the binding ability on DNA sequence that was manifested by a
shift in ratio needed for saturation of the non-telomeric substrate
N. The protein/DNA ratio was shifted to the higher values (>5:1)
in the case of duplex N than was the ratio for the telomeric duplex
R4 (Figure 2b).
The difference in DNA-sequence-dependent saturation might
be a result of different binding kinetics of AtTRB1 and AtTRB3.
To assess this possibility, direct kinetic measurements were
performed using FA.
Binding kinetics
The binding affinity of AtTRB variants to double-stranded
DNA was analysed further by FA measurements. In these
measurements, protein aliquots were added to the solution of
labelled DNA duplex, and an increase of FA was observed. The
equilibrium dissociation constants (Kd) obtained by analyses of
anisotropy curves for binding are listed in Table 1.
AtTRB1 and AtTRB3 bind telomeric DNA with high affinity
and specificity
The binding affinity of AtTRB1 to telomeric DNA is significantly
higher in comparison with the binding to non-telomeric DNA. The
titration curves obtained for AtTRB1 binding to DNA substrates
Table 1 Dissociation and association constants for binding of AtTRB1 and
AtTRB3 to DNA
Values are means +
− S.E.M. for three independent experiments in 50 mM sodium phosphate
(pH 7.5) and 100 mM NaCl measured at 25◦C.
R4 N R2
Ka Ka Ka
Protein Kd (nM) (10−6
M−1
) Kd (nM) (10−6
M−1
) Kd (nM) (10−6
M−1
)
AtTRB1 90 +
− 20 11.0 1200 +
− 300 0.83 210 +
− 30 4.8
AtTRB3 400 +
− 60 2.5 2900 +
− 300 0.35 800 +
− 100 1.3
Figure 3 Binding of AtTRB1 and AtTRB3 to DNA duplexes
(a) FA measurements of binding of AtTRB1 to telomeric duplex R4 or non-telomeric duplex N.
The binding at 20 nM DNA occurred in buffer containing 50 mM sodium phosphate (pH 7.5)
and 100 mM NaCl. (b) FA measurements of binding of AtTRB3 to R4 or N duplex. Binding
conditions were the same as in (a). (c) Binding isotherms of AtTRB1 and AtTRB3 with telomeric
duplex R2 measured by FA. Binding conditions were the same as in (a).
R4 and N are shown in Figure 3(a). As expected, AtTRB1 shows
significantly higher binding affinity to telomeric R4 than to the
non-telomeric N DNA substrate. This can be clearly seen from
the steeper rise of the curve corresponding to binding telomeric
DNA. The evaluation of binding curves revealed Kd values of
90 and 1200 nM for R4 and N substrate respectively (Table 1).
Comparison of dissociation constants thus demonstrates more
than 13-fold higher affinity and binding specificity of AtTRB1 to
DNA bearing telomeric sequences.
The binding affinity of AtTRB3 to telomeric sequence is higher
in comparison with the binding to non-telomeric sequence, but the
difference is less pronounced than in case of AtTRB1. AtTRB3
was allowed to bind either the telomeric substrate R4 or the nontelomeric
duplex N (Figure 3b). The Kd values for the binding
of AtTRB3 to R4 and N were 400 and 2900 nM respectively.
c The Authors Journal compilation c 2009 Biochemical Society
DNA-binding kinetics of SMH proteins 225
Figure 4 Dependence of the association constants for binding of AtTRB1
and AtTRB3 to substrate R4 on NaCl concentration
The inset shows the electrostatic and non-electrostatic components of the free energy of
association of AtTRB1 or AtTRB3 with substrate R4.
AtTRB3 shows more than 7-fold higher affinity to telomeric DNA
duplex than to non-telomeric DNA.
The absolute value of the dissociation constant was verified by SPR
In order to confirm the absolute values of binding constants
obtained using FA, a reverse-order experiment was performed
using SPR. In this experiment, AtTRB3 was immobilized on
the chip surface, and duplex R4 was allowed to bind. The
reverse arrangement of the SPR experiment changes interaction
stoichiometry (one DNA duplex interacts with one immobilized
protein, whereas four protein monomers bind one DNA duplex
during FA measurements). This had been considered when
the equilibrium binding constant was evaluated. The output of the
non-linear fitting of SPR curves for different concentrations
of DNA produces a Kd of 1700 nM, which agrees with the
value determined previously with a factor of 2 at a similar salt
concentration (see the Supplementary Online Data).
AtTRB1 and AtTRB3 show reduced binding affinity to R2 when
compared with binding affinity to R4
When the length of DNA duplex is shortened from four to two
telomeric repeats, the binding affinity decreases to the level of
binding affinity recorded for the non-telomeric DNA. Even though
there is one putative binding site present on the duplex R2, the
binding affinity of AtTRB1 is quite low and is characterized by a
Kd similar to that obtained for binding to duplex N. The shortening
of telomeric DNA substrate has a similar effect on binding affinity
of AtTRB3 (Table 1). The length reduction of telomeric DNA
substrate thus results in a substantial fall in the binding affinity of
both AtTRB1 and AtTRB3.
Electrostatic contribution to binding affinity
The binding of AtTRB1 or AtTRB3 to duplex R4 containing two
putative binding sites induces the formation of four or three ion
pairs respectively. Binding of both proteins to the substrate R4
was measured at different concentrations of NaCl. The change
of binding parameters is set out in the double-log-plot of the
association constants against salt concentration (Figure 4 and
Table 2). From the slope, the parameter Z was calculated. Z
denotes the number of newly formed ionic bonds between protein
and DNA. This number is 4 (after rounding) for binding of
Table 2 Salt-concentration-dependence of association constants for
binding of AtTRB1 and AtTRB3 to R4
Values are means +
− S.E.M.
Protein [NaCl] (mM) log Ka δlog Ka/δlog [NaCl] log Ka
nel
Z
AtTRB1 100 7.04 2.8 +
− 0.2 4.31 +
− 0.2 4.4
119 7.08
141 6.67
167 6.54
200 6.25
AtTRB3 100 6.41 2.0 +
− 0.1 4.4 +
− 0.1 3.2
119 6.31
141 6.06
167 6.00
200 5.81
AtTRB1, and 3 for AtTRB3. Thus approx. four ion pairs are
formed upon binding of AtTRB1 and approx. three ion pairs
upon binding of AtTRB3 to the telomeric DNA.
The binding energy is provided mainly by a non-electrostatic
component in the case of both AtTRBs
Further evaluation of the salt-dependent binding constant
was performed to obtain the non-electrostatic contribution to
the binding affinity. The electrostatic and non-electrostatic
components of the binding energy for AtTRB1 or AtTRB3 to
R4 are shown in the inset of Figure 4. It is notable that the nonelectrostatic
components of binding energy Ga
nel
for the two
proteins are identical within error range with magnitudes of 25 kJ ·
mol−1
for both AtTRB1 and AtTRB3. If this value is compared
with the values of the overall binding energy 40 kJ · mol−1
for
AtTRB1 and 37 kJ · mol−1
for AtTRB3, it can be concluded that
the non-electrostatic interactions contribute to the total energy of
binding by approx. 60% for AtTRB1 and by approx. 70% for
AtTRB3. Hence, it is apparent that the major part of the binding
energy originates from the non-electrostatic interactions.
The greater electrostatic component is responsible for a more
favourable overall binding energy of AtTRB1 compared with AtTRB3
Further inspection of calculated energetic data allowed us to
identify the main reason for different binding affinities between
these similar proteins. It is demonstrated that the kinetics of
protein–DNA interactions are different because of the electrostatic
term of the binding energy (inset in Figure 4). In other words, the
difference in the total binding energy for AtTRB1 and AtTRB3
is entirely given by the change in the electrostatic component of
binding.
DISCUSSION
Kinetics and stoichiometry of binding
The present study shows that binding of AtTRB1 and AtTRB3
with the telomeric DNA proceeds with the stoichiometry of one
protein monomer per one telomeric repeat. A higher protein/DNA
ratio was observed only in case of AtTRB3 binding to nontelomeric
DNA (Figure 2b). The shift in the ratio can be explained
by the observed lower affinity of AtTRB3 for non-telomeric DNA.
The decrease in binding affinity with the change from telomeric
to non-telomeric sequence was confirmed also by our kinetic
measurements (Table 1). All recently characterized AtTRBs form
tightly bound homo- and hetero-dimers and multimers [14,15].
Relatively strong mutual interactions of AtTRBs were also
c The Authors Journal compilation c 2009 Biochemical Society
226 C. Hofr and others
verified independently using SPR (results not shown) and their
dimerization ability was demonstrated by gel chromatography
(see the Supplementary Online Data). Therefore the feasibility of
protein dimerization and stoichiometric data of the present study
support the assumption that AtTRBs bind to DNA in dimeric
form. In this respect, the AtTRBs behave similarly to human
TRF1 and TRF2 [30–32], with the exception that TRFs do not
form heterodimers.
Surprisingly, the affinity of AtTRB1 to telomeric substrate R4
is 4-fold higher than that of AtTRB3, although AtTRB1 and
AtTRB3 are relatively similar in their primary sequences.
Interestingly, it has been found that Kd values observed in the
present study for AtTRB1 and AtTRB3 correspond very well to
Kd values obtained for the DNA-binding domain of human TRF1
and TRF2 when interacting with telomeric DNA [33]. Moreover,
similarly to AtTRB1 and AtTRB3, human TRF1 binds telomeric
DNA with a 4-fold higher affinity than that of TRF2.
In order to explain potential reasons for the different binding
manner of AtTRB1 and AtTRB3, we compared our findings with
available equilibrium kinetic data for the binding of the Myb
domain. The Kd obtained for the binding of the Myb domain
alone to telomeric DNA from NMR studies was in the range of
1 μM [18]. If we compare this value measured at physiological
salt concentration with the values for the binding of full-length
proteins measured in the present study at a corresponding NaCl
concentration, the magnitude of Kd for AtTRB3 is slightly lower
at 0.9 μM (see Table 1), and the Kd for AtTRB1 is significantly
lower (0.2 μM). Both full-length proteins showed higher binding
capacities than that reported for a Myb domain alone. Since the
Myb domain sequence is highly conserved between AtTRB1 and
AtTRB3, the higher binding affinity of AtTRB1 should originate
from another part of the protein. The domain that may contribute
to the tuning of binding affinity of AtTRBs to DNA is the H1/5
domain [13], as supported by our recent findings [14]. The
conservation of the H1/5 domain between AtTRB1 and AtTRB3
is lower than that of the Myb domain and differs in a way that
might allow the corresponding protein region to adopt a structure
with a different net charge on the surface. The surface net charge
is important for a long-range non-specific electrostatic attraction
among proteins and DNA, whereas non-electrostatic interactions
that are important for specific recognition of a DNA sequence
comprise hydrogen bonds between outer groups of DNA and
polar residues of the protein [18].
Electrostatic component of binding
Proteins controlling and regulating nucleic acid structure and
function usually show both sequence-non-specific binding
to DNA and a higher-affinity binding of their specific physiological
DNA target. In general, protein–DNA binding takes place
in two steps. In the first step, a non-specific, mainly electrostatic,
binding to the phosphate backbone occurs; in the second step, the
protein explores the DNA surface for specific non-electrostatic
interactions such as hydrogen bonds [34].
Different contributions of electrostatic and non-electrostatic
interactions to binding were observed for different classes of
DNA-binding proteins. For example, telomere-binding protein
α from Oxytricha nova induces the formation of two ion pairs
upon binding to DNA, and the electrostatic contribution to the free
energy of binding is approx. 15% [25]. On the other hand, proteins
containing a strongly positively charged scissor-grip motif for
DNA recognition induce the formation of six ion pairs with the
electrostatic contribution to the total free energy of binding being
45% [29].
The different contribution of electrostatic attraction for binding
of AtTRB1 and AtTRB3 was observed. We estimated the number
of four and three ion pairs upon AtTRB1 or AtTRB3 binding
to R4 and the corresponding electrostatic contribution to the total
free energy of binding at 40 and 30% respectively. This correlates
well with data available for electrostatic interactions of other
DNA-binding proteins. The DNA-binding event of AtTRBs is
driven mainly by non-electrostatic interactions. On the whole,
our results show that AtTRBs bind telomeric DNA primarily in a
sequence-specific manner that is essential for the recognition of
binding sites within telomeric DNA.
Kinetic data contribute to understanding of nucleoprotein
complex arrangement
Analyses of our kinetic data together with available structural data
may be also used to elucidate the arrangement of nucleoprotein
complexes of AtTRBs with telomeric DNA.
In general, one might suppose that the same binding preferences
to telomeric DNA are given primarily by the occurrence of the
recognition sequence in DNA. For this reason, one would also
expect the same binding kinetics for the telomeric DNA with one
or two putative binding sites under the consideration of a nonco-operative
independent binding. As follows from the previous
assumptions, the duplex R2, containing one binding site, should
have reached the saturation of binding sites faster (Kd would
be lower) when compared with that for duplex R4, with two
binding sites. However, our data show the opposite. The binding
affinity of both examined proteins to duplex R2 is lower (Kd
is shifted to higher values) than in the case of binding to R4.
Our quantitative kinetics results confirmed a previously reported
decrease in binding affinity of AtTRBs with the shortening of
telomeric DNA substrate [13]. Moreover, the lower affinity to
DNA containing only one putative binding site might be an
indication of an insufficient space for the binding of an active
protein. Importantly, it has been shown that the minimum length
of DNA for Myb domain binding is approx. 13 bp [18]. If AtTRBs
interacted with the DNA exclusively through the Myb domain and
binding sites were positioned suitably within the sequence, the
length of R2 duplex (14 bp) should have been sufficient for proper
binding without a change in binding affinity. Since a significant
fall in binding affinity was observed, the kinetics data suggest that
there is also another domain taking part in the interaction. As a
result, the binding affinity of AtTRBs to the 14 bp long and 28 bp
long DNA duplex differs substantially. In our recent results, the
H1/5 domain promotes interaction with DNA [14]. Presumably,
the short length might prevent the H1/5 domain from properly
interacting with the DNA. Hence, the constrained binding without
H1/5 domain might be the main reason for the reduction of the
overall binding affinity to substrate R2.
Although the picture of a molecular mechanism controlling
telomerase activity is far from complete, it is important to consider
how the protein-binding events measured in the present study
relate to structural arrangements and subsequent interactions
essential for the biology of telomeres. If we take into account the
kinetic data and the dimerization ability of AtTRBs, a speculative
protein arrangement on telomeric DNA could be considered
(Figure 5).
The model of binding arrangement considers that the protein
monomers form a dimer that binds two adjacent binding sites
simultaneously. This type of interaction mode is quite common
in the sequence-specific binding of proteins that take part in regulatory
mechanisms [35]. This model, where two recognition
sites on DNA are bound by one protein dimer, might explain
well the fall of binding activity when the DNA substrate is
c The Authors Journal compilation c 2009 Biochemical Society
DNA-binding kinetics of SMH proteins 227
Figure 5 Speculative model of interaction of AtTRBs with telomeric DNA
Both homo- and hetero-dimers of AtTRB may participate in the interaction with telomeric DNA.
shortened from 28 to 14 bp as was observed for binding to
the R4 and R2 duplex respectively. The introduced model is
supported by stoichiometric and kinetic data presented here and
it is also in accordance with our previous study demonstrating
weaker binding to DNA containing fewer telomeric repeats [13].
The binding arrangement shown in Figure 5 also takes into
consideration the multimerization ability of the H1/5 domain that
could promote the arrangement of protein monomers in the DNA
region between the binding sites. Moreover, the formation of
homo- and hetero-multimers of SMH proteins and their ability to
interact with other proteins (e.g. AtPOT1b [15,16]) contribute
to a network of protein interactions that could be employed in
the organization of telomere to form highly ordered chromatin
structures, such as t-loops, in a similar way to human TRFs
[31,32].
Thus, on the basis of results of the present study and the
data available, we suggest that interactions of the two AtTRBs
with telomeric DNA occur simultaneously with two binding
sites. Therefore the minimal length of duplex DNA required for
the proper binding of full-length AtTRB1 and AtTRB3 should
harbour at least two putative binding sites that are bound by
two dimers of AtTRBs. Consequently, SMH proteins are able to
distinguish between short (<10 bp) telomere-like sequences that
are dispersed throughout the genome, e.g. in promoter regions
[36], and longer tracts of telomere repeats occurring in telomeres.
There is still a considerable lack of general knowledge
of intracellular arrangement, molecular crowding effects,
association mechanisms and kinetics of protein–DNA-binding
events in a living cell. Nevertheless, we can draw a speculative
view of the in vivo consequences of our in vitro data, if we consider
that the behaviour of a protein would not be markedly changed in
the cellular environment. The access of AtTRBs to their telomeric
target sites is restricted in both spatial and temporal ways
by chromatin structure: the telomeric heterochromatin structure
provides low accessibility upon its tight condensation, and thus
the binding of specific proteins to DNA may occur preferentially
in a short time slot between DNA replication and chromatin
condensation [37].
AtTRBs might be first recruited by a weak non-specific binding
to multiple chromosome regions. Then, once the specific target
sites become accessible, highly specific binding occurs. On
the other hand, the AtTRB molecules which are bound only
by a highly dynamic non-specific interaction (in non-telomeric
regions) can be easily displaced by other proteins binding with a
higher affinity. Thus AtTRBs at non-telomeric sites do not impede
other functional DNA–protein interactions.
In this way, non-specific binding could serve as a tool for
increasing the local concentration of the proteins on DNA
[34]. Accumulation of SMH proteins on DNA via non-specific
electrostatic interactions may be important for their immediate
availability for functional and specific binding to their telomere
target sites.
Although further details of the binding interactions of proteins
and their biological significance have yet to be determined, these
results demonstrate the advantage of the approach employed in
the present study by using a complete protein for in vitro studies
rather than the commonly used Myb-domain-bearing fragment.
Our data imply that AtTRB1 and AtTRB3 are telomere-specific
proteins that bind telomeric DNA with distinct kinetics given by
differences in their electrostatic interactions with DNA. To our
knowledge, this is the first quantitative study of the plant-specific
SMH family of proteins. The present paper demonstrates that the
detailed quantification of protein–DNA interactions may provide
new insights into the structural dynamics of telomeres.
ACKNOWLEDGEMENTS
We are grateful to M. Chester for the critical reading of the manuscript.
FUNDING
This work was supported by the Grant Agency of the Czech Republic [grant numbers 521/
08/P452 and 204/08/H054], the Czech Ministry of Education [grant number LC06004],
the Grant Agency of the Czech Academy of Sciences [grant number IAA500040801]
and the institutional support [grant numbers MSM0021622415, MSM0021622413,
AV0Z50040702 and AV0Z50040507].
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Received 7 November 2008/18 December 2008; accepted 22 December 2008
Published as BJ Immediate Publication 22 December 2008, doi:10.1042/BJ20082195
c The Authors Journal compilation c 2009 Biochemical Society
Biochem. J. (2009) 419, 221–228 (Printed in Great Britain) doi:10.1042/BJ20082195
SUPPLEMENTARY ONLINE DATA
Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study
of telomere-binding specificity and kinetics
Ctirad HOFR*1
, Pavla ˇSULTESOV´A*, Michal ZIMMERMANN*, Iva MOZGOV´A*, Petra PROCH´AZKOV´A SCHRUMPFOV´A*,
Michaela WIMMEROV´A† and Jiˇr´ı FAJKUS*‡1
*Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Faculty of Science, Masaryk University, CZ-62500 Brno, Czech Republic, †National Centre for
Biomolecular Research and Department of Biochemistry, Faculty of Science, Masaryk University, CZ-61137 Brno, Czech Republic, and ‡Laboratory of DNA–Molecular Complexes,
Institute of Biophysics, Czech Academy of Sciences, CZ-61265 Brno, Czech Republic
EXPERIMENTAL
Gel-filtration chromatography
The molecular masses of the protein in monomeric and
dimeric forms were estimated by size-exclusion gel-filtration
chromatography through a Superdex 200 10/30 GL column
(GE Healthcare), using a gel-filtration standard (Bio-Rad
Laboratories) in a buffer containing 50 mM sodium phosphate
(pH 7.5) and 300 mM NaCl. The molecular masses of proteins
were estimated from a linear fit to the log Mr against elution
volume plot generated with the protein standards. Supplementary
Figure S1 shows the chromatograms.
Surface plasmon resonance
All SPR experiments were performed with a Biacore 3000
instrument (GE Healthcare) at 25◦
C using TBST (Tris-buffered
saline with Tween 20: 10 mM Tris/HCl, pH 7.5, 150 mM NaCl,
containing 0.005% Tween 20) and a flow rate of 5 μl/min.
AtTRB3 was immobilized on the research-grade CM5 sensor
chip in a buffer containing 10 mM Hepes, 150 mM NaCl (pH 7.5)
and 0.005% Tween 20. Sensorgrams were run in the automatic
subtraction mode using FC (flowcell) 1 as an unmodified
reference. Data were collected for FC 2, FC 3 and FC 4, which
contained various amounts of AtTRB3. Injections of DNA were
made using the ‘quickinject’ injection mode, going from lowest
to highest concentration samples, with a 5 min contact time
and a 1200 s dissociation phase in all cases. Regeneration was
achieved using several (two to five) 1 min pulses of 50 mM NaOH.
All sensorgrams were obtained at 25◦
C. Data were analysed
by equilibrium analysis in addition to the kinetic analysis. The
equilibrium response was plotted against the concentration of
DNA and fitted to:
R = Ka[DNA]Rmax(Ka[DNA] + 1)
where R is the equilibrium response at a specific concentration
of DNA substrate, Rmax is the response at saturation of the DNA
substrate on the chip, Ka is the equilibrium association constant,
which is the reciprocal of the dissociation constant Kd (Ka = 1/Kd).
When assuming a non-co-operative binding model, the apparent
Kd from SPR experiments should be divided by 4 to resemble
Figure S1 Size-exclusion chromatograms of protein AtTRB3 (continuous
line) and molecular-mass standard (broken line)
Abs, absorbance; MW standard, molecular-mass standard. The numbers next to the arrows
indicate determined molecular-mass values of monomeric and dimeric protein forms.
different binding stoichiometry of FA and SPR experiments.
The output of the non-linear fitting of SPR curves for different
concentrations of DNA produces a Kd of 6.8 μM, which, divided
by 4, gives 1.7 μM. This value agrees well with the value of Kd
determined from FA measurements considering different buffer
conditions. Supplementary Figure S2 shows the sensorgrams and
the response curve.
Purification of AtTRBs
AtTRB1 and AtTRB3 were expressed in soluble forms in
cytoplasm of E. coli. The purification strategy consisted of two
affinity steps. A capture step by IMAC (immobilized metalion-affinity
chromatography) was followed by a purification step
using HAC (heparin-affinity chromatography). To confirm final
purity, collected fractions were separated by SDS/PAGE (0.1%
SDS, 10% acrylamide). Supplementary Figure S3 shows the gelpurified
proteins.
1
Correspondence may be addressed to either of these authors (email hofr@sci.muni.cz or fajkus@sci.muni.cz).
c The Authors Journal compilation c 2009 Biochemical Society
C. Hofr and others
Figure S2 Binding of telomeric duplex R4 to immobilized AtTRB3
Response signals from the saturated region of the sensorgram have been used to calculate equilibrium dissociation constant Kd. RU, response units.
Figure S3 Analysis of purification steps using SDS/PAGE
Lane 1, collected fractions containing AtTRB3 after IMAC (immobilized metal-ion-affinity
chromatography) and subsequent HAC (heparin-affinity chromatography) (10 μg); lane 2,
collected fractions containing AtTRB3 after IMAC (15 μg); lanes M, molecular-mass markers
(sizes are indicated in kDa); lane 5, collected fractions containing AtTRB1 after IMAC and
subsequent HAC (3 μg); lane 6, clarified cytoplasmic extract with expressed AtTRB1 (35 μg).
The proteins were stained with Coomassie Brilliant Blue.
Received 7 November 2008/18 December 2008; accepted 22 December 2008
Published as BJ Immediate Publication 22 December 2008, doi:10.1042/BJ20082195
c The Authors Journal compilation c 2009 Biochemical Society
Supplement F
Peška, V., Schrumpfová, P.P., Fajkus, J., 2011. Using the telobox to search for plant telomere
binding proteins. Curr. Protein Pept. Sci. 12, 75–83
P.P.S. participated in the ms writing and editing
This journal did not provide open access, hence the article is not freely available.
Supplement G
Schrumpfová, P.P.*, Fojtová, M., Mokroš, P., Grasser, K.D., Fajkus, J., 2011. Role of HMGB
proteins in chromatin dynamics and telomere maintenance in Arabidopsis thaliana. Curr.
Protein Pept. Sci. 12, 105–111
P.P.S. was significantly involved in the experimental, data evaluation, and in the ms
writing and editing
This journal did not provide open access, hence the article is not freely available.
Supplement H
Schrumpfová, P.P.*, Vychodilová, I., Dvořáčková, M., Majerská, J., Dokládal, L., Schořová,
S., Fajkus, J., 2014. Telomere repeat binding proteins are functional components of
Arabidopsis telomeres and interact with telomerase. Plant J. Cell Mol. Biol. 77, 770–781.
P.P.S. participated in the design of experiments, significantly involved in the
experimental part, data evaluation, and in the ms writing and editing
Telomere repeat binding proteins are functional components
of Arabidopsis telomeres and interact with telomerase
Petra Prochazkova Schrumpfova1,2,
*, Ivona Vychodilova1,2
, Martina Dvorackova1,3
, Jana Majerska1,†
, Ladislav Dokladal1,3
,
Sarka Schorova1,2
and Jirı Fajkus1,2,3,
*
1
Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University,
Kamenice 5, Brno, CZ 62500, Czech Republic,
2
Functional Genomics and Proteomics, CEITEC National Centre for Biomolecular Research, Faculty of Science, Masaryk
University, Kamenice 5, Brno, CZ 62500, Czech Republic, and
3
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Kralovopolska 135, Brno, CZ 61265, Czech Republic
Received 11 October 2013; revised 6 December 2013; accepted 23 December 2013; published online 8 January 2014.
*For correspondence (e-mails fajkus@sci.muni.cz or schpetra@centrum.cz).
†
Present address: Swiss Institute for Experimental Cancer Research, Ecole Polytechnique Federale de Lausanne, Station 19, 1015, Lausanne, Switzerland.
SUMMARY
Although telomere-binding proteins constitute an essential part of telomeres, in vivo data indicating the
existence of a structure similar to mammalian shelterin complex in plants are limited. Partial characterization
of a number of candidate proteins has not identified true components of plant shelterin or elucidated
their functional mechanisms. Telomere repeat binding (TRB) proteins from Arabidopsis thaliana bind plant
telomeric repeats through a Myb domain of the telobox type in vitro, and have been shown to interact with
POT1b (Protection of telomeres 1). Here we demonstrate co-localization of TRB1 protein with telomeres in
situ using fluorescence microscopy, as well as in vivo interaction using chromatin immunoprecipitation.
Classification of the TRB1 protein as a component of plant telomeres is further confirmed by the observation
of shortening of telomeres in knockout mutants of the trb1 gene. Moreover, TRB proteins physically
interact with plant telomerase catalytic subunits. These findings integrate TRB proteins into the telomeric
interactome of A. thaliana.
Keywords: telomerase, telomere, telomere repeat binding (TRB), Arabidopsis thaliana, telomere protein
interaction, plant shelterin.
INTRODUCTION
Telomeres, nucleoprotein structures that form and protect
the ends of chromosomes, have been the subject of
intense studies for about three decades, starting with a
description of the telomere DNA component (Blackburn
and Gall, 1978) and the most common system of telomere
maintenance by the ribonucleoprotein complex of telomerase
(Greider and Blackburn, 1985, 1989). Proteins essential
for telomere functions have been described in detail in
yeasts and vertebrates. Among protein components of
telomeres, the most important is indisputably telomerase
itself, but other proteins are necessary to perform other
functions of telomeres, such as inhibiting the DNA damage
response at telomeres (de Lange, 2009), recruiting telomerase
to chromosome ends (Nandakumar et al., 2012), or
facilitating telomere replication (Sfeir et al., 2009). Current
evidence suggests that these components assemble into
two distinct complexes known as shelterin (de Lange,
2005) and CST (composed of CTC1/STN1/TEN1 proteins)
complexes (Surovtseva et al., 2009).
Human shelterin consists of six core components: telomeric
repeat-binding factor 1 (TRF1), telomeric repeatbinding
factor 2 (TRF2), represor/activator protein 1
(RAP1), TRF1-interacting protein (TIN2), TINT1/PIP1/PTOP1
(TPP1), protection of telomeres 1 (POT1). TRF1 and TRF2
anchor the complex to double-stranded telomeric DNA
using a specific Myb-like motif termed a telobox (Bilaud
et al., 1996), and recruit two other shelterin components,
RAP1 and TIN2, to the telomeres. TIN2 further interacts
with TPP1 protein, which binds the final shelterin component,
POT1. POT1 also binds the G–rich strand of telomeric
DNA from either the single-stranded G–overhang
or displacement loop (D–loop). In this way, shelterin may
bridge the double- and single-stranded parts of telomeric
DNA.
© 2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License,
which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial
and no modifications or adaptations are made.
770
The Plant Journal (2014) 77, 770–781 doi: 10.1111/tpj.12428
The CST complex, consisting of three components
(Cdc13, Stn1 and Ten1), was originally described in yeast
(Gao et al., 2007) as a telomere-specific replication protein
A-like complex that protects single-stranded chromosome
termini and regulates telomere replication. Subsequent
studies have shown that a CST-like complex also exists in
plants and humans and contributes to telomere protection
and replication (Surovtseva et al., 2009; Price et al., 2010).
According to recent studies, both complexes participate in
telomere capping, telomerase regulation and 3′ overhang
formation (Giraud-Panis et al., 2010; Pinto et al., 2011;
Chen et al., 2012; Wu et al., 2012).
In contrast to the CST complex, no functional and structural
equivalent of shelterin has been found in plants.
Although many putative shelterin-like protein components
have been found in plants (Peska et al., 2011), including
those bearing a telobox Myb-like domain at their C–terminus
(Hwang et al., 2001, 2005; Karamysheva et al., 2004) or
N–terminus (Marian et al., 2003; Schrumpfova et al., 2004),
as well as POT1 homologues (Baumann et al., 2002;
Kuchar and Fajkus, 2004; Shakirov et al., 2005; Tani and
Murata, 2005; Peska et al., 2008), none of these have been
shown to specifically associate with telomeres in situ or
in vivo.
Molecular components responsible for reversible telomerase
regulation in plant cells (Fajkus et al., 1998; Riha
et al., 1998) are an attractive target for possible biomedical
applications of telomere biology, and are sought primarily
at the levels of protein components of plant telomeres,
and regulation of the basic telomerase subunits TERT (telomerase
reverse transcriptase) and TER (telomerase RNA).
In this study, we investigated the interactions and roles
of Single myb histone (Smh) proteins at plant telomeres.
Five members of the Smh family are encoded by the A. thaliana
genome (TRB1–5). These proteins are specific to
plants, and consist of an N–terminal Myb-like domain of the
telobox type, which is responsible for specific recognition
of double/single-stranded telomeric DNA (Schrumpfova
et al., 2004; Hofr et al., 2009), a central histone-like domain,
which is involved in non-specific DNA–protein interactions
and mediates protein–protein interactions, including formation
of homo- and heteromeric complexes of TRB proteins
(Mozgova et al., 2008), and a C–terminal coiled-coil domain
to which no specific function has yet been attributed. We
previously reported that TRB proteins interact via their histone-like
domain with POT1b, an A. thaliana homologue of
the G–overhang binding protein POT1 (Kuchar and Fajkus,
2004; Schrumpfova et al., 2008; Rotkova et al., 2009). In
addition, POT1b also associates with an alternative telomerase
nucleoprotein complex in Arabidopsis (Surovtseva
et al., 2007; Cifuentes-Rojas et al., 2012). We have previously
shown that TRB1 is localized in the nucleus and
nucleolus in vivo and shows highly dynamic association
with chromatin (Dvorackova et al., 2010). Together, these
findings indicate that TRB proteins are promising candidates
for plant shelterin-like components.
Here we demonstrate that TRB proteins act as components
of a plant telomere-protection complex. Microscopic
and chromatin immunoprecipitation techniques showed
co-localization of TRB1 with telomeric tracts in vivo and
physical interaction of TRB proteins with the N–terminal
part of the catalytic subunit of telomerase. In addition, loss
of TRB1 protein leads to telomere shortening.
RESULTS
TRB1 co-localizes with telomeres
Although a possible association of GFP–TRB1 (35Spro:
GFP-TRB1) with the telomere was suggested previously
(Dvorackova et al., 2010), whether the nuclear speckles are
directly associated with telomeres remained to be deter-
mined.
Here, we took advantage of the well-established protocol
of Nicotiana benthamiana leaf infiltration and the fact that
N. benthamiana has longer telomeres that are easier to
visualize compared to Arabidopsis.
As shown in Figure 1(c), the localization of transiently
transformed TRB1 in N. benthamiana leaf is similar to that
observed in Arabidopsis cell cultures, as was shown by
Dvorackova et al. (2010), labelling the whole nucleus, with
strong nucleolar signal and relatively strong nuclear speckles.
Nuclei from transformed leaves were isolated and used
for telomere peptide nucleic acid FISH. Fluorescence from
GFP–TRB1 remained very bright during the isolation procedure;
however, a gentle denaturation step was necessary
during the FISH protocol to preserve the integrity of the
GFP signal. These FISH results showed that telomeres colocalize
or associate with TRB1 speckles in 59% and 31% of
cases, respectively, with 90% association overall (Figure 1
and Table S1). Telomeric signals sometimes appeared as
double dots connected to the TRB1 foci (Figure 1d, images
1, 2 and 3), but in other cases co-localize directly with
TRB1 (Figure 1d, images 4 and 5). These results provide in
situ evidence of telomere occupancy by TRB1.
TRB1 is associated with telomeric sequence in vivo
The observed co-localization of TRB1 with telomeric tracts,
together with our previous detailed analyses of TRB1 binding
to telomeric DNA in vitro (Schrumpfova et al., 2004;
Hofr et al., 2009), suggest the possibility that TRB1 protein
directly recognizes telomeric repeats and belongs to the
core components that shelter telomeres. We used a chromatin
immunoprecipitation assay to isolate DNA
sequences associated with TRB1 protein. As source material,
we used formaldehyde cross-linked seedlings of Arabidopsis
plants stably transformed with a TRB1–GFP
construct driven by the native promoter (TRB1pro:TRB1GFP)
(Dvorackova et al., 2010). Despite using the native
© 2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2014), 77, 770–781
TRB interactions with telomeres and telomerase 771
promoter, enhanced levels of TRB1–GFP protein were
observed (see below). TRB1–GFP protein was immunoprecipitated
from purified nuclei using GFP-Trap A matrix,
which contains a single variable antibody domain that recognizes
GFP. Non-specific binding of TRB1-GFP to the
GFP-Trap A matrix was excluded by precise detection of
GFP in all fractions (input, bound, unbound, wash, elution).
We have shown that TRB1, but not TRB1-GFP is washed
out (Figure S1). DNA co-purifying with TRB1–GFP was dotblotted
onto nylon membranes, and visualized by hybridization
with radioactively labelled telomeric probe. Figure 2
shows that TRB1 protein is indeed associated with telomeric
sequence in vivo, as telomeric sequence was repeatedly
detected in TRB1–GFP but not wild-type samples. To
demonstrate that the observed enrichment is indeed due
to sequence-specific association and not due to the high
copy number of the telomeric DNA, we hybridized DNA
co-purified with TRB–GFP with a centromeric probe. As our
previous results (Dvorackova et al., 2010) showed localization
of TRB1 protein in the nucleus and nucleolus, another
candidate sequence investigated for association with TRB1
was ribosomal DNA (rDNA). Only negligible enrichment of
the centromeric or 18S rDNA probe, in contrast to significant
enrichment of the telomeric probe, was observed when
comparing each wild-type to TRB–GFP sample (Figure S2).
Analysis of TRB1 expression in trb1 mutant, wild-type and
TRB1pro:TRB1-GFP-transformed plants
To examine the role of TRB1 in planta, we analysed T–DNA
insertion line SALK_025147 (ecotype Col–0). Three parallel
wild-type (wild-type) and homozygous trb1 (trb1À/À) lines
(A, B and C) were derived from three independent heterozygous
plants (see Figure 4a). The homozygosity of each
parallel wild-type and trb1 mutant plant line was determined
by PCR (Figure 3b). The T–DNA insertion is located
in the second intron (Figure 3a), and the absence of trb1
transcript was confirmed by RT–PCR (Figure 3c).
TRB proteins consist of three domains: Myb-like, histone-like
and a coiled-coil domain (Figure 3a). As no antibody
recognizing either TRB proteins or the plant Myb
(a) (c)
(d)
(b)
Figure 1. Co-localization of TRB protein with telomeric probe.
Nuclei isolated from N. benthamiana were transformed with 35Spro:GFP-TRB1 construct and hybridized with telomeric peptide nucleic acid (PNA) Cy3-labelled
probe.
(a) Co-localization between GFP–TRB1 nuclear speckles (green) and telomeric PNA probe (red) is detectable in most of the foci.
(b) Control experiment without telomeric probe showing very little background present in the red channel.
(c) Confocal image of GFP–TRB1 expression in an N. benthamiana leaf without any further sample processing.
(d) Details of co-localizing speckles; ImageJ (http://rsbweb.nih.gov/ij/) was used to create intensity plots for red and green channels.
© 2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2014), 77, 770–781
772 Petra Prochazkova Schrumpfova et al.
domain of the telobox type is commercially available, we
developed specific mouse monoclonal antibodies in our
laboratory. Two of them were used in this study: 1.2 (specific
to TRB1) and 5.2 (specific for the conservative part of
the Myb domain; this also recognizes other TRB proteins).
The location of antibody recognition sites within the
structure of TRB1 as determined by ELISA (Figure S3) is
shown in Figure 3(a). Although the conservative part of the
telobox Myb domain is also present in Arabidopsis TRFlike
family (TRFL) proteins (Karamysheva et al., 2004),
these proteins are not recognized by the 1.2 or 5.2 antibodies.
The anti-TRB 1.2 or 5.2 antibodies were unable to
detect in vitro expressed TRFL2 or 9 or TRP1 (telomeric
repeat binding protein 1) from the TRFL family (Figure S4,
constructs kindly provided by D.E. Shippen, Department of
Biochemistry,Texas A&M University,TX, USA).
Antibody 1.2 was used to detect native TRB1 protein in
Arabidopsis plant protein extracts. The natural level of
TRB1 protein was clearly observed on Western blots of
wild-type plants (Figure 3d), but no TRB1 protein was
observed for extracts from trb1 mutant plants (Figure 3d).
In addition, plant lines stably transformed with TRB1–GFP
construct under the control of native promoter showed a
distinct abundance of TRB1–GFP protein compared to the
native TRB1 protein. Various expression levels of native
TRB1 protein and TRB1–GFP were also apparent after
immunolocalization in vivo (Figure 3e), in which TRB1 protein
is visualized using either anti-TRB1 1.2 antibody or
anti-GFP antibody.
We tested both antibodies by indirect immunofluorescence
on trb1 mutant and GFP–TRB1-expressing plants.
These experiments showed evenly distributed nuclear and
nucleolar signals for both 1.2 and 5.2 antibodies. Antibody
1.2 did not detect any signal in trb1À/À plants, but antibody
5.2 recognizes some epitopes in trb1À/À (Figure S5).
However, the generated antibodies do not appear to be of
sufficient quality for more demanding immunolocalization
or ChIP experiments (as concluded from further testing).
Telomere shortening in trb1 null mutant plants
Derivation of independent wild-type and trb1À/À plant
lines from three heterozygous progeny (Figure 4a) provided
reliable material for phenotypic studies of the trb1
null mutation effect. All six homozygous plant lines were
propagated for five generations.
Obvious shortening of telomeres was observed by terminal
restriction fragment (TRF) analysis in all three trb1
mutant lines analysed in the fifth generation compared to
their segregated wild-type siblings. Hybridization with a
radioactively labelled telomeric probe (Figure 4b) revealed
truncation of telomeric tracts in trb1 lines by approximately
10–20% (Figure 4c). The graph represents evaluation in the
three biological replicates. Observations in earlier generations
of trb1 lines (Figure S6) show mild but progressive
shortening that continues through the generations. Despite
clear and reproducible telomere shortening in trb1, no significant
morphological differences were observed in
rosette diameter, leaf number, flowering and seed set
when analysing soil-grown wild-type and trb1À/Àplants.
TRB proteins interact with telomerase in planta
Our previous finding that TRB1 protein interacts with
POT1b and evidence presented here showing that TRB1
co-localizes with telomeric repeats and is involved in
regulation of telomere maintenance suggest its possible
association with telomerase (Kuchar and Fajkus, 2004;
Schrumpfova et al., 2008). We therefore tested the possibility
of direct interaction between TRB1 and TERT, as well as
the influence of TRB1 on telomerase activity in vitro.
As TERT is a high-molecular-weight protein (approximately
130 kDa), we used TERT fragments containing N–
terminal domains associated with distinct telomeric functions
(Sykorova and Fajkus, 2009) to detect a possible
direct interaction between TERT and TRB proteins
(Figure 5a). We tested their ability to interact using a GAL4
based yeast two-hybrid system, in which interactions take
place inside the nucleus. As shown in Figure 5(b), strong
interaction between TRB1 and the TERT 1-271 fragment
was observed on histidine-deficient plates. This interaction
was confirmed under stringent adenine selection. Clear
interactions between TRB3 and TERT 1-271 and a weak
interaction between TRB2 and TERT 1-271 were also
observed under histidine selection. Further testing TRB1
Figure 2. TRB1 proteins are associated with telomeric sequence in vivo.
DNA cross-linked with TRB1 protein was isolated by ChIP analysis using
GFP-Trap A matrix from wild-type (Wt) and TRB1pro:TRB1-GFP plants.
Hybridization of isolated DNA with radioactively labelled telomeric oligonucleotide
(CCCTAAA)4 in three biologically and technically replicated experiments
confirmed the hypothesis that TRB1 protein is associated with
telomeric sequence in vivo. As a control, telomeric oligonucleotide
(TTTAGGG)4 was dot-blotted on the same membrane and visualized
together with immunoprecipitated DNA.
© 2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2014), 77, 770–781
TRB interactions with telomeres and telomerase 773
and TRB2 with a longer fragment of TERT (amino acids 1-
582) confirmed these interactions.
To test whether the interactions observed in a yeast-two
hybrid system are reproducible in the plant cell, we used a
bimolecular fluorescence complementation assay (BiFC).
Arabidopsis protoplasts were transfected with plasmids
encoding nYFP-tagged TRB constructs and cYFP-tagged
TERT fragments, and a clear intra-nuclear interaction was
observed (Figure 5c and Figure S7). The TERT fragments
used in BiFC (TERT 1-271 and TERT 229-582) overlap with
the fragments tested in the yeast two-hybrid system.
The interaction was further verified by co-immunoprecipitation
experiments in which proteins were expressed in
rabbit reticulocyte lysate from the same vectors used in
yeast two-hybrid system. As shown in Figure S8, clear
interactions between TRB1 and all three TERT fragments
(1-271, 229-582 and 1-582) were observed. Obvious interactions
were also detected between TRB3 and TERT 1-271 or
TERT 229-582, but only weak interactions were observed
between TRB2 and TERT fragments. The generally weaker
interactions of TRB2 or TRB3 proteins with TERT fragments
in comparison to the corresponding interactions of TRB1
(a)
(b)
(d) (e)
(c)
Figure 3. Expression analysis of TRB1 protein
in mutant (trb1À/À), wild-type (Wt) and transformed
TRB1pro:TRB1-GFP plants.
(a) Schematic illustration of specific primers
and T–DNA insertion location within the trb1
gene. The domain location and antibody recognition
sites for two specific antibodies developed
in our laboratory are shown below.
(b) Three individual plant lines (A, B and C)
were derived from heterozygous progenitors
(as shown in Figure 4a). Example of PCR analysis
of genomic DNA isolated from Wt plants
(primers P3 + P2) and mutant (trb1À/À) plants
(primers P1 + P2) of line B.
(c) RT–PCR of RNA isolated from Wt and
mutant (trb1À/À) plants of line B using primers
P4 + P5.
(d) Immunodetection by Western blot analysis
of TRB1 protein in Wt and mutant (trb1À/À)
plants of line B and TRB1pro:TRB1-GFP plant
nuclear extracts using specific antibody recognizing
the Myb-like domain of TRB1 (anti-TRB1
1.2). The level of native TRB1 protein is lower
compared to the TRB1–GFP fusion protein construct
expressed under the control of the native
promoter.
(e) Immunolocalization of TRB1 protein using
anti-GFP and anti-TRB1 antibody. The level of
native TRB1 protein in the wild-type is very low.
The selected plant line does not show GFP
labelling in all cells, so some nuclei contain a
wild-type level of TRB1 and others show higher
expression due to TRB1–GFP. Thus the intensity
of signal may be clearly measured as Wt and
over-expressing nuclei are present together on
one slide and may be clearly distinguished
using specific anti-TRB1 protein antibody 1.2
and anti-GFP antibody.
© 2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2014), 77, 770–781
774 Petra Prochazkova Schrumpfova et al.
were due to lower expression of TRB2 and TRB3 proteins
in rabbit reticulocyte lysate.
To determine whether interaction between TRB proteins
and TERT directly influences telomerase activity, we used a
telomere repeat amplification protocol (TRAP). In extracts
from trb1À/À plants, no changes in telomerase activity or
processivity were observed. Correspondingly, no variations
in telomerase activity were detected in transformed plants
(TRB1pro:TRB1-GFP) expressing higher levels of protein
(Figure S9). This observation is in agreement with our previous
experiments in which Escherichia coli-expressed and
purified TRB2 and TRB3 proteins were added to the TRAP
assay (Schrumpfova et al., 2004).
DISCUSSION
The composition of plant shelterin-like complex has long
remained elusive due to the high number of candidate
proteins with apparently redundant functions (Peska
et al., 2011). These obstacles and lack of convincing evidence
raised doubts over the existence of such a complex,
and its functions have been mostly attributed to the
previously described CST complex, which is conserved
throughout eukaryotes (Nelson and Shippen, 2012b).
However, absence of evidence is not evidence of
absence. Our present data suggest the existence of a
telomere protein complex that includes plant-specific
Smh proteins (termed TRB proteins in Arabidopsis).
These proteins interact directly with the catalytic subunit
of telomerase: TRB1 protein co-localizes with telomeres,
specifically binds telomeric DNA in vitro and in vivo, and
TRB1 loss results in telomere shortening. Moreover, TRB
proteins also interact with POT1b, a POT1-like orthologue
in A. thaliana (Kuchar and Fajkus, 2004; Schrumpfova
et al., 2008).
In previous studies, we considered in detail the localization
of TRB1 protein, showing that, similar to TRB2 and
TRB3, this is a nuclear factor with markedly increased
nucleolar labelling and speckles present in the nucleus,
especially in Arabidopsis cell cultures transiently transformed
with GFP–TRB1 (Dvorackova et al., 2010). We have
previously speculated on the telomeric association of GFP–
TRB1 speckles, but the low expression of GFP–TRB1 in stably
transformed Arabidopsis plants/cultures and the short
size of Arabidopsis telomeres impeded its direct demonstration
(Dvorackova et al., 2010). In this study, we used a
plant system with longer telomeres and sufficient expression
of TRB1–GFP protein, and clearly showed that TRB1
co-localizes with telomeres in plant leaves. Close linkage
between TRB1 protein and the telomere was further supported
by the finding that plant telomeric sequence may
be isolated directly from plant seedlings together with
TRB1–GFP using the anti-GFP immunoprecipitation system.
Specific anti-TRB1 antibodies were also developed
and successfully used for detection of TRB1 alone or all
TRB proteins in the whole-protein extract by Western blot
or ELISA procedures. Using these antibodies, clear nuclear
and nucleolar localization of TRB1 protein was demonstrated.
Although TRB1 protein need not associate exclusively
with telomeres in vivo, the preferential association
of TRB1 with the telomeric tracts as described here is in
agreement with previous observations using independent
approaches (Mozgova et al., 2008; Hofr et al., 2009; Dvorackova
et al., 2010). The obvious association of TRB1 with
the nucleolus, which contains sub-telomeric clusters of
rDNA, may be due to the fact that nucleoli associate with
telomeres and telomerase at the cellular level: telomerase
assembly occurs in nucleoli in a number of model organisms
including plants (Lo et al., 2006; Brown and Shaw,
2008; Kannan et al., 2008), and nucleolus-associated telomere
clustering and pairing precede meiotic chromosome
synapsis in Arabidopsis thaliana (Armstrong et al., 2001).
The key finding of this work is that TRB proteins interact
with the N–terminal part of TERT. This part contains the
telomerase-specific motifs TEN (telomerase essential N–
terminal domain) and TRBD (N–terminal RNA-binding
domain). The most conserved motif, the T–motif, with a
high-affinity binding site for the TER subunit, is included in
the TRBD domain (Lai et al., 2001). Several distinct functions
have been proposed for the TEN domain: e.g. as an
(a)
(b) (c)
Figure 4. The telomeres are shortened in all three individually derived
trb1À/À mutant plant lines.
(a) Derivation of three independent plant lines (A, B, C) that were propagated
for five generations (G5).
(b) Terminal restriction fragment analysis, showing telomere shortening in
the trb1À/À mutant line compared with the wild-type control in the fifth
generation.
(c) Difference in mutant trb1À/À and wild-type telomere lengths in three
independent plant lines. Error bars represent standard deviation.
© 2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2014), 77, 770–781
TRB interactions with telomeres and telomerase 775
anchor during template translocation (Lue, 2005; Wyatt
et al., 2007; Sealey et al., 2010), involvement in positioning
the 3′ end of a telomeric DNA primer in the active site during
nucleotide addition (Jurczyluk et al., 2011), putative
mitochondrial localization (Santos et al., 2004), and, last
but not least, involvement in protein–protein interactions
(Sealey et al., 2011). Hence, the positioning of the region
involved in interaction between TERT and TRB proteins in
the N–terminal part of telomerase is not surprising.
Identification of TRB proteins as the interaction partner of
TERT is also supported by the observation that TRB1 protein
is present in a group of proteins that were co-purified
with the N–terminal part of TERT using tandem affinity
purification (P.P.S., J.M., L.D., E.S and J.F., unpublished
results).
The observation of TRB/telomerase interaction, together
with the previously detected interaction between TRB and
POT1b (Kuchar and Fajkus, 2004; Schrumpfova et al.,
2008), suggest that TRB proteins are part of the telomeric
interactome of A. thaliana. Interaction of POT1b protein
with the TRB1 protein is mediated by the central TRB histone-like
domain (Schrumpfova et al., 2008), but it is not
yet clear how the interaction between telomerase and TRB
is mediated. Determination of whether it occurs through
the same histone-like domain or the N–terminal Myb
domain or C–terminal coiled-coil domain would help to
(a)
(b)
(c)
Figure 5. TRB proteins interact with plant telomerase (TERT).
(a) Schematic depiction of the catalytic subunit of telomerase (TERT) showing evolutionarily conserved motifs. N-terminal fragments containing the telomerasespecific
motifs TEN (telomerase essential N–terminal domain) and TRBD (N–terminal RNA-binding domain) were used in protein–protein interaction analysis
(amino acid numbering is shown).
(b) Yeast two-hybrid system was used to assess interaction of TRB proteins with N–terminal TERT fragments. Two sets of plasmids carrying the indicated segments
of TERT fused to either the GAL4 DNA-binding domain (BD) or the GAL4 activation domain (AD) were constructed and introduced into yeast strain PJ69–
4a carrying reporter genes His3 and Ade2. Although weak interactions often fail to rescue growth under stringent adenine selection, plausible TRB–TERT interactions
were observed on histidine-deficient plates. Co-transformation with an empty vector (AD/BD/vector) served as a negative control.
(c) Bimolecular fluorescence complementation confirmed the interaction of TRB proteins with TERT fragments. Arabidopsis leaf protoplasts were co-transfected
with 10 lg each of plasmids encoding nEYFP-tagged TRB clones, cEYFP-tagged TERT fragments or Gaut10 (as negative control) and mRFP-VirD2NLS (to label
cell nuclei and to determine transfection efficiency). The cells were imaged by epifluorescence microscopy after overnight incubation. Clear nuclear interactions
of TRB proteins with TERT fragments are observed on the protoplast images: YFP fluorescence (yellow), mRFP fluorescence (red), chloroplast autofluorescence
(green pseudocolor); chloroplast autofluorescence is also visible in the YFP channel (indicated by arrows). Scale bars = 7 lm.
© 2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2014), 77, 770–781
776 Petra Prochazkova Schrumpfova et al.
determine mutual exclusion or co-existence of TERT and
POT1b association with TRB proteins.
Importantly, POT1b is also an interaction partner of TER2,
an alternative telomerase RNA subunit in Arabidopsis
(Cifuentes-Rojas et al., 2011, 2012). Together with TERT,
dyskerin and Ku, these components form telomerase ribonucleoprotein
complex that may participate in telomerase
regulation, the DNA damage response and telomere protection,
but do not substantially contribute to telomere maintenance
(Cifuentes-Rojas et al., 2011, 2012). Therefore, the
observation that TRB1 interacts with TERT but its loss or
increased expression does not change telomerase activity
is not surprising. More importantly, interaction of TRB1
with TERT, together with its affinity for telomeric DNA, indicates
a possible role of TRB1 in telomerase recruitment to
telomeres. This also explains the observed absence of any
direct effect of TRB1 on telomerase activity in the TRAP
assay, as this in vitro assay uses a non-telomeric template
oligonucleotide that is not recognized by the Myb-like
domain of TRB1 (Mozgova et al., 2008).
However, POT proteins are not the only putative singlestranded
DNA telomere-binding proteins in Arabidopsis, as
several other proteins have been identified, e.g. STEP1
(Kwon and Chung, 2004), WHY1 (Yoo et al., 2007a) or CST
complex components (Price et al., 2010). Similarly, in addition
to the TRB family of proteins, there are also other candidate
double-stranded DNA telomere-binding proteins in
Arabidopsis, such as TRFL family proteins (Karamysheva
et al., 2004). Association of these proteins with telomeres
appears not to be mutually exclusive. Presumably, dynamic
changes in the composition of telomeric nucleoprotein complexes
may reflect the different functional states of telomeres.
Two types of plant chromosome ends have been
proposed: those with G–overhangs and blunt-ended ones
that are recognized by the KU70/80 dimer (Riha et al., 2000;
Gallego et al., 2003; Kazda et al., 2012; Nelson and Shippen,
2012a). Thus, the apparently redundant proteins may operate
concurrently at telomeres with respect to cell cycle,
developmental stage or type of chromosome ends. For
example, localization of TRB1 is quite consistent but highly
dynamic during interphase; moreover, the level of nuclearassociated
TRB1 diminishes during mitotic entry, and it progressively
re-associates with chromatin during anaphase/
telophase (Dvorackova et al., 2010). Interestingly, our BiFC
assay also showed interaction of N–terminal fragments of
TERT with TRP1, a member of the TRFL I family (Figure S10)
(Hwang et al., 2001; Karamysheva et al., 2004). Importantly,
TRP1 also interacts with KU70 (Kuchar and Fajkus, 2004),
which is presumably involved in protection of blunt chromosome
ends and may also therefore be an integral part of the
plant telomere protection complex (Figure 6).
Although it is tempting to draw possible analogies
between mammalian shelterin components and the TRB
and POT1b proteins involved in a similar plant complex,
an alternative interpretation of the function of TRB is possible
when considering our data in connection with a recent
description of mammalian HOT1 protein (Kappei et al.,
2013). This protein shows strikingly similar interactions
and functions: it specifically binds double-stranded telomeric
DNA repeats, localizes to a subset of telomeres (presumably
those that are being elongated), and associates
with active telomerase. Thus, HOT1 contributes to the
association of telomerase with telomeres and to telomere
length maintenance (Kappei et al., 2013). Our findings suggest
that TRB proteins may perform similar functions in
plant telomeres, i.e. as direct telomere-binding proteins
that act as positive regulators of telomere length.
EXPERIMENTAL PROCEDURES
Primers
The sequences of all primers and probes used in this study are
provided in Table S2.
Plant material and construct generation
The 35Spro:GFP-TRB1 plants and construct have been described
previously (Dvorackova et al., 2010). The TRBpro:TRB1-GFP construct
was prepared as follows: genomic DNA from A. thaliana
Col–0 was isolated using a DNeasy plant mini kit (Qiagen, http://
www.qiagen.com/), and used as a template for PCR to amplify the
Figure 6. Schematic diagram of observed protein–protein interactions at
telomeric ends.
Half the telomeric ends in A. thaliana are blunt-ended (Riha et al., 2000; Kazda
et al., 2012). Here we show a simplified chart of interactions associated
with telomeres. Solid arrows indicate protein–protein interactions that were
verified in this study or in previous studies (Kuchar and Fajkus, 2004; Kannan
et al., 2008; Schrumpfova et al., 2008; Cifuentes-Rojas et al., 2012;
Kazda et al., 2012) using at least two independent approaches (i.e. BiFC,
pull-down or yeast-two hybrid assay). The grey arrow indicates a TERT–
TRP1 interaction observed only by BiFC. The dashed black arrow shows a
presumed interaction between POT1b and telomere single-stranded DNA
that has not yet been directly demonstrated. The interaction between POT1b
and telomerase is specific for the TER2 isoform of TER, while the other
interactions with the telomerase complex are dependent on the catalytic
TERT subunit. The diagram suggests the existence of distinct telomerase
recruitment pathways for blunt-ended telomeres and telomeres with a G–
overhang.
© 2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2014), 77, 770–781
TRB interactions with telomeres and telomerase 777
TRB1 genomic sequence including the 5′ UTR. The 3′ UTR was
amplified from BAC clone FJ10.16 (Arabidopsis Information
Resource, http://www.arabidopsis.org/). We used 0.25 units of Hot
Start Phusion polymerase (Finnzymes, http://www.thermoscienti
ficbio.com/finnzymes/) with 0.2 mM dNTPs, 19 HF reaction buffer
(Phusion Hot Start II high fidelity DNA polymerase; http://
www.thermoscientificbio.com/), 3% dimethylsulfoxide and 0.5 lM
of each primer (5′ UTRFw + TRB1 Rev or 3′ UTR Fw + 3′ UTR Rev).
The conditions used were in accordance with the manufacturer’s
instructions (Finnzymes). PCR products were precipitated using
poly(ethylene glycol), and cloned into a Gateway multi-site system
(Invitrogen, http://www.lifetechnologies.com), together with the
GFP tag (GFP in pDONR221, provided by Keke Yi, College of life
Sciences, Zhejiang University, China). pKm43GW (Karimi et al.,
2005) was used as the destination vector. A. thaliana Col–0 was
subsequently transformed by floral dipping (Clough and Bent,
1998), and transformants selected on MS medium containing
30 lg/ml kanamycin were scored for GFP expression.
PCR-based genotyping of plant lines
T–DNA insertion mutant plants of trb1 (SALK_025147) in the Col–0
background were used. To distinguish between wild-type plants
and those that were heterozygous or homozygous for the T–DNA
insertion in the trb1 gene, we isolated genomic DNA from leaves
using NucleoSpin Plant II (Machery Nagel, http://www.mn-net.
com/). The genomic DNA was used for PCR analysis with MyTaq
DNA polymerase (Bioline, http://www.bioline.com). The conditions
used were in accordance with the manufacturer’s instructions.
The primers used were specific for T–DNA (P3 + P2 primers) or
the TRB1 gene (P1 + P2 primers). Cycling conditions were 98°C for
1 min (initial denaturation), followed by 30 cycles of 94°C for 30
sec, 58°C for 45 sec and 72°C for 2 min, with a final extension at
72°C for 10 min.
Rt–pcr
Total RNA was extracted from approximately 50 mg of frozen plant
tissue using an RNeasy plant mini kit (Qiagen), and RNA samples
were treated with TURBO DNA-free (Applied Biosystems/Ambion,
http://www.lifetechnologies.com TURBO DNA-free). The quality
and quantity of RNA were determined by electrophoresis on 1%
w/v agarose gels and by measurement of absorbance using an
Implen nanophotometer (http://www.implen.de/). Reverse transcription
was performed using random hexamers (Sigma-Aldrich,
http://www.sigmaaldrich.com) with 1 lg RNA and Mu-MLV reverse
transcriptase (New England Biolabs, https://www.neb.com/). The
cDNA obtained was screened by PCR analysis for the presence of
trb1 transcripts using MyTaq DNA polymerase (Bioline) with primers
P4 and P5. Thermal conditions were 95°C for 1 min (initial denaturation),
followed by 30 cycles of 94°C for 45 sec, 55°C for 45 sec
and 72°C for 2 min, with a final extension at 72°C for 10 min.
Nicotiana benthamiana transformation, nuclei isolation
and FISH
Leaves of 5-week-old N. benthamiana plants were infiltrated with
Agrobacterium tumefaciens containing 35Spro:GFP-TRB1 (vector
pGWB6, strain LBA4404) (Dvorackova et al., 2010), and 35Spro:p19
(Silhavy et al., 2002) as described by Voinnet et al. (2003). The infiltration
medium contained 10 mM MES (pH approximately 5.7) and
10 mM MgCl2. After 3–4 days, leaf discs were checked under a fluorescence
microscope, and protoplasts were prepared as described
by Yoo et al. (2007b); the digestion medium contained also 0.25%
Pectolyase Y23 (Duchefa, http://www.duchefa-biochemie.nl/) in
addition to celullase and macerozyme and a 119 lm filter was used
for filtration. Protoplasts in W5 buffer were collected by centrifugation
at 50 g, and resuspended in NIB (Nuclei Isolation Buffer;
10 mM MES, 0.2M Sucrose, 2.5 mM EDTA,10 mM NaCl, 10 mM KCl
2.5 mM DTT, 0.1 mM Spermine, 0.5 mM Spermidine) to extract
nuclei as described by McKeown et al. (2008). Isolated nuclei were
then fixed in 4% paraformaldehyde for 15 min, resuspended in
wash buffer (50 mM Tris/Cl, pH 8.5, 5 mM MgCl2, 20% glycerol),
4°C, spun down at 300g and stored in storage buffer (50 mM Tris/
Cl, pH 8.5, 5 mM MgCl2, 50% glycerol) at -20°C until use.
Then 20 ll of nuclei were spun on the Superfrost plus microscopic
slide (http://www.menzel.de/) at 56 g, and re-fixed in 4% pformaldehyde
in 19 PBS/0.05% Triton X-100 for 15 min. Slides
were then treated with RNase (100 lg/ml) for 1 h at 37°C, and
hybridized with telomeric Cy3-labelled peptide nucleic acid probe
in 65% formamide/20% dextran sulfate/29 SSC at 37°C overnight.
Post-hybridization washes were performed at 37°C using 29 SSC.
Slides were counter-stained using 4,6–diamidino-2–phenylindole
(1 lg/ml), and observed on a Zeiss (http://www.zeiss.cz/) Axioimager
Z1 using an AHF filter set.
Immunolocalization
Arabidopsis seeds expressing TRB1pro:TRB1-GFP under the control
of the native promoter and trb1 seeds were bleach-sterilized
for 10 min, washed in water and sown onto half-strength MS
medium/1% agar plates. Seedlings grown under the constant
light, at 22°C for 2 weeks, then chopped into small pieces. Protoplasts
were prepared as described by Yoo et al. (2007b), and the
nuclei and immunolocalization protocols were adapted from those
described by McKeown et al. (2008). Slides were first blocked in a
mixture of 29 block solution (Roche, http://www.roche.cz)/19 PBS/
5% goat serum at room temperature for 30 min, then incubated
with primary antibodies [mouse anti-TRB 1.2 or 5.2 or, anti-GFP
(Abcam ab290, http://www.abcam.com/), all diluted 1:300] for 2 h
at 37°C, and visualized using secondary antibodies A11001 and
A21207 (Invitrogen) at 1:500 dilution.
Immunblot analysis
To determine the level of TRB1 protein in plants, we isolated
nuclei as described by Bowler et al. (2004). The nuclei were lysed
using SDS loading buffer (250 mM Tris/Cl, pH 6.8, 4% w/v SDS,
0.2% w/v bromophenol blue, 20% v/v glycerol, 200 mM b–mercaptoethanol),
heated at 80°C for 10 min, and protein extracts were
analysed by SDS–PAGE. Proteins were electrophoretically transferred
to a nitrocellulose membrane at 360 mA for 1 hour in
192 mM glycine, 25 mM Tris, 0.5% SDS and 10% (v/v) methanol in
a Bio-Rad Mini Trans-Blot cell. Ponceau S staining was performed
to check the quality of the extracts and to ensure equal gel loading
for immunodetection. Membranes were blocked with 5% non-fat
dry milk in Tris-buffered saline/Tween, and probed using the
monoclonal anti-TRB1 1.2 antibody and the secondary polyclonal
horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins
(DAKO, http://www.dako.com), both diluted 1:5000. Immunoreactive
bands were visualized using LumiGLO reagent and
peroxide (Cell Signaling Technology, http://www.cellsignal.com)
on a Fujifilm LAS-3000 CCD system (http://www.fujifilm.com/).
Chromatin immunoprecipitation assay
The ChIP assay was performed as described by Bowler et al.
(2004) with modifications. Chromatin extracts were prepared from
seedlings treated with 1% formaldehyde. The chromatin from isolated
nuclei was sheared to a mean length of 250–500 bp by soni©
2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2014), 77, 770–781
778 Petra Prochazkova Schrumpfova et al.
cation using a Bioruptor (Diagenode, http://www.diagenode.com)
and centrifuged (16 000 g/5 min/4°C). The matrix GFP-Trap A
(Chromtec, http://www.chromotek.com) was blocked against nonspecific
interaction using 200 mM ethanolamine, 1% BSA and the
DNA sequences TR10–24–G and TR10–24–C (Table S2), which are
not recognized by TRB proteins (Schrumpfova et al., 2004). The
pre-treated matrix was incubated with chromatin diluted with ChIP
dilution buffer (16.7 mM Tris/Cl, pH 8,0, 1.2 mM EDTA, 167 mM
NaCl, 0.1% Triton X–100, phenylmethanesulfonyl fluoride and protease
inhibitors) at 4°C for 4 h, and subsequently washed with
low-salt, high-salt, LiCl and 10 mM Tris (pH = 8,0), 1 mM EDTA (TE)
buffers. In contrast to Bowler et al. (2004), the levels of detergents
(Triton X–100, Nonidet P-40 and sodium deoxycholate) were
reduced to 0.1%. The cross-linking was reversed using 0.2 M NaCl
overnight, and was followed by treatment with proteinase K
(Serva, http://www.serva.de) treatment, phenol/chlorophorm
extraction and treatment with RNase A (Serva) as described by
Bowler et al. (2004). ChIP assays were repeated using three biological
replicates (plants grown at different times).
Dot-blot assay
DNA isolated using ChIP was diluted into 200 ll of 400 mM NaOH
and 10 mM EDTA, and samples were denatured at 95°C for 10 min
and cooled on ice. They were then spotted onto Hybond XL
membrane (GE Healthcare, http://www3.gehealthcare.com) and
subjected to hybridization with sequence-specific probe TR–4C
(Table S2). The probe was hybridized in 250 M sodium phosphate,
pH 7.5, 7% SDS and 16 mM EDTA overnight at 55°C, and washed
with 0.29 SSC + 0.1% SDS. The signal was evaluated using MultiGauge
software (Fujifilm). All experiments were performed using
three independent biological replicates. Re-hybridization with centromeric
and 18S rDNA probes was performed as described previously
(Mozgova et al., 2010).
TRAP assay
Protein extracts from 2-week-old seedlings were prepared as
described by Fitzgerald et al. (1996). These extracts were subjected
to the TRAP assay as described by Fajkus et al. (1998). TS21
was used as the substrate primer for extension by telomerase,
and TEL-PR was used as the reverse primer in the subsequent
PCR.
TRF analysis
TRF analysis was performed as described previously (Ruckova
et al., 2008) using 500 ng genomic DNA isolated from 5–7-weekold
rosette leaves using NucleoSpin Plant II (Machery Nagel).
Southern hybridization was performed using the end-labelled telomere-specific
probe TR–4C (Table S2). Telomeric signals were
visualized using an FLA7000 imager (Fujifilm), and a grey-scale
intensity profile was generated using MultiGauge software (Fujifilm).
Evaluation of fragment lengths was performed using a Gene
Ruler 1 kb DNA ladder (Fermentas, http://www.thermoscientific
bio.com/fermentas/) as the standard. Mean telomere lengths were
calculated as described by Grant et al. (2001).
Yeast two-hybrid analysis
Yeast two-hybrid experiments were performed using the Match-
makerTM
GAL4-based two-hybrid system (Clontech, http://
www.clontech.com/). cDNA sequences encoding TERT N – terminal
fragments comprising amino acids 1-271 and 1-582 were subcloned
from pDONR/Zeo entry clones (Zachova et al., 2013) into
the Gateway-compatible destination vector pGBKT7-DEST (bait
vector). The pGBKT7-DEST destination vector that was used in
this study was created by Horak et al. (2008) who introduced the
Gateway conversion cassette into the original Matchmaker system
vector pGBKT7 (Clontech). The pGADT7 prey vectors (Clontech)
carrying TRB1, TRB2 and TRB3 have been described previously
(Schrumpfova et al., 2008). Each bait/prey combination was cotransformed
into Saccharomyces cerevisiae PJ69–4a, and colonies
were inoculated into YPD medium and cultivated overnight. Successful
co-transformation was confirmed on SD medium lacking
Leu and Trp, and positive interactions were selected on SD medium
lacking Leu, Trp and His or SD medium lacking Leu, Trp and
Ade. Co-transformation with an empty vector served as a negative
control for auto-activation. Each test was performed three times
using two replicates at a time. In addition, the protein expression
levels were verified by immunoblotting.
Bimolecular fluorescence complementation
For PCR amplification of sequences encoding the tested proteins,
and to generate restriction site overhangs, Phusion HF DNA polymerase
(Finnzymes) was used. The conditions used were in accordance
with the manufacturer’s instructions. The primers used
were F–TRB1/2/3_BstBI, R–TRB1/2/3_SmaI, F–TERT_KpnI, R–
RID1+BamHI, F–F2N_KpnI and R–F2N+BamHI, and plasmids
encoding the tested proteins were used as templates. The amplified
DNA fragments were gel-purified, digested with BstBI/SmaI or
KpnI/BamHI (New England Biolabs), and ligated into vectors
pSAT1-nEYFP and pSAT1-cEYFP. As a negative control, we used
an AtGaut10-cEYFP construct. To quantify transformation efficiency
and to label cell nuclei, we co-transfected a plasmid
expressing mRFP fused to the nuclear localization signal of the
VirD2 protein of A. tumefaciens (mRFP-VirD2NLS; Citovsky et al.,
2006). The vectors and the mRFP-VirD2NLS and AtGaut10-cEYFP
constructs were kindly provided by Stanton Gelvin (Department of
Biological Sciences, Purdue University, IN, USA). Arabidopsis thaliana
leaf protoplasts were prepared and transfected as described
by Wu et al. (2009). DNA (10 lg of each construct) was introduced
into 1 9 105
protoplasts. Transfected protoplasts were incubated
in the light at room temperature overnight, and then observed for
fluorescence using a Zeiss AxioImager Z1 epifluorescence microscope
equipped with filters for YFP (Alexa Fluor 488), RFP (Texas
Red) and CY5 (chloroplast autofluorescence).
In vitro translation and co-immunoprecipitation
Proteins were expressed from the same constructs as used in the
yeast two-hybrid system with a haemagglutinin tag (pGADT7;
TRB1, 2 and 3 proteins) or a Myc tag (pGBKT7; TERT fragments)
using a TNT quick coupled transcription/translation system (Promega,
https://www.promega.com) in 50 ll reaction volumes
according to the manufacturer’s instructions. The TRB proteins
were radioactively labelled using 35
S-Met. The co-immunoprecipitation
procedure was performed as described by Schrumpfova
et al. (2011). Input, unbound and bound fractions were separated
by 12% SDS–PAGE, and analysed using an FLA7000 imager (Fuji-
film).
Accession numbers
Sequence data have been deposited in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: At1g49950 (TRB1), At5g67580 (TRB2, formerly
TBP3), At3g49850 (TRB3, formerly TBP2,), At5g16850.1 (TERT),
At2g20810 (Gaut10) and At5g59430 (TRP1).
© 2014 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2014), 77, 770–781
TRB interactions with telomeres and telomerase 779
ACKNOWLEDGEMENTS
We would thank to Dorothy E. Shippen (Department of Biochemistry,Texas
A&M University,TX, USA) for donation of TRFL
contructs, Stanton B. Gelvin (Department of Biological Sciences,
purdue University, IN, USA) for donation of mRFP-VirD2NLS and
AtGaut10-cEYFP constructs and his help with initial analysis of
BiFC experiments, and Iva Mozgova (Department of Plant Biology
and Forest Genetics, Swedish University of Agricultural
Sciences, SW) for discussion and support. The research was
supported by the Czech Science Foundation (13-06943S), by
project CEITEC (CZ.1.05/1.1.00/02.0068) of the European Regional
Development Fund, and project CZ1.07/2.3.00/30.0009
co-financed from European Social Fund and the state budget of
the Czech Republic.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version
of this article.
Figure S1. Detection of TRB1–GFP protein by specific anti-TRB1
1.2 antibody in ChIP fractions.
Figure S2. Telomeric sequence is highly enriched compared to
centromeric DNA or 18S rDNA.
Figure S3. Location of antibody recognition sites within the structure
of TRB1 protein.
Figure S4. Anti-TRB 1.2 and 5.2 antibodies were unable to detect
proteins from TRFL family.
Figure S5. Anti-TRB1 1.2 does not detect any signal on trb1À/À
plants, but 5.2 recognizes some epitopes in trb1À/Àmutant plant
lines.
Figure S6. Telomere shortening in trb1À/Àplants is progressive.
Figure S7. Whole images of protoplasts (whose segments are
shown in Figure 5C) obtained by bimolecular fluorescence com-
plementation.
Figure S8. TRB1, 2 and 3 proteins are able to pull-down TERT frag-
ments.
Figure S9. Telomerase activity or processivity in vitro is not changed
in response to TRB1 status.
Figure S10. TRP1 protein interacts with plant telomerase (TERT).
Table S1. Quantification of TRB1 foci co-localized/associated with
telomeric foci.
Table S2. Sequences of all primers and probes used in this study.
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TRB interactions with telomeres and telomerase 781
Supporting Figures and Tables
Figure S1
Detection of TRB1-GFP protein by specific anti-TRB1 1.2 antibody in ChIP fractions
20 µl of each ChIP fraction or 5 µl of GFP-TRAP_A Matrix were separated with SDS-PAGE,
transferred to nitrocellulose membrane using standard wet western blot protocol and visualised with
specific anti-TRB1 1.2 antibody (1:5 000) and secondary antibody anti-mouse HRP (DAKO) (1:10
000) 1. Input (chromatin after sonication); 2. Unbound (unbound chromatin that was previously
diluted with ChIP buffer); 3. Matrix after chromatin binding before wash procedure; 4. Matrix after
chromatin binding after whole wash procedure; 5. Matrix after elution; 6. Elution.
Figure S2
Telomeric sequence is highly enriched compared to the centromeric DNA or 18S rDNA
DNA cross-linked with TRB1 protein was isolated by ChIP analysis using GFP-TRAP_A
matrix from Wt and TRB1pro:TRB1-GFP plants. Subsequent hybridisation of isolated TRB1associated
DNA with radioactively labelled telomeric, centromeric 18S rDNA probes in three
biologically and technically replicated experiments has shown marked enrichment of
telomeric DNA contrasting with only negligible enrichment of the centromeric DNA or 18S
rDNA. The relative difference of the probed DNA co-precipitated with TRB1-GFP compared
to the Wt was measured in each replicate (1, 2, 3; enrichment was related to Wt=1) using
Multi Gauge software.
Figure S3
Location of antibody recognition sites within the structure of TRB1 protein with ELISA
Construction of vectors and protein expression/purification protocols of TRB1 - 3 proteins or TRB1
domains were previously described in (Schrumpfova, Kuchar et al. 2004, Mozgova, Schrumpfova et
al. 2008). Purified proteins as antigens were coated onto a 96-well microtiter plate (Nunc MaxiSorp)
with 500 ng / well of antigen. As primary antibody, we used supernatants of tested monoclonal
hybridomes anti-TRB 1.2 and 5.2 (1:200). Colouring intensity of secondary antibody anti-mouse HRP
(DAKO) (1:10 000) visualised with TMB (Test line) was measured as absorbance at 450 nm after
30min. Values of measured absorbance are listed in the table.
Figure S4
Anti-TRB 1.2 and 5.2 antibodies were unable to detect proteins from TRFL family
Genes coding for TRFL2, TRFL9 and TRP1 proteins were cloned into pET28 vector (Karamysheva,
Surovtseva et al. 2004). Radioactively labeled proteins were synthesized by in vitro transcription and
translation using rabbit reticulocyte system (Promega), separated with SDS-PAGE and transferred to
nitrocellulose membrane (as described in (Schrumpfova, Kuchar et al. 2008)). Proteins were visualised
using autoradiographic analysis with Typhoon FLA7000 (GE Healthcare) (35
S-labeled) or probed with
the monoclonal antibodies (anti-His (Sigma); anti-TRB1 1.2; anti-TRB1 5.2 (1:5 000)). As a second
antibody, polyclonal anti-mouse rabbit HRP (DAKO) was used. Both antibodies were diluted 1:5 000.
Immunoreactive bands were visualised with LumiGLO Reagent and Peroxide (Cell Signaling
Technology) a Fujifilm LAS-3000 CCD system. Extracts from isolated nuclei used in ChIP
experiment were used as positive control (see Figure 3D).
Figure S5
Anti-TRB1 1.2 does not detect any signal on trb1-/- plants, while 5.2 recognises some epitopes
also in trb1-/- mutant plant lines
Antibodies 1.2 and 5.2. (green) were tested on isolated nuclei from trb1-/- and TRB1pro:TRB1-GFP
plants (TRB1-GFP). Nuclei are counterstained with DAPI (magenta).
(a) 1.2 is specific to TRB1 protein since it recognises epitopes in the sample from TRB1-GFP plants,
but not in trb1-/-.
(b) 5.2 antibody recognises also other members of the SMH protein family, thus producing a
detectable signal in trb1-/- nuclei.
Figure S6
Telomere shortening in trb1-/- plants is moderately progressive.
Terminal restriction fragment analyses of trb1-/- plant lines A, B and C were performed in
their 5th
generation (G5) and in earlier generations (G2 or G4). Results are expressed
relatively to the Wt control (Wt).
Figure S7
Whole images of protoplasts (whose segments are shown in Figure 5C) obtained by bimolecular
fluorescence complementation.
Interactions of TRB proteins with TERT fragments are depicted with the arrows. Plasmids encoding
nEYFP-tagged TRB clones, cEYFP-tagged TERT fragments or Gaut10 (as negative control), and
mRFP-VirD2NLS (to mark cell nuclei and to determine transfection efficiency). [YFP fluorescence
(yellow), mRFP fluorescence (red), and chloroplasts (green pseudocolor); chloroplast autofluorescence
also visible in the YFP chanell]. Bar = 20 µm.
Figure S8
TRB1, 2 and 3 proteins are able to pull-down TERT fragments
The TNT expressed TRB1, 2 and 3 (35S-labelled*) were mixed with fragments of TERT (myc-tag)
and incubated with anti-myc antibody. In the control experiment, the TRB proteins were incubated
with myc-antibody and beads in the absence of partner protein. Input (I), unbound (U), and bound (B)
fractions were collected and run in SDS–12% PAGE gels. Interactions of TRB2 and 3 proteins with
TERT fragments appear to be relatively weaker than interaction between TRB1 and TERT fragments.
Together, these results support yeast two-hybrid or bimolecular fluorescence complementation
experiments.
Figure S9
Telomerase activity or processivity in vitro is not changed in response to TRB1 status.
Ladder of telomeric repeats produced in TRAP assay (telomere repeat amplification protocol) has
shown no differences in the trb1 mutant or TRB1-GFP plant lines compared to wild-type lines. In
negative control (-), no protein extract was used
Figure S10
TRP1 protein interacts with plant telomerase (TERT)
Bimolecular fluorescence complementation has shown interaction of TRP1 protein with TERT
fragments. Arabidopsis leaf protoplasts were transfected with 10 μg of each of plasmids encoding
nEYFP-tagged TRP1 clones, cEYFP-tagged TERT fragments or Gaut10 (as negative control), and
mRFP-VirD2NLS (to mark cell nuclei and to determine transfection efficiency). The cells were
imaged by epifluorescence microscopy after overnight incubation. Protoplast image [YFP fluorescence
(yellow), mRFP fluorescence (red), and chloroplasts (green pseudocolor); chloroplast autofluorescence
also visible in the YFP chanell] we can observe clear nuclear interactions of TRP1 proteins with TERT
fragments depicted with the arrows. Bar = 20 µm.
Table S1
Quantification of TRB1 foci co-localised/associated with telomeric foci
Isolated nuclei from leaves transformed with GFP-TRB1 were used for telomere PNA FISH.
Number of fluorescence foci of GFP-TRB1 signal co-localised with telomeric signal is
numbered in this Table. These FISH results showed that TRB1 speckles and telomere signals
are associated (31%) or co-localised (59%) cases
Table S2
The sequences of all primers and probes used in this study.
Name Sequence (5 ′→ 3 ′ )
P1 GAGAGGAGAAGATAAAGATGTCACC
P2 CGTTCTCCCTTCCTAAACAGG
P3 CAACACTCAACCCTATCTCGG
P4 AGTATTCATATGGGTGCTCCTAAGCAGA
P5 CGGGATCCTCAGGCACGGATCATCATT
TR10-24-G AGTACCAGCCATGACCAGCCATGA
TR10-24-C TCATGGCTGGTCATGGCTGGTACT
TR-4C CTAAACCCTAAACCCTAAACCCTAAACC
TS21 GACAATCCGTCGAGCAGAGTT
TEL-PR CCGAATTCAACCCTAAACCCTAAACCCTAAACCC
5´UTR Fw GGGGACAACTTTGTATAGAAAAGTTGTCCACCCATTAGAGGGACGAGTATGG
TRB1 Rev GGGGAC TGC TTT TTTGTACAA ACTTGCGGCACGGATCATCTGTCGAAT
3´UTR Fw GGGGACAGCTTTCTTGTACAAAGTGGCCggtaatggaaagcgagagaagaag
3´UTR Rev GGGGACAACTTTGTATAATAAAGTTGCTATTTTAGTATGTCA AATTTCGGATGA
F-TRB1_BstBI CCTTCGAAATGGGTGCTCCTAAGCAGAAATG
F-TRB2_BstBI CCTTCGAAATGGGTGCACCAAAGCAGAAG
F-TRB3_BstBI CCTTCGAAATGGGAGCTCCAAAGCTGAAG
R-TRB1_SmaI ATCCCGGGGGCACGGATCATCATTTTGCAG
R-TRB2_SmaI ATCCCGGGCCAAGGATGATTACGGATCCTG
R-TRB3_SmaI ATCCCGGGCCGAGTTTGGCTATGCATTCTATAC
F-TERT_KpnI atggtaccATGCCGCGTAAACCTAGACATC
R-RID1+BamHI GAGGATCCTTAGGGAGTTATACAAGGAGCATTAC
F-F2N_KpnI taggtaccGGCGAGGATGTAGACCAACAT
R-F2N+BamHI GAGGATCCCTACCAGCTCCTTTTCCGGTA
Supplement I
Schrumpfová, P.P., Vychodilová, I., Hapala, J., Schořová, Š., Dvořáček, V., Fajkus, J., 2016.
Telomere binding protein TRB1 is associated with promoters of translation machinery genes
in vivo. Plant Mol.Biol. 90, 189–206.
P.P.S. participated in the design of experiments, significantly involved in the
experimental part, data evaluation, and in the ms writing and editing
Telomere binding protein TRB1 is associated with promoters
of translation machinery genes in vivo
Petra Procha´zkova´ Schrumpfova´1,2 • Ivona Vychodilova´1,2 • Jan Hapala1,2 •
Sˇa´rka Schorˇova´1,2 • Vojteˇch Dvorˇa´cˇek1,3 • Jirˇı´ Fajkus1,2,3
Received: 29 July 2015 / Accepted: 16 November 2015 / Published online: 23 November 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Recently we characterised TRB1, a protein
from a single-myb-histone family, as a structural and
functional component of telomeres in Arabidopsis thaliana.
TRB proteins, besides their ability to bind specifically
to telomeric DNA using their N-terminally positioned
myb-like domain of the same type as in human shelterin
proteins TRF1 or TRF2, also possess a histone-like domain
which is involved in protein–protein interactions e.g., with
POT1b. Here we set out to investigate the genome-wide
localization pattern of TRB1 to reveal its preferential sites
of binding to chromatin in vivo and its potential functional
roles in the genome-wide context. Our results demonstrate
that TRB1 is preferentially associated with promoter
regions of genes involved in ribosome biogenesis, in
addition to its roles at telomeres. This preference coincides
with the frequent occurrence of telobox motifs in the
upstream regions of genes in this category, but it is not
restricted to the presence of a telobox. We conclude that
TRB1 shows a specific genome-wide distribution pattern
which suggests its role in regulation of genes involved in
biogenesis of the translational machinery, in addition to its
preferential telomeric localization.
Keywords Telomere repeat binding (TRB) Á ChIP-seq Á
Arabidopsis thaliana Á Ribosome Á snoRNA Á Translation
machinery
Introduction
Telomere binding proteins and their complexes, exemplified
by the shelterin complex in vertebrates (de Lange
2005), perform essential functions at chromosome ends.
Primarily, they inhibit DNA-damage responses at telomeres
to protect them from being mis-recognized as unrepaired
chromosome breaks, thus solving the so-called endprotection
problem (Sfeir and de Lange 2012) of linear
chromosomes. Other functions of telomere proteins
include, for example, telomerase recruitment and coordination
of telomere elongation by telomerase with lagging
strand synthesis by DNA polymerase during telomere
replication (thereby solving the end-replication problem)
(Latrick and Cech 2010; Sfeir et al. 2009; Soudet et al.
2014). However, the functions attributed to telomerebinding
proteins are presumably not the original functions
of these proteins in earlier stages of evolution, which
preceded the onset of linear chromosomes connected with
the necessity to solve the end-replication problem (Fajkus
et al. 2005; Louis and Vershinin 2005; Nosek and
Tomaska 2003; Valach et al. 2011). Examples supporting
this notion can be seen, for example, in DNA repair proteins
which paradoxically are also implicated in the control
of telomere organization and length although their
Electronic supplementary material The online version of this
article (doi:10.1007/s11103-015-0409-8) contains supplementary
material, which is available to authorized users.
& Jirˇı´ Fajkus
fajkus@sci.muni.cz
1
Mendel Centre for Plant Genomics and Proteomics,
CEITEC – Central European Institute of Technology,
Masaryk University, Kamenice 5, 625 00 Brno,
Czech Republic
2
Laboratory of Functional Genomics and Proteomics, National
Centre for Biomolecular Research, Faculty of Science,
Masaryk University, Kamenice 5, 625 00 Brno,
Czech Republic
3
Institute of Biophysics, Academy of Sciences of the Czech
Republic, v.v.i., Kra´lovopolska´ 135, 61265 Brno,
Czech Republic
123
Plant Mol Biol (2016) 90:189–206
DOI 10.1007/s11103-015-0409-8
presence at telomeres apparently contradicts the end-protective
telomere functions (Kazda et al. 2012; Weaver
1998).
One of the best known telomere-binding proteins in
budding yeast, the repressor-activator protein 1 (RAP1), is
also well known for its involvement in gene activation and
repression and in DNA replication. Further studies have
examined additional roles for RAP1 in heterochromatin
boundary-element formation, creation of hotspots for
meiotic recombination, and chromatin opening (reviewed
in Morse 2000). The TTAGGG DNA repeat-binding proteins
1 and 2 (TRF1 and TRF2) bind to mammalian
telomeres as part of the shelterin complex and are essential
for maintaining chromosome end stability. While most of
their binding sites identified in a chromatin immunoprecipitation
sequencing (ChIP-seq) study corresponded to
telomeric regions, these two proteins also localize to
extratelomeric sites (Simonet et al. 2011) of which the vast
majority contain interstitial telomeric sequences (ITSs).
However, non-ITS sites were also identified which correspond
to centromeric and pericentromeric satellite DNA,
and these TRF-binding sites are often located in the
proximity of genes or within introns. It was thus suggested
that TRF1 and TRF2 may couple the functional state of
telomeres to the long-range organization of chromosomes
and gene regulation networks by binding to extratelomeric
sequences (Simonet et al. 2011). Even the specific telomere-elongation
tool, telomerase, is involved in a number of
non-telomeric processes (reviewed in Majerska et al.
2011). These examples demonstrate that the function of
telomere-localized proteins may not be exclusively
telomere-associated and that telomere metabolism/protection
may be mediated by proteins which play more general
roles in the genome.
Understanding of the composition and function of
telomere-binding protein complexes in plants lags behind
that in animals and yeasts. Nevertheless, we have recently
characterized a key candidate shelterin-like component
belonging to the plant-specific single-myb-histone group of
proteins termed TRB (Telomere repeat binding)
(Schrumpfova et al. 2014). In addition to our earlier studies
which demonstrated specific binding of proteins from this
group to telomeric DNA in vitro (Schrumpfova et al. 2004)
and characterized their DNA-protein and protein–protein
interactions in detail (Hofr et al. 2009; Mozgova et al.
2008; Prochazkova Schrumpfova et al. 2008), our recent
study revealed preferential co-localization of a member of
this group, TRB1, with telomeres in situ and in vivo,
telomere shortening in trb1 knockout mutants, and moreover
its physical interaction with the catalytic subunit of
telomerase, TERT (Prochazkova Schrumpfova et al. 2014).
These results, together with our previous findings of TRB1
interaction with POT1b protein, one of the paralogs of
Protection Of Telomeres (POT1) protein (Kuchar and
Fajkus 2004; Schrumpfova et al. 2008) and the data on
interaction of POT1 proteins with telomerase (CifuentesRojas
et al. 2011; Rossignol et al. 2007), make this protein
currently the best-established component of a putative
plant shelterin complex.
In addition to the results demonstrating sequencespecific
binding of TRB1 to telomeric DNA and corresponding
telomere-specific functions, this protein is also
capable of binding to chromatin through protein–protein
interactions or sequence-non-specific interactions with
DNA via its H1/H5-like domain (Mozgova et al. 2008).
In vivo, the protein shows highly dynamic association with
chromatin and preferential localization to the nucleus and
the nucleolus during interphase (Dvorackova et al. 2010).
TRB1 localization is cell cycle-regulated, as the level of
nuclear-associated TRB1 diminishes during mitotic entry
and it progressively re-associates with chromatin during
anaphase/telophase. Using fluorescence recovery after
photobleaching and fluorescence loss in photobleaching,
we determined that TRB1 interaction with chromatin is
regulated at two levels at least, one of which is coupled
with cell-cycle progression with the other involving rapid
exchange (Dvorackova et al. 2010). These results strongly
suggest additional roles for TRB1 connected with chromatin
function.
In this study, we thus set out to investigate the genomewide
localization pattern of TRB1 to examine its preferential
sites of binding to DNA in vivo and its potential
functional roles in the genome-wide context. Using ChIP
followed by next generation sequencing (ChIP-seq), we
show that TRB1 associates with promoter regions of certain
genes in addition to binding long telomeric repeats.
Classification of these genes using GO analysis revealed a
strong link between TRB1 binding and promoters of
translation machinery-related genes.
Materials and methods
Plant material and construct generation
The TRBpro:GFP-TRB1 and trb1-/- plants and constructs
were described previously (Schrumpfova et al.
2014). All the A. thaliana plants used in this study have a
Col0 background.
Chromatin immunoprecipitation assay
The ChIP assay was performed as described (Bowler et al.
2004) with modifications described in (Schrumpfova et al.
2014). Chromatin extracts were prepared from seedlings
treated with 1 % formaldehyde. The chromatin from
190 Plant Mol Biol (2016) 90:189–206
123
isolated nuclei was sheared to an average length of
250–500 bp by sonication (Bioruptor, Diagenode) and
centrifuged. Anti-TRB1 5.2 antibody (Schrumpfova et al.
2014) was bound to a Protein G agarose matrix (Pierce) for
3 h at 4 °C which was subsequently washed with ChIP
dilution buffer (16.7 mM Tris–HCl, pH 8.0, 1.2 mM
EDTA, 167 mM NaCl, 0.1 % Triton X-100, 0.1 mM
PMSF and protease inhibitors). The experimental procedures
for isolating TRB-GFP protein on GFP-TRAP_A
(Chromtec) and native TRB1 protein on an Protein G
agarose matrix (Pierce) with bound anti-TRB1 antibody
were as described in (Schrumpfova et al. 2014). Both
matrices were blocked against non-specific interaction with
200 mM ethanolamine, 1 % BSA and TR10-24-G and
TR10-24-C (Schrumpfova et al. 2014), incubated with
chromatin diluted with ChIP dilution buffer, and subsequently
washed with low salt; high salt; LiCl; TE buffers as
in Bowler (Bowler et al. 2004). The cross-linking was
reversed by 0.2 M NaCl overnight followed by proteinase
K (Serva) treatment, phenol/chlorophorm extraction, and
RNase A (Serva) treatment.
ChIP-qPCR and RT-qPCR reactions
Primers used for ChIP-qPCR are described in Fig. 4 and
are listed in Supplementary Table S1a. Three ul of ChIP
DNA was added to the 20 ul reaction mix of FastStart
SYBR Green Master (Roche) and the final concentration of
each forward and reverse primer was 0.4 uM. Reactions
were done in triplicate; the PCR cycle consisted of 5 min
of initial denaturation at 95 °C followed by 40 cycles of 5 s
at 95 °C, 20 s at 60 °C and 15 s at 72 °C. At least two
biological replicates in two technical replicates were
analysed. The relative copy number of each selected gene
in Wt (-) or TRB-GFP (?) samples compared to that in
the genomic input fraction was calculated by the 2-DCt
method (Pfaffl 2004).
Total RNA was isolated from Arabidopsis seedlings
using the RNeasy Plant Mini Kit (Qiagen) followed by the
DNaseI treatment (TURBO DNA-free, Applied Biosystems/Ambion)
according to the manufacturers’ instructions.
The quality and quantity of RNA was checked by
electrophoresis on 1 % (w/v) agarose gels and by absorbance
measurement (NanoPhotometr IMPLEN). cDNA
was prepared by reverse transcription of 1 lg of RNA
using M-MuLV reverse transcriptase (NEB) and Random
Nonamers (Sigma). Quantification of the transcripts of
translation machinery related genes was related to the
ubiquitin reference gene and was done using qPCR
GreenMaster with UNG/lowROX (Jena Bioscience) by the
Rotorgene6000 (Qiagen) machine. Four ll of 129 diluted
cDNA was added to the 20 ll reaction mix; the final
concentration of each forward and reverse primer was
0.3 lM (Supplementary Table S1b). Reactions were done
in triplicates; the PCR program consisted of 5 min of initial
denaturation at 95 °C followed by 40 cycles of 15 s at
95 °C, 20 s at 58 °C and 30 s at 72 °C. Analyses were
performed for at least three biological replicates in three
technical replicates. Transcript levels of chosen genes in
the trb1-/- (Schrumpfova et al. 2014) were normalized to
Ubq10 transcript and presented ‘‘relative transcript levels’’
were calculated as the fold increase/decrease relative to
respective wild-type seedlings using (2-DDCt
) method
(Pfaffl 2004).
Library preparation and sequencing
Library preparation and sequencing was done by the
European Molecular Biology Laboratory (EMBL) Genomics
Core Facility, Heidelberg, Germany. Fifty microliters
of immunoprecipitated DNA [0.2–6 ng DNA measured by
Qubit dsDNA HS Assay Kit (Invitrogen)] was used for
library preparation using NEBNext ChIP Seq Library Prep
Master Mix (NEB), with Agencourt XP beads (Beckman)
in the ratios described in the protocol with only one
exception, dilution of the Adapter 1:17 in water. DNA
fragments of 270–300 bp were selected on 2 % e-gels
(Invitrogen). Subsequently, a PCR reaction (18 cycles)
with indexed Primers 1–11 from the NEBNext Multiplex
Oligo set (NEB) was performed.
The quality of the final libraries was checked on the
Bioanalyzer (Agilent) and the quantity with the Qubit
dsDNA HS Assay Kit (Invitrogen). The libraries were
pooled equimolar and diluted to 10 pM (denaturation in
NaOH) in Hyb Buffer 1 (HT1) from the TruSeq SR Cluster
Kit v3-cBot-HS (Illumina). Libraries were sequenced
(50 bp single-end reads) on an Illumina HiSeq 2500 using
TruSeq SBS Kit v3-HS (Illumina) sequencing reagents.
Bioinformatic methods
Sequence logo construction
The sequence logo was created using a k-mer analysis. We
counted all substrings of length k in two datasets, the 50
UTR sequences and the 50
UTR peak-covered regions only.
We decided to use 8-mers sorted by their number of
occurrence, and for fragment reconstruction used only
those whose number was at least twice as high in peak
regions as in the whole 50
UTR dataset. The fragment was
constructed by a modified version of the algorithm
described in (Macas et al. 2010), and instead of extending
the whole prefix and subsequently the whole suffix we
extended both the prefix and the suffix alternately by one
nucleotide. As fragments are composed of k-mers with
diverse frequencies, every base of each fragment was
Plant Mol Biol (2016) 90:189–206 191
123
assigned a weight representing the frequency of its k-mer
of origin. For better matching, some fragments were used
as their reverse complements with preference for C-rich
variants of DNA. Fragments were aligned with MUSCLE
(Edgar 2004). The sequence logo (Schneider and Stephens
1990) was then generated from fragments with mean
weight higher than 20. The y-axis displays position weights
totalled from the aligned fragments.
Read mapping and filtration
We mapped the Illumina fastq reads onto the reference
genome of Arabidopsis thaliana (A_thaliana_Jun_2009)
with Bowtie2 (using the very-sensitive option) (Langmead
and Salzberg 2012). Unmapped reads or reads with a low
quality mapping score (\25) were removed. Biological
replicates were merged into single files. The coverage of
the mitochondrial DNA was negligible compared to the
chromosomal DNA, while the chloroplast DNA was represented
in the data more frequently. Therefore, we separated
the reads from each dataset into three files, genomic,
mitochondrial and chloroplast sequences, and peaks were
called for all groups independently. However, we did not
detect any peaks in mitochondria and chloroplasts. We
used average profiles and heatmaps to illustrate the distribution
of ChIP-seq reads along genes (±2000 bp). The
pictures were produced by ngs.plot (Shen et al. 2014) with
parameters –R genebody, –F protein_coding.
Peak calling
We called peaks in the data files using two programs,
MACS v. 1.4.3 20131216 and PePr v. 1.0.2 (Zhang et al.
2008b, 2014). MACS is one of the most widely used programs
for peak calling in sequencing data while PePr is a
new peak-calling software which detected peaks visually
more accurately in our data.
With MACS (parameters: effective genome size—g:
90000000, band width—bw 250, keeping all duplicate
reads—keep-dup all, ChIP/control scaling ratio—ratio
1.215273 and 1.742786 for the first and second replicate,
respectively) we obtained a set of peaks for each of the two
replicates. The ChIP/control scaling ratios were calculated
with NCIS (Liang and Keles 2012). We selected peaks with
a false discovery rate (FDR) below 5 % from each replicate
and calculated an intersection of these two sets. We produced
another set of peaks with PePr (parameters: –peaktype
= sharp) which accepts two replicates (both input and
control files) and returns only peaks present in both of
them. PePr peaks with too short (\30 bp) estimated fragment
lengths in any of the two samples were removed
(1879 peaks). Finally, we made an intersection (4995) of
the PePr (24,219) and MACS (3690) peaks.
Peak analysis
We studied the relative amount of peaks in genomic
regions with different functions. We downloaded annotated
datasets provided by TAIR (ftp://ftp.arabidopsis.org/home/
tair/Sequences/blast_datasets/TAIR10_blastsets/) that contain
sequences separated with respect to their position
within or outside of genes. We converted these FASTA
files into genomic coordinate files (BED) and calculated
intersections with the 4995 TRB1 genomic peaks.
Reference datasets
We examined intersections with 50
UTR, 30
UTR (variable
length), upstream and downstream sequences (500 bp) and
intergenic regions (variable length, trimmed by 1000 bp at
both ends). In addition, we created a dataset of coding
sequences derived from a coordinate file
TAIR10_GFF3_genes.gff also provided by TAIR.
Coverage quantification
We calculated dataset coverage as the length of each reference
dataset relative to the genome size. Overlapping
base pairs of sequences of the same type (i.e. those on
opposite strands) were counted once. In addition, for each
dataset peak coverage was obtained as the total length of
the dataset-peak intersection relative to the total size (bp)
of the dataset. Finally, peak relative occurrence represents
the number of peaks intersecting with any sequence of the
particular dataset relative to the total size of the dataset
(presented in Mb). In all cases, two elements intersect each
other if they have at least 1 bp-overlap.
Quantification of telomeric, centromeric and 18SrDNA
sequences
We analysed the unprocessed Illumina files for the occurrence
of telomeric, centromeric and 18SrDNA sequences
by counting the number of reads containing their respective
sequence motifs, in particular AAACCCTAAACCC
TAAACCCT for telomeres, TATGAGTCTTTGGCTTT
GTGTCTT for centromeres, and CTAGAGCTAATACGT
GCAACAAAC for 18SrDNA.
Telo-box occupation
We examined sequences from all reference datasets for the
presence of a telobox (any of AAACCCTA, AACCCTAA,
ACCCTAAA, TAAACCCT, TAGGGTTT, TTAGGGTT,
TTTAGGGT and AGGGTTTA). Sequences containing
one or more telo-boxes were included. Next, we counted
192 Plant Mol Biol (2016) 90:189–206
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telo-boxes intersected by a TRB1 peak and calculated telobox
peak coverage as the ratio of these two numbers.
Gene ontology (GO) enrichment
We analysed the GO database using the program GoMiner
(v. 2011-01) (Zeeberg et al. 2003, 2005). Only GO categories
with a p value higher than 0.05 and categories with
size 5–500 genes were arranged graphically in CIMminer
software (http://discover.nci.nih.gov/cimminer/).
Data availability
The data have been deposited in NCBI’s Gene Expression
Omnibus (Edgar et al. 2002) and are accessible through
GEO Series accession number GSE69431 (http://www.
ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69431).
Results
TRB1 is associated with telomeric sequences in vivo
Previously, we described the co-localization of TRB1
protein with telomeres in situ and showed that TRB1 can
bind DNA containing telomeric sequences in vivo
(Schrumpfova et al. 2014). Since TRB1 is capable of
binding telomeric (via its myb-like domain) as well as nontelomeric
DNA (via its histone-like domain) (Hofr et al.
2009; Mozgova et al. 2008; Schrumpfova et al. 2014), we
pondered whether TRB1 is localized exclusively to
telomeres or has also other binding sites in the genome
in vivo. To address this question, we performed nextgeneration
sequencing of DNA isolated by ChIP of native
or GFP-tagged TRB1.
As source material, we used formaldehyde cross-linked
Arabidopsis seedlings stably transformed with a construct
driven by the native promoter TRB1pro:TRB1-GFP
[TRB1-GFP (?)] (Dvorackova et al. 2010). Wild-type
plants were used as negative controls [Wt (-)]. The
experimental setup is depicted schematically in Fig. 1a for
anti-GFP-TRB1. TRB1-GFP protein was immunoprecipitated
from purified nuclei using a GFP-Trap A matrix
which contains a single variable antibody domain that
recognizes GFP, as described in (Schrumpfova et al. 2014).
Furthermore, we performed another ChIP analysis using
anti-TRB1 (5.2) antibody that specifically recognizes the
conserved region of the myb domain of TRB proteins
(Schrumpfova et al. 2014) (Fig. 1b). Native TRB1 protein
was isolated from wild-type plants [TRB1 (?)] using
Protein G magnetic beads. In the negative control, no
antibody was used in a parallel ChIP [Wt (-)]. DNA
recovered from ChIP underwent ultra-high-throughput
Illumina sequencing.
For evaluation purposes, arrays of at least three perfect
telomeric repeats [(AAACCCT)3] were considered as
‘‘long telomeric repeats’’ and reads containing them were
counted in each sequenced sample. The relative differences
in the numbers of long telomeric repeats in TRB1-GFP (?)
or TRB1 (?) samples were expressed relative to the relevant
Wt (-) = 1 (Fig. 1c). Absolute values confirmed our
previous results (Schrumpfova et al. 2014) and demonstrated
that the observed enrichment of long telomeric
sequences in these samples is not due to the high copy
number of telomeric DNA in the Arabidopsis genome but
due to sequence-specific association of TRB1 with telomeric
sequences. No enrichment of other repetitive sequences
examined—centromeric DNA or 18S rDNA—was
found in the ChIP-seq data.
Association of TRB1 with euchromatin in vivo
To define the genomic regions that are associated with
TRB1 protein, we mapped the ChIP-seq data onto the
TAIR A_thaliana_Jun_2009 assembly (TAIR10 annotation)
and visualised the data using Integrative Genomics
Viewer (IGV). To limit falsely identified peaks in the data,
we used two independent peak calling programs, MACS
and PePr (for details see Methods). We examined peaks in
all five chromosomes of A. thaliana. Only peaks detected
by both programs in both ChIP approaches (anti-GFPTRB1
and anti-TRB1) were used in subsequent analysis.
This set contains 4995 peaks and here we term it ‘‘TRB1
genomic peaks’’. No peaks were detected in mitochondrial
or chloroplast DNA. However, mitochondrial DNA coverage
was negligible compared to the nuclear DNA.
TRB1 genomic peaks are absent in centromeric regions
or heterochromatic knobs localized on chromosomes 4 and
5, but appear associated with euchromatic genes in both
variants of ChIP (Fig. 2a, b, and Fig. S1). As the TAIR9
assembly lacks clusters of repetitive sequences, long
telomeric tracts or 45S rDNA repetitive sequences are not
visible in the IGV viewer. Figure 2c shows the detailed
enrichment of DNA regions in TRB1-GFP (?) and TRB1
(?) samples that were immunoprecipitated by ChIP and
sequenced. It can be seen that only DNA regions in TRB1GFP
(?) or TRB1 (?) samples which were highly enriched
with respect to the Wt (-) were considered as TRB1
genomic peaks (marked with an asterisk). The data have
been deposited in NCBI’s Gene Expression Omnibus
(Edgar et al. 2002) and are accessible through GEO Series
accession number GSE69431 (http://www.ncbi.nlm.nih.
gov/geo/query/acc.cgi?acc=GSE69431).
Plant Mol Biol (2016) 90:189–206 193
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Analysis of TRB1 targets
To examine preferential targeting of TRB1 protein to
specific Arabidopsis genome loci we visualized distribution
of ChIP-seq reads along protein-coding genes genes. Clear
enrichment of TRB1 was observed in the vicinity of transcription
start sites (TSS) and transcription end sites (TES)
using both purification approaches—anti-GFP-TRB1 and
anti-TRB1 (Fig. 3).
For a more detailed resolution of TRB1 association with
specific parts of genes, gene families and also non-protein
coding genes we established various datasets covering
specific parts of the A. thaliana genome according to
TAIR10 blastsets (ftp://ftp.arabidopsis.org/home/tair/
Sequences/blast_datasets/TAIR10_blastsets/). The major
division of these datasets is into Protein coding genes and
Non-protein coding genes (Fig. 4a, yellow boxes). The
group of Non-protein coding genes includes rRNA,
snoRNA, snRNA, tRNA, ncRNA, miRNA, other RNAs,
pseudogenes and transposable element genes. In the
vicinity of both Protein coding and Non-protein coding
gene categories, datasets designated as Upstream 500 bp
(from TSSs) or Downstream 500 bp (from TES) were
classified. Moreover, four extra datasets close to the Protein
coding genes sets were analysed: 50
UTR, 30
UTR,
Upstream translation start 500 bp, and Downstream
translation stop 500 bp. To clearly distinguish associations
of all datasets that closely surround coding genes with
which the TRB1 protein is associated, we designed a further
dataset named Intergenic covering regions beginning
more than 500 bp in front of the Upstream 500 bp or more
than 500 bp behind the Downstream 500 bp datasets, i.e.
1000 bp distant from the transcription start or stop site of
Protein or of Non-protein coding genes.
The relative coverage of the whole A. thaliana genome
by each dataset (e.g., the dataset Upstream 500 bp of
Fig. 1 Long telomeric tracts are tandem repeats preferentially
targeted by TRB1 genome-wide. Schematic illustration of the two
different approaches used in ChIP analysis: a first, TRB1 tagged with
GFP was isolated using a GFP-Trap A matrix from crosslinked wildtype
[Wt (-)] and TRB1pro:TRB1-GFP [TRB-GFP (?)] seedlings.
b Second, native-TRB1 [TRB1 (?)] protein was isolated from
crosslinked seedlings using anti-TRB1 antibody linked to a Protein G
matrix; wild-type seedlings [Wt (-)] were used as a negative control.
The associated DNA was subjected to next-generation sequencing
(NGS) using the Illumina platform. Two biological replicates were
analysed by both approaches. c Marked enrichment of long telomeric
DNA repeats (AAACCCT)3 compared to other repetitive sequences
(centromeric or 18S rDNA) in ChIP-seq samples showing preferred
association of TRB1 with telomeric sequence. The relative difference
in the amount of the selected repetitive sequence was measured in
each sequenced sample (biological replica) separately and enrichment
was expressed relative to the relevant [Wt (-) = 1]
194 Plant Mol Biol (2016) 90:189–206
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Protein coding genes covers 10.8 % of the whole genome)
and the coverage of individual datasets by TRB1 genomic
peaks obtained by combining both purification approaches
(see above), is listed in Fig. 4b. It can be seen that in the
vicinity of Protein coding genes, approximately 10 % of
Upstream datasets (green boxes, 9.7–9.9 %) are covered by
the TRB1 protein. This is approximately twice the value
for TRB1 association with the entire Protein coding genes
(yellow box, 3.9 %) or Downstream datasets (blue boxes,
4.8–6 %) although generally Upstream and Downstream
datasets cover roughly the same proportion of the A.
thaliana genome. The enrichment of TRB1 protein association
with the Upstream in comparison to the Downstream
genomic loci is obvious even in the Non-protein
coding genes (3.7 % for Upstream compared to 0.8 % for
Non-protein coding genes and 2.9 % for Downstream
genomic loci). In contrast, the TRB1 protein is associated
only with 1.5 % of the Intergenic sequences although these
sequences cover almost 14 % of the genome. The preferential
association of the TRB1 protein with sequences in
Upstream coding genes is shown in the graph in Fig. 4c
featuring the relative occurrence of TRB1 genomic peaks
per sequence (dataset) length (displayed per Mb). The total
bp size and the number of TRB1 genomic peaks in each
dataset are listed in the Supplemental Table S2. The overall
summary of sequences belonging to each dataset is given in
the Supplemental Table S3.
Taken together, our results demonstrate that the TRB1
protein is preferentially associated in vivo with promoter
regions of coding genes compared to gene bodies or
Downstream genomic loci. Association of TRB1 with Intergenic
sequences is very low. This detailed analysis
Fig. 2 TRB1 is preferentially associated with euchromatic regions.
a IGV view of All coding genes, i.e. Protein coding genes and Nonprotein
coding genes (transposable element genes, rRNA, snoRNA,
snRNA, tRNA, ncRNA, miRNA and pseudogenes) on chromosome 4.
Regions where TRB1 binding was enriched are depicted as TRB1
genomic peaks. The absence of TRB1 association in the centromeric
region and the heterochromatic knob (grey box) is visible. b IGV view
of a 1.4 Mb region of the short arm of chromosome 4 (position
0.9–2.3 Mb) where the heterochromatin knob is located. Only Protein
coding genes or ‘‘Transposable element genes’’ are depicted in
separate lines below (modified from GBrowse). Significant enrichment
of the TRB1-associated regions within euchromatin is visible
not only as increasing peaks in IGV viewer [boxes: TRB1-GFP (?)
and TRB1 (?)] but also in the line TRB1 genomic peaks. c The IGV
view of a 620 kb region displays regions highly associated with TRB1
proteins used for further analyses (asterisk)
Plant Mol Biol (2016) 90:189–206 195
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localizes extratelomeric TRB1-DNA association in
euchromatin, especially in promoter regions.
Validation of TRB1 binding sites by ChIP-qPCR
To verify the TRB1 binding sites identified by ChIP-seq
(Fig. 5a), we performed an independent ChIP experiment
using the same seedlings as those used for the ChIP-seq
analysis and purified DNA, and quantified the abundance
of selected loci by qPCR. Preferential association of TRB1
protein with DNA regions that were identified as TRB1
genomic peaks was verified for several selected genomic
loci (examples are shown in Fig. 5b). We confirmed by
qPCR that all selected TRB1 binding sites are enriched in
TRB1-GFP (?) samples with respect to the Wt (-) controls
(examples of ribosomal protein L34, S5 and snoRNA
Fig. 3 TRB1 specifically
associates with TSS and TES of
genes. Distribution of ChIP-seq
reads obtained using
a immunoprecipitation of
TRB1-GFP protein relative to
negative control or
b immunoprecipitation of native
TRB1 protein relative to
negative control, illustrated by
average gene profiles (upper
panels) and heatmaps (lower
panels)
196 Plant Mol Biol (2016) 90:189–206
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Fig. 4 Detailed analysis of TRB1-bound regions. a Schematic representation
of the selected ‘‘datasets’’ (coloured boxes) used in
detailed analysis of the DNA regions associated with TRB1 protein.
These datasets were designed according to the TAIR10 annotation.
The group of Non-protein coding genes contains genes depicted in
TAIR10 as rRNA, snoRNA, snRNA, tRNA, ncRNA, miRNA,
pseudogenes, transposable element genes and other RNAs. The
Intergenic dataset is a collection of regions located at least 1000 bp
from the transcription start or stop site (Upstream or Downstream
500 bp) of the Protein coding genes or of the Non-protein coding
genes to avoid interference with other datasets. b An overview shows
the percentage representation of each analysed dataset within the A.
thaliana genome: ‘‘Genome coverage by whole dataset’’. ‘‘Dataset
coverage by TRB1 genomic peaks’’ represents a portion of the dataset
overlapping with the TRB1-enriched regions. c The number of the
TRB1 genomic peaks counted per size unit (1 Mb) of each dataset
Fig. 5 Confirmation of the
TRB1 protein binding sites by
qPCR. Two ribosomal protein
coding genes and one snoRNA
coding region identified among
TRB1 genomic peaks, with
ACTIN 7 as negative control,
were chosen for quantitativePCR
(qPCR) verification of
regions enriched in ChIP-seq
and defined as TRB1 genomic
peaks. a IGV view of the TRB1
genomic peaks in Wt (-) or
TRB-GFP (?) ChIP-seq
samples where PCR amplicons
are depicted by triangles below
the simplified gene models. b Yaxis
values represent the
abundance of the DNA
recovered from each locus
related to the input DNA.
Mean ± SD of three technical
replicates are shown
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are shown). In contrast, a site that was not included in the
list of TRB1 genomic peaks (ACTIN 7) did not show any
significant difference between TRB1-GFP (?) and Wt (-)
control.
The telomeric repeat is the most conserved motif
associated with TRB1
To determine the most frequent motif associated with
TRB1 protein, we selected the dataset with the highest
relative occurrence of TRB1 genomic peaks per 1 Mb, i.e.
the 50
UTR dataset, and extracted regions associated with
the TRB1 protein for further analysis. As promoter regions
could be biased for some motifs, the TRB1-binding motif
was reconstructed as a sequence logo from 8-mers twice as
abundant in the TRB1-associated regions as in the whole 50
UTR dataset (see Table S4a, b). The sequence logo shows
that the telomeric repeat is highly represented in the
sequences recognised by the TRB1 protein (Fig. 6a). If the
plant telomeric repeat unit (CCCTAAA) and its circular
permutations are removed (Fig. 6b), a related motif (i.e.
sequences enriched for CCTA) is still clearly overrepresented
(Fig. 6c).
TRB1 is bound to short telomeric sequences
in promoters
Binding of TRB proteins to the telomeric DNA sequences
was initially shown in vitro (Schrumpfova et al. 2004) and
binding of TRB proteins expressed in E. coli to the synthetic
telomeric oligo was described in detail (Hofr et al.
2009; Mozgova et al. 2008; Schrumpfova et al. 2004). The
present study confirmed recent observation by hybridisation
with a radioactively labelled telomeric probe which
showed that the DNA immunoprecipitated with TRB1
protein is enriched for long telomeric sequences
(Schrumpfova et al. 2014).
The Arabidopsis genome contains short interspersed
segments of the telomeric sequence both in terminal and
interstitial positions, as well as the long telomeric repeats.
These short interstitial telomere motifs, termed telo-boxes,
exhibit a non-random distribution. They were described in
the promoters of genes coding for translation elongation
factor EF1a (Liboz et al. 1990), promoters of many ribosomal
protein coding genes (Tremousaygue et al. 1999),
and promoters of genes involved in the biogenesis of the
translation machinery (Gaspin et al. 2010). The occurrence
of telo-box motifs in the TRB1-associated regions from the
above mentioned datasets was examined. The motifs
analysed, AAACCCTA, AACCCTAA or ACCCTAAA
(and their reverse complements), represent the most frequent
permutations of the shortest telo-box motifs, as
described in (Gaspin et al. 2010). Only telo-boxes directly
covered by a TRB1 genomic peak were counted. Almost
28 % of telobox-sequences located in the 50
-UTR region of
the Protein coding genes are covered by TRB1 peaks
(Fig. 7 and Table S5). The total number of telo-boxes from
each dataset and the number of TRB1 genomic peaks that
Fig. 6 The most conserved DNA motif associated with the TRB1
protein. Sequence logos were constructed from the most frequent
8-mers. a The total logo was created from the most frequent 8-mers
present in the 50
UTR regions and covered by the TRB1 genomic
peaks. b Only 8-mers containing any permutation of at least one plant
telomeric repeat were used for sequence logo construction with
telomeric repeat. c The rest of the 8-mers, e.g. without any
permutation of the plant telomeric repeat, were used for sequence
logo construction without telomeric repeat
198 Plant Mol Biol (2016) 90:189–206
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overlap these telo-boxes are listed in the supplementary
Table S6. In general, it is evident that the TRB1 protein is
associated at least twice as frequently with telo-box
sequences in the Upstream datasets (22–28 %; green
boxes) in comparison with the Downstream datasets
(10–15 %; blue boxes) or with the low number (4 %) of
Interstitial sequences.
The number of telo-box genes with which the TRB1
protein is associated is presumably underestimated here,
because the two independent immunoprecipitation
approaches and the very stringent detection and refinement
of the TRB1 genomic peaks by two independent programs
limits false positives to a minimum, while also discarding
many true peaks. Therefore, although TRB1 is visibly
associated with e.g., all three promoters of eEF1 alpha-1, -
2 and -3 telo-box-containing genes (At1g07940,
At1g07930 and At1g07920) (Figure S2), a TRB1 genomic
peak was reported only in the eEF1-alpha 1 gene
(At1G07940). Our strict policy of selection of TRB1
genomic peaks is also visible in the Supplementary Figure
S3, where the genes coding for ribosomal proteins
RPS15B, RPS15C, RPS15D (At5g09490, At5g09500,
At5g09510) are shown.
TRB1 protein is associated with genes related
to ribosome biogenesis
As most of the TRB1-associated loci are located in gene
promoter regions, we wanted to know whether these genes
are related in their function, biological processes, or subcellular
localization of their products. We selected genes
with TRB1-enrichment in their upstream region (Upstream
500 bp dataset) and thus both the categories Protein coding
and also Non-protein coding genes are included in this
summary. The Arabidopsis Genome Initiative numbers
(AGI numbers) of these genes were compared to the set of
AGI numbers of all genes in the Upstream 500 bp datasets
using Gene Ontology miner (GOMiner) (Zeeberg et al.
2003, 2005). The table of GO subcategories GO:0005575
Cellular component and GO:0008150 Biological process
arranged graphically by Clustered Image Maps miner
(CIMminer) demonstrates a statistically significant
enrichment of these GO subcategories (p B 0.05) (Fig. 8).
The total output of GOMiner and also of CIMminer with
all genes and detailed description (e.g. name, gene type,
function) is shown in Supplementary Table S7.
As is clearly visible in Fig. 7, protein coding genes with
whose promoters the TRB1 protein is associated are enriched
in GO:0005575 Cellular component in categories like
GO:0005730 nucleolus/GO:0031981 nuclear lumen/
GO:0005654 nucleoplasm/GO:0016604 nuclear body, etc.
(GO categories highlighted in light orange). Also, terms
associated with small and large ribosomal subunit
(GO:0005732 small nucleolar ribonucleoprotein complex/
GO:0015934 large ribosomal subunit/GO:0022626
cytosolic ribosome) (dark orange) are highly represented.
In the category GO:0008150 Biological process, many
genes coding for ribosomal proteins were statistically
enriched in subcategories GO:0071843 cellular component
biogenesis at cellular level/GO:0042254 ribosome biogenesis/GO:0022613
ribonucleoprotein complex biogenesis
(highlighted in dark orange). Names and functions of
individual genes are visible in Supplementary Table S7, list
‘‘CIM with genes’’. Also, a significant number of intergenic
snoRNA genes are bound by TRB1 protein in their
Fig. 7 The TRB1 protein preferentially occupies telo-box sequences.
Sequences with at least one permutation of the telomeric repeat were
extracted from each dataset. The chart shows the proportion of the
sequences containing a telo-box motif that are covered by a TRB1
genomic peak. The clear preference of the TRB1 protein for telo-box
sequences is especially apparent in the 50
-UTR dataset in which
almost 28 % of the telo-box sequences located in the 50
-UTR are
recognized by the TRB1 protein
Plant Mol Biol (2016) 90:189–206 199
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Upstream 500 bp region and are included in the CIMminer
Table. These snoRNA genes are highly enriched in categories
such as e.g., GO:0034660 ncRNA metabolic process/GO:0000154
rRNA modification/GO:0006364 rRNA
processing, etc.
Discussion
After recent findings, the previously-established concept
that the shelterin proteins are located exclusively on the
chromosome ends, the telomeres, has been superseded.
Fig. 8 Schematic view of Gene Ontology (GO) classification of
categories significantly enriched in TRB1 protein. All coding genes
(Protein coding genes together with Non-protein coding genes) that
contain a TRB1 genomic peak in their 500 bp Upstream proximity
(datasets: Upstream 500 bp) were analysed by the GOMiner software.
The set of genes in which TRB1 protein is enriched in their 500 bpupstream
region was compared to the set of all coding genes from A.
thaliana. General tables of the GO subcategories ‘‘GO:0005575
Cellular components’’ and ‘‘GO:0008150 Biological processes’’ were
arranged graphically by CIMminer. The horizontal axes represent
significantly enriched GO subcategories (p B 0.05) and the vertical
axes represent individual genes, in clusters. Genes that contain a
TRB1 genomic peak within 500 bp from the transcription start site
and are significantly enriched in the GO subcategories are highlighted
as red boxes. For a detailed description of individual genes, see
Supplementary Table S7
200 Plant Mol Biol (2016) 90:189–206
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Emerging evidence indicates that the shelterin components
also possess non-telomeric functions such as transcriptional
regulation (Martinez et al. 2010; Zhang et al. 2008a), DNA
repair (Bradshaw et al. 2005), NF-jB activation (Teo et al.
2010), Epstein-Barr virus replication (Deng et al. 2002), or
regulation of mitochondrial oxidative phosphorylation
(Chen et al. 2012). Some of these non-telomeric functions
could be explained, at least partially, by their binding to
ITSs (Bosco and de Lange 2012; Krutilina et al. 2003;
Mignon-Ravix et al. 2002; Simonet et al. 2011) or even to
unrelated DNA sequences which remain to be identified in
future studies.
Association of TRB1 with telomeric sequences
Besides the terminally located long telomeric repeats, the
A. thaliana genome contains two long interstitial telomeric
tracts that consist of degenerate telomeric repeats with
islands of perfect plant telomeric sequences (Uchida et al.
2002). In a previous study we speculated on the telomeric
association of GFP–TRB1 speckles in A. thaliana (Dvorackova
et al. 2010), and using a plant system with longer
telomeres, N. benthamiana, we showed clear in situ colocalization
of TRB1-GFP with telomeres in leaves
(Schrumpfova et al. 2014). However, localization of TRB1
in many speckles in A. thaliana nuclei, together with the
fact that the TAIR9 assembly, used as the reference genome,
lacks not only telomeres but also interstitial telomeric
regions, did not allow us to determine whether the TRB1associated
telomeric tracts are terminally or interstitially
located. In the current study we describe a significant
enrichment of perfect long plant telomeric repeats associated
with the TRB1 protein using ChIP-seq.
TRB1 specific binding to telomeric sequence
Many different telomere-binding proteins possess a single
myb-related DNA-binding domain with Helix-Turn-Helix
(HTH) organization (reviewed in Peska et al. 2011). In the
case of human TRF1 and TRF2, the X-ray crystal structures
of complexes with telomeric DNA show that they
recognize the same AACCCTA binding site by means of
homeodomains, as does the yeast telomeric protein Rap1p;
TRF dimers specifically recognize two of the three G-C
base pairs in the major groove that characterize telomeric
repeats (Court et al. 2005; Hanaoka et al. 2005; Nishikawa
et al. 2001). Our search for a sequence-specific motif recognized
by TRB1 revealed that the telo-box sequence is
highly abundant in promoter regions, and moreover the
core of this sequence (CCTA) containing two G-C base
pairs is still present if we focus only on sequences without
the complete telo-box motif. Thus we conclude that
potential TRB1-binding motifs are associated with short
interstitial telomere sequences as well as with long
telomeric tracts, and furthermore the TRB1 protein binds to
the core segment of telomeric repeats (CCTA) through its
myb-domain in the same manner as human TRF proteins.
Preferential association of TRB1 with promoter
regions
The landscape of the A. thaliana epigenome can be classified
into four main chromatin states (Roudier et al. 2011)
which have been further subdivided in a recent more
detailed study, providing a total of nine chromatin states
(Sequeira-Mendes et al. 2014). Only a small proportion of
the TRB1 protein is associated with two heterochromatin
states, mostly located in the centromeric regions or knobs
on the short arm of chromosome 4 or the long arm of
chromosome 5. In contrast, most of the TRB1 protein
shows a preferential euchromatic localization, especially
with sequences in the Upstream regions, mostly 50
UTR
regions. Association of TRB1 protein with entire gene
bodies or intergenic regions is markedly lower. A similar
distribution of the TRB1 protein preferring DNA Upstream
from TSSs was observed in both Protein coding and Nonprotein
coding genes. The partial increase in TRB1 association
with the genomic loci behind the transcription stop,
Downstream, may be due to the fact that neighbouring
downstream genes transcribed in the same direction are
frequent at a distance of 100–700 bp (Alexandrov et al.
2006), and thus an overlap of the genomic regions located
Upstream from TSSs or Downstream from the transcriptional
stop may result in unspecific enrichment in one of
these datasets. However, association of TRB1 with the
upstream regions is markedly higher.
In accordance with the localization of TRB1 in
euchromatin, we found that the overlap between the list of
the chromosome 4 genes associated with marks of silent
euchromatin (H3K9me3) and Polycomb-regulated chromatin
(H3K27me3) (Turck et al. 2007) and the genes with
a TRB1 genomic peak in their 500 bp upstream vicinity
(Upstream 500 bp of Protein and Non-protein coding
genes datasets) is considerably higher (20 and 9 %,
respectively) than the overlap between the genes associated
with the heterochromatin mark H3K9me2 and the genes
with a TRB1 genomic peak in proximity to their 500 bp
upstream region (0.3 %) (Turck et al. 2007).
TRB1 target genes are connected with ribosome
biogenesis
The distribution of short interspersed telomeric repeats or
telo-boxes within the Arabidopsis genome is not uniform
and their frequency is higher within 50
flanking regions
(Gaspin et al. 2010). In this study we show that telo-boxes
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located Upstream from TSSs are more likely to be associated
with the TRB1 protein than those located Downstream
from the transcription stop region or even in entire
Protein coding or Non-protein coding gene bodies or
within the Intergenic range. The occurrence of telo-boxes
is enriched in promoter regions of genes participating in
translation, e.g. genes coding for ribosomal proteins,
translation elongation factors (EF1 a), eukaryotic initiation
factors (eIFs), and small nucleolar RNAs (snoRNAs), or in
the promoters of genes involved in rRNA processing
(Gaspin et al. 2010; Liboz et al. 1990; Tremousaygue et al.
1999). The majority of telo-boxes of plant translation-related
genes are located within a narrow window located
between -50 and ?50 relative to the TSS (Gaspin et al.
2010). Telo-boxes are not able, by themselves, to activate
gene expression in transgenic plants but act in synergy with
other cis-acting elements like site II motifs or TEF boxes
that show conservative topological association with teloboxes
in Arabidopsis and O. sativa (Gaspin et al. 2010;
Manevski et al. 2000; Tremousaygue et al. 2003). In
agreement with this observation, we found that TRB1
shows a statistically significant enrichment in 500 bp
Upstream regions of the genes connected in GO with terms
describing small and large subunit of the ribosome,
nucleolus, or rRNA modification.
Since many snoRNA genes that are involved in the
processing of rRNA are transcribed in clusters or are
located in introns (Brown et al. 2008), the total number of
snoRNA genes with TRB1-binding sites in their promoter
is underestimated because the distal snoRNA gene in a
transcribed cluster may be farther than 500 bp away from
the transcription start site and, consequently, is not included
in our analysis. Another similar case is intronic
snoRNA genes, which are indistinguishable from the protein
coding genes. For details, see supplementary Figure S4
(snoRNA clusters) which displays the IGV view of
snoRNA clusters significantly associated with TRB1 protein
in their shared promoter. We proved that only the
snoRNAs category is associated with the TRB1 protein in
its 500 bp Upstream proximity (Fig. 9a). Other categories
from the Non-protein coding dataset e.g., rRNA, snRNA,
tRNA, ncRNA, miRNA, other RNA, pseudogenes and
transposable element do not exhibit increased association
with TRB1. We found that more than 80 % of intergenic
orphan snoRNA genes (not transcribed in clusters) or
intergenic snoRNAs with a telo-box in proximity to their
their 500 bp Upstream are covered by TRB1 genomic peaks
(Fig. 9b, c).
Our analysis of 192 ribosomal protein coding genes,
used for a transcription level study (Savada and BonhamSmith
2014), revealed that almost 85 % of them contain a
telo-box in proximity to their 500 bp Upstream translation
start and 65 % of these telo-box sequences are covered by
TRB1 genomic peaks. At least nine of ten promoters of the
most frequently transcribed ribosomal genes in A. thaliana
seedlings (Savada and Bonham-Smith 2014) are recognised
by the TRB1 protein. By contrast, promoters of the ribosomal
protein-coding genes (which are transcribed at the
lowest level in A. thaliana seedlings (Savada and BonhamSmith
2014)) showed either lower or a zero level of TRB1
binding.
Analogous estimation of eIFs with telo-boxes which are
associated with the TRB1 protein was derived from a list of
eIFs (https://www.arabidopsis.org/browse/genefamily/eIF.
jsp). Almost 60 % of eIFs contain a telo-box sequence in
proximity to their 500 bp Upstream translation start and
50 % of these telo-box sequences are covered by TRB1
genomic peaks.
The pattern of TRB1 association with chromatin
suggests its role as a transcription factor
In humans, some core shelterin subunits are documented to
modulate gene expression outside of telomeres; for example,
TRF2 interacts with the repressor element 1-silencing
transcription factor (REST), a repressor of genes devoted to
neuronal functions (Zhang et al. 2008a). Another example
is mammalian RAP1 which is known to play a role in
repression of subtelomeric genes, and more than 70 % of
its binding sites are found at intragenic positions or in the
vicinity of gene-coding chromatin (Martinez et al. 2010).
Strong nucleolar localization of the TRB1 protein,
besides weaker nuclear and cytoplasmic localization, has
been already shown (Dvorackova et al. 2010). Genes
coding for 45S ribosomal RNA (rDNA) which organize
nucleoli are close neighbours of telomeres in chromosomes
of many eukaryotes including Arabidopsis, and also show a
number of functional associations (Dvorackova et al. 2015;
Fransz et al. 2002). A proteomic analysis has revealed that
a significant portion of the nucleolar protein pool consists
of ribosomal proteins (RPSs or RPLs), RNA modifying
factors (snRNA or snoRNA binding), and proteins participating
in translation (EF1s, eIFs) (Brown et al. 2005;
Pendle et al. 2005). The majority of non-ribosomal nucleolar
proteins occur in the nucleolus only transiently, since
many of these factors fluctuate between the nucleus and
nucleolus (Dundr et al. 1997; Snaar et al. 2000; Sprague
and McNally 2005), and similar behaviour was observed
for the TRB1 protein (Dvorackova et al. 2010) that is
largely dispersed at prophase, coinciding with nucleolar
disassembly, and re-localized in early anaphase after
cytokinesis.
TRB1 association was detected in the promoters of the
H2AX A and H2AX B genes, which also contain telo-box
sequences in their promoter region. Phosphorylated products
of these genes are involved in response to DNA double
202 Plant Mol Biol (2016) 90:189–206
123
strand breaks. All these connections indicate the importance
to examine possible involvement of the TRB1 protein
in regulation of transcription.
Whole-genome duplication events in Arabidopsis phylogeny
often resulted in increased number of genes of the
same family (Mandakova and Lysak 2008; Nelson et al.
2014). TRB1 protein belongs to the single-myb-histone
family with five members (Marian et al. 2003; Schrumpfova
et al. 2004, 2014). Although these proteins slightly differ in
binding properties (Hofr et al. 2009; Schrumpfova et al.
2004, 2014) their partial functional redundancy in vivo
cannot be excluded. Therefore, no observation of significant
Fig. 9 Analysis of individual categories of the Upstream 500 bp of
Non-protein coding genes dataset. The group of Non-protein coding
genes includes rRNA, snoRNA, snRNA, tRNA, ncRNA, miRNA, other
RNA, pseudogenes and transposable element genes categories. a The
pie chart shows the proportional representation of individual
categories. The most represented categories transposable elements,
pseudogenes, pre-tRNA, other RNA cover nearly 64, 15, 10, and
6.5 %, respectively of the Non-coding genes dataset. By contrast,
categories miRNA, snoRNA, snRNA, rRNA occupy only 2.9, 1.2, 0.2,
and 0.1 %, respectively. b Dark grey represents the proportion of the
genes in each category where any TRB1 genomic peak interferes with
the genomic region located in region 500 bp upstream from the
transcription start site of the relevant non-protein coding gene. c The
dark grey part represents the proportion of the genes in each category
that harbour at least one telo-box in their 500 bp upstream region. The
hatched part represents the subset of the telobox-containing genes that
are covered by TRB1 genomic peaks
Plant Mol Biol (2016) 90:189–206 203
123
changes in transcript levels of chosen genes in trb1-/mutant
plants (Fig. S5) is not surprising and points to the
need for using multiple trb mutants in further analyses.
In conclusion, this report demonstrates that the TRB1
protein from A. thaliana, whose N-terminal myb-domain
shows high sequence homology with the human TRF1 or
TRF2 C-terminal myb-domain, is associated in vivo with a
subset of interstitial sites in the Arabidopsis genome
besides its major location at telomeres (Schrumpfova et al.
2014). Our finding of an association of TRB1 with the 50
flanking region of protein or non-protein coding regions,
especially with sequences located upstream of the transcription
start site of the ribosomal protein coding genes,
snoRNA genes, and genes coding for elongation factors
(eEF-1) and eukaryotic initiation factors (eIFs), correlated
with a higher frequency of short telomere-like sequences
(telo-boxes) in their promoters, together with its cell-cycle
regulated localization (Dvorackova et al. 2010), suggest
that the TRB1 protein may be functional also as a transcription
factor (see concluding overview in Fig. 10). In
this feature, TRB1 resembles other analogous shelterin
proteins from diverse organisms which, besides their
telomeric localization and functions, can bind to nontelomeric
regions and have extra-telomeric functions in
gene regulation networks.
Acknowledgments We thank Vladimir Benes from the European
Molecular Biology Laboratory (EMBL), Genomics Core Facility,
Heidelberg, Germany, for valuable comments and recommendations
on our ChIP procedure. We also thank Ronald Hancock, Hoˆtel-Dieu
de Que´bec for critical reading of the manuscript. Access to computing
and storage facilities owned by parties and projects contributing to the
National Grid Infrastructure MetaCentrum, provided under the programme
‘‘Projects of Large Infrastructure for Research, Development,
and Innovations’’ (LM2010005), is greatly appreciated. This research
was supported by the Czech Science Foundation (13-06943S) and by
project CEITEC (CZ.1.05/1.1.00/02.0068) of the European Regional
Development Fund.
Fig. 10 Concluding overview of telomeric and non-telomeric locations
and roles of TRB1 protein. TRB1 protein from A. thaliana,
whose myb-domain shows high sequence similarity to the human
TRF1 or TRF2 myb-domains, acts as a component of plant telomereprotection
complex. TRB1 is co-localised with telomeric tracts
in vivo, interacts with POT1b protein, telomerase reverse transcriptase
(TERT) subunit and the loss of TRB1 protein leads to evident
telomere shortening (Kuchar and Fajkus 2004; Schrumpfova et al.
2008, 2014). Furthermore, our present finding demonstrated
association of TRB1 also to the 50
flanking region of protein- or
non-protein coding regions related to the translation machinery genes
e.g.: ribosomal protein coding genes, snoRNA genes, and genes
coding for eEF-1 and eIFs, correlated with a higher frequency of short
telomere-like sequences (telo-boxes) in their promoters. Physical
association of telomeres to nucleoli (depicted in the upper part) may
hypothetically correspond to functional relevance of TRB1 for
coordinated regulation of translation machinery genes
204 Plant Mol Biol (2016) 90:189–206
123
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Plant Molecular Biology
Telomere Binding Protein TRB1 is Associated with Promoters of
Translation Machinery Genes in vivo
Petra Procházková Schrumpfová1,2
, Ivona Vychodilová1,2
, Jan Hapala1,2
, Šárka Schořová1,2
, Vojtěch
Dvořáček and Jiří Fajkus1,2,3§
1
Mendel Centre for Plant Genomics and Proteomics, CEITEC – Central European Institute of Technology, Masaryk
University, Kamenice 5, CZ-625 00 Brno, Czech Republic; 2
Laboratory of Functional Genomics and Proteomics, National
Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech Republic;
3
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, CZ-61265 Brno, Czech
Republic
Coresponding author:
e-mail: fajkus@sci.muni.cz
tel: +420 549 49 4003
fax: +420 549 492 654
Supplementary Figures and Tables
Figure S1- Overview of all TRB1 genomic peaks in each chromosome
The IGV view of all five chromosomes from A. thaliana visualizes the preference of the TRB1 protein binding
outside of heterochromatic regions (centromeres and heterochromatin knobs). The All coding genes category
represents both Protein coding genes and Non-protein coding genes. TRB1 genomic peaks represents TRB1enriched
regions. The heterochromatin knobs on chromosome 4 and 5 are indicated by grey boxes.
Figure S2 - TRB1 association with the EF1-alpha genes
Three EF1-alpha 1, 2 and 3 genes that contain telo-box sequence in Upstream 500 bp distance (At1g07940,
At1g07930 and At1g07920) are visualised with IGV viewer. Telo-box sequences are highlighted in separate
rows. Although the pattern of TRB1 occupancy would visually suggest that TRB1 is occupying regions around
TSSs of all three loci, only one (EF1-alpha 1 gene (At1G07940)) has been detected as a TRB1 genomic peak
under our stringent threshold criteria. So the number of the TRB1 associated regions should be actually higher.
Figure S3 – TRB1 association with genes coding for ribosomal proteins
Three genes coding for ribosomal proteins RPS15B, RPS15C, RPS15D (At5g09490, At5g09500, At5g09510)
are visualised with IGV viewer. Telo-box sequences are highlighted in separate rows. Although the pattern of
TRB1 occupancy would visually suggest that TRB1 is occupying regions around TSSs of all three loci, only one
(RPS15D gene (At5g09510)) has been detected as TRB1 genomic peak under our stringent threshold criteria. So
the number of the TRB1 associated regions should be actually higher.
Figure S4 – Schematic view of the snoRNA clusters
An IGV view of three examples of association of the TRB1 protein with promoter sequences of the snoRNA
genes that are clustered into one transcript (blue boxes). TRB1 genomic peaks are highlighted.
Figure S5 – Effect of TRB1 on transcription of selected TRB1-associated genes
Several genes with promoters visibly associated with TRB1 protein, coding for proteins of large or small
ribosome subunits, and genes coding for eEFs and H2AX proteins were chosen for analysis of transcript levels in
trb1-/- plants and compared to the respective wt control. For comparison, genes coding for Pheres (PHE) and
RuvB-like AAA ATPase 1 (RuvBL1), lacking association with TRB1 protein (light grey), were also analysed.
Y-axis values represent relative transcript levels (Wt=1).
Table S1 - List of primers used in the qChIP-qPCR and RT-PCR analysis.
List of primers used for ChIP-qPCR described in (a) Figure 4 and (b) Figure S5
A Actin 7 (AT5G09810) Forward GGAAACATCGTTCTCAGTGGT
Reverse CTTGATCTTCATGCTGCTAGGT
Ribosomal protein L34 (AT3G28900) Forward AGGTGATCTCTTGAGCCCTAG
Reverse AAAGATAAAAAACACTACAATCAATCTGAG
Ribosomal protein S5 (AT2G41840) Forward AGGCCTTGTTGGGTTTGT
Reverse TTGTTCTGATTAACGTGTGACATTAG
snoRNA (AT4G15258 ) Forward CGAAACCTTATAAATACACAGACACAG
Reverse TTGGGCCGAGAACCTAAAATAG
B RPL37aC (at3g60245) Forward AGGTTGGAATCGTCGGCAAA
Reverse TCACTCCGTACTTGCCACAG
RPL10C (at1g66580) Forward ACCGTGCTGAGTACACGAAG
Reverse TCATTCTTATTCGCTAGTGGCTGA
RPL23C (at3g04400) Forward GCCACTGTGAACTGTGCTGA
Reverse CAACACACGCTGATGGCAAA
RPL17B (at1g67430) Forward GTACTCGCAAGAACCCGACA
Reverse TGGTAGCTTCCTGATTGCGT
RPL18aB (at2g34480) Forward GGACAGATGCTCGCCATCAA
Reverse CATCTGCTCAACAGCTCCGT
eEF1-alpha 2 (at1g07930 ) Forward GTCTGTTGAGATGCACCACG
Reverse CCCTCTCTTAAGATCCTTCACGG
eEF1-alpha 3 (at1g07920) Forward GCTGCTAACTTCACCTCCCA
Reverse TCTCCTTACCAGAACGCCTG
eEF1a-4 (at5g60390) Forward ACAAGCGTGTCATCGAGAGG
Reverse TCACGCTCGGCCTTAAGTTT
γH2AX A (at1g08880) Forward ATGAGTACAGGCGCAGGAAG
Reverse CTAGCGATTCTTCCGACGGG
γH2AX B (at1g54690) Forward ACAACTAAAGGTGGCAGAGGA
Reverse TCGGCGTATTTACCGGCTTTA
eEF1-alpha 3 (at1g07920) Forward GCTGCTAACTTCACCTCCCA
Reverse TCTCCTTACCAGAACGCCTG
eEF1a-4 (at5g60390) Forward ACAAGCGTGTCATCGAGAGG
Reverse TCACGCTCGGCCTTAAGTTT
γH2AX A (at1g08880) Forward ATGAGTACAGGCGCAGGAAG
Reverse CTAGCGATTCTTCCGACGGG
γH2AX B (at1g54690) Forward ACAACTAAAGGTGGCAGAGGA
Reverse TCGGCGTATTTACCGGCTTTA
PHE (At1g65330) Forward GATCGCCAAAGAAACAGAACG
Reverse ATCTCAACCCTACGAATAACACC
RuvBL1 (At5g22330) Forward CGGATTGCTACTCACACCCA
Reverse GCTGCCTCTCTAGCCTCAAG
UBQ10 (At4g05320) Forward AACGGGAAAGACGATTAC
Reverse ACAAGATGAAGGGTGGAC
Table S2 – Computation of the TRB1 protein coverage dataset per size unit of 1 Mb
The size of each dataset analysed in this study (bp) is listed in the column “whole dataset size”. Overlapping
base pairs were counted once. The proportional representation of these numbers is shown in Figure 3b as
“Genome coverage by whole dataset”. The section “Number of TRB1 genomic peaks in dataset” represents the
absolute number of DNA sequences from each dataset that were covered at least partly by one (or more) TRB1
genomic peak. The “TRB1 genomic peaks per size unit of 1 Mb” column is illustrated graphically in the Figure
3c and represents the frequency of TRB1 genomic peaks per one megabase of each dataset.
Table S3 - Overall summary of sequences belonging to each dataset
(In separate Supplementary .xls file)
A table of DNA sequences in each dataset that are significantly covered with the TRB1 protein (TRB1 genomic
peaks).
Table S4 - List of 8-mers and List of fragments used in the logo construction
(In separate Supplementary .xls file)
(A) A list of every unique substring of the length k (8) in the dataset 5’UTR covered by TRB1 genomic peaks.
The proportion of purple 8-mers is twice as high in the dataset 5’UTR covered by TRB1 genomic peaks as in the
whole 5’UTR dataset. Only these purple 8-mers were used for fragment construction of the total sequence logo
(Fig. 5a). The green boxes highlight 8-mers with a permutation of at least one plant telomeric repeat and used for
partial sequence logo construction with telomeric repeat (see Fig. 5b). The rest of the purple 8-mers do not
contain any telomeric repeat and were used for partial sequence logo construction without telomeric repeat (Fig.
5c).
(B) Here are listed fragments constructed from 8-mers separately for each sequence logo (total, with telomeric
repeat, without telomeric repeat (Fig. 5)). Fragments in their forward or reverse complementary version (orange)
were aligned by MUSCLE. The mean weight of each fragment or the relative weight for each base in the
fragment is included.
Table S5 - Table of Telo-box sequences in each dataset
The number of all sequences in the datasets which contain at least one telo-box are listed in the column “All telobox
containing sequences”. The column “Telo-box covered by TRB1 genomic peaks” lists the number of
sequences where at least one telo-box is preferentially recognized by the TRB1 protein and thus termed a TRB1
genomic peak. The last column corresponds to the graphical illustration in Figure 6 where the percentages of all
sequences from each dataset covered by TRB1 genomic peak are shown.
Table S6 - List of telo-box sequences belonging to each dataset
(In separate Supplementary .xls file)
Genomic coordinates of all telo-boxes and telo-boxes with TRB1 genomic peaks.
Table S7- Detailed reports from GOMiner and CIMminer
(In separate Supplementary .xls file)
Two main GO categories, GO:0008150 Biological process and GO:0005575 Cellular component, were
individually analyzed in GOMiner and CIMminer. The list “CIM” contains GO subcategories of these GO
categories which are statistically enriched (p ≤ 0.05). For this table, only GO subcategories (listed in horizontal
rows) containing 5−500 members were selected. Separate genes are listed vertically (AGI gene codes), so this
table corresponds to Figure 7 in the main text.
The list “CIM with genes” contains highly enriched GO subcategories (p ≤ 0.05), ordered vertically, and selected
genes where AGI gene codes are presented with detailed description of the gene names and function in rows.
The list “Total vs. Total Report” contains a comparison of All coding genes from A. thaliana (Total file; Protein
coding genes together with Non-protein coding genes) together with the set of genes where the TRB1 protein is
enriched in the region 500 bp upstream from the transcription start site ("Changed" file). All coding genes
involved in each GO subcategory are listed and marked whether they were enriched in the region 500 bp
upstream from the transcription start site or not ("no change/changed"). GO subcategories are ordered according
to the number of members.
The list “Gene Category Report” contains a detailed analysis of individual genes involved in each GO
subcategory. GO subcategories are ordered according to the p-value so the GO subcategories where the amount
of genes associated with TRB1 protein in their Upstream 500 bp proximity is highest are listed in the upper part
of the Table.
The list “Category Summary Report” summarizes the results for all GO subcategories (Gene Category Report).
GO subcategories are ordered by p-value.
Supplement J
Schrumpfová, P.P.*, Schořová, Š., Fajkus, J., 2016. Telomere- and Telomerase-Associated
Proteins and Their Functions in the Plant Cell. Front. Plant Sci. 7:851
P.P.S. wrote the ms and was significantly involved the ms editing
REVIEW
published: 28 June 2016
doi: 10.3389/fpls.2016.00851
Edited by:
Anne-Catherine Schmit,
Institut de Biologie Moléculaire des
Plantes, CNRS UPR2357, France
Reviewed by:
Takashi Murata,
National Institute for Basic Biology,
Japan
Franziska Katharina Turck,
Max Planck Society, Germany
*Correspondence:
Petra Procházková Schrumpfová
schpetra@centrum.cz
Specialty section:
This article was submitted to
Plant Cell Biology,
a section of the journal
Frontiers in Plant Science
Received: 12 November 2015
Accepted: 31 May 2016
Published: 28 June 2016
Citation:
Procházková Schrumpfová P,
Schoˇrová Š and Fajkus J (2016)
Telomereand
Telomerase-Associated Proteins
and Their Functions in the Plant Cell.
Front. Plant Sci. 7:851.
doi: 10.3389/fpls.2016.00851
Telomere- and
Telomerase-Associated Proteins and
Their Functions in the Plant Cell
Petra Procházková Schrumpfová1,2
*, Šárka Schoˇrová2 and Jiˇrí Fajkus1,2,3
1
Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Brno,
Czech Republic, 2
Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty
of Science, Masaryk University, Brno, Czech Republic, 3
Institute of Biophysics, Academy of Sciences of the Czech Republic,
v.v.i., Brno, Czech Republic
Telomeres, as physical ends of linear chromosomes, are targets of a number of specific
proteins, including primarily telomerase reverse transcriptase. Access of proteins to
the telomere may be affected by a number of diverse factors, e.g., protein interaction
partners, local DNA or chromatin structures, subcellular localization/trafficking, or
simply protein modification. Knowledge of composition of the functional nucleoprotein
complex of plant telomeres is only fragmentary. Moreover, the plant telomeric repeat
binding proteins that were characterized recently appear to also be involved in nontelomeric
processes, e.g., ribosome biogenesis. This interesting finding was not totally
unexpected since non-telomeric functions of yeast or animal telomeric proteins, as well
as of telomerase subunits, have been reported for almost a decade. Here we summarize
known facts about the architecture of plant telomeres and compare them with the
well-described composition of telomeres in other organisms.
Keywords: telomere, telomerase, telomeric proteins, shelterin, telomeric repeat binding (TRB), plant
TELOMERES AS NUCLEOPROTEIN STRUCTURES
Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that protect linear
chromosomes against damage by endogenous nucleases and erroneous recognition as unrepaired
chromosomal breaks. It is now known that telomeric structures are formed by telomeric DNA,
histone octamers, and a number of proteins that bind telomeric DNA, either directly or indirectly,
and together, form the protein telomere cap (Fajkus and Trifonov, 2001; de Lange, 2005; Bianchi
and Shore, 2008; Sfeir, 2012). The telomeric cap proteins of diverse organisms are less conserved
than one might expect. Even within a single taxonomic class, such as mammals, telomeric proteins
display less conservation than other chromosomal proteins (Linger and Price, 2009). On the other
hand, in many plant families, whole-genome duplication events have occurred, resulting in a
multitude of genomic changes, such as deletions of large fragments of chromosomes, silencing
of duplicate genes, and recombining of homologous chromosomal segments, as was shown, e.g.,
in crucifer species (Mandakova and Lysak, 2008). Polyploidy can result in increased numbers
of genes of the same family (Taylor and Raes, 2004; He and Zhang, 2005; Freeling, 2009),
which may show sub-functionalization, neo-functionalization, and partial or full redundancy and
complicates assignment of an actual and specific function for individual proteins in vivo. Gene
duplications and losses in plant phylogeny can be traced also in telomere associated protein families
Frontiers in Plant Science | www.frontiersin.org 1 June 2016 | Volume 7 | Article 851
Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
(e.g., in Arabidopsis thaliana: single myb histone (SMH) family,
TRF-like (TRFL) family, or Pot1-like family) (Nelson et al., 2014;
Beilstein et al., 2015).
In land plants, the telomere is mostly composed of
Arabidopsis-type TTTAGGG repeats (Richards and Ausubel,
1988; Figure 1A). Known exceptions are species in the order
Asparagales, starting from divergence of the Iridaceae family,
which shares the human-type telomeric repeat (TTAGGG;
probably caused by a mutation that altered the RNA template
subunit of telomerase ∼80 Mya; Adams et al., 2001; Weiss and
Scherthan, 2002; Sykorova et al., 2003). The human-type telomere
is also shared by species of the Allioideae subfamily, except for
the Allium genus (Sykorova et al., 2006), where novel telomeric
sequence (CTCGGTTATGGG) was recently described (Fajkus
et al., 2016). An unusual telomeric motif (TTTTTTAGGG) was
found in the family Solanaceae, in Cestrum elegans and related
species (Peska et al., 2015). Also some of the species from the
carnivorous genus Genlisea display, instead normal Arabidopsistype
of telomere, two intermingled sequence variants (TTCAGG
and TTTCAGG; Tran et al., 2015).
Moreover, across the Plantae kingdom, outside of land plants
but including red algae, green algae, and Glaucophytes (Koonin,
2010), telomere types also vary (Figure 1B). For example, in
algae, in addition to the Arabidopsis-type of telomeric repeat, the
Chlamydomonas-type (TTTTAGGG), human-type (TTAGGG),
and a novel TTTTAGG repeat have been described (Fulnecková
et al., 2013; Fulneˇcková et al., 2015).
The length of plant telomeric DNA at a single chromosomal
arm can be as small as 500 base pairs (bp) in Physcomitrella
patens (Shakirov et al., 2010; Fojtova et al., 2015), as long as
160 kb in Nicotiana tabacum (Fajkus et al., 1995), or 200 kb in
Nicotiana sylvestris (Kovarik et al., 1996). Besides the remarkable
variation in telomere lengths among diverse plant genera or
orders, telomere lengths can also vary at the level of the species
or ecotypes: e.g., Arabidopsis telomeres range from 1.5 to 9 kb,
depending on the ecotype. Also in the long-living organism
Betula pendula, telomeres in different genotypes varied from a
minimum length of 5.9–9.6 kb to a maximum length of 15.3–
22.8 kb (Shakirov and Shippen, 2004; Maillet et al., 2006; Aronen
and Ryynanen, 2014).
Since telomeric DNA serves as a landing pad for a set of
proteins, the total length or composition of telomeric tracts could
markedly affect the number or selection of telomere-associated
proteins and subsequently influence telomere packaging,
structural transitions, or launch various biochemical pathways
(see below).
NUCLEAR LOCALIZATION AND
DYNAMICS OF TELOMERES
In some species during interphase, telomeres, and centromeres
could be located at opposite sides of the nucleus, at the nuclear
periphery, in limited regions or clusters; this is known as the
Rabl organization (Rabl, 1885; for review, see Cowan et al., 2001).
The Rabl organization (Wen et al., 2012) was observed in wheat,
rye, barley, and oats. Other plant species, such as maize (Zea
mays) and sorghum (Sorghum bicolor), despite having fairly large
genomes, are not known to exhibit the Rabl configuration (Dong
and Jiang, 1998). A recent study among Brachypodium species
revealed a positive correlation between Rabl configuration and
an increase in DNA content (resulting from replication) and a
negative influence of increasing nuclear elongation (Idziak et al.,
2015). A rosette-like organization of chromosomes in interphase
nuclei was observed in Arabidopsis: telomeres show persistent
clustering at the nucleolus while centromeres do not cluster
(Armstrong et al., 2001; Tiang et al., 2012). Moreover, during
early meiotic prophase, at the leptotene–zygotene transition,
telomeres of most plant species cluster to form a bouquet (Bass
et al., 1997; Martinez-Perez et al., 1999; Cowan et al., 2002;
Corredor and Naranjo, 2007; Higgins et al., 2012; Phillips et al.,
2012). Arabidopsis belongs to a small group of species that do not
form telomeric bouquets (Armstrong et al., 2001).
Chromatin attachment to the inner nuclear membrane in
plants, as well as in other species, is mediated by a well conserved
multi-protein complex gathered around SUN (Sad1-UNC-84
homology)-KASH (Klarsicht, ANC-1, and Syne homology)
proteins [respectively AtSUN-AtSINE (SUN domain-interacting
NE proteins) in A. thaliana; Starr et al., 2001; Zhou et al.,
2014; Tamura et al., 2015]. In fission and budding yeasts,
interactions during meiosis between telomeres and the nuclear
envelope, via interactions between SUN domain proteins and
telomere-binding proteins, was described: in Saccharomyces
cerevisiae SUN-domain protein yMps3 (monopolar spindle
protein 3) is needed for yKu80-mediated telomeric chromatin
anchoring (Schober et al., 2009), while in Schizosaccharomyces
pombe, interactions between telomeric protein pRap1 (repressor
activator protein 1) and pSUN proteins are mediated by pBqt1
and pBqt2 (telomere bouquet protein 1 and 2; Chikashige et al.,
2006). The tethering of human telomeres to the nuclear matrix
was proposed to depend on an isoform of telomere repeat binding
factor 1 (TRF1) interacting partner (hTIN2), named hTIN2L
(Kaminker et al., 2009), or an A-type lamin (Ottaviani et al.,
2008; for review, see Giraud-Panis et al., 2013). Various homologs
of SUN domain proteins were identified in Arabidopsis or in
maize. In Arabidopsis, they are also localized to the inner nuclear
membrane in somatic cells (Graumann et al., 2010; Tamura et al.,
2015), however, homologs of Bqt proteins or TIN2 proteins
have not been found in plants and their sequences are poorly
conserved.
Telomeres are processed by a telomere-specific machinery that
includes telomerase and its regulatory units, as well as nucleases,
as exemplified by the exonuclease 1 (AtEXO1) ortholog in
Arabidopsis (Kazda et al., 2012; Derboven et al., 2014). In
plants, as well as in most of other kingdoms, replication of
chromosomal ends results in single-stranded 3 DNA protrusions
(G-overhangs) after degradation of the last RNA primer at
the 5 terminus of a nascent strand. In Silene latifolia or
A. thaliana, relatively short (20–30 nucleotides) G-overhangs
were detected. Moreover, half of the Silene and Arabidopsis
telomeres showed no overhangs or overhangs less than 12
nucleotides in length (Riha et al., 2000; Kazda et al., 2012). These
G-overhangs are also thought to be required for chromosome
end protection by forming secondary DNA structures such as
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
FIGURE 1 | Summary of current knowledge on telomere DNA diversity in land plants (A) and green algae (B). The prevalent plant telomeric sequence motif
TTTAGGG was first described in A. thaliana (Richards and Ausubel, 1988). Divergent telomeric sequences have been observed in Asparagales (Sykorova et al.,
2003), in Cestrum spp. (Peska et al., 2015), in Genlisea (Tran et al., 2015), or in Allium (Fajkus et al., 2016). While Arabidopsis-type telomeric sequence is dominant in
“green lineages” of algae Chlorophyta and Streptophyta, this ancestral motif was replaced several times with a novel motifs (reviewed in Fulne ˇcková et al., 2015).
(The relationships between families and genera are adapted from the schematic phylogenetic tree presented in Fulne ˇcková et al., 2015 and Fajkus et al., 2016.)
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
t-loops (reviewed in Tomaska et al., 2009). Although formation
of t-loop structures was demonstrated among plants only in
the garden pea (Pisum sativum; Cesare et al., 2003), it is
believed that excision from a t-loop in Arabidopsis may result
in t-circle formation and in telomere rapid deletion (Watson
and Shippen, 2007). In tobacco cell culture, knockdown of
one of three human hnRNP homologs, named NgGTBP1
(G-strand specific single-stranded telomere-binding protein
1), led to frequent formation of extrachromosomal t-circles,
inhibition of single-stranded invasion into double-stranded
telomeric DNA and the loss of protection of telomeres against
inter-telomeric recombination (Lee and Kim, 2010, 2013).
As well as in humans, mouse, or Caenorhabditis (Uringa et al.,
2011; Vannier et al., 2012), the regulator of telomere elongation
helicase 1 (AtRTEL1) plays a putative role in Arabidopsis in
the destabilization of DNA loop structures such as t-loops or
d-loops (Recker et al., 2014). However, a substantial portion of
telomeres in Arabidopsis does not apparently undergo nucleolytic
resection, and 3 ends produced by leading-strand replication
remain blunt-ended (Riha et al., 2000). It is believed that bluntends
in Arabidopsis are specifically recognized and protected by
the AtKu70/80 heterodimer although in situ localization of Ku to
telomeres remains elusive (Kazda et al., 2012).
PROTEINS ASSOCIATED WITH
TELOMERIC DNA
Telomere-associated proteins can regulate lengths of telomere
tracts by modulating access of telomerase or affecting
conventional DNA replication machinery. In mammals,
telomeric DNA associates with a six-protein complex called
shelterin. The specific telomeric dsDNA binding is mediated
by TRF1 and TRF2 (Broccoli et al., 1997; Court et al., 2005),
through their Myb-like domain with an LKDKWRT amino acid
motif that is also conserved in other telobox binding proteins,
not only in mammals but also in plants (Bilaud et al., 1996;
Feldbrugge et al., 1997). A bridge between proteins directly
associated with DNA—TRF1, TRF2, and ssDNA binding
protein Pot1 (Protection of telomeres 1)—is mediated by TIN2
and the oligosaccharide/oligonucleotide binding (OB)-fold
domain of TPP1 (TINT1, PTOP, PIP1) protein (for review see
Schmidt and Cech, 2015; Lazzerini-Denchi and Sfeir, 2016).
Moreover, protein Rap1, the last component of shelterin,
interacts with TRF2 (Arat and Griffith, 2012) and modulates
its recruitment to telomeric DNA (Janouskova et al., 2015).
A schematic model of mammalian telomere-associated proteins
(Figure 2A) and a proposed model of the telomeric complex
in A. thaliana (Figure 2B) summarizes recent knowledge
in mammalian and plant telomere biology and provides a
clear comparison of conserved structures at chromosome
termini. In addition, a general overview of telomere-associated
proteins that have been described in plants is given in Table 1.
Detailed description of telomeric and putative telomeric dsDNA
and ssDNA binding proteins from A. thaliana is shown in
Table 2.
Telomeric dsDNA-Associated Proteins
Myb-like Proteins
In plants, telomeric dsDNA sequence binding proteins with a
Myb-like domain of a telobox (short telomeric motif) type can
be classified into three main groups: (i) with a Myb-like domain
at the N-terminus (SMH family), (ii) with a Myb-like domain at
the C-terminus (TRFL family), and (iii) with a Myb-like domain
at the C-terminus (AID family; reviewed in Peska et al., 2011; Du
et al., 2013).
The first group of proteins, with a Myb-like domain at the
N-terminus, also contains a central histone-like domain with
homology to the H1 globular domain found in the linker
histones H1/H5, and is therefore called the SMH family (Marian
et al., 2003; Schrumpfova et al., 2004). Proteins with an SMH
motif are plant-specific but are well conserved throughout the
plant kingdom (e.g., eudicots, monocots, moss, or red algae;
Du et al., 2013). In A. thaliana, there are five members of
the SMH family, named telomere repeat binding (AtTRB)
proteins (Marian et al., 2003; Schrumpfova et al., 2004). AtTRB1
protein specifically binds plant telomeric repeats through a
Myb-like domain in vitro (Mozgova et al., 2008), co-localizes
with telomeres in situ, and physically interacts with AtTERT
(Figure 2B). Moreover shortening of telomeres was observed in
attrb1 knockout mutants (Schrumpfova et al., 2014). Also other
members of this family, AtTRB2 and AtTRB3 (previously named
AtTBP3 and AtTBP2, respectively; Schrumpfova et al., 2004),
bind telomeric dsDNA as well as telomeric ssDNA in vitro as
homo- or heteromultimers (Schrumpfova et al., 2004; Mozgova
et al., 2008; Hofr et al., 2009; Lee W.K. et al., 2012; Yun
et al., 2014). In Arabidopsis, AtTRB1 protein physically interacts
via its histone-like domain with AtPot1b (Schrumpfova et al.,
2008), an A. thaliana homolog of the G-overhang binding
protein Pot1, and a component of an alternative telomerase
holoenzyme complex (Tani and Murata, 2005; He et al., 2006;
Surovtseva et al., 2007). Also other members of SMH family
proteins in land plants show telomeric dsDNA binding capability:
e.g., Oryza sativa OsTRBFs (Byun et al., 2008) or Z. mays
ZmSMHs (Marian et al., 2003). In addition, proteins with Myblike
domain of a telobox type in plants, adopt distinct nontelomeric
functions, e.g., PcMYB1 from Petroselinum crispum
acts only as a transcription factor (Feldbrugge et al., 1997).
Recently it was shown that AtTRB1 from A. thaliana was not
only telomere- and telomerase-binding but was also associated,
in vivo, with promoters, mostly with a telo box motif of
translation machinery genes (Figure 3; Schrumpfova et al.,
2016). The AtTRB1 association with telo box motif was then
proven by Zhou et al. (2016). Moreover AtTRB proteins seem
to have a new role as chromatin modulators: AtTRB1 competes
with LIKE HETEROCHROMATIN PROTEIN 1 (AtLHP1) to
maintain downregulation of polycomb group (PcG) target genes
(Zhou et al., 2016) and protein AtTRB2 directly interacts
with histone deacetylases, HDT4 and HDA6, in vitro and
in vivo (Lee and Cho, 2016). Deacetylase activity of HDT4
(Lee and Cho, 2016) and HDA6 (To et al., 2011) against
H3K27ac, could be important for subsequent methylations of
H3K27me3, that is among others target also for AtLHP1.
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
FIGURE 2 | Nucleoprotein complexes associated with mammalian and A. thaliana telomeres. (A) Mammalian shelterin proteins (TRF1/2, Rap1, TIN2, TPP,
and Pot1) modulate access to the telomerase complex and the ATR/ATM dependent DNA damage response pathway. The CST complex (CTC1–STN1–TEN1)
affects telomerase and DNA polymerase α recruitment to the chromosomal termini, and thus coordinates G-overhang extension by telomerase with fill-in synthesis of
the complementary C-strand (blue dashed line; figure adopted from Chen and Lingner, 2013). (B) Arabidopsis TRB1, 2 and 3 interact with the telomeric sequence
due to the same Myb-like binding domain as mammalian TRF1/2 (Marian et al., 2003; Kuchar and Fajkus, 2004; Schrumpfova et al., 2004). TRB proteins interact
with TERT and Pot1b and when localized at chromosomal ends they are eligible to function as components of the plant shelterin complex, mainly at telomeres with a
G-overhang (Schrumpfova et al., 2008, 2014). An evolutionary conserved CST complex is suggested to coordinate the unique requirements for efficient replication of
telomeric DNA in plants as well as in other organisms (Derboven et al., 2014). Blunt-ended telomeres are specifically recognized and protected by the KU70/80
heterodimer that directly interacts with TRP1, and by extension, with TERT (Kuchar and Fajkus, 2004; Kazda et al., 2012; Schrumpfova et al., 2014).
Taken together, two lines of evidence classify the AtTRB
proteins as novel epigenetic regulators that potentially impact
transcription status of thousands of genes: (i) association
of AtTRB1 with telo box DNA motif (Schrumpfova et al.,
2016; Zhou et al., 2016) that is linked with PcG protein
pathway (Deng et al., 2013; Wang et al., 2016; Zhou et al.,
2016), (ii) involvement of AtTRB proteins in control of
H3K27 epigenetic modifications (Lee and Cho, 2016; Zhou
et al., 2016), that are also connected with PcG chromatin
remodelers.
The second group of proteins, with a Myb-like domain at
the C-terminus, is also named TRFL. However a TRFL Myblike
domain alone is not sufficient for telomere binding and
requires a more extended domain—Myb-extension (Myb-ext)—
for telomeric dsDNA interactions in vitro (Karamysheva et al.,
2004; Ko et al., 2008). Consequently, two families of TRFL
can be distinguished: TRFL family 1 with a Myb-ext, whose
protein members bind telomeric dsDNA in vitro, and TRFL
family 2 without a Myb-ext, whose protein members do not bind
telomeric dsDNA specifically in vitro and they are usually not
considered as telomeric proteins (Karamysheva et al., 2004). The
first identification of a TRFL family protein from O. sativa—
telomere-binding protein 1 (OsRTBP1; Yu et al., 2000) was soon
followed by numerous other TRFL members: e.g., Nicotiana
glutinosa (NgTRF1; Yang et al., 2003), Solanum lycopersicum
(LeTBP1; Moriguchi et al., 2006), A. thaliana (AtTBP1, AtTRP1,
AtTRFL2-10; Chen et al., 2001; Hwang et al., 2001; Karamysheva
et al., 2004), Cestrum parqui (CpTBP; Peska et al., 2011). Even
though O. sativa or N. glutinosa mutants for TRFL members
exhibited markedly longer telomeres (Yang et al., 2004; Hong
et al., 2007), in A. thaliana, a knockout of AtTRP1, member of
TRFL family 1 with a Myb-ext, did not change telomere length
significantly (Chen et al., 2005). Even multiple knock-out plant,
deficient for all six proteins from TRFL family 1 in A. thaliana
(AtTBP1, AtTRP1, AtTRFL1, AtTRFL2, AtTRFL4, and AtTRF9)
did not exhibit changes in telomere length, or phenotypes
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
TABLE1|Ageneraloverviewoftelomere/telomeraseassociatedproteinsdescribedinplants.
A.thalianaO.sativaZ.maysN.glutinosa/
N.tabacum/
N.sylvestris
H.vulgareS.lycopersicum/
S.tuberosum
C.parquiP.crispum
TelomericdsDNA-associatedproteins
Myb-like
proteins
Myb-likedomain
attheN-terminus
(SMHfamily)
AtTRB1-3(Schrumpfovaetal.,2004)OsTRBF1-3
(Byunetal.,2008;
Heetal.,2013)
ZmSMHs
(Marianetal.,2003)
PcMYB1
(Feldbruggeetal.,
1997)
Myb-likedomain
attheC-terminus
(TRFL-family)
AtTBP1(Hwangetal.,2001)
AtTRP1(Chenetal.,2001)
AtTRFL2-10(Karamyshevaetal.,2004)
OsRTBP1
(Yuetal.,2000)
ZmIBP1
(LugertandWerr,1994)
ZmIBP2
(Moore,2009)
NgTRF1
(Yangetal.,2003)
LeTBP1
(Moriguchietal.,
2006)
CpTBP1
(Peskaetal.,
2011)
PcBPF-1
(daCostaeSilva
etal.,1993)
Myb-likedomain
attheC-terminus
(AIDfamily)
OsAID1
(Zhuetal.,2004;
Heetal.,2013)
ZmTacs1
(MarianandBass,
2005)
CSTcomplexAtStn1(Songetal.,2008)
AtTEN1(Leehyetal.,2013)
AtCTC1(Surovtsevaetal.,2009)
TelomericssDNA-associatedproteins
OB-foldPot-likeAtPot1a-c(KucharandFajkus,2004;Tani
andMurata,2005;Rossignoletal.,2007)
ZmPot1a(Shakirov
etal.,2009b)
ZmPot1b
(Shakirovetal.,2009b)
HvPot1(Shakirov
etal.,2009b)
StPot1
(Shakirovetal.,
2009b)
CpPot1
(Peskaetal.,
2008)
Non-OBfoldWHYAtWhy1(Yooetal.,2007)HvWhy1
(Grabowskietal.,
2008)
RRM-motifAtSTEP1(KwonandChung,2004)Os08g0492100
(Heetal.,2013)
Os08g0320100
(Heetal.,2013)
NtGTBP1-3
(Hirataetal.,
2004;Leeand
Kim,2010)
Telomeraseassociated
TERTsubunitAtTERT(Fajkusetal.,1996)OsTERT
(Oguchietal.,
2004)
ZmTERT(Sykorova
etal.,2009)
NtTERT,NsTERT
(Sykorovaetal.,
2012)
TERT/TR
associated
proteins
Myb-likedomain
attheN-terminus
(SMHfamily)
AtTRB1-3(Schrumpfovaetal.,2014)
Myb-likedomain
attheC-terminus
(TRFLfamily)
AtTRP1(Schrumpfovaetal.,2014)
Dyskerin-likeAtCBF5(Lermontovaetal.,2007)
Pot-likeAtPot1a(Rossignoletal.,2007)
RRM-motifAtRRM(LeeL.Y.etal.,2012)
ARM-motifAtARM(LeeL.Y.etal.,2012)
RNA-bindingAtG2p(Dokládaletal.,2015)
Metallothionein-
like
AtMT2A(Dokládaletal.,2015)
(Continued)
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
TABLE1|Continued
A.thalianaO.sativaZ.maysN.glutinosa/
N.tabacum/
N.sylvestris
H.vulgareS.lycopersicum/
S.tuberosum
C.parquiP.crispum
DNAprocessingandrepair-associatedproteinsattelomerases
HelicaseAtRTEL1(Reckeretal.,2014)
ExonucleaseEXO1(Kazdaetal.,2012)
KuAtKu70/80(Bundocketal.,2002;
Rihaetal.,2002)
OsKu70
(Hongetal.,2010)
PI3kinaseAtATM(Amiardetal.,2011)
AtATR(Amiardetal.,2011)
MRNComplexAtRad50(GallegoandWhite,2001)
AtMre11(BundockandHooykaas,2002)
AtNbs1(NajdekrovaandSiroky,2012)
Ku-independentEJpathwayAtRad1(Vannieretal.,2009)
AtERCC1(Vannieretal.,2009)
AtXRCC1(Amiardetal.,2014)
Backup-NHEJKU-independentpathwayAtPARP1,2(Boltzetal.,2014)
ProteinsthatwereprovenastelomericDNA-ortelomerase-associatedproteinsarelistedabove.Theirhomologuesinvolvedinnon-telomericprocesses,whiletheirassociationswithtelomeresortelomerasehavenot
beenobserved,areshowningreen.
SingleMybHistonefamily(SMH);TRF-like(TRFL-family);AntherIndehiscencefamily(AID-family);Cdc13/CTC1-Stn1-Ten1(CST);Oligonucleotide/OligosaccharideBindingfold(OB-fold);proteinswithout
Oligonucleotide/OligosaccharideBindingfold(non-OB-fold);Protectionoftelomeres–like(Pot-like);Whirly(WHY);RNARecognitionMotifs(RRM-motif);TelomeraseReverseTranscriptase(TERT);TelomeraseRNA
template(TR);Armadillo/β-catenin-likeRepeat-containingprotein(ARM);Phosphoinositide3-kinase(PI3kinase);Mre11/Rad50/Nbs1(MRN);End-joining(EJ);Non-homologousend-joining(NHEJ).
Arabidopsisthaliana(At):TelomericRepeatBinding1-3(AtTRB1-3);TelomereBindingProtein1(AtTBP1);TelomereRepeatbindingFactor1(AtTRF1);TRF-like2-10(AtTRFL2-10);Suppressorofcdcthirteenhomolog
(AtStn1);TelomericpathwaysinassociationwithStn1(AtTen);Conservedtelomeremaintenancecomponent1(AtCTC1);Protectionoftelomeres1a,b,c(AtPot1a,b,c);Whirly1(AtWhy1);Single-strandedTelomere-
bindingProtein1(AtSTEP1);TelomeraseReverseTranscriptase(AtTERT);CajalBodiesFactor5(AtCBF5);RNARecognitionMotifs(AtRRM);Armadillo/β-catenin-likeRepeat-containingprotein(AtARM);Gene2(AtG2p);
Metallothionein-like2A(AtMT2A);RegulatorofTelomereElongationHelicase1(AtRTEL1);Exonuclease1(AtEXO1);Ataxiatelangiectasiamutatedkinase(AtARM);ATM-andRAD3-relatedkinases(AtATR);DNArepair
protein50(AtRad50);Meioticrecombination11(AtMre11);DNArepairprotein1(AtRad1);Excisionrepaircross-complementation1(AtERCC1);X-rayrepaircross-complementing1(XRCC1);Nijmegenbreakage
syndrome1(AtNbs1);Poly(ADP-Ribose)polymerase1,2(AtPARP1,2).
Orizasativa(Os):TelomereRepeatBindingFactor1(OsTRBF1);RiceTelomereBindingProtein1(RTBP1);AntherIndehiscence1(OsAID1);TelomeraseReverseTranscriptase(OsTERT).
Zeamays(Zm):SingleMybHistone(ZmSMHs);Initiator-bindingprotein1(ZmIBP1);Initiator-bindingprotein2(ZmIBP2);TerminalacidicSANT1(ZmTacs1);Protectionoftelomeres1a,b(ZmPot1a,b);Telomerase
ReverseTranscriptase(ZmTERT).
Nicotianaglutinosa/tabacum/sylvestris(Ng/Nt/Ns):TelomereRepeatbindingFactor1(NgTRF1);TelomericssDNAbindingprotein1-3(NtGTBP1-3);TelomeraseReverseTranscriptase(Ng/NtTERT).
Hordeumvulgare(Hv):Protectionoftelomeres1(HvPot1);Whirly1(HvWhy1).
Solanumlycopersium/tuberosum(Le/St):TelomereBindingProtein1(LeTBP1);Protectionoftelomeres1(StPot1).
Cestrumparqui(Cp):TelomereBindingProtein1(CpTBP1);Protectionoftelomeres1(CpPot1).
Petroselinumcrispum(Pc):Myb-likeprotein1(PcMYB1);BoxP-bindingFactor1(PcBPF-1).
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
TABLE2|TelomericandputativetelomericdsDNA-andssDNA-bindingproteinsfromArabidopsisthaliana.
Protein
abbreviation
Accession
number
SchematicDepictionof
ConservedDomains
Telomeric-
Sequence-
Binding
invitro
Localization
on
Telomeres
insitu
Telomere
length
regulation
Reference
TelomericdsDNA-associatedproteins
Myb-like
proteins
Myb-like
domainatthe
N-terminus
(SMHfamily)
AtTRB1At1g49950(+)(+)(+)Schrumpfovaetal.,2004:Schrumpfovaetal.,2014
AtTRB2At5g67580(+)(+/−)Schrumpfovaetal.,2004;Dvoráckováetal.,2010
AtTRB3At3g49850(+)(+/−)(+/−)Schrumpfovaetal.,2004;Dvoráckováetal.,2010;
Zhouetal.,2016
Myb-like
domainatthe
C-terminus
(TRFL-familyI)
AtTBP1At5g13820(+)(−)Hwangetal.,2001;FulcherandRiha,2016
AtTRP1At5g59430(+)(−)Chenetal.,2001;FulcherandRiha,2016
AtTRFL1At3g46590(+)(−)Karamyshevaetal.,2004;Hwangetal.,2005;
FulcherandRiha,2016
AITRFL2At1g07540(+)(−)Karamyshevaetal.,2004;FulcherandRiha,2016
AtTRFL4At3g53790(+)(−)Karamyshevaetal.,2004;FulcherandRiha,2016
AtTRFL9At3g12560(+)(−)Karamyshevaetal.,2004;Hwangetal.,2005;
FulcherandRiha,2016
Myb-like
domainatthe
terminus
(TRFL-familyII)
AtTRFL3At1g17460(−)Karamyshevaetal.,2004
AtTRFL5At1g15720(−)Karamyshevaetal.,2004
AtTRFL6At1g72650(−)Karamyshevaetal.,2004
AtTRFL7At1g06910(−)Karamyshevaetal.,2004
AtTRFL8At2g37025(−)Karamyshevaetal.,2004
AtTRFL10At5g03780(−)Karamyshevaetal.,2004
CST
complex
AtStn1At1g07130(+)(+)Songetal.,2008
AtTen1At1g56260(+/−)(+)Leehyetal.,2013
AtCTC1At4g09680(+)(+)Surovtsevaetal.,2009
TelomericssDNA-associatedproteins
OB-foldPot-likeAtPot1aAt2g05210(+/−)(+)KucharandFajkus,2004;TaniandMurata,2005;
Shakirovetal.,2005,2009b;Surovtsevaetal.,
2007
AtPot1bAt5g06310(+/−)(+)KucharandFajkus,2004;TaniandMurata,2005;
Shakirovetal.,2005,2009b
AtPot1cAt2g04395(+)Rossignoletal.,2007;Nelson,2012
Non-OB
fold
WHYAtWhy1At1g14410(+)(+)Yooetal.,2007
RRM-motifAtSTEP1At4g24770(+)KwonandChung,2004
Symbol(+)indicatespublishedevidenceofproteinbindingtotelomericsequence“Telomeric-sequenceBindinginvitro,”protein“LocalizationonTelomeresinsitu”,orthatproteinregulatestelomerelengthinplants
“TelomereLengthRegulation.”Symbol(+/−)meansthatonlyindirectevidenceorambiguousresultsexist.Symbol(−)indicatesthatproteindoesnotspecificallyinteractwithtelomericDNA“Telomeric-sequenceBinding
invitro,”proteinisnotlocalizedontelomeres“LocalizationonTelomeresinsitu”,orthatproteindoesnotregulatetelomerelengthinplants“TelomereLengthRegulation.”Myb-likedomain(Myb);Myb-extension(-ext);
Histone-likedomain(Hl/5);CoiledCoilDomain(CCD);Oiigonucleotide/Oligosaccharide-BindingFolddomain(OB);Whirlydomain(Whirly);RNA-bindingdomain(RB);A.thaliana(At);TelomericRepeatBinding(AtTRB);
TRF-likefamily(TRFLfamily);Suppressorofcdcthirteenhomolog(AtStn1);Conservedtelomeremaintenancecomponent1(AtCTC1);(CTC1-Stn1-Ten1)complex(CST);RNArecognitionmotifs(RRM);Protectionof
telomeres1a,b,c(AtPot1a,b,c);Whirly1(Why1);Single-strandedtelomere-bindingprotein1(STEP1)
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
FIGURE 3 | Association of shelterin proteins with extra-telomeric sequences. (A) Mammalian telomere-binding proteins TRF1/2, TIN2, or Rap1 associate not
only with terminally localized telomeric repeats but also with interstitial telomeric sequences (ITS) and satellite repeats (Krutilina et al., 2001; Mignon-Ravix et al.,
2002; Simonet et al., 2011; Yang et al., 2011). Mouse Rap1, together with TRF2, acts as a gene transcription regulator, including subtelomeric-gene (S-gene)
silencing, and also binds to non-coding genomic regions enriched with TTAGGG repeats (Martinez et al., 2010). Moreover, TRF2 regulates neuronal genes by
interaction of TRF2 with repressor element 1-silencing transcription factor (REST; Zhang et al., 2008). (B) Arabidopsis TRB1 protein was found not only as a
component of the telomeric interactome (Schrumpfova et al., 2014), but also as a factor associated with 5 flanking regions (mostly comprising the telo box) of
translation machinery genes (Schrumpfova et al., 2016; Zhou et al., 2016).
associated with telomere dysfunction (Fulcher and Riha, 2016).
Thus, although the AtTRFL proteins from A. thaliana specifically
bind telomeric DNA in vitro and an interaction between AtTRP1
and AtKu70 was observed, suggesting a putative telomere
function (Figure 2B; Kuchar and Fajkus, 2004), no functional
evidence exists for their role at telomeres. Another member of
this family—ZmIBP2 (initiator-binding protein) protein—binds
not only telomeric repeats (Moore, 2009), but was originally
identified as a promoter binding ligand (Lugert and Werr, 1994).
Moreover, some members of this Myb-like family were identified
exclusively based on their ability to bind promoter regions of
certain genes: ZmIBP1 (Lugert and Werr, 1994), PcBPF-1 (box
P-binding factor) from P. crispum (da Costa e Silva et al.,
1993) or CrBPF from Catharanthus roseus (van der Fits et al.,
2000).
The third group with a Myb-like domain at the C-terminus
(AID family) contains only a few members. The AID family is
named according to anther indehiscence 1 (AID) protein from
O. sativa—OsAID1 (Zhu et al., 2004). OsAID1 was initially
identified as being involved in anther development. Another
member of this family—ZmTacs1 (terminal acidic SANT) from
Z. mays—may function in chromatin remodeling within the
meristem (Marian and Bass, 2005).
In an affinity pull-down technique, 80 proteins from O. sativa
were identified for their ability to bind to a telomeric repeat
(He et al., 2013). Among them, two of three previously
reported proteins from the SMH family–OsTRBF1 and OsTRBF2
(Byun et al., 2008), and one protein with a Myb-domain
at the C-terminus (AID family)—OsAID1 (Zhu et al., 2004;
Du et al., 2013) were demonstrated, while no member with
a Myb-domain at the C-terminus of the TRFL family could
be found. From other ribonucleoproteins or RNA-binding
proteins with putative telomere association, two homologs
of N. tabacum telomeric ssDNA binding protein NtGTBP1
(Os08g0492100 and Os08g0320100), with RNA recognition
motifs (RRM; see below; Lee and Kim, 2010), were also
identified.
Telomere-binding proteins in budding yeast (yRap1) or in
mammals (TRF1, TRF2, Rap1, and TIN2) are associated with
extra-telomeric sequences and thus participate in additional
roles, e.g., gene activation and repression, DNA replication,
heterochromatin boundary-element formation, creation of
hotspots for meiotic recombination and chromatin opening
(Figure 3A; Morse, 2000; Smogorzewska et al., 2000; Krutilina
et al., 2001; Zhang et al., 2008; Martinez et al., 2010; Simonet
et al., 2011; Yang et al., 2011; Maï et al., 2014; Ye et al., 2014).
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
CST Complex
An evolutionary conserved trimeric protein complex named CST
(Cdc13/CTC1–Stn1–Ten1) is, similarly to Myb-like proteins,
involved in several stages of telomere end formation. In yeast,
these OB-fold proteins are required for recruitment of telomerase
and DNA polymerase α to the chromosomal termini, and thus
coordinate G-overhang extension by telomerase with the fillin
synthesis of the complementary C-strand (Qi and Zakian,
2000; Grossi et al., 2004; Giraud-Panis et al., 2010; Wellinger and
Zakian, 2012). In mammals, CST is primarily involved in the
rescue of stalled replication forks at the telomere and elsewhere in
the genome, and limits telomerase action at individual telomeres
to approximately one binding and extension event per cell cycle
(Figure 2A; Chen et al., 2012; Stewart et al., 2012; Chen and
Lingner, 2013; Kasbek et al., 2013).
In A. thaliana, a mutation in any CST subunits leads
to severe morphological defects and is accompanied by
a decrease in telomere length, single-strand G-overhang
elongation, mostly subtelomere–subtelomere chromosomal
fusions and the appearance of extra-chromosomal telomeric
circles. Plants lacking Suppressor of cdc thirteen homolog
(AtStn1) or Conserved telomere maintenance component 1
(AtCTC1) exhibit no change in telomerase activity whereas
telomerase activity was elevated in atten1 mutants (Song et al.,
2008; Surovtseva et al., 2009; Leehy et al., 2013). Although
circumstantial evidence indicates that CST in plants is needed for
telomere integrity, clear evidence is absent that would show any
direct physical interaction of any component of the CST complex
with plant telomeric DNA. As Arabidopsis AtCTC1 interacts
with the catalytic subunit of DNA polymerase α (ICU2) in vitro
(Price et al., 2010) and atstn1 mutant phenotypes can be partially
phenocopied by impairment of DNA polymerase α, it was
recently suggested that seemingly specific function(s) of CST in
telomere protection may rather represent unique requirements
for efficient replication of telomeric DNA (Figure 2B; Derboven
et al., 2014). It seems that the CST complex controls access of
telomerase, end-joining recombination and the ATR-dependent
(ATM and Rad3-related) DNA damage response pathway at the
chromosomal ends in wild-type plants (Boltz et al., 2012; Leehy
et al., 2013; Amiard et al., 2014).
Telomeric ssDNA-Associated Proteins
Proteins with OB-fold
The telomeric G-rich overhang is evolutionarily conserved and
is a substrate for ssDNA binding proteins. The majority of
ssDNA binding proteins bind through OB motifs (OB-fold)
and are required for both chromosomal end protection and
regulation of telomere length, e.g., telomere-binding protein
subunit alpha/beta (TEBPαβ) from Oxytricha nova (telomere end
binding protein; Price and Cech, 1987), Cell division cycle 13
(Cdc13p) from S. cerevisiae (Garvik et al., 1995) and Pot1, are
present in diverse organisms including human, mouse, chicken,
or S. pombe (Figure 2A; Baumann and Cech, 2001; Lei et al.,
2002; Wei and Price, 2004; Wu et al., 2006). In A. thaliana, three
Pot-like proteins have been named AtPot1a, AtPot1b, AtPot1c
(Kuchar and Fajkus, 2004; Rossignol et al., 2007; previously
also named as AtPOT1-1, AtPOT1-2 (Tani and Murata, 2005)
or AtPot1, AtPot2 (Shakirov et al., 2005; see Rotkova et al.,
2009 for an overview). However, descriptions of their functions
and binding properties are not unanimously agreed. While a
very weak, but specific affinity of AtPot1a and AtPot1b for
plant telomeric ssDNA was originally described (Shakirov et al.,
2005), later these authors could not demonstrate AtPot1a and
AtPot1b binding to telomeric ssDNA in vitro (Shakirov et al.,
2009a,b). Nevertheless, stable telomeric ssDNA binding was
observed for two full-length plant Pot1 proteins: OlPot1 from
the green alga Ostreococcus lucimarinus as well as for ZmPot1b
from Z. mays (Shakirov et al., 2009b). Although Pot1 proteins
from plant species as diverse as Hordeum vulgare (HvPot1;
barley), Populus trichocarpa (poplar), Helianthus argophyllus
(sunflower), Selaginella moellendorffii (spikemoss), Gossypium
hirsutum (cotton), Pinus taeda (pine), Solanum tuberosum
(StPot1; potato), Asparagus officinalis and Z. mays (ZmPot1a)
failed to bind telomeric DNA when expressed in a rabbit
reticulocyte lysate expression system in vitro and subjected
to an electrophoretic mobility shift assay (Shakirov et al.,
2009b), binding of plant Pot1 proteins to telomeric DNA under
native conditions cannot be excluded. Plants expressing AtPot1a
truncated by an N-terminal OB-fold, showed progressive loss of
telomeric DNA. In contrast, telomere length was unperturbed
in plants expressing analogously trimmed AtPot1b, although
overexpression of C-terminally truncated AtPot1b resulted in
telomere shortening (Shakirov et al., 2005).
AtPot1a binds AtStn1 and AtCTC1 proteins (Figure 2B;
Renfrew et al., 2014), associates with an N-terminally spliced
variant of AtTERT (AtTERT-V(I8)) (Rossignol et al., 2007),
TER1, one of the RNA subunits of Arabidopsis telomerase, and is
required for maintenance of telomere length in vivo (Surovtseva
et al., 2007). AtPot1b directly interacts with Myb-like proteins
AtTRB1-3 from the SMH family (Schrumpfova et al., 2008),
and associates with TER2 and TER2s, putative alternative RNA
subunits of telomerase that negatively regulate the function of
active telomerase particles (TER1-AtTERT; Cifuentes-Rojas et al.,
2012). Nevertheless, AtPot1b does not seem to substantially
contribute to telomere maintenance (Cifuentes-Rojas et al.,
2012). Pot1-like proteins were also identified in plants with
unusual telomeres (e.g., CpPot1 protein in C. parqui; Peska et al.,
2008).
Non-OB-fold Telomeric ssDNA Binding Proteins
The transcriptional activator protein Whirly 1 (Why1), from a
small protein family found mainly in land plants (Desveaux et al.,
2000, 2002; Krause et al., 2005), was also identified in a fraction
of telomere-binding proteins in A. thaliana, and an atwhy1
knockout mutant appeared to have shorter telomeres (Yoo et al.,
2007). While proteins from A. thaliana (AtWhy1; Yoo et al.,
2007) and from H. vulgare (HvWhy1; Grabowski et al., 2008)
were found to bind plant telomeric repeat sequences in vitro,
diverse organelle localization of other Why family members
from O. sativa, A. thaliana, S. tuberosum (Krause et al., 2005;
Schwacke et al., 2007) and proposed binding to ssDNA of melted
promoter regions (Desveaux et al., 2002), rather indicate a role
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
in communication between plastid and nuclear genes encoding
photosynthetic proteins (Foyer et al., 2014; Comadira et al., 2015).
A truncated derivative of chloroplast RNA-binding protein
(AtCP31) with RRMs from A. thaliana, named AtSTEP1 (singlestranded
telomere-binding protein 1), localizes exclusively to
the nucleus, specifically binding single-stranded G-rich plant
telomeric DNA sequences and inhibiting telomerase-mediated
telomere extension (Kwon and Chung, 2004).
A protein identified by gel mobility shift assay that specifically
binds the G-strand of telomeric ssDNA from N. tabacum
(NtGTBP1) also contains a tandem pair of RRMs (Hirata
et al., 2004). NtGTBP1 is not only associated with telomeric
sequences, as well as two additional GTBP paralogs (NtGTBP2
and NtGTBP3), but also inhibits telomeric strand invasion
in vitro and leaves of knockdown tobacco plants contained
longer telomeres with frequent formation of extrachromosomal
t-circles (Lee and Kim, 2010). These observations correspond
to a previously detected protein from tobacco nuclei that binds
G-rich telomeric strands and reduces accessibility to telomerase
or terminal transferase (Fulneckova and Fajkus, 2000).
In addition to the above described proteins, various telomeric
ssDNA binding proteins have also been reported in nuclear
extracts from Glycine max, A. thaliana, O. sativa, or Vigna
radiata (Zentgraf, 1995; Kim et al., 1998; Lee et al., 2000; Kwon
et al., 2004). However, precise characterization of these proteins,
identified by gel mobility shift assay, is mostly missing.
DNA Repair Proteins and Telomeres
Ku in plants, as well as in other eukaryotes, is a highly
conserved complex, consisting of two polypeptides (Ku70 and
Ku80; Mimori et al., 1981). Due to its high affinity for DNA ends,
Ku has a generally conserved role across species in protecting
DNA from nucleolytic degradation. Ku is important for several
cellular mechanisms: the DNA double-stranded break (DSB)
repair pathway by the Ku-dependent non-homologous endjoining
(NHEJ) pathway, the DNA damage response machinery,
or protection of telomere ends from being recognized as
DSBs, thereby preventing their recombination and degradation
(reviewed in Fell and Schild-Poulter, 2015). Human Ku directly
interacts not only with the shelterin proteins hTRF1, hTRF2, and
hRap1, but also with telomerase subunits hTERT and hTR (RNA
template; reviewed in Fell and Schild-Poulter, 2015). In contrast
to a massive loss of telomeric DNA that was observed in human
cells (Wang et al., 2009), mutations in Ku70 and Ku80 in the
dicotyledonous A. thaliana, as well as in the monocotyledonous
O. sativa, resulted in longer telomeres, suggesting their conserved
role in the negative regulation of plant telomerase (Bundock et al.,
2002; Riha et al., 2002; Gallego et al., 2003; Hong et al., 2010).
On the other hand, severe developmental defects were observed
in O. sativa osku70 knockout mutants, but a similar mutation
in A. thaliana atku70 showed no effect on plant development
(Bundock et al., 2002; Hong et al., 2010). In S. latifolia and
A. thaliana, Ku contributes to the integrity of blunt-ended
telomeres by protecting them from nucleolytic resection (Kazda
et al., 2012). AtKu specifically interacts with AtTRP1 protein (see
above; Figure 2B; Kuchar and Fajkus, 2004) and also assembles
with TER2 and TER2S into alternative telomerase complexes
that cannot sustain telomere repeats on chromosomal ends
(Cifuentes-Rojas et al., 2012).
The mammalian shelterin complex is involved in the
repression of the primary signal transducers of DNA breakage,
two phosphatidylinositol-3-kinase-like (PI3K) protein kinases:
ataxia telangiectasia mutated (ATM) and ATM- and RAD3related
(ATR) kinases. Mice TRF2 acts mainly to protect
telomeres against ATM activation (Celli and de Lange, 2005) and
POT1 is principally involved in repression of the ATR pathway
(Denchi and de Lange, 2007; Guo et al., 2007). Short telomeres in
telomerase-deficient plants activate both the AtATM and AtATR,
whereas absence of members of the CST complex initiates only
AtATR-dependent, but not AtATM-dependent DNA damage
response (Amiard et al., 2011; Boltz et al., 2012). In mammals as
well as in other organisms, DSBs activate ATM kinase in a manner
dependent on the meiotic recombination 11 (Mre11), DNA
repair protein 50 (Rad50), and Nijmegen breakage syndrome 1
(Nbs1) named MRN complex. The MRN complex has been found
to associate with telomeres and contributes to their maintenance
(reviewed in Lamarche et al., 2010). A. thaliana AtRad50 mutant
plant cells show a progressive shortening of telomeric DNA
(Gallego and White, 2001), while in AtMre11 mutant plants,
telomere lengthening was observed (Bundock and Hooykaas,
2002). Contrary to these observations, the absence of the third
MRN subunit, AtNbs1, does not affect the length of telomeres
(Najdekrova and Siroky, 2012).
A. thaliana plants mutated in XPF (xeroderma pigmentosum
group F-complementing) and ERCC1 (excision repair crosscomplementation
group 1) orthologs that form a structurespecific
endonuclease essential for nucleotide excision repair
(known as AtRad1 and AtERCC1), develop normally and show
wild-type telomere length. However, in the absence of telomerase,
mutations in either of these genes induce a significantly earlier
onset of chromosomal instability, thus indicating a protective role
of AtERCC1/AtRad1 against a 3 G-strand overhang invasion of
interstitial telomeric repeats (Vannier et al., 2009). In addition
to the Ku proteins that are involved in Ku-dependent NHEJ,
an alternative Ku-independent NHEJ pathway was described
(reviewed in Decottignies, 2013). Members of the poly(ADPribose)
polymerase family play a role not only in the base
excision repair pathway and the backup-NHEJ KU-independent
pathway (Decottignies, 2013) but were also studied in the context
of telomere maintenance, association with shelterin proteins or
modulation of telomerase activity (Smith et al., 1998; Cook et al.,
2002; Beneke et al., 2008). However, analysis of Arabidopsis
orthologs AtPARP1/AtPARP2 (poly(ADP-Ribose) polymerase)
has revealed that, unlike in humans, AtPARPs play a minor
role in telomere biology (Boltz et al., 2014). It was proposed
that DSB repair pathways in A. thaliana are hierarchically
organized and the Ku-dependent NHEJ restricts access and
action of other DSB repair processes (Charbonnel et al., 2010,
2011). Furthermore the end-joining recombination proteins
(AtKU80, AtXRCC1, AtRad1) restrict telomerase activity at
deprotected telomeres (Amiard et al., 2014). It was found
recently that structure-specific endonucleases AtMUS81 (MMS
and UV-sensitive protein 81) and AtSEND1 (single-strand DNA
endonuclease 1), which presumably act to repair potentially toxic
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
structures produced by DNA replication and recombination, are
essential for telomere stability in Arabidopsis. Combined absence
of these endonucleases results in increased occurrence of histone
γ-H2AX foci in S-phase and in loss of telomeric DNA (Olivier
et al., 2016).
PLANT TELOMERASE
Telomere length in plants and various other organisms is
maintained by telomerase, a specialized reverse transcriptase
which, in addition to its catalytic subunit (TERT), carries its
own RNA template (TR) and elongates telomeric tracts at the
chromosomal terminus (Blackburn and Gall, 1978; Fajkus et al.,
1996).
TERT subunits consist of an N-terminal portion with
telomerase-specific motifs important for binding the telomerase
RNA subunit, catalytic domains with the telomerase reverse
transcriptase (RT) motifs essential for enzyme activity, and the
C-terminal extension, which is highly conserved among plants as
well as vertebrates (Sykorova and Fajkus, 2009). Although most
eukaryotes harbor only a single TERT gene, in the allotetraploid
N. tabacum there are three NtTERT gene variants inherited
from its diploid progenitor species N. sylvestris and Nicotiana
tomentosiformis. All three NtTERT gene variants are transcribed.
Alternative splicing provides a major source of protein
diversity within a given organism. Alternatively spliced variants
of TERT transcripts with out-of-frame and/or in-frame
mutations were identified not only in humans, mouse, chicken,
or Xenopus (reviewed in Hrdlickova et al., 2006), but also
in many plant species, e.g., A. thaliana, Z. mays (ZmTERT),
O. sativa (OsTERT), Iris tectorum, and tobacco [with human-type
(TTAGGG) telomere motif; reviewed in Sykorova and Fajkus,
2009; Sykorova et al., 2012]. Isoforms generated by alternative
splicing may show changes or loss of specific function(s) or
subcellular localization of the respective product, or could be
functionally important, as was suggested for the A. thaliana
variant AtTERT V(I8) that exclusively interacts with AtPot1a
(Rossignol et al., 2007).
It has been proposed that human telomerase is subjected
to posttranslational regulation such as phosphorylation (Kang
et al., 1999). Putative phosphorylation sites were detected in
the OsTERT sequences from O. sativa (Oguchi et al., 2004) or
N. tabacum BY-2 cells (Yang et al., 2002) but not in AtTERT from
A. thaliana (Oguchi et al., 2004).
Telomerase-Associated Proteins
Rich protein interactomes of yeast, mammalian or Ciliate TERT
have been described, including the Ku heterodimer (Chai et al.,
2002), HSP90 (heat-shock protein of 90 kDa; Holt et al., 1999;
Grandin and Charbonneau, 2001), ATPases pontin and reptin
(Venteicher et al., 2008), TEP1 (telomere protein 1; Harrington
et al., 1997), and many others, in a broad study (Fu and Collins,
2007) and reviewed in a constantly updated telomerase database
(Podlevsky et al., 2008).
In AtTERT, a mitochondrial targeting signal, multiple nuclear
localization signals or a nuclear export signal have been reported
(Zachova et al., 2013). As AtTERT protein and its domains
localize mainly within the nucleus and the nucleolus (Zachova
et al., 2013), it can be assumed that most interacting protein
partners relevant to telomeric functions will be found among
nuclear or nucleolar proteins.
In plants, a limited number of proteins that directly
interact with TERT have been described. It was demonstrated
by various direct methods that AtTRB proteins, a group
of plant homologs of human TRF proteins with a Mybdomain
at the N-terminus (see above), physically interact with
N-terminal domains of AtTERT (Figure 2B; Schrumpfova et al.,
2014). A mediated interaction between AtTRP1 protein that
belongs to the TRFL family, and AtTERT, was also observed
(Schrumpfova et al., 2014). Moreover, the N-terminal part of
AtTERT exclusively interacts with AtPot1a but not AtPot1b
(Rossignol et al., 2007). Also various proteins with an RRMmotif
(AtRRM), an ARM-motif (armadillo/β-catenin-like repeatcontaining
protein; AtARM), metallothionein-like (AtMT2A), or
RNA-binding (AtG2p) motifs were found as AtTERT interacting
partners in A. thaliana (Lee L.Y. et al., 2012; Dokládal et al.,
2015).
Indirect regulation of TERT by various proteins or
hormones was further described in plants. In tobacco cell
culture, phytohormones such as auxin or abscisic acid regulate
phosphorylation of telomerase protein, which is required for the
generation of a functional telomerase complex (Tamura et al.,
1999; Yang et al., 2002). In A. thaliana, reduced endogenous
concentrations of auxin in telomerase activator 1 (AtTAC1)
mutant plants blocks the ability of this zinc-finger protein
to induce AtTERT. However, AtTAC1 does not directly bind
the AtTERT promoter (Ren et al., 2004, 2007). A minimal
promoter region for AtTERT was proposed using a set of
T-insertion mutant lines in the protein-coding region of the
AtTERT gene or in lines with insertions at the 5 end of
AtTERT (Fojtova et al., 2011). Moreover T-DNA insertions in
the region upstream of the ATG start of AtTERT also led to
the activation of putative regulatory elements (Fojtova et al.,
2011).
In vertebrates, only one TR per organism was described. The
folding of the TR molecule offers interaction sites for various
associating cofactors such as dyskerin, Ku, nucleolar protein 10
(NOP10), H/ACA ribonucleoprotein complex subunit 1 (GAR1),
or subunit 2 (NHP2; Ting et al., 2005; for review, see Kiss
et al., 2010). A single TR was also described among Brassicaceae
family plants. However, in A. thaliana, two TRs were detected—
TER1 and TER2, and the latter may be alternately spliced to a
TER2s form (Beilstein et al., 2012). The Arabidopsis homolog
of human dyskerin, named AtCBF5 (alias AtNAP57), is located
within nucleoli and Cajal bodies (Lermontova et al., 2007),
associates with active telomerase, and weakly with AtPOT1a, but
not AtTERT or AtKu70 (Kannan et al., 2008).
Telomerase-Independent Processes in
Plant Telomere Dynamics
Compared to the human model, knowledge of individual
protein contributions to the maintenance of telomere length/
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Procházková Schrumpfová et al. Telomere- and Telomerase-Associated Proteins
accessibility/folding in plants or telomerase biogenesis/regulation
is still very limited. The process of telomere maintenance is
complicated by the fact that besides the widespread system of
telomere maintenance by telomerase (Fajkus et al., 1996; Heller
et al., 1996) in plants as well as in other organisms, in the absence
of telomerase, telomeres can be elongated by recombinationdependent
and telomerase-independent alternative telomere
lengthening (ALT) mechanisms (Fajkus et al., 2005). Moreover,
in plants, the ALT events appear to participate in early
plant development (Ruckova et al., 2008). It was shown that
AtKu70 deficiency facilitates engagement of ALT lengthening
in A. thaliana (Zellinger et al., 2007) and that ALT was
suppressed in the absence of ATM protein (Vespa et al.,
2007).
Telomeric DNA of higher eukaryotes, including plants,
is associated not only with specific proteins, but also with
histone complexes that form nucleosomes (Figure 2; reviewed
in Dvoˇráˇcková et al., 2015). In various organisms, as well
as in plants, telomeric nucleosomes display an unusually
short periodicity (157 bp in length), usually 20–40 bp
shorter than bulk nucleosomes of the corresponding organism
(Fajkus et al., 1995; Fajkus and Trifonov, 2001; reviewed in
Pisano et al., 2008). Moreover, the plant telomeric repeat
(CCCTAAA) is a natural target for plant-specific asymmetric
methylation (Cokus et al., 2008) that was shown to be mediated
by an siRNA pathway (Vrbsky et al., 2010). Analysis of
telomeres in A. thaliana (Vrbsky et al., 2010) and N. tabacum
(Majerova et al., 2011) has demonstrated that telomeric
histones were associated with both heterochromatin- and
euchromatin-specific marks. Recent data strongly support
the involvement of various epigenetic mechanisms (DNA
methylation, posttranslational modifications of histones,
nucleosome assembly or levels of telomere-repeat containing
RNA) in maintenance of telomere stability (reviewed in
Dvoˇráˇcková et al., 2015) thus demonstrating complexity of
telomere regulation.
CONCLUSION
The need for protection of chromosomal termini remains
conserved across most species. Nevertheless, an extraordinary
plasticity of mechanisms protecting telomeres has been described
among various organisms (reviewed in Giraud-Panis et al., 2013).
While individual capping proteins can differ greatly, common
features such as homologous binding domains, structures, or
interacting partners exist between seemingly different capping
systems. Plant systems show certain distinct features of telomere
maintenance, including the reversible regulation of telomerase
in somatic cells and the absence of developmental telomere
shortening (Fajkus et al., 1998; Riha et al., 1998). These
distinctions promote further efforts to elucidate plant telomere
interactomes. Only recently the first complexes of telomerebinding
proteins were demonstrated and meanwhile it seems
that the plant telomere-maintenance system shares similarities
with that described in mammals. For example, in A. thaliana,
one of the most studied plant model systems: (i) the core
plant telomeric dsDNA binding proteins (AtTRBs, AtTRP, etc.)
contain similar Myb-domains which are also present in human
TRF1 or TRF2 proteins; (ii) homologs of human telomeric
ssDNA binding hPot1 (AtPOT1a-c) were described; (iii) crossspecies
conserved CST complexes (AtCTC1/AtTen1/AtStn1)
retain its function in plants. The similarities between plant
and mammalian telomeric DNA-associated proteins apply also
to their roles in regulation of gene expression, which are
independent of their roles in telomere capping (Lee and Cho,
2016; Schrumpfova et al., 2016; Zhou et al., 2016), as was
previously described in their mammalian counterparts (reviewed
in Maï et al., 2014; Ye et al., 2014). Elucidation of the composition
of the plant version of shelterin and molecular dissection of
its components and their roles will be important in the near
future to assess the conservation and mechanisms of endprotection
and end-replication processes in yeasts, plants and
animals.
AUTHOR CONTRIBUTIONS
PPS contributed substantially to the writing of the manuscript,
tables and drawing the figures; ŠS participated in preparation of
tables; JF edited the manuscript. All authors read and approved
the manuscript for publication.
FUNDING
This research was supported by the Czech Science Foundation
(13-06943S and 16-01137S) and by the Ministry of Education,
Youth and Sports of the Czech Republic under the project
CEITEC 2020 (LQ1601).
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
The handling Editor declared a current collaboration as co-Topic Editor in a
Frontiers Research Topic with one of the authors, JF, and states that the process
nevertheless met the standards of a fair and objective review. This was also
confirmed by the Specialty Chief Editor of section Plant Cell Biology, Simon Gilroy.
Copyright © 2016 Procházková Schrumpfová, Schoˇrová and Fajkus. This is an openaccess
article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) or licensor are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these
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Frontiers in Plant Science | www.frontiersin.org 19 June 2016 | Volume 7 | Article 851
Supplement K
Schrumpfová, P.P., Majerská, J., Dokládal, L., Schořová, Š., Stejskal, K., Obořil, M., Honys,
D., Kozáková, L., Polanská, P.S., Sýkorová, E., 2017. Tandem affinity purification of AtTERT
reveals putative interaction partners of plant telomerase in vivo. Protoplasma 254, 1547–1562
P.P.S. participated in the design of experiments, was significantly involved in the
experimental part, data evaluation and participated in the ms writing
This journal did not provide open access, hence the article is not freely available.
Supplement L
Schrumpfová, P.P., Majerská, J., Dokládal, L., Schořová, Š., Stejskal, K., Obořil, M., Honys,
D., Kozáková, L., Polanská, P.S., Sýkorová, E., 2018. Correction to: Tandem affinity
purification of AtTERT reveals putative interaction partners of plant telomerase in vivo.
Protoplasma 255, 715
In the published online version, P.P. Schrumpfová was not recognized as the first co-author,
and the affiliations were mixed up. Corrections were made to both the first co-authorship
and the affiliation section.
CORRECTION
Correction to: Tandem affinity purification of AtTERT reveals putative
interaction partners of plant telomerase in vivo
Jana Majerská1,2,3
& Petra Procházková Schrumpfová2
& Ladislav Dokládal1
& Šárka Schořová2
& Karel Stejskal2
&
Michal Obořil2
& David Honys4
& Lucie Kozáková2
& Pavla Sováková Polanská2
& Eva Sýkorová1
Published online: 14 February 2018
# Springer-Verlag GmbH Austria, part of Springer Nature 2018
Correction to: Protoplasma (2017) 254:1547–1562
https://doi.org/10.1007/s00709-016-1042-3
In the published online version, the affiliations were mixed up.
Corrected affiliation section is shown below. Also, the update
has also been reflected in the author group section above.
Jana Majerská and Petra Procházková Schrumpfová are both co-authors
The online version of the original article can be found at https://doi.org/
10.1007/s00709-016-1042-3
* Eva Sýkorová
evin@ibp.cz
1
Institute of Biophysics, Academy of Sciences of the Czech Republic,
v.v.i, Královopolská 135, CZ-61265 Brno, Czech Republic
2
Central European Institute of Technology and Faculty of Science,
Masaryk University, Kotlářská 2, CZ-61137 Brno, Czech Republic
3
Present address: Swiss Institute for Experimental Cancer Research
(ISREC), School of Life Sciences, École Polytechnique Fédérale de
Lausanne (EPFL), 1015 Lausanne, Switzerland
4
Institute of Experimental Botany, Academy of Sciences of the
Czech Republic v.v.i, Rozvojová 263,
CZ-16502 Prague, Czech Republic
Protoplasma (2018) 255:715
https://doi.org/10.1007/s00709-018-1224-2
Supplement M
Schrumpfová, P.P., Fojtová, M., Fajkus, J., 2019. Telomeres in Plants and Humans: Not So
Different, Not So Similar. Cells 8
P.P.S. was significantly involved in the ms writing and editing
cells
Review
Telomeres in Plants and Humans: Not So Different,
Not So Similar
Petra Procházková Schrumpfová 1, Miloslava Fojtová 1,2,3 and Jiˇrí Fajkus 1,2,3,*
1 Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of
Science, Masaryk University, CZ-61137 Brno, Czech Republic; petra.proch.schrumpfova@gmail.com (P.P.S.);
miloslava.fojtova@gmail.com (M.F.)
2 Mendel Centre for Plant Genomics and Proteomics, CEITEC, Masaryk University, CZ-62500 Brno,
Czech Republic
3 Institute of Biophysics of the Czech Academy of Sciences, CZ-61265 Brno, Czech Republic
* Correspondence: tttaggg@gmail.com; Tel.: +420-5494-94003
Received: 17 December 2018; Accepted: 7 January 2019; Published: 16 January 2019
Abstract: Parallel research on multiple model organisms shows that while some principles of telomere
biology are conserved among all eukaryotic kingdoms, we also find some deviations that reflect
different evolutionary paths and life strategies, which may have diversified after the establishment of
telomerase as a primary mechanism for telomere maintenance. Much more than animals, plants have
to cope with environmental stressors, including genotoxic factors, due to their sessile lifestyle. This is,
in principle, made possible by an increased capacity and efficiency of the molecular systems ensuring
maintenance of genome stability, as well as a higher tolerance to genome instability. Furthermore,
plant ontogenesis differs from that of animals in which tissue differentiation and telomerase silencing
occur during early embryonic development, and the “telomere clock” in somatic cells may act as
a preventive measure against carcinogenesis. This does not happen in plants, where growth and
ontogenesis occur through the serial division of apical meristems consisting of a small group of stem
cells that generate a linear series of cells, which differentiate into an array of cell types that make
a shoot and root. Flowers, as generative plant organs, initiate from the shoot apical meristem in
mature plants which is incompatible with the human-like developmental telomere shortening. In this
review, we discuss differences between human and plant telomere biology and the implications for
aging, genome stability, and cell and organism survival. In particular, we provide a comprehensive
comparative overview of telomere proteins acting in humans and in Arabidopsis thaliana model plant,
and discuss distinct epigenetic features of telomeric chromatin in these species.
Keywords: telomere; telomerase; human; Arabidopsis; aging; chromatin; epigenetics; review
1. Introduction
Telomere biology, whose foundations were laid out in maize and Drosophila at the end of the
1930s and which developed at the molecular level in the 1980s, has flourished enourmously in the last
30 years. This interest in telomere biology follows from the generally attractive links between telomere
functions, cell aging mechanisms, and the genesis of severe diseases in humans. Research in recent
decades has elucidated the principles of protection of the ends of linear eukaryotic chromosomes from
progressive shortening due to the incomplete replication (end-replication problem) [1] and from their
erroneous recognition as unrepaired chromosome breaks (end-protection problem) [2–4]. In addition
to these basic functions, other potential roles of telomeres have been suggested, such as a trap for
reactive oxygen species [5,6]. Telomeres are composed of non-coding repetitive tandem repeats of
(TTAGGG)n in humans and the other vertebrates, and (TTTAGGG)n in most plants. During human
Cells 2019, 8, 58; doi:10.3390/cells8010058 www.mdpi.com/journal/cells
Cells 2019, 8, 58 2 of 31
aging, telomeres in most somatic cells are shortened at each cell division and it is generally assumed
that when telomeres reach a critical length, cells enter a senescent state and cell division ceases [7,8].
However, most human individuals do not reach this critical telomere length brink during their life
course [8,9], e.g., the mean leukocyte telomere length (LTL) in newborns is 9.5 kb [10] whereas a
length of ~5 kb was defined as the ‘telomeric brink’, which denotes a high risk of imminent death,
but only 0.78% of people younger than 90 years display an LTL ≤ 5 kb [9]. So it is obvious, that the
link between shortened telomeres and human longevity is more complex than mere reaching the
critical telomere length. For instance, age-dependent telomere shortening might alter gene expression
in sub-telomeric regions (telomere position effect, TPE) or double strand DNA breaks in telomeres
might be inefficiently repaired and initiate cell senescence [11,12]. Furthermore, it has been suggested
that even a single critically short telomere in a cell can induce cellular senescence, which potentially
contributes to organismal senescence [13,14]. In humans, five short telomeres were reported to predict
the onset of cell senescence [15].
Although the principles of protection and replication of telomeres are conserved and point to
common evolutionary roots of eukaryotes, their implications for cell and organism survival, senescence,
and aging are not shared among kingdoms. In particular, plants show specific features of their growth
and development, which lead to confusion of terms like lifespan or aging as commonly used and
understood in animals. First, a plant’s body plan is not fully established during embryogenesis
and all tissues and organs are formed from proliferating meristem cells throughout the adult life.
Second, plant growth is modular. Individual modules of the body (branches, flowers, leaves) are
dispensable for survival, and their functions can be replaced by tissues newly differentiated from
indefinitely proliferating meristems. This results in the enormous developmental plasticity of plants.
Moreover, the vegetative meristems can give rise to a new organism, which will be a somatic clone,
genetically indistinguishable from the parental organism. Since these general aspects distinguishing
plant from animal development and aging have been well-reviewed [16], we will focus here on a more
detailed view of peculiarities of plant telomere biology, including its latest developments.
2. Telomerase Core Components
The requirement to finish the incomplete replication of chromosome ends is common for all
organisms with linear chromosomes. In eukaryotes, this requirement is commonly solved by a
specific nucleoprotein enzyme complex called telomerase, which is considered as an ancestral telomere
maintenance system that solves the end-replication problem of linear chromosomes. In humans,
telomerase activity is detected in all early developmental stages from oocytes through to blastocyst
stage embryos, and increases progressively with advancing embryo stage. Telomerase reaches its
highest level in morula and blastocyst stage embryos and then decreases in the inner cell mass
stage. In human fetuses—when the embryonic period and organogenesis are finished—telomerase is
expressed in tissue-specific stem cells. However, just after birth, telomerase activity in somatic cells is
downregulated with the exception of dividing cells (e.g., proliferating cells, T-lymphocytes) [17,18]
(Figure 1A).
As seen in mammals, telomeres in plants are maintained by telomerase [19]. Active telomerase is
detected in organs and tissues containing highly dividing meristem cells such as seedlings, root tips,
young and middle-age leaves, flowers, and floral buds [20,21]. In terminally differentiated tissues
(stems, mature leaves), telomerase activity is suppressed (Figure 1B). In some groups of organisms
(in particular insects), telomerase has been lost and replaced by telomere-specific retrotransposons
(in Drosophila) or tandem arrays of satellite repeats elongated by a gene conversion mechanism
(reviewed in References [22,23]). Based on a long-term systematic search, no telomerase-independent
exception has been found among vertebrates or land plants despite the variability of telomere DNA
observed in land plants [24–27]. Besides the telomerase-based mechanism of telomere elongation,
alternative lengthening of telomeres (ALT), which is based on homologous recombination (HR)
Cells 2019, 8, 58 3 of 31
and may become active upon the loss of telomerase was described in humans as well as in plants
(see below).Cells 2019, 8, x FOR PEER REVIEW 3 of 31
Figure 1. Telomerase activity in human and plant tissues. (A) During human embryonic development,
high telomerase activity is detected in the blastocyst, but not in mature spermatozoa or oocytes.
Highly active telomerase is detected in 16 to 20-week-old human fetuses in most somatic tissues with
the exception of brain tissue [18,28]. In adults, low telomerase activity is detected in hair follicule
bulbs [29], basal cells of crypt and villi or muconasal basal cells of the gastrointestinal tract, basal
keratinocytes of the skin [30], lymphocytes, blood bone marrow, and stem cells [31–33], and
urothelium [34]. High telomerase activity is detected in prostate tissues and endometrium [30,35]. (B)
High telomerase activity is detected in plant pollen, seedling, young rosette leaves, and silliques
[21,36–39]. Likewise, both apical meristems—shoot and root—show high telomerase activity [36–38].
Figures adopted from human and Arabidopsis eFP browsers [40].
In yeasts, animals, and plants, telomerase consists of the telomerase reverse transcriptase (TERT)
protein subunit providing the catalytic activity, and the telomerase RNA (TR) subunit whose short
region provides a template for reverse transcription [41,42]. Besides these two core subunits, the
telomerase complex comprises several other accessory proteins with diverse roles in telomerase
assembly, trafficking, localization, recruitment to telomeres, or the processivity of telomere synthesis
[43,44]. During movement of the maturing human telomerase complex through the nucleolus to Cajal
bodies and to the telomeres, the TERT catalytical subunit is associated with e.g., HSP90, p23, or
pontin. Assembly of human TR, as well as other box C/D or H/ACA small nucleolar RNAs
(snoRNAs), is governed by conserved scaffold proteins: dyskerin, NHP2, NOP10, NAF1 in the
nucleoplasm, where NAF1 is replaced by GAR1 before the hTR RNP complex reaches the nucleolus.
Several orthologues of these conserved scaffold have been identified in plants, e.g., CBF5 (dyskerin),
RuvBL1 (pontin), RuvBL2a (reptin), and NAF1. The nucleolar localization of these orthologues
suggests potential conservation of the trafficking pathway during telomerase maturation ([45–47];
Schorova et al., submitted). Human and plant homologues of proteins associated either with the
telomerase protein subunit TERT (Table 1) or the telomerase RNA subunit (Table 2) are listed below.
Figure 1. Telomerase activity in human and plant tissues. (A) During human embryonic development,
high telomerase activity is detected in the blastocyst, but not in mature spermatozoa or oocytes.
Highly active telomerase is detected in 16 to 20-week-old human fetuses in most somatic tissues with the
exception of brain tissue [18,28]. In adults, low telomerase activity is detected in hair follicule bulbs [29],
basal cells of crypt and villi or muconasal basal cells of the gastrointestinal tract, basal keratinocytes
of the skin [30], lymphocytes, blood bone marrow, and stem cells [31–33], and urothelium [34].
High telomerase activity is detected in prostate tissues and endometrium [30,35]. (B) High telomerase
activity is detected in plant pollen, seedling, young rosette leaves, and silliques [21,36–39]. Likewise,
both apical meristems—shoot and root—show high telomerase activity [36–38]. Figures adopted from
human and Arabidopsis eFP browsers [40].
In yeasts, animals, and plants, telomerase consists of the telomerase reverse transcriptase
(TERT) protein subunit providing the catalytic activity, and the telomerase RNA (TR) subunit
whose short region provides a template for reverse transcription [41,42]. Besides these two core
subunits, the telomerase complex comprises several other accessory proteins with diverse roles in
telomerase assembly, trafficking, localization, recruitment to telomeres, or the processivity of telomere
synthesis [43,44]. During movement of the maturing human telomerase complex through the nucleolus
to Cajal bodies and to the telomeres, the TERT catalytical subunit is associated with e.g., HSP90, p23,
or pontin. Assembly of human TR, as well as other box C/D or H/ACA small nucleolar RNAs
(snoRNAs), is governed by conserved scaffold proteins: dyskerin, NHP2, NOP10, NAF1 in the
nucleoplasm, where NAF1 is replaced by GAR1 before the hTR RNP complex reaches the nucleolus.
Several orthologues of these conserved scaffold have been identified in plants, e.g., CBF5 (dyskerin),
RuvBL1 (pontin), RuvBL2a (reptin), and NAF1. The nucleolar localization of these orthologues
suggests potential conservation of the trafficking pathway during telomerase maturation ([45–47];
Schorova et al., submitted). Human and plant homologues of proteins associated either with the
telomerase protein subunit TERT (Table 1) or the telomerase RNA subunit (Table 2) are listed below.
Cells 2019, 8, 58 4 of 31
Table 1. Comparative overview of proteins associated with the telomerase catalytic subunit TERT.
Telomerase Calytic Subunit (TERT) Associated Proteins.
Human TERT
Associated Proteins
Protein Function and Direct
Interactions
References
Arabidopsis TERT
Associated Proteins
Protein Function and Direct Interactions References
TERT Catalytic subunit of telomerase [48] TERT Catalytic subunit of telomerase [49]
POT1
Shelterin. Int.: telomeric ssDNA,
TPP1 and CTC1.
[50–54] POT1a
Shelterin-like. Int.: TERT, telomeric
ssDNA, TER1, TRFL9, CBF5, RuvBL1,
CTC1 and STN1.
[47,55–58]
TRF1
Shelterin. Int.: telomeric dsDNA,
TIN2, TANK1, PINX1, and ATM.
[59–63] TRB1-3
Shelterin-like. Int.: TERT, telomeric
dsDNA, POT1b, RuvBL1 and RuvBL2a.
[64–71]
TRF2
Shelterin. Int.: telomeric dsDNA;
TIN2, NBS1, RAD50, Apollo, Ku70,
PARP1, XPF-ERCC1, BLM, FEN1,
POLB, ORC, RTEL1, ATM and HP1.
[61,72–80] TRP1
Possible non-telomeric functions of
telomerase. Int.: TERT, telomere dsDNA
in vitro, ARM, Ku70 and TRFL9.
[66,69,81–83]
TRFL2
Possible non-telomeric functions of
telomerase. Int.: TERT, telomere dsDNA
in vitro and ARM.
[69,83]
TRFL11 Associates with TERT. [84]
KPNA1 Promotes nuclear import of the TERT. [85] ImpA4 Associates with TERT. [84]
NCL
Involves nucleolar localization of
TERT.
[86] NUC-L1
Role in telomere maintenance and
telomere clustering.
[87,88]
pontin
Telomerase assembly. Int.: TERT and
dyskerin.
[89] RuvBL1
Associates with TERT via TRBs, regulates
telomerase activity.
[84,90]
reptin Telomerase assembly. Int.: dyskerin. [89] RuvBL2a
Associates with TERT via TRBs, regulates
telomerase activity.
[84]
ARMC6 Int.: TRF2, telomerase. [69,91] ARM
May reflect posible non-telomeric
functions of telomerase. Int.: TERT, TRP1,
TRFL2, TRFL9 and CHR19.
[69,92]
TPP1
Shelterin, mediates telomerase
recruitment. Int.: TERT, POT1, TIN2,
CTC1 and STN1.
[51–54,75] n.a.
Cells 2019, 8, 58 5 of 31
Table 1. Cont.
PINX1
Potent telomerase inhibitor. Int.:
TERT and TRF1.
[62] n.a.
HOT1
Int.: telomeric dsDNA, active
telomerase.
[93] n.a.
Ku70/80 Int.: TERT, TR, TRF2 and RAP1. [94,95] Ku70/80
Role in telomere length regulation, may
protect blunt-ended telomeres Int.: TRP1,
TER2 and TER2s.
[82,96–101]
Hsp90 TERT assembly. Int.: TERT. [102] Hsp90 NP_194150.1 [103]
p23 TERT assembly. Int.: TERT. [102] p23 CAC16575, NP_683525 [104]
Purα
p.h. Unwinds dsDNA telomeric
oligonucleotides.
[105] PURα1 Associates with TERT. [84]
SMARCAD1
p.h. SWI/SNF-like protein that
presumably associates with
telomeres.
[106,107] CHR19
May reflect possible non-telomeric
functions of telomerase. Int.: TERT, ARM,
TRB1 and TRFL9.
[69]
PABPN1
Promotes poly(A)-dependent TR 3
end maturation.
[108] RRM Associates with TERT. [92,109]
MT2A p.h. Int.: HOT1. [110,111] MT2A Associates with TERT. [84,109]
PA2G4 NP_006182.2 [112] G2p Associates with TERT. [84,109]
The proteins depicted in grey are involved in telomere maintenance, however, their association with telomerase has not been described. The proteins in green are structural
homologous to their human/plant counterparts, however, any involvement in telomere maintenance or association with telomerase has not been described so far. Direct interaction
partners (Int.) of TERT-associated proteins are enumerated. Cases with not yet identified sequence homologues are denoted with n.a. ATP-dependent DNA helicase 2 subunit 1 and
2 (Ku70/80); Origin recognition complex (ORC); RuvB-like 2 (reptin); TIN2- and POT1-organizing protein (TPP1); TRF1-interacting nuclear protein 2 (TIN2); TRF1-interacting
protein 1 (PINX1); 5 exonuclease Apollo (Apollo); Armadillo repeat-containing protein 6 (ARMC6); Armadillo/β-catenin-like repeat-containing protein (ARM); Ataxia telangiectasia
mutated kinase (ATM); Bloom syndrome protein (BLM); Centromere-binding factor (CBF5); Conserved telomere maintenance component 1 (CTC1); DNA polymerase beta (POLB);
DNA repair protein RAD50 (RAD50); Double strand DNA (dsDNA); Excision repair cross-complementation 1 (ERCC1); Flap endonuclease 1 (FEN1); H/ACA ribonucleoprotein
complex subunit DKC1 (dyskerin); Heterochromatin protein 1 (HP1); Homeobox telomere-binding protein 1 (HOT1); Hsp90 co-chaperone (p23); Chromatin remodeling 19 (CHR19);
Importin-α5 (KPNA1); Importin subunit alpha-4 (ImpA4); Metallothionein-like 2A (MT2A); Nijmegen breakage syndrome protein 1 (NBS1); Nucleolin (NCL); Nucleolin like
1 (NUC-L1); Heat shock protein HSP 90 (Hsp90); Poly(ADP-ribose) polymerase 1 (PARP1); Polyadenylate-binding protein (PABPN1); Proliferation-associated 2G4 (PA2G4);
Proliferation-associated protein (G2p); Protection of telomeres 1 (POT1); Protection of telomeres 1a, b (POT1a, b); Pur-alpha 1 (Purα1); Regulator of telomere elongation helicase 1
(RTEL1); RNA recognition motif (RRM); RuvB-like 1 (pontin); RuvB-like 1, 2a (RuvBL1, 2a); Single strand DNA (ssDNA); Suppressor of cdc thirteen homolog (STN1); SWI/SNF-related
matrix-associated actin-dependent regulator of chromatin subfamily A containing DEAD/H box 1 (SMARCAD1); Tankyrase 1 (TANK1); Telomerase reverse transcriptase (TERT);
Telomerase RNA (TR); Telomerase RNA subunit 1 (TER1); Telomere repeat-binding factor 1, 2, 3 (TRB1, 2, 3); Telomere repeat-binding protein 1 (TRP1); Telomeric repeat binding
factor 1-like 2, 9, 11 (TRFL 2, 9, 11); Telomeric repeat-binding factor 1, 2 (TRF1, 2); Xeroderma pigmentosum group F (XPF1); putative homolog according to NCBI blastp (p.h.).
Cells 2019, 8, 58 6 of 31
Table 2. Comparative overview of proteins associated with the RNA component of telomerase.
Telomerase RNA Associated Proteins
Human TR
Associated Proteins
Protein Function and Direct
Interactions
References
Arabidopsis TR
Associated Proteins
Protein Function and Direct Interactions References
TR RNA subunit of telomerase [113] TER1, TER2, TER2s Putative RNA subunit of telomerase [56,100]
TERT Catalytic subunit of telomerase [48,114] TERT Catalytic subunit of telomerase [100]
Dyskerin
H/ACA snoRNPs, associated with
nucleolus. Int.: TR, GAR1, NHP2,
NOP10 and TCAB1.
[44,115] CBF5
H/ACA snoRNPs,
Ath orthologue of Dyskerin, associated
with nucleolus, subnuclear bodies and
Cajal bodies, associated with telomerase
RNP complex. Direct interaction with
either of putative TERs not demonstrated.
Int.: NAF1.
[45,57]
NOP10
H/ACA snoRNPs, associates with
nucleolus. Int.: TR and dyskerin.
[44,116] NOP10
H/ACA snoRNPs,
Ath orthologue of NOP10, associates with
nucleolus.
[45,46]
NHP2
H/ACA snoRNPs, associates with
nucleolus. Int.: TR, dyskerin and
TCAB1.
[117,118] NHP2
H/ACA snoRNPs,
Ath orthologue of NHP2, associates with
nucleolus.
[45,46]
GAR1
H/ACA snoRNPs,
associated with nucleolus. Int.:
dyskerin and TCAB1.
[44,118] GAR1, 2
H/ACA snoRNPs,
Ath orthologues of GAR1, associate with
nucleolus.
[45,46]
NAF1
H/ACA snoRNPs, nucleolar shuttle NAF1
is substituted by GAR1 during
maturation of telomerase. Int.:
dyskerin.
[119] NAF1
H/ACA snoRNPs,
Ath orthologue of NAF1, associates with
nucleolus and Cajal bodies. Int.: CBF5.
[45]
Ku70/80 Int.: TR, TERT, TRF2 and RAP1. [95,120] Ku70/80
Role in telomere length regulation, may
protect blunt-ended telomeres Int.: TRP1,
TER2 and TER2s.
[100]
Cells 2019, 8, 58 7 of 31
Table 2. Cont.
pontin
Telomerase assembly. Int.: TERT and
dyskerin.
[89] RuvBL1
Associates with TERT via TRBs, regulates
telomerase activity.
Schorova et al.,
submitted
reptin Telomerase assembly. Int.: dyskerin. [89] RuvBL2a
Associates with TERT via TRBs, regulates
telomerase activity.
Schorova et al.,
submitted
RHAU
RNA helicase, unwinds a
G4-quadruplex in human telomerase
RNA. Int.: TR.
[121] RHAU
NP_850255.1, NP_175298.2, NP_680142.2,
NP_178223.2
n.a.
PARN
Poly(A)-specific ribonuclease, 3 -end
maturation of the TR. Int.: TR
[122] PARN
Poly(A) degradation activity, essential
gene first required during early
development.
[123]
TCAB1
H/ACA snoRNPs, driving telomerase
to Cajal bodies. Int.: TR, dyskerin,
NHP2 and GAR1.
[124] TCAB1 NP_193883.2 n.a.
The proteins in green are structural homologues to their human counterparts, however, any involvement in telomere maintenance or association with RNA component of telomerase
has not been described so far. Direct interaction partners (Int.) of TR-associated proteins are enumerated. Cases when reference is not available are denoted n.a. H/ACA
ribonucleoprotein complex subunit DKC1 (dyskerin); RuvB-like 2 (reptin); Arabidopsis (Ath); ATP-dependent DNA helicase 2 subunit 1 and 2 (Ku70/80); box H/ACA small nucleolar
RNA-protein complexes (H/ACA snoRNPs); Centromere-binding factor (CBF5); Glycine arginine rich 1, 2 (GAR1, 2); Non-histone protein 2 (NHP2); Nuclear assembly factor
1 (NAF1); Nucleolar protein 10 (NOP10); Repressor-activator protein 1 (RAP1); RNA helicase (PARN); RNA helicase (RHAU); RuvB-like 1 (pontin); RuvB-like 1, 2a (RuvBL1,
2a); Telomerase Cajal body protein 1 (TCAB1); Telomerase reverse transcriptase (TERT); Telomere repeat-binding factors (TRBs); Telomere repeat-binding protein 1 (TRP1);
Telomerase RNA subunit 1, 2, 2s (TER1, 2, 2s); Telomeric repeat-binding factor 2 (TRF2); Telomerase RNA (TR).
Cells 2019, 8, 58 8 of 31
Considerable homology in TERT sequences and domain organization exists among organisms,
and this homology has frequently been used to identify novel TERTs in genomic or transcriptomic data
(reviewed in Reference [125]). Human TERT, as well as the plant TERTs, can be split into the N-terminal
part, the central catalytic reverse transcriptase (RT) motifs, and the C-terminal extension (CTE) which
is highly conserved among vertebrates as well as among plants. The N-terminal part comprises regions
of both low and high similarity, e.g., the structural domains TEN (telomerase essential N-terminal
domain) or TRBD (RNA-binding domain). Although most eukaryotes, including humans, harbor a
single TERT gene, in the allotetraploid Nicotiana tabacum plant, three transcribed variants of the TERT
gene were described, which were inherited from its diploid progenitor species [126].
Compared to the conserved structure of the TERT subunit, TRs show high sequence diversity
among more distant organisms, as exemplified by the length differences of TRs in protozoa (159 nt
in ciliate Tetrahymena, 2200 nt in Plasmodium), zebrafish (317 nt), mouse (397 nt), human (451 nt),
and budding yeasts (1160 nt). Even within yeasts, the homology among TRs is rather low and their
lengths range from 930 to more than 2000 nt [42,113,127–133]. Analogous variance of TR within the
plant kingdom is still questionable, since only putative TRs have been predicted in A. thaliana so
far [56].
However, several secondary structure motifs in TRs which are essential for telomerase activity
are conserved in fungi and animals. Starting from the 5 -end of TR, these include a core-enclosing
helix (CEH) formed by pairing the 5’-terminus of TR with the complementary internal TR region,
a template boundary element (TBE)—a hairpin defining the end of the sequence recognized by TERT
as a template, the template sequence itself, and a pseudoknot [133]. Except for the template sequence,
none of these structural elements has been recognized in TER1 in Arabidopsis thaliana, which is the
only reported candidate TR among plants so far [56]. With respect to the above-mentioned sequence
diversity of plant telomere repeats, it will be interesting to learn whether and how these evolutionary
changes are reflected by the corresponding TR subunits. For example, when assuming the phylogeny of
Asparagales plants, telomeres switched first from Arabidopsis-like repeats (TTTAGGG)n to human-like
repeats (TTAGGG)n in the divergence of the Iridaceae family, and this repeat survived all downstream
speciation events until the divergence of the genus Allium, when the human-type repeat was replaced
with the unusual (CTCGGTTATGGG)n repeat [24,134,135]. The molecular basis underlying these
evolutionary switches in telomere DNA sequences should be sought primarily in the corresponding
TRs. We can consider the following possible scenarios. (i) TR remained essentially the same across
Asparagales phylogeny and the observed switches in telomere synthesis occurred either as a result of
mutations in the template region of TR or in its vicinity, which could have changed the boundaries of
the region used as a template, (ii) a different RNA molecule took over the TR function. Experiments are
in progress in our laboratory to provide a clear answer to this question.
3. Telomere Chromatin Composition
While the end-replication problem of telomeres is most commonly solved by telomerase, the other
essential function of telomeres—their end-protection role (i.e., to distinguish natural chromosome
ends from DNA breaks, and to eliminate unwanted repair events at telomeres)—is performed
by other proteins associated with telomeres. In humans, these include proteins directly binding
telomere DNA either in its double strand part (TRF1, TRF2) or at the single strand overhang (POT1).
The other proteins bind telomeres via protein-protein interactions with these proteins (RAP1, TIN2,
TPP1), which together form a complex termed shelterin [136,137]. Shelterin components and their
interaction partners can inhibit the DNA damage response [138–141]. In addition to the end-protective
function, shelterin components also play other roles as, e.g., the recruitment of telomerase to
telomeres, facilitating replication fork movement through telomeres, or formation of telomere loops
(t-loops) [142–149]. In particular, t-loops exist as a “closed-state” telomere conformation both in
mammalians and plants [146,150]. While t-loop is considered as a structure inaccessible to telomerase,
it may provide a template for telomerase-independent ALT (see below).
Cells 2019, 8, 58 9 of 31
The composition of shelterin-like complexes shows differences in individual components among
vertebrates, while the overall functions remain conserved. Human proteins associated with double
and single strand telomeric DNA, together with their plant orthologues, are listed in Tables 3
and 4, respectively.
In plants, knowledge of a shelterin-like complex is incomplete. The only proteins with
confirmed in vivo telomere localization and function are members of the single-myb-histone
family, telomere repeat binding (TRB) proteins, which have been characterised in Arabidopsis
thaliana [66,82,151] and their orthologues were identified in other plants ([152]; Schorova et al.,
submitted). TRB proteins bind specifically telomeric double strand DNA through their myb-like
domain of a telobox type [153,154], as well as the human core components of shelterin—TRF1 and
TRF2 proteins. While the myb-like domain in TRF1 and TRF2 is localized at the C-terminus, that of TRB
proteins occupies the N-terminus. Additionally, TRB proteins contain the centrally located histone-like
domain (H1/5) involved in DNA sequence-unspecific DNA-protein interactions, multimerization,
and interaction with POT1b (one of the plant POT1 paralogues) [65,151]. This plant-specific
protein-domain organization has not been described in animals. TRB proteins bind telomeric DNA
in vitro and in vivo, localize to the telomeres in vivo, interact directly with the telomerase TERT
subunit, and the deregulation of telomeres was observed in mutant plants [66,68].
TRB proteins are not only components of the terminal complex associated with
telomeres/telomerase, but they are also associated in vivo with promoters of translation machinery
genes, which mostly contain a short telomeric sequence [67]. It seems that TRB proteins serve as
epigenetic regulators that potentially affect the transcription status of thousands of genes by playing a
role of recruiting subunits of multiple epigenetically active multi-protein complexes [68–71,155,156].
These findings are consistent with the observations from yeast or mammals where telomeric proteins
(e.g., TRF1, TRF2, and RAP1) are able to localize outside telomeric regions and regulate the transcription
of genes involved in metabolism, immunity, and differentiation [157–164].
Surprisingly, no functions in telomere maintenance were found in Arabidopsis orthologues
of mammalian TRF proteins (TRFL proteins) where a myb-domain of the telobox type is located
C-terminally as in human TRF1 and TRF2 [165]. However, a recent study revealed protein-protein
interactions between TRFL2 and TRP1, members of the TRFL family, and TERT from A. thaliana [66,69].
Plant TRFL2 and TRP1 proteins interact with armadillo/β-catenin-like repeat-containing protein
(ARM). ARM directly interacts with plant TERT [70] and might be involved in translation initiation or
in regulation of recombination-related genes [69]. Moreover, ARM interacts with the chromatin
remodeling protein CHR19 (Table 1). ARM, TRB1, POT1a, and CHR19 (but none of the TRFL
proteins) were found among proteins that co-purified with Arabidopsis TERT using tandem affinity
purification [84]. Association of TERT with proteins that are not essential for telomere maintenance
may reflect possible non-telomeric functions of telomerase.
A dual function for telomerase, both telomeric and non-telomeric, is not unique to plants, as
mammalian telomerase is involved not only in elongation of telomeres but also non-telomeric activities
have been described, including involvement in regulating cellular processes such as apoptosis,
proliferation, and cell cycle progression ([166]; reviewed in Reference [167]). Human telomerase
and human ARM proteins play a role in the Wnt/APC/β-catenin signaling pathway [168].
A putative human homologue of ARM, ARMC6, interacts with the shelterin protein TRF2 and
immuno-precipitates telomerase activity [69].
Cells 2019, 8, 58 10 of 31
An additional telomere maintenance component is—somewhat paradoxically—Ku70/80
heterodimer, a DNA repair factor with a high affinity for DNA ends, that plays essential roles in
the maintenance of genome integrity in both human and plants cells. In human cells, Ku70/80
heterodimer interacts with the RNA component of telomerase hTR [120] and with catalytic subunit
hTERT [94]. In plants, Ku proteins, as well POT1b protein, are associated with TER2. This is a
candidate plant TR that is not required for telomere maintenance in A. thaliana [56]. Ku70/80 is,
however, important for protection of blunt-ended telomeres and for suppression of ALT (see below).
An integrative updated schematic view based on these and previous studies is depicted in Figure 2.
It is obvious that the number of plant telomere-associated and telomerase-associated orthologues
(where they exist) is larger in comparison to their mammalian counterparts. The phenomenon
of the multiplication of genes of the same family is not surprising, since in many plant families,
polyploidy (i.e., whole genome duplication) resulting in retention of multiple gene paralogues may
lead to their sub-functionalization, neo-functionalization, or partial or full redundancy [169,170].
In association with the previously mentioned evolutionary divergence of plant telomere DNA repeats
toward human-like repeats or unusual telomeric repeats, it will be of interest to learn whether
pre-existing components of plant shelterin-like complexes have adapted to the change in DNA sequence
(this will be particularly interesting in proteins directly recognizing DNA sequences, such as the TRB
or POT1 proteins), or whether some other proteins have replaced their function.
Besides the shelterin complex in mammals and its emerging equivalents in plants, there is
yet another complex termed CST (CTC1-STN1-TEN1), which is involved in telomere maintenance.
This tripartite complex binds the 3 -overhang of the G-rich strand of telomeric DNA and its function in
telomere maintenance is conserved in both mammals and plants, and a similar complex exists also in
yeast (with Cdc13 instead of CTC1 subunit) [171]. Recently, the roles of individual components of the
human CST complex in telomere maintenance were elucidated: while CTC1-STN1 limits telomerase
action to prevent G-overhang over-extension, TEN1 is essential for CST function in C-strand fill-in
synthesis due to its stabilizing effect on binding the whole CST complex to telomeres and DNA
polymerase α engagement in telomere synthesis [172,173]. CST functions, at least in humans, are not
limited only to telomeres. CST is also required to avoid replication problem at G-rich sites throughout
the genome, likely resolving replication fork stalling [174].
In addition to the telomere-specific proteins, the major part of telomeres is assembled into the
nucleosomal chromatin structure which shows a shorter nucleosome periodicity (spacing) than that
in the other parts of the chromosomes of the same organism [175–179]. Since shorter telomeres in
cultured human cells show a lower nucleosome density than that in cells with longer telomeres,
a close relationship was hypothesized between histone density, heterochromatin protein associations,
telomere length, and TPE [180]. Interestingly, this feature of telomeric chromatin is conserved at least
in vertebrates and plants, and may reflect the specific columnar structure of telomeric chromatin
with stacked nucleosomes and weak determination of nucleosome positions by telomeric DNA
sequence [181].
Cells 2019, 8, 58 11 of 31
Table 3. Comparative overview of proteins associated with telomeric double strand DNA (dsDNA).
Telomeric dsDNA Associated Proteins
Human Telomeric
dsDNA Associated
Proteins
Protein Function and Direct
Interactions
References
Arabidopsis Telomeric
dsDNA Associated
Proteins
Protein Function and Direct
Interactions
References
TRF1
Shelterin. Int.: telomeric dsDNA, TIN2,
TANK1 and PINX1.
Non-telomeric: binding to ITS and
chromatin and satellite DNA and
modulation of their chromatin structure.
Control of a common fragile site
containing ITS.
[59–62]
[162,182]
TRB1, 2, 3
Shelterin-like. Int.: telomeric dsDNA,
TERT, POT1b, RuvBL1 and RuvBL2a.
Non-telomeric functions - a recruitment
subunit of protein complexes involved in
epigenetic regulations. Binding to ITSs.
[64–66]; Schorova et
al., submitted
[67–71]
TRF2
Shelterin. Int.: telomeric dsDNA; TIN2,
RAP1, NBS1, RAD50, Apollo, Ku70,
PARP1, XPF-ERCC1, BLM, FEN1, POLB,
ORC, RTEL1 and ATM.
[61,72–80,
183–187]
TRP1
Possible non-telomeric functions of
telomerase. Int.: telomere dsDNA
in vitro, TERT, ARM, Ku70, TRFL1 and
TRFL9.
[66,69,81–83]
Non-telomeric function: transcriptional
regulator. Binding to ITSs and satellite
III.
[155,163] TRFL2
Possible non-telomeric functions of
telomerase. Int.: telomere dsDNA
in vitro, TERT and ARM.
[69,83]
TRFL9
Possible non-telomeric functions of
telomerase. Int.: telomere dsDNA
in vitro, TRP1 and POT1a.
[69,83]
TBP1, TRFL1, TRFL4 Int.: telomere dsDNA in vitro. [83,188]
HOT1 Int.: telomeric dsDNA, active telomerase. [93] n.a.
Ku70/80
The way of association with telomeric
dsDNA is not fully elucidated. Int.:
TRF2, RAP1, TR and TERT.
[95] Ku70/80
Role in telomere length regulation, may
protect blunt-ended telomeres Int.: TRP1,
TER2 and TER2s.
[82,96–99,101]
The proteins depicted in grey are involved in telomere maintenance, however, their association with telomeric dsDNA has not been fully proven yet. Direct interaction
partners (Int.) interacting with telomeric dsDNA-associated proteins and concerning their telomeric functions are enumerated. No sequence homologue has been identified yet
(n.a.). Double-strand DNA (dsDNA); 5 exonuclease Apollo (Apollo); Armadillo/β-catenin-like repeat-containing protein (ARM); Ataxia telangiectasia mutated kinase (ATM);
ATP-dependent DNA helicase 2 subunit 1 and 2 (Ku70/80); Bloom syndrome protein (BLM); DNA polymerase beta (POLB); DNA repair protein RAD50 (RAD50); Excision repair
cross-complementation 1 (ERCC1); Flap endonuclease 1 (FEN1); Homeobox telomere-binding protein 1 (HOT1); Interstitial telomeric sequences (ITSs); Nijmegen breakage syndrome
protein 1 (NBS1); Origin recognition complex (ORC); Poly(ADP-Ribose); polymerase 1 (PARP1); Protection of telomeres 1b (POT1b); Regulator of telomere elongation helicase 1
(RTEL1); Repressor-activator protein 1 (RAP1); Telomerase RNA (TR); RuvB-like 1, 2a (RuvBL1, 2a); Tankyrase 1 (TANK1); Telomerase reverse transcriptase (TERT); Telomerase RNA
subunit 2, 2s (TER2, TER2s); Telomere binding protein 1 (TBP1); Telomere repeat-binding factor 1, 2, 3 (TRB1, 2, 3); Telomere repeat-binding protein 1 (TRP1); Telomeric repeat
binding Factor 1-like 1, 2, 4, 9 (TRFL1, 2, 4, 9); Telomeric repeat-binding factor 1 (TRF1); Telomeric repeat-binding factor 2 (TRF2); TRF1-interacting nuclear protein 2 (TIN2);
TRF1-interacting protein 1 (PINX1); Xeroderma pigmentosum group F (XPF1).
Cells 2019, 8, 58 12 of 31
Table 4. Comparative overview of proteins associated with telomeric single strand (ssDNA).
Telomeric ssDNA Associated Proteins
Human Telomeric
ssDNA Associated
Proteins
Protein Function and Direct
Interactions
References
Arabidopsis Telomeric
ssDNA Associated
Proteins
Protein Function and Direct Interactions References
POT1
Shelterin. Int.: telomeric ssDNA, TPP1
and CTC1.
[50–54] POT1a
Shelterin-like. Int.: TERT, telomeric ssDNA, TER1,
TRFL9, CBF5, RuvBL1, CTC1 and STN1.
[47,55–58,69,105,189]
POT1b Shelterin-like. Int.: TRB1, TER2, TER2s. [56,82,100]
POT1c POT1 paralogue of unknown function. [47]
TERT Catalytic subunit of telomerase. [190] TERT Catalytic subunit of telomerase.
STN1
CST complex subunit, prevents
G-overhang overextension. Int.: CTC1,
TEN1, TPP1 and POLA.
[54,172,191,192] STN1
CST complex subunit, controls access of
telomerase and DDR, together with POLA may be
involved in C-strand synthesis. Int.: CTC1, TEN1
and POT1a. Non-telomeric function. Facilitates
re-replication at non-telomeric loci.
[189,193–195]
TEN1
CST complex subunit, involves C-strand
fill-in synthesis. Int.: STN1.
[172,192] TEN1
CST complex subunit, controls access of
telomerase and DDR, coordinating synthesis of
the C-strand. Int.: STN1.
[194]
CTC1
CST complex subunit, prevents
G-overhang overextension. Int.:
telomeric ssDNA, STN1, TPP1 and
POT1.
[54,192] CTC1
CST complex subunit, controls access of the
telomerase and DDR, coordinating synthesis of
the C-strand. Int.: STN1, POT1a and POLA.
[171,189,196]
Purα
p.h. Unwinds dsDNA telomeric
oligonucleotides.
[105] PURα1 Associates with TERT. [84]
n.a. Why1
Regulates telomere-length homeostasis. Int.:
telomeric ssDNA.
[197]
n.a. STEP1
Truncated derivative of chloroplast RNA-binding
protein, role in plant telomere biogenesis. Int.:
telomeric ssDNA.
[198]
The proteins depicted in grey are involved in telomere maintenance, however, their association with telomeric ssDNA has not been fully proven yet. The proteins in green are
structural homologues of their human/plant counterparts, however, any involvement in telomere maintenance or association with telomeric sequences has not been described so far.
Direct interaction partners (Int.) interacting with telomeric ssDNA associated proteins are enumerated. Cases with not yet identified sequence homologues are denoted with n.a.
Single strand DNA (ssDNA); Double-strand DNA (dsDNA); Cajal bodies factor 5 (CBF5); Conserved telomere maintenance component 1 (CTC1); CST complex (CTC1, STN1 and
TEN1 subunits); DNA damage response (DDR); DNA polymerase alpha (POLA); Protection of telomeres 1 (POT1); Protection of telomeres 1a, b, c (POT1a, b, c); Pur-alpha 1 (Purα1);
RuvB-like 1 (RuvBL1); Single-stranded telomere-binding protein 1 (STEP1); Suppressor of cdc thirteen homolog (STN1); Telomerase reverse transcriptase (TERT); Telomerase RNA
subunit 1, 2, 2s (TER1, 2, 2s); Telomeric pathways in association with STN1 (TEN1); Telomeric repeat binding factor 1 -like 9 (TRFL9); TIN2- and POT1-organizing protein (TPP1);
Whirly 1 (Why1); putative homolog according to NCBI blastp (p.h.).
Cells 2019, 8, 58 13 of 31
Cells 2019, 8, x FOR PEER REVIEW 14 of 31
Figure 2. An integrative schematic view of the human and plant terminal telomeric complex. (A)
Human active telomerase is associated with Hsp90 and p23 chaperones as well as with TR associated
conserved scaffold proteins of box H/ACA small nucleolar RNAs (dyskerin, NHP2, NOP10, GAR1).
Mammalian shelterin proteins (TRF1/2, RAP1, TIN2, TPP1, and POT1) modulate access to the
telomerase complex and the ATR/ATM-dependent DNA damage response pathway. The CST
complex (CTC1-STN1-TEN1) affects telomerase and DNA polymerase α recruitment to the
chromosomal termini, and, thus, coordinates G-overhang extension by telomerase with fill-in
synthesis of the complementary C-strand (blue dashed line). G-quadruplexes, D-loops, and t-loops
during telomere replication are resolved by RTEL helicase. HOT1 directly binds double strand
telomere repeats and associates with the active telomerase. Telomere nucleosomes show a shorter
periodicity than that in the other parts of chromosomes. For human telomere histone modifications,
see Figure 3. (B) Arabidopsis telomerase is associated with TRB proteins as well as with POT1a that
interacts with the dyskerin orthologue CBF5. Plants possess all orthologue proteins of conserved
scaffold box H/ACA of small nucleolar RNAs (CBF5, GAR1, NOP10, NHP2). Moreover, TRB proteins
interact with the telomeric sequence due to the same myb-like binding domain as that in mammalian
TRF1/2. TRB proteins interact with TERT and POT1b, and, when localized at chromosomal ends, they
are eligible to function as components of the plant shelterin complex. An evolutionarily conserved
CST complex is suggested to coordinate the unique requirements for efficient replication of telomeric
DNA in plants as well as in other organisms. In addition, plant RTEL contributes to telomere
homeostasis. For the sake of clarity, only the situation in telomere with 3′ overhang is depicted. For
plant telomere histone modifications, see Figure 3.
4. Telomere Epigenetics
Figure 2. An integrative schematic view of the human and plant terminal telomeric complex.
(A) Human active telomerase is associated with Hsp90 and p23 chaperones as well as with TR
associated conserved scaffold proteins of box H/ACA small nucleolar RNAs (dyskerin, NHP2, NOP10,
GAR1). Mammalian shelterin proteins (TRF1/2, RAP1, TIN2, TPP1, and POT1) modulate access to the
telomerase complex and the ATR/ATM-dependent DNA damage response pathway. The CST complex
(CTC1-STN1-TEN1) affects telomerase and DNA polymerase α recruitment to the chromosomal termini,
and, thus, coordinates G-overhang extension by telomerase with fill-in synthesis of the complementary
C-strand (blue dashed line). G-quadruplexes, D-loops, and t-loops during telomere replication are
resolved by RTEL helicase. HOT1 directly binds double strand telomere repeats and associates with
the active telomerase. Telomere nucleosomes show a shorter periodicity than that in the other parts of
chromosomes. For human telomere histone modifications, see Figure 3. (B) Arabidopsis telomerase is
associated with TRB proteins as well as with POT1a that interacts with the dyskerin orthologue CBF5.
Plants possess all orthologue proteins of conserved scaffold box H/ACA of small nucleolar RNAs
(CBF5, GAR1, NOP10, NHP2). Moreover, TRB proteins interact with the telomeric sequence due to
the same myb-like binding domain as that in mammalian TRF1/2. TRB proteins interact with TERT
and POT1b, and, when localized at chromosomal ends, they are eligible to function as components of
the plant shelterin complex. An evolutionarily conserved CST complex is suggested to coordinate the
unique requirements for efficient replication of telomeric DNA in plants as well as in other organisms.
In addition, plant RTEL contributes to telomere homeostasis. For the sake of clarity, only the situation
in telomere with 3 overhang is depicted. For plant telomere histone modifications, see Figure 3.
Cells 2019, 8, 58 14 of 31
4. Telomere Epigenetics
As chromatin structures, telomeres are natural targets for epigenetic modifications. At the
DNA level, methylation at carbon 5 of cytosine represents the dominant mark in eukaryotic cells.
Methylated cytosines (mCs) are generally enriched in heterochromatic regions of the genome and
silenced promoters. Important differences in the sequence contexts, in which mCs are located,
exist between animals and plants. In mammalian cells, they are predominantly located in CG doublet
motifs, with the symmetry of the sequence crucial for the maintenance of the methylation pattern
during DNA replication (reviewed in Reference [199]). A fraction of mCs in non-CG contexts was
found in human embryonic cells. This fraction disappears after differentiation and is restored in
induced pluripotent stem cells, which shows involvement of distinct methylation patterns in the
regulation of gene expression [200]. Also in plants, cytosines in the CG motif are most frequently
methylated, but mCs are also commonly placed in non-CG sequences, symmetrical CHG triplets
(H=C or A or T), or non-symmetrical CHH motifs (reviewed in Reference [201]). In telomeres,
cytosines in non-symmetrical sequence contexts are present in the telomeric C-rich strand, i.e.,
in CCCTAA repeats in animals and CCCTAAA repeats in plants. Using shotgun bisulfite genomic
sequencing, mCs were detected in A. thaliana telomeric repeats with the inner cytosine most frequently
methylated [202]. This pattern was confirmed by an independent approach, with high reliability
at least in the proximal part of the telomere [203,204], and methylated telomeric cytosines were
detected in cultured Nicotiana tabacum (tobacco) cells [205] and other plants [206]. Disruption of
telomere homeostasis as a consequence of decreased genomic DNA methylation was observed in A.
thaliana [203,207] but not in tobacco cells [205], which shows differences in the involvement of DNA
methylation in regulation of telomere homeostasis between these model plants (for a more detailed
review see Reference [208]).
Telomeres formed by mini-satellite repeats were traditionally considered as heterochromatic
regions, and, thus, associated with heterochromatin-specific histone marks. Certain differences
in histone modifications in heterochromatin have been described between animals and plants.
In animals, constitutive heterochromatin is defined by the presence of H3K9me3 (trimethylation
of lysine 9 of histone H3) (reviewed in Reference [209]) while in plants, this mark decorates silenced
euchromatic genes, and constitutive heterochromatin is associated with H3K9me2 modification [210].
Facultative heterochromatin is enriched in H3K27me3 in cells of representatives of both kingdoms.
In agreement with the hypothesis of the heterochromatic character of telomeres, the importance
of heterochromatin-specific epigenetic marks for telomere maintenance and genome stability
was demonstrated in numerous studies using human and mouse cells as models (reviewed in
Reference [211]). On the other hand, data showing a low level of heterochromatin-specific modifications
and an abundance of active marks on human telomeric histones have been presented [212–214],
which shows certain dynamics of the human telomeric chromatin structure. Based on these and other
reports, distinct differences exist in telomeric chromatin composition between the most important
mammalian models, human and mouse cells, because H3K9me3 density and HP1 enrichment were
significantly higher in mouse compared to humans [215,216]. Nevertheless, according to a study
utilizing quantitative locus purification [217] the heterochromatic histone modification H4K20me3
is underrepresented at mouse telomeres even though it was previously detected by others at
mouse [218,219] and also human [220] telomeres in analyses based on chromatin immuno-precipitation.
Further research is necessary to draw final conclusions on the epigenetic nature of mammalian
telomeres, especially considering other factors mentioned below.
Plant telomeric chromatin was shown to be associated with both heterochromatin-specific
H3K9me2 and euchromatic H3K4me3 marks, with the latter less abundant [204,206,221]. Therefore,
the plant telomeric chromatin exhibits a dual epigenetic character. Identification of the H3K27me3
modification, which is typical for facultative heterochromatin, in telomeric histones of A.
thaliana [221,222] and N. tabacum [206] was rather surprising. However, it correlates with its presence at
human telomeres [215], and with the recent observation that polycomb repressor complex 2-dependent
Cells 2019, 8, 58 15 of 31
loading of H3K27me3 at human telomeres is essential for the proper establishment of H3K9me3 and
H4K20me3 modifications [220]. Nevertheless, H3K27me3 was not detected at mouse telomeres [217].
Thus, although significantly fewer results are available on the epigenetics of telomeric chromatin
in plants compared to mammals, interesting similarities as well as differences have already been
described and hopefully others will be elucidated based on future studies using different model
organisms, including plants with non-canonical telomere sequences [24,25,27,134].
When discussing telomeric chromatin, it is necessary to mention that analysis of epigenetic
modifications may be complicated by the presence of non-terminally located telomeric repeats forming
interstitial telomeric sequences (ITSs). ITSs are relatively abundant in subtelomeric, pericentromeric,
and centromeric regions of most eukaryotic organisms and represent fragile parts of chromosomes,
which are prone to rearrangements and recombinations. The detailed compositions of telomeres
and ITSs are different. In contrast to telomeres consisting of long tracts of perfect telomeric repeats,
ITSs are often degenerated and/or disrupted by non-telomeric sequences. However, ITSs may still
contribute to the telomere-specific signal in epigenetic studies, mainly those based on hybridization of
membrane-bound DNA. Frequently-used genome-wide sequencing analyses (ChIP-seq and bisulfite
sequencing) do not completely solve this problem because telomeres, like other tandem repeats,
are difficult to analyze, and even direct analysis of respective read counts (i.e., those comprising
perfect telomeric repeats versus those formed by degenerated repeats and non-telomeric sequences)
may be ambiguous due to the non-linearity of PCR amplification of repetitive sequences [223].
Both mammalian and plant telomeres are transcribed to long non-coding RNA called TERRA [204,224]
and this transcriptional potency could reflect the relatively lower level of compactness of telomeric
chromatin compared with heterochromatin. The apparent discrepancy between the association
of heterochromatic marks with telomeric histones and the transcriptional activity of telomeres is
weakened by the facts that a mechanistic relationship between TERRA transcription and loading of
heterochromatic modifications to human telomeres has been described [220], and that in Arabidopsis
a certain—maybe dominant—fraction of TERRA is transcribed from ITSs [204], which are purely
heterochromatic [225].
At this stage of knowledge, it is difficult or even impossible to formulate any general conclusion
on the epigenetic nature of telomeric chromatin (Figure 3). Without any doubt, the specific structure
of telomeres is crucial for the maintenance of genome integrity. Telomeres are rigid enough to
prevent repair and recombination at chromosome ends and to restrict telomere accessibility for
telomerase, but open enough to be transcribed and, at least in a specific time window of the cell
cycle, accessible to telomerase. Moreover, in disagreements about telomeric “heterochromatin”
or “euchromatin”, contribution of non-histone players, mainly shelterin proteins, to the telomeric
chromatin compaction should be reflected (reviewed in Reference [226]). Why not admit, that telomeric
chromatin is so specific that it does not fit into the existing criteria and that these should be widened?
This suggestion is strengthened by the finding that other non-genic parts of the human genome,
originally thought to be uniformly heterochromatic, are associated with different combinations of
histone marks [213]. It is well possible that the epigenetic state of telomeres is more dynamic than
previously thought and shows tissue-specific, cell-cycle specific, and developmental stage-specific
changes. This would not only explain the diverse results of the above studies, but would be consistent
with our current understanding of the epigenetics of other chromosome regions.
Cells 2019, 8, 58 16 of 31Cells 2019, 8, x FOR PEER REVIEW 17 of 31
Figure 3. Modifications of mammalian and plant telomere (telo.) and pericentromere (peric.) histones.
The relative enrichments of selected epigenetic modifications of telomeric and pericentromeric
histones in human, mouse and Arabidopsis are schematically depicted according to data presented in
References [204,212,213,215,217–222,225].
5. Telomere 3′-Overhangs, Blunt Ends, and Loops
Telomeres in vertebrates, in particular humans, possess 3′-overhangs at both chromosome ends.
These overhangs are of different sizes on lagging versus leading strands [227]. In human telomeres a
G-overhang is prevalent whose length varies from several tens to 280 nt [228–230]. Likewise, a 5′ Crich
overhang is present at the telomeres of human chromosomes, being far more prevalent in tumor
cells using ALT (see below) [231]. This is not the case in Arabidopsis thaliana, Silene latifolia, and other
angiosperm plants, which lack telomere overhangs or possess only short 1–3 nt overhangs at about
half of their telomeres [232,233]. The telomere whose 3′- end is being synthesized in a given cell cycle
by leading strand synthesis remains blunt-ended likely due to protection against end-processing by
a specific exonuclease. This protection is dependent on the Ku70/80 heterodimer [233]. The role of the
Ku complex in plant telomere protection was also suggested by our earlier studies, which indicated
Ku as an interaction partner of AtTRP1, one of the TRF-like proteins in A. thaliana ([82]; see Reference
[155] for a review). An analogous interaction between the shelterin components TRF2 and Ku70 was
observed earlier in human cells [77]. Due to the asymmetry (non-equivalence) of plant telomeres, a
different set of proteins may protect the telomere whose 3′-end serves as a template in “incomplete”
lagging strand synthesis and can be elongated by telomerase. Protection of blunt-ended telomeres in
Arabidopsis by the Ku70/80 complex seems paradoxical considering the presumed end-protective
function of telomeres on one hand, and a key role of the Ku complex in non-homologous end-joining
repair of double strand DNA breaks on the other hand. A possible solution of this enigma was
suggested recently by a study which indicated different binding modes of the Ku complex to dsDNA
breaks and to telomeres. Both functions were dissected using Ku mutants with impaired ability to
translocate along DNA. While Ku sliding is not required for its association with plant telomeres, it is
essential for its involvement in the non-homologous end joining pathway of DNA repair [101]. The
presence of blunt-ended telomeres is, however, not common to all plants. For example, in the moss
Physcomitrella patens, both telomeres of a chromosome possess overhangs and, correspondingly, lack
of the Ku complex components shows no effect on telomere maintenance or end protection [234]. The
Ku70/80 complex was also reported to be a negative regulator of telomerase function in Arabidopsis
[99]. In addition to telomere elongation by telomerase, an extension of telomere G-strand overhangs
was observed in Ku mutants, which suggests a role of Ku70/80 in C-rich telomeric strand
maintenance [235].
Figure 3. Modifications of mammalian and plant telomere (telo.) and pericentromere (peric.)
histones. The relative enrichments of selected epigenetic modifications of telomeric and pericentromeric
histones in human, mouse and Arabidopsis are schematically depicted according to data presented in
References [204,212,213,215,217–222,225].
5. Telomere 3 -Overhangs, Blunt Ends, and Loops
Telomeres in vertebrates, in particular humans, possess 3 -overhangs at both chromosome
ends. These overhangs are of different sizes on lagging versus leading strands [227]. In human
telomeres a G-overhang is prevalent whose length varies from several tens to 280 nt [228–230].
Likewise, a 5 C-rich overhang is present at the telomeres of human chromosomes, being far more
prevalent in tumor cells using ALT (see below) [231]. This is not the case in Arabidopsis thaliana,
Silene latifolia, and other angiosperm plants, which lack telomere overhangs or possess only short
1–3 nt overhangs at about half of their telomeres [232,233]. The telomere whose 3 - end is being
synthesized in a given cell cycle by leading strand synthesis remains blunt-ended likely due to
protection against end-processing by a specific exonuclease. This protection is dependent on the
Ku70/80 heterodimer [233]. The role of the Ku complex in plant telomere protection was also
suggested by our earlier studies, which indicated Ku as an interaction partner of AtTRP1, one of
the TRF-like proteins in A. thaliana ([82]; see Reference [155] for a review). An analogous interaction
between the shelterin components TRF2 and Ku70 was observed earlier in human cells [77]. Due to the
asymmetry (non-equivalence) of plant telomeres, a different set of proteins may protect the telomere
whose 3 -end serves as a template in “incomplete” lagging strand synthesis and can be elongated
by telomerase. Protection of blunt-ended telomeres in Arabidopsis by the Ku70/80 complex seems
paradoxical considering the presumed end-protective function of telomeres on one hand, and a key role
of the Ku complex in non-homologous end-joining repair of double strand DNA breaks on the other
hand. A possible solution of this enigma was suggested recently by a study which indicated different
binding modes of the Ku complex to dsDNA breaks and to telomeres. Both functions were dissected
using Ku mutants with impaired ability to translocate along DNA. While Ku sliding is not required
for its association with plant telomeres, it is essential for its involvement in the non-homologous end
joining pathway of DNA repair [101]. The presence of blunt-ended telomeres is, however, not common
to all plants. For example, in the moss Physcomitrella patens, both telomeres of a chromosome possess
overhangs and, correspondingly, lack of the Ku complex components shows no effect on telomere
maintenance or end protection [234]. The Ku70/80 complex was also reported to be a negative
regulator of telomerase function in Arabidopsis [99]. In addition to telomere elongation by telomerase,
Cells 2019, 8, 58 17 of 31
an extension of telomere G-strand overhangs was observed in Ku mutants, which suggests a role of
Ku70/80 in C-rich telomeric strand maintenance [235].
Besides telomerase, eukaryotic cells can also utilize a back-up mechanism of
telomere maintenance—ALT—which is based on homologous recombination (HR) [236].
This telomerase-independent mechanism is activated in a number of human tumors, in human cells
immortalized in culture, and also in normal somatic tissues [237]. In plants, the ALT mechanism is
activated in mutants with telomerase dysfunction and possibly also during the earliest stages of normal
plant development [238]. ALT relies on the formation of terminal telomeric loops (t-loops) [146],
which parallels the first steps of HR. The eventual resolution of these t-loops and aberrant HR at
telomeres generates not only telomeres of highly heterogeneous lengths but also extrachromosomal
t-circles, which are the known hallmarks of ALT. In mutant plants that are deficient for components
of the Ku70/80 complex, induction of t-circle formation was observed at telomeres but not at other
regions rich in DNA repeats. Despite ongoing terminal deletions arising from excision of t-circles
in mutant plants, the telomeres remain functional, which indicates an efficient telomere healing by
telomerase [239].
Another interesting protein connecting telomeric loops and circles with DNA recombination and
telomere replication is RTEL1. This was originally described in Caenorhabditis elegans as a functional
homologue of the yeast Srs2 protein, which removes Rad51 from single strand DNA. Therefore,
it prevents the homology search step of HR and helps to protect the cell from inappropriate HR (for
review, see Reference [240]). Furthermore, in C. elegans, the RTEL1 helicase suppresses inappropriate
recombination events by promoting disassembly of D-loop recombination intermediates, and the loss
of its function results in increased genome instability [241]. In addition to its regulatory role in HR,
RTEL1 acts in telomere maintenance in mammalian telomerase-positive cells [242]. This function was
explained by the function of RTEL1 in opening t-loops, which blocked inappropriate excision of large
telomere regions—the process known as telomere rapid deletion. To promote this t-loop unwinding,
RTEL1 is recruited to telomeres in the S-phase by the telomeric protein TRF2 [186].
In addition to its role in t-loop stability, mouse RTEL1 can dissolve G4-DNA structures,
which otherwise block replication fork progression and the extension of telomeres by telomerase [243].
Importantly, the role of RTEL1 in telomere dynamics was clearly confirmed by the finding that its
mutation is causative for Hoyeraal-Hreidarsson syndrome, which is a severe form of dyskeratosis
congenita, predisposing to bone-marrow failure and cancer. This disease is characterised by short
telomeres and genome instability [244–246]. A recent report revealed that reversed replication forks
occurring in telomeres of RTEL1-deficient cells is due to compromised telomere replication aberrantly
recruiting telomerase, which prevents the restart of reversed replication forks at telomeres and leads
to critically short telomeres [247]. In this context, telomerase paradoxically contributes to telomere
shortening by stabilizing stalled replication forks at chromosome ends.
In addition, the A. thaliana RTEL1 homolog suppresses HR and is involved in processing
DNA replication intermediates and interstrand and intrastrand DNA cross-links. Deficiency of
the Arabidopsis RTEL1 triggers a SOG1-dependent replication checkpoint in response to DNA
crosslinks [248]. Similarly to the situation in mammals, the Arabidopsis RTEL1 contributes to telomere
homeostasis. The concurrent loss of RTEL1 and TERT accelerates telomere shortening, which results in
a developmental arrest after four generations [249] compared to 10 generations in single-mutant
tert plants [250]. This observation indicates a role of RTEL1 in ALT, which otherwise partially
compensates for the loss of TERT [238]. In agreement with these results, it was recently demonstrated
that RAD51-dependent homologous recombination participates in ALT in A. thaliana [251]. This is
not surprising when considering the essential role of RAD51 in HR, and HR as a major molecular
mechanism of ALT. However, the authors further showed that this role of RAD51 is dependent on
RTEL1 helicase, which possibly functions in dissolution of the D-loop after telomere replication.
In P. patens, RTEL1 has been found among genes, which are up-regulated after γ-irradiation.
RTEL1 knockout resulted in a severe growth deficiency, which was independent of the presence
Cells 2019, 8, 58 18 of 31
of bleomycin [252], and the authors hypothesized that this growth phenotype might be the result
of telomere deficiency. Thus, the functions of RTEL1 seem widely conserved. In conclusion,
the requirement for RTEL1 in multiple pathways to preserve plant genome stability can be explained by
its putative role in the destabilization of DNA loop structures such as D-loops and t-loops, which aligns
with previous studies in mammalian systems.
6. Cellular Aging and the Immortal DNA Strand Hypothesis
Cellular aging is characterized by progressive loss of physiological integrity that leads to impaired
function and genomic instability and ultimately to a functional decline at the tissue and organ level.
Telomere attrition during cell aging is classified as one of the several major hallmarks of aging—together
with, e.g., genomic instability, epigenetic alterations, loss of proteostasis, mitochondrial dysfunction,
cellular senescence, or altered intercellular communication [7]. In Metazoa, there is no universal pattern
of telomere erosion [253], and, in some animals, the progressive telomere shortening with age has
not been observed [254]. Nevertheless, telomere length is typically inversely correlated with lifespan,
while telomerase expression co-evolved with body size [255]. A connection between cellular aging
and replicative telomere shortening is widely accepted and experimentally validated in both humans
and plants. Importantly, under normal conditions (in wild type plants) this type of cellular aging is
prevented by telomerase activity in dividing cells [20,21,38]. The associations between telomere length
and age-related disease and mortality in humans have been proven in several studies (reviewed in
References [8,256,257]). However, telomere length of humans is not a determinant of aging but rather
a marker able to explain life expectancy and disease risk.
In animals, the distribution of cellular age varies among tissues and cell compartments,
including progenitor cell compartments, depending on the influx of stem cells and the dynamics
of self-renewal and differentiation of progenitor cells. In particular, the mode of cell division of
progenitor cells may be: (i) symmetric self-renewal, in which progenitor cell division results in
two daughter progenitor cells (one generation older) remaining in the compartment, (ii) symmetric
differentiation, resulting in two differentiated cells which leave the progenitor cell compartment,
or (iii) asymmetric division resulting in one progenitor and one differentiated cell. Importantly,
cellular age distributions between healthy and cancerous tissues may inform dynamic changes
within the hierarchical tissue structure, i.e., an acquired increased self-renewal capacity in certain
tumors [258]. In this connection, it is of interest to mention the hypothesis of the immortal DNA
strand [259]. This hypothesis proposes that adult stem cells segregate their template and newly
synthesized DNA strands non-randomly, preferentially retaining parental DNA strands in each
division. This way, adult stem cells pass mutations resulting from replication errors onto non-stem
cell daughter cells that differentiate and terminate division. Adult stem cells could thus reduce the
accumulation of mutations and the associated deterioration of gene functions with each cell cycle.
Moreover, this strategy would also slow down replicative telomere shortening. Thus, two major factors
of cellular and organismal aging could be substantially limited if immortal DNA strand segregation
operates in progenitor cells. Several studies have supported this hypothesis up to now. For example,
using sequential pulses of halogenated thymidine analogues, high frequencies of segregation of
older and younger template strands during proliferative expansion of mouse muscle stem cells was
observed [260]. Template strand co-segregation was strongly associated with asymmetric cell divisions
yielding daughters with divergent fates. Daughter cells inheriting the older templates retained a
more immature phenotype, whereas daughters inheriting the newer templates acquired a more
differentiated phenotype. It will be of interest to learn if the validity of this hypothesis is more general,
and specifically to elucidate the molecular mechanism of non-random DNA segregation in asymmetric
cell division. This principle may also be functional in meristem cell division and differentiation.
While replicative telomere shortening is efficiently counteracted by telomerase in wild type plants
(see above), reduction of accumulation of mutations would be extremely beneficial when considering
e.g., trees sustaining their growth for centuries. Low telomere loss per plant generation has been
Cells 2019, 8, 58 19 of 31
found in telomerase-deficient Arabidopsis mutants [250], which indicates a possible involvement of
non-random DNA strand segregation in addition to ALT [238]. Unfortunately, the application of
sequential pulse labeling in planta is technically too demanding, and any direct evidence for the
immortal DNA strand hypothesis is, thus, missing in plants.
7. Concluding Remarks
Currently available data show remarkably conserved principles in telomere biology across
eukaryotes, which is consistent with an association of telomere and telomerase emergence with
the earliest steps of their evolution. At the same time, however, a number of specific features and
exceptions cannot be ignored since they point to limitations of our wider understanding of these
principles. Among a number of open questions to be answered, elucidation of the structure of
telomeric chromatin (telochromatin), including its epigenetic and higher-order dynamics, with high
spatial and temporal resolution is needed in various model systems. Furthermore, the biological
relevance of non-canonical structures formed by telomeric DNA should be addressed mainly under
in vivo conditions. Such studies are timely due to recent fast progress in adequate technical tools,
including e.g., super-resolution and cryo-electron microscopy.
Studies of repair processes at telomeres and of telomerase regulation belong to the hot topics
in this field, since this knowledge can clearly be applied to promote protection of genome stability.
In this respect, plants are indispensable due to the natural telomerase-competent character of their
cells which allows us to examine mechanisms of repression and activation of telomerase in association
with proliferation, differentiation, and dedifferentiation of plant cells. This knowledge is essential for
understanding carcinogenesis and is potentially applicable to tumor therapy and cell rejuvenation.
Author Contributions: P.P.S., M.F. and J.F. contributed to this paper with a literature review, drafting the paper,
and approval of the final version.
Funding: This work was supported by the Czech Science Foundation (projects 16-01137S and 17-09644S), by the
project SYMBIT, reg. number: CZ.02.1.01/0.0/0.0/15_003/0000477 financed by the ERDF, and by the Ministry of
Education, Youth and Sports of the Czech Republic under the projects CEITEC 2020 (LQ1601) and INTER-COST
(LTC17077).
Acknowledgments: We thank to Ladislav Dokládal and Ronald Hancock for reviewing and discussing the MS
prior to submission.
Conflicts of Interest: The authors declare no conflict of interest.
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Supplement N
Schořová, Š., Fajkus, J., Drábková, L.Z., Honys, D., Schrumpfová, P.P.*, 2019. The plant
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volume 98 | number 2 | April 2019
http://www.theplantjournal.com | issn 1365-313X
tpj_v98_i2_issueinfo.indd 1tpj_v98_i2_issueinfo.indd 1 13-Apr-19 7:36:20 PM13-Apr-19 7:36:20 PM
Lee Sweetlove
Department of Plant Sciences,
University of Oxford,
South Parks Road,
Oxford OX1 3RB, UK
Tel: +44 (0) 1865 275137
Email: lee.sweetlove@plants.
ox.ac.uk
Research Interests:
Central metabolism; Metabolic flux;
Metabolic modeling; Metabolic
engineering; Metabolons and
metabolite channelling
Previous Editors-in-Chief
Dianna Bowles (Founding EiC),
1990-2001
Harry Klee, 2002-2009
Christoph Benning, 2009-2017
The Plant Journal Editorial Office,
Wiley Blackwell,
9600 Garsington Road, Oxford,
OX4 2DQ, UK
E-mail: tpj-general@wiley.com
Jim Ruddock Managing Editor
E-mail: tpj-editor@wiley.com
Lauren Dawson Editorial Assistant
E-mail: tpj-submissions@wiley.com,
tpj-manuscripts@wiley.com
Rajalakshmi Sundararamanujam
Production Editor
E-mail: tpj@wiley.com
Editor-in-Chief Editorial Office Production Office
Senior Editors
Editors
Asaph Aharoni
Weizmann Institute of Science, Department of
Plant Sciences, P.O.B 26, Rehovot 76100, Israel
Tel: +972 8 934 3643; Fax: +972 8 934 4181
Email: asaph.aharoni@weizmann.ac.il
Research Interests
Fruit metabolism and ripening control,
The plant surface (epidermis and the cuticle),
Secondary metabolism, Functional genomics and
metabolomics, Metabolic engineering,
Metabolism – development interface
Jose Alonso
Department of Genetics,
North Carolina State University, 2548 Gardner Hall,
Box 7614, Raleigh, North Carolina, 27695-7614, USA
Tel: +1 919 515-5729; Fax: +1 919 515-3355
Email: jmalonso@unity.ncsu.edu
Research Interests
Ethylene signaling and response pathway; Hormone
interactions and signal integration; Arabidopsis functional
genomics and tool development; Auxin biosynthesis; Posttranscriptional
control of hormonal responses
Bonnie Bartel
Department of Biosciences, Rice University, 6100 Main Street,
MS-140, Houston,Texas 77005, USA
Tel: +1 713 348 5602; Fax: +1 713 348 5154
Email:bartel@rice.edu
Research Interests
Cell biology; Peroxisome biology; Autophagy;
Protein trafficking; Arabidopsis genetics
Jörg Bohlmann
Michael Smith Laboratories, University of British Columbia,
321-2185 East Mall, Vancouver V6T 1Z4
British Columbia, Canada
Tel: +1 604 822 0282; Fax: +1 604 822 2114
Email: bohlmann@interchange.ubc.ca
Research Interests
Secondary metabolism;Terpenoid synthases, cytochrome P450;
Plant-herbivore interactions; Chemical ecology;
Conifers, Poplar, Grapevine
Federica Brandizzi
Michigan State University,
Plant Research Laboratory, East Lansing, MI 48824, USA
Tel: +1517 353-7872; Fax: +1 517 353-9168
Email: fb@msu.edu
Research Interests
Cell biology; Protein trafficking; Plant endomembranes;
Endoplasmic reticulum stress; Fluorescent protein technology
Jorge J. Casal
Faculty of Agronomy, University of Buenos Aires,
Av. San Martín 4453, 1417-Buenos Aires, Argentina
Tel: +5411 4524 8070/71; Fax: +5411 4514 8730
Email: casal@ifeva.edu.ar
Research Interests
Light signaling circuitry, its architecture and functional
implications; Functional genomics, transcriptome patterns
during plant development; Photomorphogenesis in crops
John Cushman
University of Nevada, Department of Biochemistry,
Mail Stop 330, 1664 N. Virginia St., Reno89557-0330, USA
Email: jcushman@unr.edu
Research Interests
Abiotic stress responses; Metabolic adaptation to stress;
Crassulacean acid metabolism; Biosystem engineering
Brendan Davies
Centre for Plant Sciences, University of Leeds, Leeds
LS2 9JT, UK
Tel: +44 113 343 3123; Fax: +44 113 343 3144
Email: b.h.davies@leeds.ac.uk
Research Interests
Plant development; Reproduction;Transcription factors;
Nonsense-mediated mRNA decay
Katherine Denby
University ofYork, Department of BiologyYork,YO10 5DD, UK
Tel: +44 1904 328 670
Research Interests
Plant systems biology; Plant pathogen interaction; Functional
genomics; Gene regulation; Gene regulatory networks; Crop
breeding
Alisdair R. Fernie
Molecular Physiology, Max-Planck Institute for Molecular Plant
Physiology,Golm 14476, Germany
Tel: +49 331 567 8211
Email:fernie@mpimp-golm.mpg.de
Research Interests
Carbohydrate metabolism; Metabolomics; Primary metabolism
Jaume Flexas
Universitat de les Illes Balears, Biologia, Carretera de
Valldemossa Km 7.5, Palma de Mallorca, 07122, Spain
Tel: +34 971 172365; Fax: +34 971 173184
Email: jaume.flexas@uib.es
Research Interests
Photosynthesis; Ecophysiology; Abiotic stress;
Mesophyll conductance; Drought
Crisanto Gutierrez
Centro de Biologia Molecular Severo Ochoa, Consejo Superior
de Investigaciones Cientificas, Nicolas Cabrera 1, Cantoblanco,
Madrid, 28049, Spain
Tel: +34 911964638; Fax: +34 911964420
Email: cgutierrez@cbm.uam.es
Research Interests
Cell cycle regulation, DNA replication/endoreplication,
Chromatin/Gene expression, Retinoblastoma/E2F, Cell fate, cell
differentiation – Meristems, cell proliferation in organs
Jose Gutierrez-Marcos
Life Sciences, University of Warwick, Coventry, CV4 7AL, UK
Tel: +44 247 6 57 5077
Email: j.f.gutierrez-marcos@warwick.ac.uk
Research Interests
Epigenetics; Seed development
Scott Jackson
Institute for Plant Breeding, Genetics and Genomics,
University of Georgia , 111 Riverbend Rd, Athens, Georgia,
GA 30602, USA
Tel: +1 706.242.4021; Fax: +1 706-583-8120
Email: sjackson@uga.edu
Research Interests
Genomics; Cytogenetics; Evolutionary genomics; Legumes;
Polyploidy
Kerstin Kaufmann
Humboldt-Universität zu Berlin, Chair for Plant Cell and
Molecular Biology, Philippstr. 13, 10115 Berlin, Germany
Tel: +49(0)30209349740; Fax: +49(0)30209349741
Email: kerstin.kaufmann@hu-berlin.de
Research Interests
Gene regulatory networks; Flower development;
Plant transcription factors; Epigenetics;
Transcriptional regulation; Flowering
Tracy Lawson
University of Essex, School of Biological Siences,
Wivenhoe Park, Colchester, Essex CO4 3SQ, UK
Tel: +44 1206 873327; Fax: +44 1206 872552
Email: tlawson@essex.ac.uk
Research Interests
Plant physiology; Plant phenotyping; Conductance;
Gas exchange; Water use efficiency
Olivier Loudet
INRA - IJPB, Route de Saint Cyr, 78000 Versailles, France
Email: olivier.loudet@versailles.inra.fr
Research Interests
Natural variation; Quantitative genetics; Abiotic stress
tolerance; High-throughput phenotyping; Arabidopsis genetics
Jianxin Ma
Purdue University, 915 S. University St, West Lafayette, Indiana
47907, USA
Tel: 765-496-3662; Fax: 765-496-7255; Email: maj@purdue.edu
Research Interests
ROS; Comparative genomics; Genome evolution;Transposable
elements; Soybean genetics; Crop domestication
Hiroshi Maeda
University of Wisconsin – Madison, Department of Botany,
430 Lincoln Drive , Madison, Wisconsin 53706, USA
Tel: +1 608-262-5833; Fax: +1 608-262-7509;
Email: maeda2@wisc.edu
Research Interests
Amino acid metabolism; Specialized metabolism; Interaction of
metabolism and plant development and physiology
Ron Mittler
University of NorthTexas College of Arts and Sciences,
Department of Biological Sciences, College of Science,
1155 Union Circle #305220, Denton,Texas 76203-5017, USA
Tel: +1 940 293 7170; Fax: +1 940 565 3821
Email: ron.mittler@unt.edu
Research Interests
ROS; Abiotic stress; Gene expression; Systems biology;
Cancer biology
Eiji Nambara
University ofToronto, Cell & Systems Biology, 25 Willcocks
Street,Toronto, Ontario M5S 3B2, Canada
Email: eiji.nambara@utoronto.ca
Research Interests
Plant hormone; Biosynthesis; Catabolism; Abscisic acid; Seed;
Stress response
Holger Puchta
Plant Molecular Biology and Biochemistry Karlsruhe Institute
ofTechnology (KIT), Hertzstr. 16 Geb. 06.35, Karlsruhe D-76187,
Germany
Tel: +49 721 608 8894; Fax: +49 721 608 4874
Email: holger.puchta@kit.edu
Research Interests
DNA repair; Meiotic recombination; DNA processing enzymes;
Genome evolution; Gene technology
Dominique Roby
Laboratory of Plant Microorganism Interactions (LIPM),
UMR CNRS-INRA 2594/441, BP 52627, F-31326,
Castanet-Tolosan cedex, France
Tel: +33 05 61 28 55 11; Fax: +33 05 61 28 50 61
Email: dominique.roby@toulouse.inra.fr
Research Interests
Plant-microbe interactions; Genetic analyses of plant
disease resistance pathways; Defence regulatory proteins;
Programmed cell death and transcriptional regulators;
Pathogen effector activities; Quantitative resistance to
pathogens
Ian Small
University of Western Australia,
ARC Centre of Excellence in Plant Energy Biology, 35 Stirling
Highway, Crawley, Perth, 6009 Western Australia
Tel: +61 (0)8 6488 4499; Fax: +61 (0)8 6488 4401
Email: iansmall@cyllene.uwa.edu.au
Research Interests
Organelle biogenesis; Gene expression in mitochondria
and chloroplasts; Protein and RNA targeting;
Post-transcriptional regulation of gene expression;
Energy metabolism; Functional genomics,
bioinformatics and systems biology
Edgar Spalding
Department of Botany, University of Wisconsin-Madison,
430 Lincoln Drive Madison WI 53706, USA
Tel: +1 608 265 5294
Email: spalding@wisc.edu
Research Interests
Ion transport and electrophysiology Growth;
control by light and auxin
Phenotype platforms; computer vision-based
Dorothea Tholl
Department of Biological Sciences, Virginia Polytechnic
Institute and State University,408 Latham Hall, Agquad
Lane,Blacksburg, VA 24061, USA
Tel: +1 540 231 4567; Fax: +1 540 231 3347
Email: tholl@vt.edu
Research Interests
Isoprenoid biochemistry, Secondary metabolism,
Chemical diversity, Plant volatiles, Plant chemical
interactions, Root biology, Metabolic pathways,
Evolution, Engineering
Michael F. Thomashow
Michigan State University, MSU-DOE Plant Research
Laboratory, Plant Biology Building, 312 Wilson Rd,
East Lansing, MI 48824, USA
Tel: 1 517 355 2299; Fax: 1 517 353 9168
Email: thomash6@msu.edu
Research Interests
Abiotic stress; Biotic stress
Yves Van de Peer
Department of Plant Biotechnology and Genetics,
University of Gent,Technologiepark 927,
9052 Zwijnaarde, Belgium
Tel: +09 331 38 07; Fax: +09 331 38 09
Email: yves.vandepeer@ugent.be
Research Interests
Comparative genomics, systems biology, evolutionary
analysis, genome evolution, gene and genome
duplication
Saskia van Wees
Utrecht University, Biology, Padualaan 8, Utrecht, 3584 CH,
Netherlands
Tel: +31-302536861; Fax: +31-302532837;
Email:s.vanwees@uu.nl
Research Interests
Plant-microbe interactions; Plant-insect interactions;
Defenses to pathogens and insects; Gene expression
profiling; Gene expression regulation; Hormonal regulation
of plant immunity
Daoxin Xie
School of Life Sciences,Tsinghua University,
Beijing 100084, China
Email: daoxinlab@tsinghua.edu.cn
Research Interests
Hormone perception; Jasmonate signal transduction;
Strigolactone signaling; Hormonal crosstalk
Bin Yu
Center for Plant Science Innovation, & School of
Biological Sciences,
University of Nebraska, Lincoln,
1901 Vine St, Lincoln, NE, USA
Tel: 402-472-2125;
Email: byu3@unl.edu
Research Interests
Small RNA metabolism; Epigenetics; RNA silencing;
Non-coding RNAs; Arabidopsis genetics
http://www.theplantjournal.com
ISSN 1365-313X (online)
Federica Brandizzi
Michigan State University, Plant Research
Laboratory,
East Lansing, MI 48824, USA
Tel: (517) 353-7872; Fax: (517) 353-9168
Email: fb@msu.edu
Research Interests
Cell biology; Plant endomembranes;
Endoplasmic reticulum stress; Fluorescent
protein technology; Protein trafficking
Alisdair R. Fernie
Molecular Physiology,
Max-Planck Institute for Molecular Plant
Physiology,
Golm 14476, Germany
Tel: +49 331 567 8211
Email: fernie@mpimp-golm.mpg.de
Research Interests
Carbohydrate metabolism; Metabolomics;
Primary metabolism
Lyza Maron (Research highlights Editor)
BoyceThompson Institute,
USDA/ARS Robert W. Holley Center for
Agriculture & Health,
Ithaca, NY 14853, USA
Research Interests
Abiotic stress responses; Root biology; Natural
variation; Gene expression regulation
Sheila McCormick (Consultant Editor)
Plant and Microbial Biology, 111 Koshland
Hall, UC-Berkeley,
Berkeley, CA 94720, USA
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tpj_v98_i2_issueinfo.indd 3tpj_v98_i2_issueinfo.indd 3 13-Apr-19 7:36:21 PM13-Apr-19 7:36:21 PM
RESEARCH HIGHLIGHT
193 The journey to the end of the chromosome: delivering active telomerase to telomeres in plants. L. Sweetlove and
C. Gutierrez
ORIGINAL ARTICLES
195 The plant Pontin and Reptin homologues, RuvBL1 and RuvBL2a, colocalize with TERT and TRB proteins in vivo, and
participate in telomerase biogenesis. S. Schorˇová, J. Fajkus, L. Záveská Drábková, D. Honys and P. P. Schrumpfová
213 A newly identified cluster of glutathione S-transferase genes provides Verticillium wilt resistance in cotton. Z.-K. Li,
B. Chen, X.-X. Li, J.-P. Wang, Y. Zhang, X.-F. Wang, Y.-Y. Yan, H.-F. Ke, J. Yang, J.-H. Wu, G.-N. Wang, G.-Y. Zhang, L.-Q. Wu,
X.-Y. Wang and Z.-Y. Ma
228 Lotus SHAGGY-like kinase 1 is required to suppress nodulation in Lotus japonicus. C. Garagounis, D. Tsikou, P. K. Plitsi,
I. S. Psarrakou, M. Avramidou, C. Stedel, M. Anagnostou, M. E. Georgopoulou and K. K. Papadopoulou
243 Extreme variation in rates of evolution in the plastid Clp protease complex. A. M. Williams, G. Friso, K. J. van Wijk and
D. B. Sloan
260 Differential alternative polyadenylation contributes to the developmental divergence between two rice subspecies,
japonica and indica. Q. Zhou, H. Fu, D. Yang, C. Ye, S. Zhu, J. Lin, W. Ye, G. Ji, X. Ye, X. Wu and Q. Q. Li
277 ICE1 and ZOU determine the depth of primary seed dormancy in Arabidopsis independently of their role in endosperm
development. D. R. MacGregor, N. Zhang, M. Iwasaki, M. Chen, A. Dave, L. Lopez-Molina and S. Penfield
291 Comparative analysis of the reactive oxygen species-producing enzymatic activity of Arabidopsis NADPH oxidases.
H. Kaya, S. Takeda, M. J. Kobayashi, S. Kimura, A. Iizuka, A. Imai, H. Hishinuma, T. Kawarazaki, K. Mori, Y. Yamamoto,
Y. Murakami, A. Nakauchi, M. Abe and K. Kuchitsu
301 Targeted exome sequencing of unselected heavy-ion beam-irradiated populations reveals less-biased mutation
characteristics in the rice genome. H. Ichida, R. Morita, Y. Shirakawa, Y. Hayashi and T. Abe
315 OsSHOC1 and OsPTD1 are essential for crossover formation during rice meiosis. Y. Ren, D. Chen, W. Li, D. Zhou, T. Luo,
G. Yuan, J. Zeng, Y. Cao, Z. He, T. Zou, Q. Deng, S. Wang, A. Zheng, J. Zhu, Y. Liang, H. Liu, L. Wang, P. Li and S. Li
329 Suppression of tryptophan synthase activates cotton immunity by triggering cell death via promoting SA synthesis.
Y. Miao, L. Xu, X. He, L. Zhang, M. Shaban, X. Zhang and L. Zhu
346 Enhancing microRNA167A expression in seed decreases the α-linolenic acid content and increases seed size in Camelina
sativa. G. Na, X. Mu, P. Grabowski, J. Schmutz and C. Lu
TECHNICAL ADVANCE
359 sRNA-FISH: versatile fluorescent in situ detection of small RNAs in plants. K. Huang, P. Baldrich, B. C. Meyers and
J. L. Caplan
370 Rapid and reproducible phosphopeptide enrichment by tandem metal oxide affinity chromatography: application to
boron deficiency induced phosphoproteomics. Y. Chen and W. Hoehenwarter
CONTENTS OF VOL. 98, NO. 2, APRIL 2019
Front cover: The cover image shows an overlay and individual layers of Arabidopsis
thaliana protoplast used for Bimolecular Fluorescence Complementation assay. In this case,
interaction between AtCBF5 and AtTRB3 proteins was analysed. The biggest protoplast
represents the final overlay of three layers, where the green one shows only chloroplasts, the
red coloured protoplast symbolizes nuclear signal delivered by mRFP-VirD2NLS and finally, the
yellow dot shows a specific interaction between selected proteins AtCBF5 and AtTRB3 localized
in the nucleolus. For details, see article by Schorˇová et al. (pp.195–212).
tpj_v98_i2_issueinfo.indd 4tpj_v98_i2_issueinfo.indd 4 13-Apr-19 7:36:21 PM13-Apr-19 7:36:21 PM
RESEARCH HIGHLIGHT
The journey to the end of the chromosome: delivering active
telomerase to telomeres in plants
Lee Sweetlove and Crisanto Gutierrez
Linked article: This is a Research Highlight about Sarka Schorova et al. To view this article visit https://doi.org/10.1111/tpj.
14306.
Linear chromosomes offer many advantages over circular
DNA for transcription and replication of large genomes,
hence their prevalence in eukaryotes. But the linear
arrangement of the DNA has a massive Achilles heel: the
terminal ends, or telomeres, are unstable and prone to
mutation. Moreover, DNA replication cannot proceed to the
end of a linear DNA molecule because the synthesis of Okazaki
fragments needs RNA primers to bind ahead of the lagging
strand. Eukaryotes deal with both of these problems
by adding repetitive DNA sequences to the telomeres that
act as a disposable buffer, protecting terminal genes from
being truncated during replication and from mutation.
Because the telomere is shortened during each DNA replication,
it is necessary to resynthesise telomere DNA using
an enzyme, telomerase reverse transcriptase.
Our understanding of telomere biology is dominated by
research into human telomeres. This is understandable
due to the links between telomere biology and cellular
mortality, ageing and a range of diseases including cancer.
However, telomere biology in plants shows some specific
differences to humans which may be crucial in our understanding
of telomere biology in general. For example,
telomerase activity in plant cells is well balanced with the
cellular proliferation rate. The reversible regulation of
telomerase activity is thought to be important in this context:
its activity is turned off in differentiated tissues and
turned on during cell periods of active cell replication, for
example, during regeneration of plant tissues. Understanding
the mechanism for this reversible regulation of telomerase
activity could be beneficial in biomedical
applications of telomere biology in humans.
But where to start? From protozoans and humans, it was
known that telomerase was a ribonucleoprotein, carrying
its own RNA molecules that are complementary to the
telomere repeats and are used as a template for telomere
elongation, catalysed by the reverse transcriptase activity
of the enzyme. But, in addition, a number of accessory proteins
are required to deliver functional telomerase to the
telomeres, to regulate its activity and to protect the elongated
telomere from DNA repair enzymes. These
components assemble into two distinct complexes known
as shelterin and CST. Functional homologues of the CST
complex have been identified in plants, but the same is not
true for the shelterin complex. In plants, not all of the
homologues of the six core shelterin components exist,
and only some of them seem to be associated with telomeres
in vivo. The goal, therefore was to identify undiscovered
telomerase accessory proteins in plants and to
establish how active telomerase is formed and regulated.
Jirı Fajkus and his research group at Masaryk University,
have been working on plant telomeres for over 20 years. A
key member of his team in the hunt for plant telomeraseassociated
proteins has been Petra Prochazkova Schrumpfova,
first as a Ph.D. student and then through several
postdoc periods. Working in Arabidopsis, the group had
already established that Telomere Repeat Binding proteins
(TRB) were involved in recruitment of telomerase to the
telomeres. These proteins are specific to plants and contain
an N–terminal Myb-like domain which is responsible
for specific recognition of telomeric DNA. Attention turned
to the plant homologues of two human telomere associated
proteins called Pontin and Reptin after they turned up
in a pull-down of TERT, the catalytic subunit of Arabidopsis
telomerase, in an experiment done in collaboration
with Eva Sykorova’s group at the Institute of Biophysics in
Brno.
The plant Pontin and Reptin homologues are encoded
by RuvBL1 and RuvBL2a, respectively. But despite the fact
that RuvBL proteins were isolated from plant cells as
TERT-associated, Jirı and his team were not able to prove
a direct interaction between TERT and RuvBL as had
already been described in mammals. Serendipity then
intervened. During their characterisation of RuvBL interactions
with TERT, they used several proteins as negative
controls. Surprisingly, one of the supposed negative controls
showed reproducibly positive interaction with RuvBL
proteins. It was in this way that they discovered that TRB
proteins interact with RuvBL. Knowing that TRB proteins
directly interact with TERT they started to closely characterise
the trimeric complex TERT-TRB-RuvBL and that is
© 2019 The Authors
The Plant Journal © 2019 John Wiley & Sons Ltd
193
The Plant Journal (2019) 98, 193–194 doi: 10.1111/tpj.14328
the focus of the highlighted paper which is drawn from the
MSc and PhD research of Sarka Schorova with Petra
Prochazkova Schrumpfova and Jirı Fajkus as joint
corresponding authors. The work also involved Lenka
Zaveska Drabkova, a postdoc from David Honys’s group at
the Institute of Experimental Botany in Prague who did
phylogenetic analysis of the RuvBL family in plants. That
collaboration started late one afternoon during a shortterm
visit of Petra Prochazkova Schrumpfova to David Honys’s
lab that was focused on a completely different scientific
topic. Such is the nature of science and scientists!
In this highlighted paper (Schorova et al., 2019), a combination
of BiFC, yeast-two hybrid and pull-down assays
confirmed that there is no direct interaction between
RuvBLs and TERT, but that the interaction is mediated by
TRBs as an intermediary. It was also shown that RuvBL proteins
form hetero- and homo-oligomers in vivo. Proof of
the importance of RuvBL1/2 for telomerase biogenesis was
provided by analysis of Arabidopsis knockout lines which
had substantially reduced telomerase activity in flower
buds (a rapidly proliferating tissue with high telomerase
requirement). This crucial experiment turned out to be the
hardest part of the research, with identification of knockout
alleles a real struggle. Jirı and Petra say that they had to
genotype hundreds of individual plants from several lines
and were only able to identify a few heterozygous individuals
of each gene with homozygous mutants being lethal.
Further protein interaction experiments identified
another protein in the complex: CBF5, a homologue of
mammalian dyskerin, a known telomerase-associated protein.
Cell biological analyses were able to place all of these
proteins in the nucleolus and some of them in Cajal bodies
and, combined with previous studies, the authors were
able to put together the most complete picture of the plant
telomerase complex to date, as shown in Figure 1.
One of the most interesting facets of this picture is the
similarities and divergence between plants and humans.
On the one hand, identification of Reptin and Pontin in
Arabidopsis and their conservation in humans, shows that
the factors involved in telomerase biogenesis and function
are evolutionary ancient. On the other, the interactions and
mechanism of action of plant Reptin and Pontin is different
than in human cells. The TERT subunit of Arabidopsis
telomerase does not interact directly with Reptin and Pontin
but through TRBs which in human cells are telomereassociated
proteins but not TERT-accessory factors. This
reveals that different mechanisms have evolved although
using basically the same set of factors, a finding that
would justify a similar study in other eukaryotic lineages to
define the evolutionary history of complex formation
between telomeric repeats, TERT, accessory factors and
shelterin proteins.
One possible reason to explain the variety of mechanisms
suggested by this study is the specific organization,
and possibly the 3D structure, of TERT RNA (TER) molecules
which may limit the ability of TERT to interact
directly with them or require other bridging proteins, as it
occurs in Arabidopsis. Differences in the subnuclear localization
of telomeric sequences may be also important. For
Jirı and his team, work will continue to unpick the regulation
of synthesis of both basic subunits of telomerase, TER
and TERT, their intracellular trafficking and assembly into
the holoenzyme complex, together with a number of associated
factors.
REFERENCE
Schorova, S., Fajkus, J., Zaveska Drabkova, L., Honys, D. and
Prochazkova Schrumpfova, P. (2019) The plant Pontin and Reptin
homologues, RuvBL1 and RuvBL2a, colocalize with TERT and TRB
proteins in vivo, and participate in telomerase biogenesis. Plant J. 98,
195–212.
Figure 1. RuvBL1 and RuvBL2a, are orthologues
of human Pontin and Reptin, respectively, in
Arabidopsis.
Besides their mutual interactions, RuvBL1 associates
with the catalytic subunit of telomerase (TERT)
in the nucleolus in vivo. In contrast to mammals,
interactions between TERT and RuvBL proteins in
Arabidopsis are not direct but are mediated by one
of the Telomere Repeat Binding (TRB) proteins. The
plant orthologue of human dyskerin, named CBF5,
is indirectly associated with TRB proteins but not
with the RuvBL proteins in the plant nucleus/nucleolus,
and interacts with the Protection of telomere 1
(POT1a) in the nucleolus or cytoplasmic foci.
© 2019 The Authors
The Plant Journal © 2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 98, 193–194
194 Lee Sweetlove and Crisanto Gutierrez
The plant Pontin and Reptin homologues, RuvBL1 and
RuvBL2a, colocalize with TERT and TRB proteins in vivo, and
participate in telomerase biogenesis
Sarka Schorova1
, Jirı Fajkus1,2,3,
* , Lenka Zaveska Drabkova4
, David Honys4
and Petra Prochazkova
Schrumpfova1,
*
1
Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of Science,
Masaryk University, Brno, Czech Republic,
2
Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Brno,
Czech Republic,
3
Institute of Biophysics of the Czech Academy of Sciences, v.v.i., Brno, Czech Republic, and
4
Laboratory of Pollen Biology, Institute of Experimental Botany of the Czech Academy of Sciences, v.v.i., Prague, Czech
Republic
Received 26 November 2018; revised 8 February 2019; accepted 26 February 2019; published online 4 March 2019.
*For correspondence (e-mails petra.proch.schrumpfova@gmail.com; fajkus@sci.muni.cz).
SUMMARY
Telomerase maturation and recruitment to telomeres is regulated by several telomerase- and telomereassociated
proteins. Among a number of proteins, human Pontin and Reptin play critical roles in telomerase
biogenesis. Here we characterized plant orthologues of Pontin and Reptin, RuvBL1 and RuvBL2a, respectively,
and show association of Arabidopsis thaliana RuvBL1 (AtRuvBL1) with the catalytic subunit of telomerase
(AtTERT) in the nucleolus in vivo. In contrast to mammals, interactions between AtTERT and AtRuvBL
proteins in A. thaliana are not direct and they are rather mediated by one of the Arabidopsis thaliana Telomere
Repeat Binding (AtTRB) proteins. We further show that plant orthologue of dyskerin, named AtCBF5, is
indirectly associated with AtTRB proteins but not with the AtRuvBL proteins in the plant nucleus/nucleolus,
and interacts with the Protection of telomere 1 (AtPOT1a) in the nucleolus or cytoplasmic foci. Our genomewide
phylogenetic analyses identify orthologues in RuvBL protein family within the plant kingdom. Dysfunction
of AtRuvBL genes in heterozygous T-DNA insertion A. thaliana mutants results in reduced telomerase
activity and indicate the involvement of AtRuvBL in plant telomerase biogenesis.
Keywords: telomerase assembly, Pontin, Reptin, AtTERT, AtTRB, AtRuvBL, AtPOT1a, nucleolus, Arabidopsis.
Linked article: This paper is the subject of a Research Highlight article. To view this Research Highlight article
visit https://doi.org/10.1111/tpj.14328.
INTRODUCTION
Telomeres are nucleoprotein structures at the ends of
eukaryotic chromosomes that protect linear chromosomes.
Telomeric structures are formed by telomeric DNA, RNA,
histones, and a number of other proteins that bind telomeric
DNA, either directly or indirectly, together forming the protein
telomere cap (Fajkus and Trifonov, 2001; de Lange,
2005; Schrumpfova et al., 2016a). The core component of
the telomere cap in mammals is a six-protein complex called
shelterin. The specific telomeric double-stranded DNA binding
of the shelterin is mediated by its TRF1 and TRF2 (Telomere
Repeat Binding Factors 1 and 2) components through
their Myb-like domain of a telobox type (Bilaud et al., 1996;
Peska et al., 2011). In Arabidopsis thaliana Telomere Repeat
Binding (AtTRB) proteins, that contain Myb-like domain of a
telobox type and bind plant telomeric repeats in vitro
(Schrumpfova et al., 2004; Mozgova et al., 2008), were
found to colocalize with telomeres in situ and in vivo
(Dvorackova et al., 2010; Schrumpfova et al., 2014; Dreissig
et al., 2017), directly interacted with the telomerase reverse
transcriptase (AtTERT) (Schrumpfova et al., 2014) and physically
interacted with AtPOT1b (Protection Of Telomeres 1)
(Schrumpfova et al., 2008). Moreover, shortening of telomeres
was observed in attrb knockout mutants (Schrumpfova
et al., 2014, 2019; Zhou et al., 2018).
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Telomere- or telomerase-associated proteins can regulate
lengths of telomere tracts by affecting the assembly of
active telomerase complex or by modulation of the accessibility
of telomeres to telomerase. The process of maturation
and recruitment of human telomerase is partially
understood (Schmidt and Cech, 2015; MacNeil et al., 2016;
Schmidt et al., 2016). However, a similar description
of telomerase assembly and recruitment to the telomeres
in plants is still missing which would allow to distinguish
between general and specific features of these processes.
Among a number of proteins, which were identified as
associated with human telomerase, proteins named
RuvBL (RuvB-like), that share limited sequence similarity
to the bacterial RuvB helicase, were also identified. RuvBL
proteins belong to the evolutionarily highly conserved
AAA+ family (ATPase Associated with various cellular
Activities) that are involved in ATP binding and hydrolysis
(Matias et al., 2006). Eukaryotic RuvBL1 (Pontin, TIP49a,
Rvb1, TAP54a) and RuvBL2 (Reptin, TIP48, TIP49b, Rvb2,
TAP54b) participate in many diverse cellular activities like
chromatin remodeling (Jha et al., 2008), transcriptional
regulation (Ohdate et al., 2003; Gallant, 2007), oncogenic
transformation (Osaki et al., 2013), epigenetic regulations
(Gallant, 2007) or DNA-damage signaling (Rosenbaum
et al., 2013). RuvBL1 and RuvBL2 can also play a role in
the assembly of box C/D or H/ACA of small nucleolar
RNAs (snoRNAs) with specific proteins to form functional
ribonucleoprotein particles (RNPs) (Watkins et al., 2004;
McKeegan et al., 2007; Boulon et al., 2008; Zhao et al.,
2008). Participation of RuvBL1 and RuvBL2 proteins in
diverse cellular processes, as well as their association
with specific interactors, can vary among cytoplasm,
nucleus and nucleolus (Izumi et al., 2012). RuvBL1 and
also RuvBL2 monomers can assemble into different oligomeric
forms, including hexameric structure with a central
channel, or dodecamer composed of two hetero-hexameric
rings with alternating RuvBL1 and RuvBL2 monomers
(Torreira et al., 2008; Niewiarowski et al., 2010). RuvBL
structure suggests that these proteins can act as scaffolding
proteins, which explains their appearance in various
cellular protein complexes (Matias et al., 2006; Mao and
Houry, 2017).
Mammalian RuvBL1 and RuvBL2, also termed as Pontin
and Reptin, respectively, were found to play a critical
role in telomerase biogenesis. Telomerase is a ribonucleoprotein
enzyme complex composed of two core subunits:
the catalytic telomerase reverse transcriptase (TERT)
protein subunit and the telomerase RNA (TR) subunit
(containing a box H/ACA motif). It performs the addition
of telomeric DNA repeats onto the telomeres (Greider,
1996; Zhang et al., 2011). Proper assembly of TERT with
TR into a functional complex is a stepwise regulated process
governed also by multiple associated proteins (Schmidt
and Cech, 2015; MacNeil et al., 2016). Human TR
(hTR), as well as other box H/ACA snoRNPs, is associated
with conserved scaffold proteins: dyskerin, NHP2, NOP10,
NAF1 in the nucleoplasm, where NAF1 is replaced by
GAR1 before the hTR RNP complex reaches the nucleolus.
Association of hTR RNP with hTERT is proceeded in
the nucleolus and the subsequent formation of catalytically
active telomerase holoenzyme is localized into the
Cajal bodies (CBs) (MacNeil et al., 2016) that are evolutionary
conserved mobile nuclear substructures involved
in the RNA modification and the RNP assembly processes
(Cioce and Lamond, 2005). Venteicher et al. (2008)
demonstrated that hRuvBL1 (Pontin) and hRuvBL2 (Reptin)
are interdependent proteins and are recruited to
hTERT complexes through the association between
hTERT and hRuvBL1. Additionally, they showed that both
hRuvBL1 and hRuvBL2 directly interact with dyskerin and
may help to assemble or remodel a nascent hTERT/hTR/
dyskerin complex. The scaffold proteins, including dyskerin,
together with hRuvBL1 and hRuvBL2, are required
for a proper assembly of hTR RNP and are involved in
the biogenesis of telomerase.
A homologue of human RuvBL1 from A. thaliana has
been already described by Holt et al. (2002). They observed
that plants with reduced AtRuvBL1 (AT5G22330) mRNA
levels had morphological defects and suggested that
AtRuvBL1 was required in meristem development. Moreover,
they observed that T-DNA insertion mutation in
AtRuvBL1 gene was lethal. In our laboratory, AtRuvBL1
protein and also one of two AtRuvBL2 homologues, named
AtRuvBL2a (AT5G67630), were purified together with
AtTERT using Tandem Affinity Purification (TAP) from
A. thaliana suspension cultures (Majerska et al., 2017).
In this study, we examined a mutual interaction of
AtRuvBL1-AtRuvBL2a proteins and demonstrated that
AtRuvBL proteins are associated with AtTERT in the nucleolus
in vivo. However, in contrast to mammalian counterparts,
interactions between AtTERT and AtRuvBL proteins
are not direct and are likely to be mediated by one of the
AtTRB proteins. We prove that AtTRB3 protein physically
interacts with AtRuvBL1 and simultaneously with AtTERT.
We further show that in plants, similarly to mammals,
telomerase assembly is a dynamic process, as is supported
by our observation that AtCBF5, a plant orthologue of dyskerin,
is in the plant nucleus/nucleolus indirectly associated
with three of AtTRB proteins, but not with the AtRuvBL
proteins, and interacts with the AtPOT1a in the cytoplasmic
or nucleolus foci. Heterozygous T-DNA insertion mutants
in AtRuvBL1 or AtRuvBL2a genes show reduced telomerase
activity indicating the potential involvement of
AtRuvBL proteins in telomerase assembly in A. thaliana.
To identify new homologues of RuvBL protein family and
elucidate their evolutionary relationships, we performed a
survey of 83 plant species (80 angiosperms, one gymnosperm
and two bryophytes).
© 2019 The Authors
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196 Sarka Schorova et al.
RESULTS
AtRuvBL proteins form homomers or mutually interact
RuvBL1 and RuvBL2 proteins from mammals and yeast
can co-exist in different monomeric or oligomeric complexes
comprising dimers, trimers, hexamers or doublehexamers
that can be composed as mixed multimers (Torreira
et al., 2008; Niewiarowski et al., 2010; Queval et al.,
2014). Each RuvBL monomer contains three basic
domains (DI, DII, DIII) (Figure 1a). Domain I (DI) together
with domain III (DIII) represent the AAA+ core and are sufficient
to form hexameric rings. In the AAA+ domain, the
Walker A/B motifs are responsible for ATP binding and
hydrolysis, while sensor I/II motifs sense whether the protein
is bound to di- or triphosphates. Domain II (DII) corresponds
to an insertion that is unique to RuvBL in
comparison with other AAA+ family members (Silva-Martin
et al., 2016).
Figure 1. AtRuvBL proteins can form homo- or hetero-oligomers.
(a) Schematic representation of the conserved motifs of the RuvBL proteins from Arabidopsis thaliana. DI, DII, DIII, Domain I, II, III; Walker A/B, Walker motifs;
Sensor I/II, sensors; Arg finger, arginine finger. AtRuvBL2a and AtRuvBL2b form closely related sequence pairs.
(b) Y2H system is used to assess homo- or heteromerization of AtRuvBL proteins. Two sets of plasmids carrying the indicated protein fused to either the GAL4
DNA-binding domain (BD) or the GAL4 activation domain (AD) are constructed and introduced into yeast strain PJ69-4a carrying reporter genes His3 and Ade2.
Clear AtRuvBL1 and also AtRuvBL2a homomerization is detected on histidine-deficient plates. Mutual interaction between AtRuvBL1 and AtRuvBL2a is detected
not only on histidine-deficient plates but also under stringent adenine selection. Co-transformation with an empty vector (AD, BD) serves as a negative control.
(c) Co-IP is performed with the TNT-RRL expressed AtRuvBL1* and AtRuvBL2a* (35
S-labelled*, prey) mixed with their protein counterparts AtRuvBL1 and AtRuvBL2a,
fused with Myc-tag (anchor) and incubated with anti-Myc antibody. In the control experiment, the AtRuvBL* proteins are incubated with Myc-antibody
and protein G-coupled magnetic beads in the absence of partner protein. Input (I), Unbound (U) and Bound (B) fractions are collected and run in 12% SDS-PAGE
gels. Mutual AtRuvBL1 and AtRuvBL2a interactions appear to be stronger than entirely homo-interactions between AtRuvBL proteins.
(d) BiFC confirms homo- and also mutual heteromerization of AtRuvBL1 and AtRuvBL2a proteins. A. thaliana leaf protoplasts are co-transfected with 10 lg of
each of the plasmids encoding nYFP-tagged or cYFPÀtagged AtRuvBL1, AtRuvBL2a or AtGAUT10 clones (as negative control) and simultaneously with
mRFPÀVirD2NLS clone. Bright Field (left); RFP, mRFPÀVirD2NLS (red fluorescent protein fused with nuclear localization signal) labels cell nuclei and determines
transfection efficiency; YFP, yellow fluorescent protein signals indicate specific protein–protein interactions (PPI) also marked by white arrows; Chl, chloroplast
autofluorescence is marked by green pseudocolor, chloroplast autofluorescence is also visible in the YFP channel. Scale bars = 10 lm.
© 2019 The Authors
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Characterization of plant Pontin and Reptin 197
To examine whether plant homologues of RuvBL proteins
form homomers or mutual heteromers as their mammalian
counterparts, or exist only as monomers, we performed several
assays for protein–protein interactions (PPIs): yeast
two-hybrid system (Y2H), co-immunoprecipitation (Co-IP)
and bimolecular fluorescence complementation (BiFC).
First, we tested AtRuvBL1 and AtRuvBL2a homo-interactions.
BiFC assay performed in A. thaliana leaf protoplasts,
which enables direct visualization of protein interactions in
living cells, demonstrated that AtRuvBL1 and AtRuvBL2a
form homodimers or homomultimers in vivo. These
results were confirmed using a GAL4 based Y2H assay, in
which interactions took place inside the nucleus. We
observed a clear homomeric interaction of AtRuvBL1 proteins
as well as of AtRuvBL2a proteins in Y2H mating
assay. The homomerization was further verified by Co-IP
experiments in which proteins were expressed in the Coupled
Transcription/Translation Rabbit Reticulocyte Lysate
(TNT-RRL) System using the same vectors as in Y2H (Figure
1b, c).
Additionally, we expanded our BiFC study (Majerska
et al., 2017) and tested heteromerization of AtRuvBL1
and AtRuvBL2a not only in Nicotiana tabacum BY-2
protoplasts, but also in A. thaliana leaf protoplasts (Figure
1d). Analysis of subcellular localization of the
AtRuvBL1-AtRuvBL2a interactions further showed that one
reciprocal interaction of nYFP/AtRuvBL1 and cYFP/AtRuvBL2a
was negative, and cYFP/AtRuvBL1 and nYFP/AtRuvBL2a
showed nuclear, but not nucleolar localization,
maybe due to the presence of a tag that may induce conformational
changes of the AtRuvBL proteins (Cheung et al.,
2010). Using Y2H assay, we confirmed clear interaction not
only on histidine-deficient (ÀHis) plates but also under
stringent adenine (ÀAde) selection. Both Y2H and Co-IP
experiments revealed that mutual AtRuvBL1ÀAtRuvBL2a
interaction seemed stronger than pure homomerization of
Figure 2. AtRuvBL1 interacts indirectly with N-terminal part of Arabidopsis thaliana catalytic subunit AtTERT. The analyses were performed as described in
Figure 1.
(a) Schematic depiction of the plant catalytic subunit of telomerase (AtTERT) showing functional motifs. The regions of structural domains TEN (telomerase
essential N-terminal domain), TRBD (RNA-binding domain), RT (reverse transcriptase domain) and CTE (C-terminal extension) are depicted above the conserved
RT motifs (1, 2, A, B0
, C, D and E), telomerase-specific motifs (T2, CP, QFP and T) and a NLS (nucleus localisation-like signal). All the depicted AtTERT fragments
were used in protein–protein interaction analysis (amino acid numbering is shown). All AtTERT fragments were fused with activation domains (AD/BD or nYFP/
cYFP) and used for further BiFC, Y2H and Co-IP analysis.
(b) BiFC in A. thaliana leaf protoplasts were used to detect the interaction between AtRuvBL1 and all AtTERT fragments from schematic depiction. Here we show
PPI interaction (white arrows) of two N-terminal fragments of AtTERT (AtTERT 1À233 and AtTERT 1À271) and AtRuvBL1 located in the nucleolus. AtGAUT10,
negative control; RFP, nucleus marker; YFP, detects PPI; Chl, Chloroplast autofluorescence. Scale bars = 10 lm.
(c) Y2H system fails to detect the interactions between AtRuvBL1 protein and N-terminal fragments of AtTERT (AtTERT 1À233 and AtTERT 1À271). BD, GAL4
DNA-binding domain; AD, GAL4 activation domain.
(d) Co-IP analysis does not detect interactions between AtTERT fragments and AtRuvBL1 protein which were demonstrated by BiFC. I, Input; U, Unbound; B,
Bound fractions; asterisks*, 35
S-labelling.
© 2019 The Authors
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198 Sarka Schorova et al.
AtRuvBL proteins. These results showed that RuvBL1 and
RuvBL2a proteins from A. thaliana are able to form both
homo- and heteromers, as well as their homologues in
diverse organisms, although they preferably form hetero-
mers.
AtRuvBL1 and AtTERT colocalize in the nucleus but
contrary to mammalian homologues do not interact
directly
Human RuvBL proteins are involved in the biogenesis
and maturation of human telomerase complex. Human
hRuvBL1 directly interacts with hTERT catalytic subunit.
hRuvBL2 does not exhibit direct interaction with hTERT
and seems to be recruited to an hTERT complex through
bridging hRuvBL1 molecules (Venteicher et al., 2008). To
gain a deeper insight whether direct RuvBL-TERT interaction
is conserved throughout the higher eukaryotes, we
applied the above described Y2H, Co-IP and BiFC techniques.
As TERT is a high-molecular-weight protein (approximately
130 kDa), we used the Gateway-compatible
donor vectors carrying the AtTERT fragments that were
described in Lee et al. (2012) and Zachova et al. (2013)
(Figure 2a). We observed a clear nuclear interaction
between AtRuvBL1 protein and AtTERT N-terminal fragments
covering AtTERT domains localized in positions 1-
233 and 1-271 in the A. thaliana leaf protoplasts using
BiFC (Figure 2b). These results supported the observation
from tobacco BY2 culture protoplasts where N-terminal
fragments of AtTERT interact with AtRuvBL1 (Majerska
et al., 2017). As the central reverse transcriptase (RT)
domain of hTERT is implicated in hRuvBL1 binding (Venteicher
et al., 2008), we expanded our interest to the
other AtTERT fragments. However, no interactions were
detected between AtRuvBL1 protein and AtTERT fragments
localized in positions 229À582, 597À987 and
972À1123, therefore covering RT or C-terminal domains
of AtTERT. Likewise, no interaction was observed
between any of AtTERT fragments and AtRuvBL2a protein
(Figure S1).
Notably, interactions of the N-terminal fragments
between AtTERT domains and AtRuvBL1 were not confirmed
in Y2H or Co-IP (Figure 2c, d). This discrepancy can
be caused by the fact that the BiFC analysis detects the
presence of proteins within the same macromolecular
complex even in the absence of a direct contact between
the proteins fused to the cYFP and nYFP fragments. The
presence of proteins within the visualized macromolecular
complex generally indicates that they participate in the
same biological process (Kerppola, 2009). Our data show
the interaction between AtRuvBL1 and AtTERT is localized
in the nucleus and supports the suggestion of Majerska
et al., that AtRuvBL1-AtTERT interaction is mediated by an
unknown partner and occurs in plant cells but not in RRL
lysate or yeast system.
AtRuvBL proteins physically interact with AtTRB proteins
Previously, we have described that members of plant-specific
group of AtTRB proteins physically interact with the Nterminal
part of AtTERT and colocalized with telomeres
in situ (Schrumpfova et al., 2004, 2014; Mozgova et al.,
2008; Dreissig et al., 2017; Zhou et al., 2018). AtTRB1 interaction
with double-stranded telomeric DNA is mediated by the
Myb-like domain, while the H1/5 domain is involved in DNA
sequence-non-specific DNA-protein interactions, interaction
with AtPOT1b (Schrumpfova et al., 2008) and in the multimerization
of AtTRB1 (Mozgova et al., 2008) (Figure 3a).
According to these findings, AtTRB proteins might be
components of a putative shelterin-like complex in plants
that modulates access of the telomerase to telomeres
(Schrumpfova et al., 2016a, 2019). Our BiFC analysis
revealed the AtTRB3 protein interaction with both
AtRuvBL1 and AtRuvBL2a proteins in the nucleus (Figure
3b). These interactions were confirmed by Y2H (Figure
3c) and also by Co-IP (Figure 3d), in which AtTRB3
Figure 3. AtTRB3 proteins directly interact with AtRuvBL1 and AtRuvBL2a
proteins. The methods are performed as is described in Figure 1.
(a) Schematic representation of the conserved motifs of the AtTRB3 protein
from Arabidopsis thaliana. Myb-like, Telobox-containing Myb domain;
H1/H5, histone-like domain; coiled-coil, C-terminal domain.
(b) BiFC shows interaction between AtTRB3 and both AtRuvBL proteins.
PPIs marked by white arrows are localized in the nucleus. AtGAUT10, negative
control; RFP, nucleus marker; YFP, detects PPI; Chl, Chloroplast autofluorescence.
Scale bars = 10 lm.
(c) Y2H results show interactions between AtTRB3 and both AtRuvBL proteins
on His- deficient plates. BD, GAL4 DNA-binding domain; AD, GAL4
activation domain; asterisks*, 5 mM 3-aminotriazol.
(d) Co-IP results confirm direct interactions between radioactively labelled
AtTRB3 and Myc-tagged AtRuvBL proteins. I, Input; U, Unbound; B, Bound
fractions; asterisks*, 35S-labelling.
© 2019 The Authors
The Plant Journal © 2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 98, 195–212
Characterization of plant Pontin and Reptin 199
protein was radioactively labelled by 35
S-methionine and
mixed with its putative protein partners AtRuvBL1 or
AtRuvBL2a fused with Myc-tag, and incubated with antiMyc
antibody. Clear nuclear interaction of AtTRB2 and
AtRuvBL2a, but not of AtTRB2 with AtRuvBL1, was
detected by BiFC and verified by Y2H and Co-IP. However,
the nuclear interaction of AtTRB1 with AtRuvBL1 observed
in BiFC seems to be indirect, as it was not proven by Y2H
or Co-IP assays, but indicates that both proteins are present
in the same macromolecular complex (Figure S2). Collectively,
direct interactions of AtTRBs with AtTERT, as well
as with AtRuvBL proteins, imply the role of AtTRB proteins
as mediators of the interactions between AtRuvBL proteins
and AtTERT telomerase subunit in vivo.
AtTRB3 protein mediates interaction between AtRuvBL1
and AtTERT
Our data, showing the indirect interaction between the Nterminal
part of AtTERT and AtRuvBL1, suggested that this
interaction could be mediated by AtTRB3 protein. We performed
Co-IP assay with all three proteins of interest (Figure
4). Two prey proteins AtRuvBL1 and AtTRB3, were
labelled with 35
S-methionine during the expression in
TNTÀRRL system. N-terminal fragment of AtTERT (AtTERT
1-271), fused with Myc-tag as an anchor, was expressed in
TNTÀRRL system in non-radioactive form ensuring a better
resolution of the prey proteins in the 12% SDS-PAGE separation.
Radioactively labelled AtTERT fragment was
expressed in parallel tube to affirm the proper AtTERT 1-
271 expression. The complex was captured with anti-Mycantibody
and protein G-coupled magnetic beads. Several
negative controls were performed, where some of the
monitored proteins were not present, to ensure specificity
of the AtRuvBL1ÀAtTRB3ÀAtTERT complex. From these
negative controls, it is evident that AtRuvBL1 protein neither
directly interacts with the AtTERT 1À271 fragment nor
is non-specifically bound to the magnetic beads. Conversely,
the presence of AtTRB3 in immunoprecipitation
mixture resulted in reproducible and significant increase of
the AtRuvBL1 in the immunoprecipitated complex. So, it is
evident that AtRuvBL1 is recruited to the AtTERT complex
through an interaction with AtTRB3 protein, which mediates
interaction of both proteins, AtTERT and AtRuvBL1.
Plant homologue of mammalian dyskerin AtCBF5
associates with AtTRB proteins in the plant nucleus
Mammalian protein dyskerin is a core component of
mature and functional telomerase complex (He et al., 2002;
Schmidt and Cech, 2015; MacNeil et al., 2016). Dyskerin
binds the H/ACA box of small nuclear and nucleolar RNAs
(sn- and sno-RNAs) and belongs to conserved scaffold proteins
of human hTR (MacNeil et al., 2016). Plant homologue
AtCBF5 (also named AtNAP57) is localized within
nucleoli and CBs (Lermontova et al., 2007) and associates
with enzymatically active telomerase RNP particles in an
RNA-dependent manner (Kannan et al., 2008).
Here we observed a clear indirect interaction of AtCBF5,
fused with cYFP, with all three examined nYFP/AtTRB proteins
using BiFC technique (Figure 5). As has already been
discussed above, BiFC analysis can detect the presence of
proteins within the same macromolecular complex even
without a direct contact between the proteins fused with
cYFP/nYFP (Kerppola, 2009). We assume that the interactions
between AtCBF5 and AtTRBs are indirect because we
Figure 4. AtTRB3 protein is mediator of AtRuvBL1 and AtTERT interaction.
(a) Co-Immunoprecipitation of the three proteins of interest. Two proteins AtRuvBL1 and AtTRB3 are radioactively labelled by 35
S-methionine (marked with
asterisks) during the expression in TNT-RRL lysate and subsequently incubated with non-radioactive Myc-tagged AtTERT 1À271 fragment and anti-Myc antibody.
In the control experiments, the proteins are incubated with Myc-antibody and protein G-coupled magnetic beads in the absence of one or both partner
proteins. Radioactively labeled AtTERT fragment is expressed in parallel tube as a control of the expression. From penult column it is evident that the presence
of AtTRB3 results in significant increase of the AtRuvBL1 in the immunoprecipitated complex. I, Input; U, Unbound; B, Bound fractions were collected and run in
12% SDS-PAGE gels.
(b) Schematic depiction of the putative protein complex formed by proteins AtRuvBL1, AtTRB3 and AtTERT. AtRuvBL1 is depicted in its presumed hexameric
form and AtTRB3 in its dimeric form.
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200 Sarka Schorova et al.
were not able to confirm the AtCBF5ÀAtTRBs interactions
observed by BiFC in Y2H mating assay. Also Co-IP did not
reveal any iteraction between proteins expressed in
TNTÀRRL system, fused with Myc-tag (AtRuvBL1, AtRuvBL2a,
AtTRB1, AtTRB2 and AtTRB3) and with radioactively
labelled AtCBF5 as a prey. Additionally, no interaction was
detected between AtCBF5 and any of AtRuvBL proteins
neither in BiFC nor in Y2H or Co-IP. As a positive control
we used the interaction between AtCBF5 and AtPOT1a.
Here we show that the AtCBF5 interacts with AtPOT1a not
only in Y2H and Co-IP, as was shown in Kannan et al.
(2008), but also in the plant nucleus using BiFC assay. In
addition to the nucleolar localization of AtPOT1a–AtCBF5
interactions, we also observed this interaction in several
Figure 5. AtCBF5 associates with AtTRB proteins indirectly. The methods are done in the same manner as in Figure 1.
(a) BiFC assay shows indirect interaction between AtCBF5 and three proteins from AtTRB family (AtTRB1-3). AtCBF5 interacts also with AtPOT1a protein. PPIs
are marked by white arrows. AtGAUT10, negative control; RFP, nucleus marker; YFP, detects PPI; Chl, Chloroplast autofluorescence. Scale bars represent 10 lm.
(b) Y2H assay analysis does not detect the interaction between AtCBF5 and AtTRB proteins which was found by BiFC. AtCBF5 protein interacts only with
AtPOT1a on histidine deficient plate (-His). BD, GAL4 DNA-binding domain; AD, GAL4 activation domain.
(c) Co-IP analysis shows interaction only between AtCBF5 and AtPOT1a protein, fused with Myc-tag and incubated with Myc-antibody and protein G-coupled
magnetic beads. I, Input; U, Unbound; B, Bound fractions; asterisks*, 35
S-labelling.
© 2019 The Authors
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Characterization of plant Pontin and Reptin 201
nuclear and cytoplasmic foci (Figure S3). Further, we
observed a weak interaction between AtPOT1aÀAtRuvBL1
proteins in Y2H and Co-IP assays but not in BiFC system
(Figure S4). As a negative control in BiFC assay, we cotransfected
protoplasts with cYFP/AtGAUT10. AtGAUT10
protein did not interact with any of the proteins of interest
fused with nYFP: AtRuvBL1, AtRuvBL2a, AtTRB1, AtTRB2,
AtTRB3 or AtPOT1a. Co-transformation with an empty vector
(AD, BD) served as a negative control in Y2H experiments.
In Co-IP experiment, the AtCBF5 proteins were
incubated with Myc-antibody and protein G-coupled magnetic
beads in the absence of partner protein as negative
control. Together, we conclude that AtTRB proteins are
associated in very close proximity with AtCBF5, the plant
homologue of mammalian dyskerin, in the plant nucleus.
However, at the same time, AtCBF5 is not localized in the
nearby complex with the AtRuvBL proteins in vivo.
Association of AtRuvBLs, AtTRBs and AtTERT indicates
the formation of their complex in the nucleolus
During the assembly of a fully functional complex of the
human telomerase, the mature hTR gets recruited to the
nucleolus where it binds the hTERT complex. Both of the
core telomerase components, hTR and also hTERT, are
previously processed by several proteins, including
hRuvBL1 and hRuvBL2. It has already been published that
in the interphase, the AtTRB proteins showed preferential
localization to the nucleus and specially to the nucleolus
(Dvorackova et al., 2010). In comparison with the mammalian
nucleoli, plant nucleoli are larger, more frequently
undergo fusions, and sometimes have a central clear
region, often called the nucleolar vacuole, the size of which
depends on nucleoli transcriptional activity (Shaw and
Brown, 2012; Stepinski, 2014).
We analyzed the subcellular localizations of the interactions
of our proteins of interest: AtTERT 1-271, AtRuvBL1,
AtRuvBL2a, AtTRB3 and AtCBF5 fused with nYFP- or cYFPtag
in routinely performed BiFC experiments. The nucleoli
were marked by control plasmid mRFPÀAtFibrillarin 1 (Pih
et al., 2000). Figure 6 shows interactions between
AtRuvBL1ÀAtTERT, AtTRB3ÀAtTERT, AtRuvBL1ÀAtTRB3
and AtRuvBL2aÀAtTRB3, which occupy distinct areas
within the plant nucleus that match to the plant nucleolus.
The number of the PPIs foci localized exclusively in the
nucleolus is listed in the Table S1. Similar patterns of
nuclear or nucleolar PPI localization is visible also in Figure
S5 where the whole nucleus was marked by
mRFPÀVirD2NLS. However, the AtCBF5ÀAtTRB3 interaction
showed different localization pattern than the other
examined PPIs. AtCBF5ÀAtTRB3 interaction seems to be
localized in nucleoli and sometimes in additional nuclear
bodies at the periphery or outside the nucleoli, which is
consistent with localization of free AtCBF5 (Lermontova
et al., 2007). Together, our data indicate formation of
AtRuvBLsÀAtTRBsÀAtTERT complex in the nucleolus.
Dysfunction of AtRuvBL genes reduces telomerase activity
In human cells, the hRuvBL1 and hRuvBL2 proteins associate
with a significant population of hTERT molecules that
do not yield high-level telomerase activity, measured by
Telomere Repeat Amplification Protocol (TRAP). The depletion
of hRuvBL1 and hRuvBL2 markedly impaired telomerase
RNP accumulation and diminished human
telomerase activity (Venteicher et al., 2008). To assess
whether mutations in AtRuvBL genes have any impact on
telomerase activity in A. thaliana, we set to perform TRAP
assay on telomerase extracts isolated from T-DNA insertion
mutant lines. Extensive search of several T-DNA
Figure 6. Association of AtRuvBLs, AtTRBs and
AtTERT in the nucleolus in A. thaliana leaf protoplasts.
Protoplasts are co-transfected with mRFPÀAtFibrillarin
1 encoding RFP that labels nucleolus and
simultaneously with each of the plasmids encoding
nYFP-tagged or cYFP-tagged AtRuvBL1, AtRuvBL2a,
AtTERT 1À271, AtTRB3 or AtCBF5 to determine PPI
localization. AtRuvBL1ÀAtTERT, AtTRB3ÀAtTERT,
AtRuvBL1ÀAtTRB3 or AtRuvBL2aÀAtTRB3 interactions
show nucleolar localization. Plant homologue
of mammalian dyskerin, AtCBF5, is associated with
AtTRB3 in the nucleolus and in additional nuclear
bodies at the periphery of the nucleolus. RFP,
marked nucleus; YFP, detects PPI; Scale
bars = 10 lm.
© 2019 The Authors
The Plant Journal © 2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 98, 195–212
202 Sarka Schorova et al.
insertion lines, which are available from several plant databases,
revealed only two suitable plant lines with a limited
number of heterozygous mutant plants but with no
homozygous mutant plants: SAIL_397_C11 (AtRuvBL1) and
GK-543F01 (AtRuvBL2a). In an additional seven tested TDNA
insertion plant lines we did not detect any viable
mutant or heterozygous plants for AtRuvBL1 or AtRuvBL2a
genes (Table S2). Furthermore, genotypic ratios of offspring
of individual heterozygous plants did not follow the
expected Mendelian genotypic ratio. The observed ratio for
AtRuvBL1+/À
and for AtRuvBL2a+/À
plants was (51:21:0) and
(91:10:0), respectively, instead of (1:2:1) (Figure 7a) and the
cause of this phenomenon will be further investigated.
Quantitative TRAP assay performed with telomerase
extract isolated from flower buds of individual AtRuvBL2a
heterozygous plants demonstrated that relative telomerase
activity showed apparent reduction in comparison with
telomerase extract from wild-type A. thaliana (Col-0+/+
)
buds (Figure 7b). T-DNA insertion mutation in AtRuvBL1
gene was lethal (Holt et al., 2002) but we detected viable
heterozygous AtRuvBL1 plants. These plants showed a
milder reduction of telomerase activity than AtRuvBL2a+/À
plants, which supports the assumption that AtRuvBL1 protein
is essential for meristem development (Holt et al.,
2002).
Human RuvBL proteins are direct interactors of transcription
factor MYC that is required for expressing many
genes involved in cell-cycle transition events and proliferation
(Wood et al., 2000). hRuvBL2 regulates MYC-dependent
transcription of TERT via targeting the hTERT
promoter (Wood et al., 2000; Li et al., 2010; Flavin et al.,
2011; Zhao et al., 2014). We analyzed the levels of AtTERT
transcripts in AtRuvBL1 and AtRuvBL2a heterozygous
plants to detect whether the decrease of telomerase activity
was caused by the negative regulation of AtTERT promoter
i.e. the decrease of the abundance of AtTERT
transcripts. We did not observe significant changes in transcripts
of AtTERT gene in AtRuvBL1 heterozygous mutant
plants compared with the wild-type A. thaliana. Instead,
we observed very slight, though significant, increase in
AtTERT transcripts in AtRuvBL2a heterozygous mutant
plant lines (Figure S6).
Due to the difficulties in maintaining the heterozygous
AtRuvBL plant lines for several subsequent generations,
we were not able to analyze the transgenerational effects
of reduced telomerase activity on telomere lengths in
plants heterozygous in AtRuvBL1 and AtRuvBL2a genes.
However, in the analyzed generation of AtRuvBL1+/À
and
AtRuvBL2a+/À
plants that were descendants of heterozygous
predecessors, we did not detect any significant
changes in telomere lengths compared with the wild-type
plants using Terminal Restriction Fragment analysis (TRF)
(Figure S7).
Together, we conclude that the depletion of AtRuvBL1
and especially of AtRuvBL2a proteins reduces telomerase
activity which suggests a conserved role of AtRuvBL
proteins in maturation of functional telomerase complex
across the mammals and also plants.
Identification and phylogenetic analysis of the RuvBL
family in plants
RuvBL proteins, showing association with TERT in human
cells, represent a group of proteins well conserved across
all eukaryotic kingdoms, including Fungi, Animalia or
Plantae.
Here, we present a genome-wide analysis of RuvBL proteins
in 80 vascular plant species, one gymnosperm and
two bryophytes, totally 83 taxa, that were analyzed for the
presence of all three basic domains (DI, DII, DIII). The evolutionary
relationships among the RuvBL proteins were
Figure 7. Reduction of relative telomerase activity in heterozygous AtRuvBL
mutant plants.
(a) Genotypic ratio of the offspring of heterozygous AtRuvBL1 and AtRuvBL2a
T-DNA insertion mutant plants. Homozygous mutant plants in
AtRuvBL genes are fully absent and even the number of heterozygous
plants does not follow the Mendelian genotypic ratio.
(b) Samples isolated from AtRuvBL1+/À
and AtRuvBL2a+/À
buds are analyzed
in three technical replicates by quantitative TRAP. Data are related to wildtype
Col-0 sample (telomerase activity in Col-0 buds are arbitrarily chosen
as 1). Relative telomerase activity is reduced in both AtRuvBL1+/À
and
especially in AtRuvBL2a+/À
samples. P < 0.05 are considered as significant.
Single asterisk denotes 0.01 < P < 0.05. Three asterisks denote
0.01 < P < 0.001.
© 2019 The Authors
The Plant Journal © 2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 98, 195–212
Characterization of plant Pontin and Reptin 203
determined using maximum likelihood analyses based on
multiple alignments producing a phylogenetic tree depicting
the relationships among all currently accessible RuvBL
sequences. The evolutionary hypotheses from these analyses
were highly congruent. RuvBL protein family was
divided in two distinct groups based on the similarity of
sequences and branch length. Sequence similarity
between RuvBL1 and RuvBL2 is generally low, about 35–
40% while the sequence similarity within RuvBL subfamilies
is about 80%. For instance, in A. thaliana AtRuvBL2a
and AtRuvBL2b share 82% similarity. On the other hand,
AtRuvBL1 with AtRuvBL2a or AtRuvBL1 with AtRuvBL2b
share 37.5 and 38.8% similarity, respectively. However,
only a subset of RuvBL1 was clearly separated (100% BS;
blue branch in Figure 8). Surprisingly, based on BLAST
search, RuvBL1 was found only in dicots and basal angiosperms
(Amborella trichopoda) up to now, RuvBL2 was represented
in both, dicots and monocots from angiosperms,
but also in gymnosperms (Picea sitchensis) and bryophytes
(Physcomitrella patens and Marchantia polymorpha).
The number of the homologues varied from 1 to 8
(Data S1 and S2).
Figure 8. Phylogenetic analysis of the RuvBL family in plants.
Unrooted phylogenetic tree of 190 proteins sequences of RuvBL family with enumerated plant species. Numbers above branches means bootstrap support values.
Orthologues from Arabidopsis thaliana and Nicotiana tabacum are in bold letters.
© 2019 The Authors
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204 Sarka Schorova et al.
DISCUSSION
The formation of functional and enzymatically active
telomerase, a multisubunit RNP complex, is a dynamic
process governed by number of cofactors. In mammals,
hRuvBL1 and hRuvBL2 proteins, Pontin and Reptin, respectively,
are present in early steps of telomerase RNP biogenesis.
We characterized plant homologues of RuvBL
proteins: AtRuvBL1 and AtRuvBL2a, previously co-purified
together with telomerase protein subunit AtTERT from
A. thaliana suspension cultures (Majerska et al., 2017).
Here we show that AtRuvBL1 protein colocalizes with Nterminal
part of AtTERT subunit of plant telomerase also
in vivo. However, in contrast with the AtRuvBL mammalian
counterparts, their interaction in plants seems to be indirect.
Association of AtRuvBL proteins with AtTERT in the
plant nucleolus appears to be bridged by telomeric AtTRB
proteins. Requirement of AtRuvBL proteins for a proper
telomerase assembly is endorsed by the fact that depletion
of AtRuvBL1 and especially of AtRuvBL2a protein, reduces
telomerase activity in plants heterozygous for AtRuvBL1 or
AtRuvBL2a genes. Moreover, AtTRB proteins are associated
in the plant cell with a homologue of mammalian dyskerin,
AtCBF5, that plays a role in telomerase RNP
biogenesis and directly interacts with AtPOT1a protein.
AtTRB proteins thus play a role of interaction hubs not
only in telomere chromatin structure but also in telomerase
biogenesis. AtRuvBL proteins are able to multimerize,
which is analogous to the situation in mammalian
cells, and our data show preference to form mutual heteromers.
Detailed summary of protein–protein interactions
between AtRuvBLs, AtTRBs, AtTERT fragments, AtPOT1s
and AtCBF5 proteins, that have been detected using BiFC,
Y2H or Co-IP assays in this and other relevant publications,
are given in the Table 1.
Our detailed phylogeny proved that RuvBL proteins
are evolutionarily conserved in land plants and implied
plausible functional conservation of the RuvBL proteins.
However, further biochemical validation of the possible
conservation of mutual RuvBLÀTRB interaction across
the plant kingdom can be limited by the fact that the
number of paralogues varies from 1 to 8 members in
between RuvBL proteins. The multiplication of genes of
the same family is not surprising as, in many plant families,
the polyploidy (i.e. whole-genome duplication,
WGD), resulting in retention of multiple gene paralogs
may lead to their sub-functionalization, neo-functionalization
or partial or full redundancy (Mandakova and Lysak,
2008; Freeling, 2009). These limitations might be deteriorated
by the fact that the AtRuvBL proteins can be
involved in a similar biochemical pathway but their
interaction partners might slightly differ (this paper; Venteicher
et al., 2008).
RuvBL proteins are involved in various cellular processes
The exact function even of mammalian RuvBL proteins is
still quite unknown as they interact with many molecular
complexes with vastly different downstream effectors
(Mao and Houry, 2017). Among others, hRuvBL2 was
shown to regulate hTERT promoter likely through the regulation
of MYC (c-myc), the transcription factor for TERT
(Wood et al., 2000; Li et al., 2010; Flavin et al., 2011; Zhao
et al., 2014). We observed no significant changes in transcripts
of AtTERT gene in AtRuvBL1 heterozygous mutant
plants, however we detected a very slight increase in transcripts
of AtTERT gene in AtRuvBL2a heterozygous plants.
Although the transcript levels of AtTERT gene were slightly
increased in AtRuvBL2a heterozygous plant lines, we
observed a very significant reduction of telomerase activity
Table 1 A summary table of protein–protein interactions
Summary table shows all interactions between AtRuvBLs, AtTRBs, AtTERT fragments, AtPOT1s and AtCBF5 proteins that are detected
using BiFC, Y2H or Co-IP assays.
© 2019 The Authors
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Characterization of plant Pontin and Reptin 205
in these plants. Telomerase activity was reduced also in
AtRuvBL1 heterozygous T-DNA insertion plant lines. To
verify whether the regulation of telomerase activity was
affected due to the compromised assembly of telomerase
complex rather than due to regulation of transcript levels
of AtTERT gene in AtRuvBL-dependent manner, however,
needs further investigation.
The participation of RuvBL proteins in heterogeneous
cellular process as well as their association with specific
interactors can vary between cytoplasm, nucleus and
nucleolus (Mao and Houry, 2017). It seems that, also in
A. thaliana, the function of AtRuvBL proteins is not specific
only to the telomerase assembly, as they were suggested
as regulators of disease resistance (R) genes (Holt et al.,
2002). It has already been published that AtRuvBL1 is
essential in meristem development (Holt et al., 2002), the
function consistent with its function in telomerase assembly
observed in this work. Our extensive, but unsuccessful,
Figure 9. Comparative model of telomerase assembly in human and Arabidopsis.
(a) Human TR binds dyskerin, NHP2, NOP10, and GAR1 and human TERT associates with the chaperones Hsp90 and p23. The telomerase RNP is retained into
the nucleoli through the interaction between TERT and nucleolin. Assembly of TR and TERT into catalytically active telomerase is aided by Pontin (hRuvBL1)
and Reptin (hRuvBL2) AAA+ ATPases. Telomerase is recruited to Cajal bodies (CBs) by its interaction with TCAB1. The CBs will colocalize with telomeres, and
telomerase is recruited to telomeres by the interaction with the shelterin component TPP1 (MacNeil et al., 2016; Lim et al., 2017).
(b) Arabidopsis CBF5, GAR1, NOP10, NHP2, but in contrast with human cells also NAF1, were localized into the plant nucleolus (Pendle et al., 2005; Lermontova
et al., 2007). In the plant nucleolus, we observe colocalization of TERT with RuvBL AAA+ ATPases complex bridged by telomeric TRB proteins, as well as the
interaction of telomeric protein POT1a with CBF5. Arabidopsis telomeres cluster at the periphery of the nucleolus which is mediated by the presence of nucleolin.
Recruitment of the mature telomerase complex to telomeres with or without commitment of CBs in Arabidopsis needs further investigation. Proteins that
were already proven as associated with CBs are highlighted in color in Cajal bodies. Proteins that have not yet been experimentally proven as CBs associated
are marked with black and white.
© 2019 The Authors
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206 Sarka Schorova et al.
effort to receive homozygous mutant plants in several
T-DNA insertion lines mutant in the AtRuvBL1 and AtRuvBL2a
genes also indicated the essentiality of AtRuvBL proteins
in various cellular processes in plants. Furthermore,
genotypic ratio of offspring of individual heterozygous
plants does not follow the Mendelian genotypic ratio, indicating
that both AtRuvBL proteins are essential regulators
of plant development. Therefore, we suggest to investigate
the function of AtRuvBLs in plant sporophyte or female
gametophyte development in future studies.
Nucleolus localization of telomerase assembly complex
Telomere maintenance requires a proper assembly of the
TERT and TR components of telomerase into RNP as well
as a number of cofactors involved in maturation, stability
and subcellular localization of telomerase. In mammals,
the association of hTR RNP with hTERT proceeds in the
nucleolus during the early S-phase (Lee et al., 2014).
Assembled and catalytically active telomerase RNP separates
from the nucleoli and is transported to CBs during
the S-phase for subsequent recruitment to telomeric chromatin
and telomere extension (Figure 9a) (MacNeil et al.,
2016). Association of hTERT with human RuvBL proteins,
Pontin and Reptin, peaks in S-phase, which may reflect
cell-cycle regulation of total TERT and/or assembly of
telomerase on telomeres (Venteicher et al., 2008). RuvBL1
and RuvBL2a proteins, together with, for example, Fibrillarin
1 and many other proteins, were purified and identified
in nucleoli isolated from A. thaliana cell culture
protoplasts (Pendle et al., 2005). Our data indicated that
plant homologues of human Pontin and Reptin, the
AtRuvBL proteins, are associated in the plant nucleolus
with AtTERT, together with AtTRB proteins (Figure 9b).
AtTRB proteins are highly dynamic and during the interphase,
they are preferentially localized to the nucleolus or
nuclear bodies of different size (Dvorackova, 2010). AtTRBs
behave as typical nucleolar resident proteins, being largely
dispersed at prophase, coinciding with nucleolar disassembly.
However, a small but detectable amount of the
protein remains associated with the chromatin throughout
mitosis (Azum-Gelade et al., 1994; Dvorackova et al.,
2010). Similarly, to the AtTRB proteins, also the N-terminal
part of AtTERT was detected in the nucleoli in A. thaliana
(Rossignol et al., 2007; Zachova et al., 2013).
In mammals, the telomerase RNP is retained in nucleoli
through the interaction between hTERT and nucleolin in
the dense fibrillar component (Khurts et al., 2004; Lee
et al., 2014). In A. thaliana, null mutants for the nucleolar
protein NUCLEOLIN 1 cause telomere shortening on all
chromosome arms (Pontvianne et al., 2016). Telomeres in
A. thaliana do not form a Rabl conformation, as in some
other species, but telomeres and their flanking regions
strongly associated with the nucleolus in a rosette-like
organization (Armstrong et al., 2001; Fransz et al., 2002;
Roberts et al., 2009; Pontvianne et al., 2016; Schrumpfova
et al., 2016a). Our data indicated the presence of
AtTERTÀAtTRBÀAtRuvBL complex in the nucleolus. Nucleolar
localization of the AtTERTÀAtTRBÀAtRuvBL complex
together with the close proximity of telomeres to the
nucleolus, suggested the conservation of the recruitment
of the maturating telomerase to the nucleolus during the
telomerase assembly. Figure 9 shows a comparative
model of the assembly and localization of telomerase in
mammalian and plant cells.
Plausible conservation of the telomerase trafficking
pathway
Cajal bodies are spherical suborganelles localized in the
nucleoplasm either in the vicinity of the nucleolus and/or
they are present free. The function of CBs is not completely
understood, but they were implicated mainly in snRNAs
synthesis and processing. CBs also contribute to the biogenesis
of telomerase. In S-phase, CBs colocalize with
telomeres and facilitate recruitment of the mature mammalian
telomerase complex to the telomeres. Human dyskerin,
hNHP2, hNOP10 and hGAR1, that displaces hNAF1 in
the hTR RNP, belong to conserved scaffold proteins, which
colocalize with CBs and are involved in hTR RNP assembly
(MacNeil et al., 2016). Expression of putative AtGAR1,
AtNOP10, AtNHP2 genes encoding protein components of
the H/ACA box snoRNP complex correlate with that of
AtCBF5, a plant homologue of dyskerin (Lermontova et al.,
2007). AtCBF5 directly interacts with AtNAF1 (Lermontova
et al., 2007) and has been identified as a component of the
enzymatically active A. thaliana telomerase RNP (Kannan
et al., 2008). AtCBF5 localizes in nucleoli and sometimes in
additional nuclear bodies at the periphery or outside the
nucleoli, but AtCBF5 also colocalizes with TMG-capped
snRNA, a marker for CBs (Lermontova et al., 2007).
Here we show that plant dyskerin, AtCBF5, indirectly
interacts with AtTRB proteins in the plant nucleolus or in
other nuclear bodies. It has already been published that
AtTRBs are located not only in the nucleolus but also in
nuclear bodies of different size, some of which might be
CBs (visualized by a marker protein Coilin) (Dvorackova,
2010). Dvorackova detected significant colocalization of
AtTRB1 with Coilin present in the CBs adjacent to the
nucleolus. However, no colocalization was detected
between signals corresponding to the AtTRB1 and free
CBs in the nucleoplasm. Presence of AtTRB1 protein
entirely in the CBs adjacent to the nucleoli implies a
potential conservation of the trafficking pathway during
the telomerase maturation, which comprises movement
of maturating telomerase complex through nucleolus to
CBs and finally to the telomeres. Notably, not all the
organisms (e.g. budding yeast and ciliates) rely on the
CBs trafficking since telomerase RNAs from these species
do not have H/ACA or CAB box motifs, and further studies
© 2019 The Authors
The Plant Journal © 2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 98, 195–212
Characterization of plant Pontin and Reptin 207
are needed to prove this hypothesis. We observe that
interaction between AtCBF5 and AtPOT1a is localized
mostly in the nucleolus but in few cases also in cytoplasmic
foci. The cytoplasmic localization is not surprising as
it has already been shown that plant AtPOT1a and
AtPOT1b, as well as their human homologue hPot1, are
localized in the nucleus, as well in the cytoplasm (Chen
et al., 2007; Rossignol et al., 2007).
The assembly of hTR RNP to the telomerase holoenzyme
is not fully elucidated and it is highly complex multistep
process. Therefore, the absence of the interaction between
AtCBF5 and AtRuvBLs in the plant nucleus in our experiments
is also not surprising. For example, Machado-Pinilla
et al. (2012) showed that dyskerin was sandwiched between
two hSHQ1 domains in the first steps of the biogenesis of
telomerase. C-terminal tail of hCBF5 was essential for
hSHQ1 release mediated by hRuvBLs. However, a stable
interaction with the tails is not a part of the process because
hRuvBLs bind to hCBF5 in a pull-down assay, even in the
absence of its tail. Assembly of functional AtTER RNP, as
well as the assembly of mammalian hTR RNP, is certainly a
multistep process that may include AtTER, AtCBF5, AtTRBs,
AtRuvBLs, AtPOT1a and many other factors, whose presence/participation/mutual
interactions will be the subjects
of our future research. Dynamics and complexity of mutual
interactions can be demonstrated by the fact that we detect
the interacting complex of AtCBF5ÀAtPOT1a in the nucleolus
or in the cytoplasmic and nuclear foci using BiFC assay,
while AtCBF5ÀAtTRBs interactions are localized entirely to
the nucleoli and additional nuclear bodies. Moreover, association
of AtTRB3 with AtTERT and AtRuvBLs is entirely
localized to the nucleolus.
Concluding remarks
Homologues of the mammalian Pontin and Reptin, named
RuvBL proteins, as well as TRB proteins, might be involved
in diverse processes in the plant cell. AtTRB proteins are
not only components of terminal complex associated with
telomeres and catalytic subunit of telomerase, AtTERT
(Schrumpfova et al., 2016a, 2019), but they also serve as
epigenetic regulators that potentially impact the transcription
status of thousands of genes as subunits of epigenetically
active multiprotein complexes (Lee and Cho, 2016;
Schrumpfova et al., 2016b; Zhou et al., 2016; Dokladal
et al., 2018; Tan et al., 2018). AtRuvBL1 protein has been
assumed as a regulator of R genes so far and is essential
in meristem development (Holt et al., 2002). Here we suggest
involvement of AtRuvBL proteins in telomerase
assembly pathway in A. thaliana. We detected new interactions
of AtTRB proteins with AtRuvBL proteins, localized
the AtTERTÀAtTRBÀAtRuvBL complex exclusively in the
nucleolus and observed that heterozygous T-DNA insertion
mutants in AtRuvBL1 or AtRuvBL2a genes showed reduced
telomerase activity. Further, our results showed
interactions of AtCBF5, the plant orthologue of dyskerin,
with AtTRB and AtPOT1, but not with the AtRuvBL proteins,
which expanded our knowledge on the telomerase
assembly process. Indispensability of the AtRuvBL proteins
for the plant development was supported by our finding
that homozygous atruvbl1 and atruvbl2a mutant plants
were not viable. Furthermore, we identified new homologues
RuvBL proteins and analyzed their evolutionary
relationships in plants. Altogether, our data show that the
plant homologues of Pontin and Reptin, AtRuvBLs, and
also AtTRB are involved in telomerase assembly and suggest
conservation of telomerase trafficking pathway via the
nucleolus to the telomeres in plants.
EXPERIMENTAL PROCEDURES
Searching transcriptomes and genomes for RuvBL
homologues
RuvBL homologues were identified by BLASTP searches using
A. thaliana proteins from the TAIR database (https://www.arabid
opsis.org/) to query NCBI protein databases (http://www.ncbi.nlm.
nih.gov/). The BLASTP searches used default parameters,
adjusted to the lowest E-value. The duplicates from all searches
were eliminated. We conducted an iterative search of the UniProt
database (http://www.uniprot.org/) and the Phytozome version 11
database (https://phytozome.jgi.doe.gov) was next searched for
proteins not found by BLASTP. We analyzed all sequences independently
of their annotations, with no prior assumptions. Information
summary of accession numbers for RuvBL are in Data S1
and S2.
Sequence alignment
Amino acid sequences were aligned using the Clustal Omega algorithm
(Sievers et al., 2011) in the Mobyle platform (Neron et al.,
2009), with homology detection by HMM-HMM comparisons (Soding,
2005). Protein isoforms with the same length were also used,
because the differential expression patterns producing protein isoforms
from various tissues suggested that isoforms could have different
biological functions in vivo (Chen et al., 2014).
Phylogenetic reconstruction
Maximum likelihood (ML) analyses of the matrices were performed
in RAxML 8.2.4 (Stamatakis, 2014) to examine differences
in optimality between alternative topologies. Using the Akaike
information criterion as implemented in Modeltest 3.8 (Posada
and Crandall, 1998), a GTR+I+Γ model was chosen as the best-fitting
model, and 1000 replications were run for bootstrap values.
The final data set for RuvBL contained 190 proteins of different
species and length 576 bp. Phylogenetic trees were constructed
and modified with iTOL v3.4 (Letunic and Bork, 2016).
Transgenic constructs
The Gateway-compatible donor and destination vectors carrying
the AtTERT (AtTERT 1-233, 1-271, 229-582, 597-987, 958-1123) fragments
were described in Zachova et al. (2013). The Gateway-compatible
donor vectors carrying AtRuvBL1, AtRuvBL2a, AtPOT1a,
AtPOT1b and AtGAUT10 were described in Majerska et al. (2017).
The AtTRB1, 2 and 3 constructs have described previously
(Schrumpfova et al., 2014).
© 2019 The Authors
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208 Sarka Schorova et al.
The cloned cDNA sequence of AtCBF5 (GC105080 from Arabidopsis
Information Resource (http://www.arabidopsis.org/)) in
pENTR223 was used as entry vector. For preparation of yeast twohybrid
(Y2H) and/or BiFC constructs, DNA fragments were introduced
into the destination Gateway vectors pGBKT7-DEST,
pGADT7-DEST (Horak et al., 2008) and/or the pSAT5-DEST-c(175end)EYFPÀC1(B),
pSAT4-DEST-n(174)EYFPÀCl (Lee et al., 2012)
using the LR recombinase reaction (Invitrogen, Carlsbad, CA, USA).
PCR-based genotyping of plant lines
Plants with annotated T-DNA insertion within AtRuvBL1 gene
(SAIL_397_C11, WiscDsLoxHs027_03G, WiscDsLoxHs117_06F,
WiscDsLoxHs168_06D) and AtRuvBL2a gene (GK-543F01,
SALK_071103, SALK_144539, SALK_144540, SAIL_500_C04) in the
Col-0 background were used (Figure S8). To distinguish between
wild-type plants and those that were heterozygous for the T-DNA
insertion in the AtRuvBL1 or AtRuvBL2a genes, we isolated genomic
DNA from leaves by the standard protocol of Dellaporta et al.
(1983). The genomic DNA was used for PCR analysis using MyTaq
DNA polymerase (Bioline, http://www.bioline.com). The conditions
used were in accordance with the manufacturer’s instructions. The
primers used were specific for T-DNA and AtRuvBL1 or AtRuvBL2a
genes (Table S3, Figure S9). Thermal conditions were 95°C for 1 min
(initial denaturation), followed by 30 cycles of 95°C for 30 sec, 60°C
for 30 sec and 72°C for 1 min 20 sec, with a final extension at 72°C
for 10 min.
RT-PCR
Total RNA was extracted from approximately 100 mg of frozen
young leaves using an RNeasy plant mini kit (Qiagen, Venlo, Netherlands)
and RNA samples were treated with TURBO DNA-free
(Applied Biosystems/Ambion, http://www.lifetechnologies.com
TURBO DNA-free). The quality and quantity of RNA were
determined by electrophoresis on 1% w/v agarose gels and by measurement
of absorbance using NanoDropTM
2000/2000c
spectrophotometer (https://www.thermofisher.com/). Reverse transcription
was performed using random nonamers (Sigma-Aldrich,
http://www.sigmaaldrich.com) with 1 lg RNA and Mu-MLV RT
(New England Biolabs, https://www.neb.com/). Quantification of
transcript levels of the AtRuvBL1, AtRuvBL2a (Figure S10) and
AtTERT genes (Fojtova et al., 2011) was carried out by FastStart l
SYBR Green Master (Roche, Basel, Switzerland), a Rotorgene 6000
cycler (Qiagen) and using the Ubiquitin-10 gene as suitable references
for quantitative analyses in A. thaliana. A 2 ll aliquot of
cDNA, from two biological replicates, were added to the 20 ll reaction
mix; the final concentration of each forward and reverse primer
(sequences are given in Table S3) was 0.25 lM. Three technical replicates
were done for each reaction that was measured in triplicates;
the PCR cycle consisted of 15 min of initial denaturation followed by
40 cycles of 15 sec at 95°C, 20 sec at 56°C and 30 sec at 72°C. SYBR
Green I fluorescence was monitored consecutively after the extension
step (Fojtova et al., 2011) sequences of primers are given in
Table S3. Statistical analysis was performed using unpaired Student’s
t-test.
Quantitative TRAP assay
Protein extracts from buds were prepared as described by Fitzgerald
et al. (1996). qTRAP analysis was performed as described in
Herbert et al. (2006) using FastStart SYBR Green Master (Roche)
and TS21 and TEL-PR primers. Samples were analyzed in triplicate.
A 1 ll aliquot of extract diluted to 50 ng llÀ1
protein concentration
was added to the 20 ll reaction mix. Ct values were
determined using the Rotorgene 3000 (Qiagen) machine software,
and relative telomerase activity was calculated by the DCt method
(Pfaffl, 2004).
TRF analysis
TRF analysis was performed as described previously (Ruckova
et al., 2008) using 500 ng genomic DNA isolated from 5 to 7 weeks
old rosette leaves using NucleoSpin Plant II (Machery Nagel).
Hybridized samples (Hybond XL, GE Healthcare, Chicago, IL, USA)
by Southern hybridization method were radioactively marked by
random priming, in which the telomeric probe was prepared
according to a modified protocol from Ijdo et al. (1991). Telomeric
signals were visualized using an FLA7000 imager (Fujifilm, Tokyo,
Japan). Evaluation of fragment lengths was performed using a
Gene Ruler 1 kb DNA ladder (Fermentas, http://www.thermoscien
tificbio.com/fermentas/) as the standard. Mean telomere lengths
were calculated as described by Grant et al. (2001).
Yeast two-hybrid assay
Yeast two-hybrid experiments were performed using the Matchmaker
TM GAL4-based two-hybrid system (Clontech, Kyoto,
Japan) as described in Schrumpfova et al. (2014). AtRuvBL1 and
AtRuvBL2a constructs from pDONR/221 entry clones were subcloned
into the Gateway-compatible destination vector pGBKT7DEST
(bait vector) and pGADT7-DEST (prey vector). cDNA
sequences encoding AtTERT fragments from pDONR/221 entry
clones and AtCBF5 from PENTR223 entry clone were subcloned
into the Gateway-compatible destination vector pGBKT7-DEST
(bait vector). AtPOT1a constructs were subcloned from pDONR/
221 entry clones into the Gateway-compatible destination vector
pGADT7-DEST (prey vector). The pGADT7 prey vectors (Clontech)
carrying AtTRB1-3 and AtPOT1a have been described previously
(Schrumpfova et al., 2008). Successful co-transformation of each
bait/prey combination into Saccharomyces cerevisiae PJ69-4a was
confirmed on SD plates lacking Leu and Trp, and positive interactions
were selected on SD medium lacking Leu, Trp and His (with
or without 3-aminotriazol (3-AT)) or SD medium lacking Leu, Trp
and Ade. Co-transformation with an empty vector and homodimerization
of the AtTRB1 protein served as negative and positive
control, respectively (Schrumpfova et al., 2014). Protein expression
was verified by immunoblotting in equal amounts of protein
extracts separated by SDS-PAGE (12%), blotted onto nitrocellulose
membrane, and probed with mouse anti-Myc (1:1000; SigmaAldrich)
and mouse anti-HA (1:1000) primary antibodies binding
to specific protein epitope tags of AD- and BD-fusion proteins, followed
by an anti-mouse HRP-conjugated secondary antibodies
(1:8000; Sigma-Aldrich) for chemiluminescence detection.
In vitro translation and co-immunoprecipitation
Additionally, the Y2H constructs were employed for verification in
assay as described in Schrumpfova et al. (2008). Briefly, radioactively
(35
S-Met) labelled proteins with hemagglutinin tag (HA)
(pGADT7, pGADT7-DEST), as well as non-radioactively labelled
protein partners with a Myc-tag (pGBKT7, pGBKT7-DEST) were
separately expressed in the TNT Quick Coupled Transcription/
Translation System (TNT-RRL) (Promega, Fitchburg, WI, USA) in
50 ll of each reaction according to the manufacturer’s instructions.
The co-immunoprecipitation procedure was performed as
described by Schrumpfova et al. (2008) with 1 lg anti-Myc-tag
polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) and incubated
overnight at 4°C with 10 ll protein G magnetic particles
(Dynabeads, Invitrogen-Dynal).
© 2019 The Authors
The Plant Journal © 2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 98, 195–212
Characterization of plant Pontin and Reptin 209
During the co-immunoprecipitation with three proteins of interest,
two radioactively labeled proteins with HA-tag (AtRuvBL1,
AtTRB3) and one non-radioactively labeled AtTERT 1-271 fragment
with Myc-tag were expressed separately in TNT-RRL and incubated
in the same manner as previous Co-IP together with protein
G magnetic particles (Dynabeads, Invitrogen-Dynal) and 1 lg antiMyc-tag
polyclonal antibody (Sigma). Input, Unbound and Bound
fractions were separated by 12% SDS-PAGE and analyzed by
FLA7000 imager (Fujifilm).
Bimolecular fluorescence complementation
Arabidopsis thaliana leaf protoplasts were prepared and co-transfected
with DNA (10 lg of each construct) as was described in Lee
et al. (2012). The same entry vectors (pDONR/221, PENTR223),
already used for AtRuvBL1, AtRuvBL2a, AtTERT fragments,
AtCBF5 and AtPOT1a Y2H constructs cloning (Majerska et al.,
2017) or entry vectors used for cloning AtTRB1-3 (Schrumpfova
et al., 2008) were ligated into pSAT5-DEST-c(175-end)EYFPÀC1(B),
pSAT4-DEST-n(174)EYFPÀCl vectors. As a negative control, we
used the cYFP/AtGAUT10 construct. To control transformation efficiency
and to label cell nuclei, we co-transfected a plasmid
expressing mRFP fused to the nuclear localization signal of the
VirD2 protein of A. tumefaciens (mRFPÀVirD2NLS; Citovsky et al.,
2006). To label nucleolus we co-transfected a plasmid expressing
mRFP fused to the to AtFibrillarin 1 (Pih et al., 2000). Transfected
protoplasts were incubated in the light, at room temperature overnight,
and then observed for fluorescence using a Zeiss AxioImager
Z1 epifluorescence microscope equipped with filters for
YFP (Alexa Fluor 488), RFP (Texas Red) and CY5 (chloroplast autofluorescence).
The mRFPÀVirD2NLS and AtGAUT10ÀcEYFP constructs
for BiFC experiments were kindly provided by Prof.
Stanton B. Gelvin (Purdue University, USA).
ACCESSION NUMBERS
AtRuvBL1 (AT5G22330); AtRuvBL2a (AT5G67630); AtTERT
(AT5G16850); AtTRB1 (AT1G49950); AtTRB2, formerly TBP3
(AT5G67580); AtTRB3, formerly TBP2 (AT3G49850); AtCBF5
(AT3G57150); AtPOT1a (AT2G05210); GAUT10
(AT2G20810); AtFibrillarin 1 (AT5G52470).
ACKNOWLEDGEMENTS
This work was supported by the Czech Science Foundation (16-
1137S), by the project SYMBIT, reg. number: CZ.02.1.01/0.0/0.0/
15_003/0000477 financed by the ERDF, and by the Ministry of Education,
Youth and Sports of the Czech Republic under the project
CEITEC 2020 (LQ1601). We thank Inna Lermontova (IPK Gatersleben)
for AtCBF5 constructs. We further thank Eva Sykorova and
Vratislav Peska for providing us with the collection of TERT fragments
and Fibrillarin 1-RFP vectors. We thank to Miloslava Fojtova
for help with qTRAP assay. Jennifer McMahon is gratefully
acknowledged for proofreading. The access to the computing and
storage facilities owned by parties and projects contributing to the
National Grid Infrastructure MetaCentrum provided under the program
‘Projects of Large Infrastructure for Research, Development,
and Innovations’ (LM2010005) was highly appreciated, as was the
access to the CERIT-SC computing and storage facilities provided
under the program ‘Center CERIT Scientific Cloud’, part of the
Operational Program Research and Development for Innovations,
reg. no. CZ.1.05/3.2.00/08.0144. Core Facility Plant Sciences of CEITEC
MU is acknowledged for the cultivation of experimental
plants used in this paper.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
AUTHORS’ CONTRIBUTIONS
PPS designed the study, supervised the project and wrote
the manuscript with support from SS, JF, LZD and DH.SS
performed the experiments. LZD designed the phylogeny
analysis. JF helped to supervise the project and wrote the
manuscript. All authors discussed the results and contributed
to the final manuscript.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article.
Figure S1. AtRuvBL1 does not interact with either the RT-domain
or the C-terminus of AtTERT.
Figure S2. AtTRB2 proteins directly interact with AtRuvBL2a protein
and AtTRB1 is associated with AtRuvBL1 in BiFC assay.
Figure S3. Nucleolar or cytoplasmic localization of AtPOT1a–
AtCBF5 interactions.
Figure S4. Weak interaction between AtPOT1a and AtRuvBL1 pro-
teins.
Figure S5. Association of AtRuvBLs, AtTRBs and AtTERT in the
nucleolus in A. thaliana leaf protoplasts where the whole nucleus
is marked.
Figure S6. Relative transcript levels of AtTERT gene in AtRuvBL1
and AtRuvBL2a heterozygous mutant plants.
Figure S7. Terminal restriction fragment analysis.
Figure S8. Schematic illustration of specific primers and T-DNA
insertion location within the AtRuvBL1 and AtRuvBL2a genes.
Figure S9. Example of PCR analysis of genomic DNA isolated from
wild-type (Wt) plants and heterozygous AtRuvBL1 and AtRuvBL2a
plants.
Figure S10. Relative AtRuvBL1 and AtRuvBL2a transcript levels in
heterozygous AtRuvBL1 and AtRuvBL2a plants.
Table S1. The number of PPIs foci with exclusively nucleolar local-
ization.
Table S2. List of T-DNA insertion lines.
Table S3. List of primers.
Data S1. List of the analyzed plant species sorted by phylogenetic
system with number of homologues.
Data S2. List of the analyzed plant species for AtRuvBL homologues
and their accession numbers.
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212 Sarka Schorova et al.
SUPPORTING INFORMATION
Figure S1. AtRuvBL1 Does Not Interact with either the RT-domain or the C-terminus of AtTERT. The
analyses are performed as described in Figure 1.
(a) Schematic depiction of the catalytic subunit of plant telomerase - AtTERT.
(b) BiFC assay detects interaction between cYFP/AtRuvBL1 and the N-terminal part of AtTERT
(nYFP/AtTERT 1-271). No interactions are detected between AtRuvBL1 protein and AtTERT
fragments covering AtTERT domains localized in regions 229-582, 597-987 and 972-1123. No
interactions are observed between any of AtTERT fragments and AtRuvBL2a protein in A. thaliana
leaf protoplasts. PPIs are marked with white arrows. AtGAUT10, negative control; RFP, nucleus
marker; YFP, detects PPI; Chl, Chloroplast autofluorescence. Scale bars = 10 μm.
(c) Y2H system results show no interaction between AD/AtRuvBL1 nor AD/AtRuvBL2a proteins and
fragments of AtTERT fused with GAL4 DNA-binding domain (BD). Vectors with AD or BD without
gene of interest are used as negative control.
(d) Co-IP analysis does not detect interaction between AtTERT fragments and AtRuvBL1 or
AtRuvBL2a protein as is demonstrated by BiFC. I, Input; U, Unbound; B, Bound fractions;
asterisks*,35
S-labelling.
Figure S2. AtTRB2 Proteins Directly Interact with AtRuvBL2a Protein and AtTRB1 is Associated with
AtRuvBL1 in BiFC Assay. The methods are performed as is described in Figure 1.
(a) Schematic representation of the conserved motifs of the AtTRB1 and AtTRB2 proteins from A.
thaliana. Myb-like, Telobox-containing Myb domain; H1/H5, histone-like domain; coiled-coil, Cterminal
domain.
(b) BiFC assay reveals interactions between nYFP/AtTRB1 and cYFP/AtRuvBL1 and between
nYFP/AtTRB2 and cYFP/AtRuvBL2a. PPIs are marked with white arrows. AtGAUT10, negative
control; RFP, Red Fluorescent Protein, positive control; YFP, Yellow Fluorescent Protein, PPI; Chl,
Chloroplast autofluorescence, control. Scale bars = 10 μm.
(c) Y2H results confirm the interaction between AD/AtTRB2 and BD/AtRuvBL2 on His- deficient
plates, but not the interaction between AD/AtTRB1 and BD/AtRuvBL1. BD, GAL4 DNA-binding
domain; AD, GAL4 activation domain; asterisks*, 5mM 3-aminotriazol.
(d) Co-IP results confirm interactions between radioactively labelled AtTRB2 and Myc-tagged
AtRuvBL2 protein. I, Input; U, Unbound; B, Bound fractions; asterisks*,35S-labelling.
Figure S3. Nucleolar or Cytoplasmic Localization of AtPOT1a-AtCBF5 Interactions.
BiFC performed in A. thaliana leaf protoplasts show either clear nucleolar AtPOT1a-AtCBF5 foci or
cytoplasmic and nuclear foci. The examples of AtPOT1a-AtCBF5 interactions localized in the
nucleolus are given in the first four columns. The following four columns show AtPOT1a-AtCBF5
interactions localized in the cytoplasmic and nucleus foci. The whole nucleus was marked by mRFP-
VirD2NLS.
Figure S4. Weak Interaction Between AtPOT1a and AtRuvBL1 Proteins. The analyses are performed as
is described in Figure 1.
(a) Y2H results show weak but reproducible interactions between AtPOT1a and AtRuvBL1 protein
on His- deficient plates. BD, GAL4 DNA-binding domain; AD, GAL4 activation domain.
(b) Co-IP results confirm direct interactions between radioactively labelled AtRuvBL1 and Myctagged
AtPOT1a protein. I, Input; U, Unbound; B, Bound fractions; asterisks*, 35S-labelling
(c) BiFC does not detect any interaction between AtPOT1a and AtRuvBL1 proteins. AtGAUT10,
negative control; RFP, nucleus marker; YFP, detects PPI; Chl, Chloroplast autofluorescence. Scale
bars = 10 μm
Figure S5. Association of AtRuvBLs, AtTRBs and AtTERT in the nucleolus in A. thaliana Leaf Protoplasts
where the Whole Nucleus is Marked.
A. thaliana leaf protoplast are co-transfected with mRFP-VirD2NLS encoding RFP that labels the
whole nucleus and simultaneously with each of the plasmids encoding nYFP-tagged or cYFP-tagged
AtRuvBL1, AtRuvBL2a, AtTERT 1-271, AtTRB3 or AtCBF5 to determine PPI localization. AtRuvBL1AtTERT,
AtTRB3-AtTERT, AtRuvBL1-AtTRB3 or AtRuvBL2a-AtTRB3 interactions show nucleolar
localization. Plant homologue of mammalian dyskerin, AtCBF5, is associated with AtTRB3 in
nucleolus and in additional nuclear bodies at the periphery of nucleolus. RFP, marked nucleus; YFP,
detects PPI; Scale bars = 10 μm.
Figure S6. Relative Transcript Levels of AtTERT Gene in AtRuvBL1 and AtRuvBL2a Heterozygous Mutant
Plants.
No significant differences are detected in the transcript levels of AtTERT gene in the heterozygous
mutant plants in AtRuvBL1 gene. We detect a slight increase of transcripts of AtTERT gene in the
plants heterozygous in AtRuvBL2a genes compared to the wild-type. Levels or AtTERT transcripts
from WT Col-0 are arbitrarily set as 1. P values<0.05 are considered significant. Single star denotes
0.01<P<0.05.
Figure S7. Terminal Restriction Fragment Analysis
TRF analysis does not reveal any changes in telomere lengths in the analyzed AtRuvBL1+/- and
AtRuvBL2a+/- plants. Genomic DNA either before (b.c.) or after cleavage (a.c.) with MseI enzyme
was hybridized with radioactively labeled telomeric probe.
Figure S8. Schematic Illustration of Specific Primers and T-DNA Insertion Location within the atruvbl1
and atruvbl2a Genes.
Schematic illustration of locations of various T-DNA insertion lines available from several plant databases
(see also Table S2). T-insertion lines with limited number of heterozygous AtRuvBL1 and AtRuvBL2a
plants are marked with dark grey triangles (SAIL_397_C11, GABI-Kat_543F01, respectively). The other Tinsertion
lines are marked by light triangles, since they did not provide either homozygotes or
heterozygotes plants. Primers used for PCR analysis of genomic DNA (T-ins.) or analysis of relative
transcript levels (RT-PCR) are marked by black arrows.
Figure S9. Example of PCR Analysis of Genomic DNA Isolated from wild-type (Wt) Plants and
Heterozygous AtRuvBL1 and AtRuvBL2a Plants.
We used PCR analysis to distinguish heterozygous AtRuvBL1 and AtRuvBL2a plant lines from wildtype
AtRuvBL1and AtRuvBL2aplants. As positive control was used genomic DNA isolated from wildtype
Col-0 plants.
Figure S10. Relative AtRuvBL1 and AtRuvBL2a Transcript Levels in Heterozygous AtRuvBL1 and
AtRuvBL2a Plants.
RT-PCR was used to detect transcript levels in the heterozygous AtRuvBL1 and AtRuvBL2a plant
lines. The transcript levels of AtRuvBL1 and AtRuvBL2a genes were reduced compare to the wildtype
Col-0 plants in both heterozygous AtRuvBL1and AtRuvBL2a plants, respectively. Levels or
AtRuvBL1 and AtRuvBL2a transcripts from wild-type Col-0 are arbitrarily set as 1.
Table S1. The Number of PPIs Foci with Exclusively Nucleolar Localization.
Total
A. thaliana
protoplasts
Exclusively
nucleolar
localization
Nuclear and at the same time
nucleolar localization
AtRuvBL1-AtTRB3 30 27 3
AtRuvBL2a-AtTRB3 30 28 2
AtTRB3-AtTERT 30 28 2
AtRuvBL1-AtTERT 30 19 11
To quantify the numbers of the PPIs between AtRuvBL1-AtTRB3, AtRuvBL2a-AtTRB3, AtTRB3-AtTERT
and AtRuvBL1-AtTERT with exclusively nucleolar localization we performed at the same day several
BiFC analysis. Total 30 A. thaliana protoplasts from each protein-protein combination, with positive
PPIs, were analyzed for either exclusively nucleolar or nucleolar and at the same time nuclear
localization of these PPIs interactions.
Table S2. List of T-insertion Lines
AtRuvBL1 T-insertion lines AtRuvBL2a T-insertion lines
Wiscseq_DsLoxHs027_03G SALK_071103
WiscDsLoxHs117_06F SALK_144539
WiscDsLoxHs168_06D SALK_144540
SAIL_397_C11* SAIL_500_C04
GK - 543F01*
Accession numbers of T-insertion lines that are tested for the presence of mutation in the AtRuvBL1 or
AtRuvBL2a genes. Lines marked with asterisks provided limited number of heterozygous mutant
plants, that are analyzed.
Table S3. List of Primers
The name of primer Sequence (5'→3') Method
AtRuvBL1_F AAAAAGCAGGCTACATGGAGAAAGTAAAGATTGAAGAA Gateway cloning
AtRuvBL1_R AGAAAGCTGGGTGCTATGAGATGTATTTTTCTTGTTGCC Gateway cloning
AtRuvBL2a_F AAAAAGCAGGCTACATGGCGGAACTAAAGCTATCA Gateway cloning
AtRuvBL2a_R AGAAAGCTGGGTCTCAGATCTGCATAGCATCTTG Gateway cloning
attB1 adapter primer GGGACAAGTTTGTACAAAAAAGCAGGCT Gateway cloning
attB2 adapter primer GGGGACCACTTTGTACAAGAAAGCTGGGT Gateway cloning
AtRuvBL1_T-ins_F (SAIL_397_C11) TTTTTGTTTCGCCCTTTTCTC Genotyping
AtRuvBL1_T-ins_R (SAIL_397_C11) AGAGATTCCAAGAGCCAAAGC Genotyping
SAIL_T-ins_LB1 GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC Genotyping
AtRuvBL2a-T-ins_F (GK-543F01) ACAACTTGTTCGACCAAATGC Genotyping
AtRuvBL2a-T-ins_R (GK-543F01) TTTGATCCTAACACCAATCGC Genotyping
GK-T-ins_8474_F ATAATAACGCTGCGGACATCTACATTTT Genotyping
R1_Wisc_027_03G.6_LP CGGTTAATTCTTCAACTTGCG Genotyping
R1_Wisc_027_03G.6_RP TCAGTTTGTTCCAAAACCTGC Genotyping
R1_Wisc_117_06F_LP AAACCGCAAATAGCAACACAC Genotyping
R1_Wisc_117_06F_RP GCGTTGCAAACTCAATAAAGC Genotyping
R1_Wisc_168_06D_LP ATTTGCTGCATCCAGATCTTG Genotyping
Wisc_T-ins AACGTCCGCAATGTGTTATTAAGTTGTC Genotyping
R1_Wisc_168_06D_RP TAAAATTGGCAGCTGGATTTG Genotyping
R2a_SAIL_500_C04.2_LP CTTTTCGTAAAGCGATTGGTG Genotyping
R2a_SAIL_500_C04.2_RP ACGTCCGATCAATGTCAAAAG Genotyping
R2a_SALK_071103.2_LP CGGGTCGGGCTATTCTAATAG Genotyping
R2a_SALK_071103.2_RP ACAAGGATTGGTGACATTTCG Genotyping
R2a_SALK_144539_LP AATTGGGGATTGCAGAGAAAC Genotyping
R2a_SALK_144539_RP CTATTAGAATAGCCCGACCCG Genotyping
R2a_SALK_144540_LP AATTGGGGATTGCAGAGAAAC Genotyping
R2a_SALK_144540_RP CTATTAGAATAGCCCGACCCG Genotyping
SALK_T-ins_LBb1.3 ATTTTGCCGATTTCGGAAC Genotyping
TS21 GACAATCCGTCGAGCAGAGTT qTRAP assay
TEL-PR CCGAATTCAACCCTAAACCCTAAACCCTAAACCC qTRAP assay
Fw_RuvBL1_SAIL_397_C11_RT-
PCR
CAACGGATTGCTACTCACAC RT-PCR
Rev_RuvBL1_SAIL_397_C11_RT-
PCR
AAGAGCCAAAGCTGTTTTCC RT-PCR
Fw_GK_543F01_RuvBL2a_RT-PCR TATGGTCGGTCAAGTGAAGG RT-PCR
Rev_GK_543F01_RuvBL2a_RT-PCR GGGTTGACCCGCTATTAGAA RT-PCR
Fw_AtTERT_ex1 CCGATGATCCCATTCACTACCGTAAACT RT-PCR
Rev_AtTERT_ex1 TCTCTGTGACCACCAAGATGTTGGAGA RT-PCR
Data S1. List of the Analyzed Plant Species Sorted by Phylogenetic System with Number of Homologues.
Order Family Species No.RuvBL homologues
BRYOPHYTES Marchantiales Marchantiaceae
Marchantia polymorpha ssp.
polymorpha 1
Funariales Funariaceae Physcomitrella patens 1
GYMNOSPERMS Pinales Pinaceae Picea sitchensis 1
BASAL ANGIOSPERMS Amborellales Amborellaceae Amborella trichopoda 3
MONOCOTS
Alismatales Araceae Anthurium amnicola 3
Arecales Arecaceae
Elaeis guineensis 2
Phoenix dactylifera 3
Zingiberales Musaceae Musa acuminata ssp. malaccensis 1
Poales
Bromeliaceae Ananas comosus 1
Poaceae
Aegilops tauschii 1
Brachypodium distachyon 1
Dichanthelium oligosanthes 1
Hordeum vulgare ssp. vulgare 1
Oryza brachyantha 1
Oryza sativa var. japonica 1
Setaria italica 1
Zea mays 1
DICOTS
Proteales Nelumbonaceae Nelumbo nucifera 3
Vitales Vitaceae Vitis vinifera 4
Fabales Fabaceae
Arachis duranensis 1
Arachis ipaensis 2
Cajanus cajan 1
Cicer arietinum 1
Lupinus angustifolius 4
Medicago truncatula 3
Phaseolus vulgaris 2
Trifolium subterraneum 1
Vigna angularis 2
Vigna radiata ssp. radiata 2
Rosales
Moraceae Morus notabilis 1
Rhamnaceae Ziziphus jujuba 2
Rosaceae
Fragaria vesca ssp. vesca 3
Malus domestica 1
Prunus persica 2
Pyrus x bretschneideri 4
Fagales Juglandaceae Juglans regia 2
Cucurbitales Cucurbitaceae
Cucumis melo 1
Cucumis sativus 2
Oxalidales Cephalotaceae Cephalotus follicularis 2
Malphigiales Euphorbiaceae Jatropha curcas 4
Manihot esculenta 3
Ricinus communis 1
Salicaceae
Populus euphratica 3
Populus trichocarpa 3
Myrtales Myrtaceae Eucalyptus grandis 2
Sapindales Rutaceae
Citrus clementina 3
Citrus sinensis 1
Malvales Malvaceae
Corchorus capsularis 2
Corchorus olitorius 1
Glycine max 3
Gossypium arboreum 4
Gossypium hirsutum 5
Gossypium raimondii 5
Theobroma cacao 4
Cleomaceae Tarenaya hassleriana 3
Brassicaceae
Arabidopsis lyrata ssp. lyrata 2
Arabidopsis thaliana 3
Arabis alpina 2
Brassica napus 8
Brassica oleracea var. oleracea 4
Brassica rapa 4
Camelina sativa 6
Capsella rubella 3
Eutrema salsugineum 2
Noccaea caerulescens 3
Raphanus sativus 3
Caryophyllales Amaranthaceae Beta vulgaris spp. vulgaris 1
Gentianales Rubiaceae Coffea canephora 2
Solanales
Convolvulaceae Ipomoea nil 3
Solanaceae
Capsicum annuum 2
Nicotiana attaenuata 2
Nicotiana sylvestris 2
Nicotiana tabacum 1
Nicotiana tomentosiformis 2
Solanum lycopersicum 2
Solanum tuberosum 2
Spinacia oleracea 1
Lamiales
Gesneriaceae Dorcoceras hygrometricum 2
Phrymaceae Erythranthe guttata 2
Lentibulariacea
e Genlisea aurea 1
Pedaliaceae Sesamum indicum 2
Asterales Asteraceae Cynara cardunculus ssp. scolymus 2
Apiales Apiaceae Daucus carota var. sativus 3
Data S2. List of the Analyzed Plant Species for RuvBL Homologues and Their Accession Numbers.
Species Codes Acc. Nos.
Aegilops tauschii Aegilops tauschii RuvBL2 EMT28256.1
Amborella trichopoda Amborella trichopoda hypot ERN15972.1
Amborella trichopoda RuvBL1 XP_006854505.2
Amborella trichopoda RuvBL2 XP_006852153.1
Ananas comosus Ananas comosus RuvBL1 OAY71163.1
Anthurium amnicola Anthurium amnicola RuvBL1 JAT57168.1
Anthurium amnicola RuvBL1 JAT60842.1
Anthurium amnicola RuvBL2 JAT67104.1
Arabidopsis lyrata ssp. lyrata Arabidopsis lyrata lyrata hypot a XP_002864989.1
Arabidopsis lyrata lyrata hypot b XP_002874064.1
Arabidopsis thaliana Arabidopsis thaliana RuvBL1 NP_197625.1
Arabidopsis thaliana RuvBL2 a NP_201564.1
Arabidopsis thaliana RuvBL2 b NP_190552.1
Arabis alpina Arabis alpina hypot a KFK23151.1
Arabis alpina hypot b KFK28446.1
Arachis duranensis Arachis duranensis RuvBL2 XP_015972776.1
Arachis ipaensis Arachis ipaensis RuvBL1 XP_016172459.1
Arachis ipaensis RuvBL2 XP_016166745.1
Beta vulgaris ssp. vulgaris Beta vulgaris vulgaris RuvBL2 XP_010679660.1
Brachypodium distachyon Brachypodium distachyon RuvBL2 XP_003562823.1
Brassica napus Brassica napus hypot a CDX88801.1
Brassica napus hypot b CDX98571.1
Brassica napus hypot c CDY09131.1
Brassica napus hypot d CDY35680.1
Brassica napus RuvBL1 a XP_013667200.1
Brassica napus RuvBL1 b XP_013680083.1
Brassica napus RuvBL1 c XP_013715810.1
Brassica napus RuvBL1 d XP_013723522.1
Brassica oleracea var. oleracea Brassica oleracea oleracea RuvBL1 a XP_013612873.1
Brassica oleracea oleracea RuvBL1 b XP_013621188.1
Brassica oleracea oleracea RuvBL1 c XP_013625320.1
Brassica oleracea oleracea RuvBL2 XP_013630404.1
Brassica rapa Brassica rapa RuvBL2 XP_009150681.1
Brassica rapa RuvBL1 a XP_009120666.1
Brassica rapa RuvBL1 b XP_009126536.1
Brassica rapa RuvBL1 c XP_009131872.1
Cajanus cajan Cajanus cajan RuvBL2 KYP76213.1
Camelina sativa Camelina sativa RuvBL1 XP_010454496.1
Camelina sativa RuvBL1 XP_019084763.1
Camelina sativa RuvBL2 XP_010463807.1
Camelina sativa RuvBL2 XP_010484449.1
Camelina sativa RuvBL2 a XP_010426510.1
Camelina sativa RuvBL2 b XP_010444617.1
Capsella rubella Capsella rubella hypot a XP_006282280.1
Capsella rubella hypot b XP_006287714.1
Capsella rubella hypot c XP_006292981.1
Capsicum annuum Capsicum annuum RuvBL1 XP_016548155.1
Capsicum annuum RuvBL2 XP_016570371.1
Cephalotus follicularis Cephalotus follicularis hypot a GAV57713.1
Cephalotus follicularis hypot b GAV85060.1
Cicer arietinum Cicer arietinum RuvBL1 NP_001265936.1
Citrus clementina Citrus clementina hypot a XP_006426097.1
Citrus clementina hypot b XP_006430524.1
Citrus clementina hypot c XP_006430525.1
Citrus sinensis Citrus sinensis RuvBL2 XP_006466461.1
Coffea canephora Coffea canephora hypot a CDP02620.1
Coffea canephora hypot b CDP07117.1
Corchorus capsularis Corchorus capsularis hypot a OMO56475.1
Corchorus capsularis hypot b OMO91824.1
Corchorus olitorius Corchorus olitorius hypot OMP01809.1
Cucumis melo Cucumis melo RuvBL1 XP_008443365.1
Cucumis sativus Cucumis sativus RuvBL2 XP_004143406.1
Cucumis sativus RuvBL1 XP_004136684.1
Cynara cardunculus ssp. scolymus Cynara cardunculus scolymus hypot a KVI07257.1
Cynara cardunculus scolymus hypot b KVI09360.1
Daucus carota var. sativus Daucus carota sativus hypot KZN05222.1
Daucus carota sativus RuvBL2 XP_017215222.1
Daucus carota sativus RuvBL1 XP_017231242.1
Dichanthelium oligosanthes Dichanthelium oligosanthes RuvBL2 OEL27319.1
Dorcoceras hygrometricum Dorcoceras hygrometricum RuvBL1 KZV49640.1
Dorcoceras hygrometricum RuvBL2 KZV33016.1
Elaeis guineensis Elaeis guineensis RuvBL1 XP_010922426.1
Elaeis guineensis RuvBL2 XP_010943144.1
Erythranthe guttata Erythranthe guttata RuvBL1 XP_012836027.1
Erythranthe guttata RuvBL2 XP_012854107.1
Eucalyptus grandis Eucalyptus grandis RuvBL2 XP_010028079.1
Eucalyptus grandis RuvBL1 XP_010031536.1
Eutrema salsugineum Eutrema salsugineum hypot a XP_006384768.1
Eutrema salsugineum hypot b XP_006400738.1
Fragaria vesca ssp. vesca Fragaria vesca vesca RuvBL1 XP_004294694.1
Fragaria vesca vesca RuvBL1 XP_011458673.1
Fragaria vesca vesca RuvBL2 XP_004288244.1
Genlisea aurea Genlisea aurea hypot EPS69881.1
Glycine max Glycine max RuvBL2 a XP_014626670.1
Glycine max RuvBL2 b XP_014634945.1
Glycine max RuvBL1 XP_006583068.1
Gossypium arboreum Gossypium arboreum RuvBL1 a XP_017603372.1
Gossypium arboreum RuvBL1 b XP_017608006.1
Gossypium arboreum RuvBL1 c XP_017608007.1
Gossypium arboreum RuvBL2 XP_017638900.1
Gossypium hirsutum Gossypium hirsutum RuvBL1 a XP_016669038.1
Gossypium hirsutum RuvBL1 b XP_016669039.1
Gossypium hirsutum RuvBL1 c XP_016750696.1
Gossypium hirsutum RuvBL2 a XP_016697338.1
Gossypium hirsutum RuvBL2 b XP_016740101.1
Gossypium raimondii Gossypium raimondii RuvBL1 a XP_012447317.1
Gossypium raimondii RuvBL1 b XP_012485133.1
Gossypium raimondii RuvBL1 c XP_012485134.1
Gossypium raimondii RuvBL2 a XP_012470737.1
Gossypium raimondii RuvBL2 b XP_012491995.1
Hordeum vulgare ssp. vulgare Hordeum vulgare vulgare hypot BAK05068.1
Ipomoea nil Ipomoea nil RuvBL1 XP_019167132.1
Ipomoea nil RuvBL2 a XP_019173339.1
Ipomoea nil RuvBL2 b XP_019182975.1
Jatropha curcas Jatropha curcas RuvBL1 a XP_012068093.1
Jatropha curcas RuvBL1 b XP_012068094.1
Jatropha curcas RuvBL1 c XP_012068095.1
Jatropha curcas RuvBL2 XP_012079431.1
Juglans regia Juglans regia RuvBL1 XP_018844889.1
Juglans regia RuvBL2 XP_018817409.1
Lupinus angustifolius Lupinus angustifolius RuvBL1 a XP_019436439.1
Lupinus angustifolius RuvBL1 b XP_019454631.1
Lupinus angustifolius RuvBL2 a XP_019433128.1
Lupinus angustifolius RuvBL2 b XP_019449887.1
Malus domestica Malus domestica RuvBL1 XP_008378810.1
Manihot esculenta Manihot esculenta hypot a OAY33600.1
Manihot esculenta hypot b OAY35617.1
Manihot esculenta hypot c OAY51378.1
Marchantia polymorpha ssp. polymorpha Marchantia polymorpha hypot OAE28740.1
Medicago truncatula Medicago truncatula hypot a XP_003600480.1
Medicago truncatula hypot b XP_003603117.1
Medicago truncatula hypot c XP_003618820.1
Morus notabilis Morus notabilis RuvBL2 XP_010106923.1
Musa acuminata ssp. malaccensis Musa acuminata malaccensis RuvBL1 XP_009397499.1
Nelumbo nucifera Nelumbo nucifera RuvBL1 XP_010248994.1
Nelumbo nucifera RuvBL1 XP_010250729.1
Nelumbo nucifera RuvBL2 XP_010273117.1
Nicotiana attaenuata Nicotiana attaenuata RuvBL1 XP_019255439.1
Nicotiana attaenuata RuvBL2 XP_019251608.1
Nicotiana sylvestris Nicotiana sylvestris RuvBL1 XP_009798172.1
Nicotiana sylvestris RuvBL2 XP_009785904.1
Nicotiana tabacum Nicotiana tabacum RuvBL1 XP_016434317.1
Nicotiana tomentosiformis Nicotiana tomentosiformis RuvBL1 XP_009592138.1
Nicotiana tomentosiformis RuvBL2 XP_009615360.1
Noccaea caerulescens Noccaea caerulescens RuvBL1 JAU05222.1
Noccaea caerulescens RuvBL1 b JAU35471.1
Noccaea caerulescens RuvBL2 JAU89129.1
Oryza brachyantha Oryza brachyantha RuvBL2 XP_006657884.2
Oryza sativa var. japonica Oryza sativa japonica RuvBL2 XP_015644421.1
Phaseolus vulgaris Phaseolus vulgaris a XP_007135667.1
Phaseolus vulgaris b XP_007146707.1
Phoenix dactylifera Phoenix dactylifera RuvBL1 XP_008776327.1
Phoenix dactylifera RuvBL1 XP_008788050.1
Phoenix dactylifera RuvBL2 XP_008806201.1
Physcomitrella patens Physcomitrella patens hypot XP_001779312.1
Picea sitchensis Picea sitchensis hypot ABR17735.1
Populus euphratica Populus euphratica RuvBL1 XP_011006555.1
Populus euphratica RuvBL2 a XP_011015752.1
Populus euphratica RuvBL2 b XP_011018846.1
Populus trichocarpa Populus trichocarpa XP_002323491.1
Populus trichocarpa XP_006381348.1
Populus trichocarpa Ruv XP_006384768.1
Prunus persica Prunus persica a XP_007205168.1
Prunus persica b XP_007223107.1
Pyrus x bretschneideri Pyrus bretschneideri RuvBL1 a XP_009374971.1
Pyrus bretschneideri RuvBL1 b XP_009377811.1
Pyrus bretschneideri RuvBL2 a XP_009362554.1
Pyrus bretschneideri RuvBL2 b XP_009379332.1
Raphanus sativus Raphanus sativus RuvBL1 a XP_018442937.1
Raphanus sativus RuvBL1 b XP_018471784.1
Raphanus sativus RuvBL2 XP_018493256.1
Ricinus communis Ricinus communis RuvBL1 XP_002523847.1
Sesamum indicum Sesamum indicum RuvBL1 XP_011071718.1
Sesamum indicum RuvBL2 XP_011073680.1
Setaria italica Setaria italica RuvBL2 XP_004957042.1
Solanum lycopersicum Solanum lycopersicum RuvBL1 XP_004241955.1
Solanum lycopersicum RuvBL2 XP_004234506.1
Solanum tuberosum Solanum tuberosum RuvBL1 XP_006365976.1
Solanum tuberosum RuvBL2 XP_006343323.1
Spinacia oleracea Spinacia oleracea hypot KNA25984.1
Tarenaya hassleriana Tarenaya hassleriana RuvBL1 a XP_010520446.1
Tarenaya hassleriana RuvBL1 b XP_010535704.1
Tarenaya hassleriana RuvBL2 XP_010536617.1
Theobroma cacao Theobroma cacao hypot a EOY33372.1
Theobroma cacao hypot b EOY33373.1
Theobroma cacao RuvBL1 XP_017983833.1
Theobroma cacao RuvBL2 XP_007047571.1
Trifolium subterraneum Trifolium subterraneum GAU13448.1
Vigna angularis Vigna angularis RuvBL1 XP_017407371.1
Vigna angularis RuvBL2 XP_017436661.1
Vigna radiata ssp. radiata Vigna radiata radiata RuvBL1 XP_014515071.1
Vigna radiata radiata RuvBL2 XP_014519124.1
Vitis vinifera Vitis vinifera b CBI16308.3
Vitis vinifera RuvBL1 XP_002285127.1
Vitis vinifera RuvBL2 CAN80826.1
Vitis vinifera RuvBL2 XP_003635231.2
Zea mays Zea mays RuvBL2 NP_001148563.1
Ziziphus jujuba Ziziphus jujuba RuvBL1 XP_015895701.1
Ziziphus jujuba RuvBL2 XP_015890340.1
Supplement O
Schrumpfová, P.P.* and Fajkus, J. 2020. Composition and Function of Telomerase - a
polymerase associated with the origin of eukaryotes. Review. Biomolecules, 10(10):1425
P.P.S. was significantly involved in the ms writing and editing
Biomolecules 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/biomolecules
Review1
Composition and Function of Telomerase – a2
polymerase associated with the origin of eukaryotes3
Petra Procházková Schrumpfová 1,2,*, Jiří Fajkus 1,2,34
1 Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of5
Science, Masaryk University, Kotlářská 2, CZ-61137, Brno, Czech Republic.6
2 Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk7
University, Kamenice 5, CZ-62500, Brno, Czech Republic.8
3 The Czech Academy of Sciences, Institute of Biophysics, Královopolská 135, 612 65, Brno, Czech Republic.9
petra.proch.schrumpfova@gmail.com (P.P.S), fajkus@sci.muni.cz (J.F)10
* Correspondence: petra.proch.schrumpfova@gmail.com11
Received: date; Accepted: date; Published: date12
Abstract: Canonical DNA polymerases involved in replication of the genome are unable to fully13
replicate the physical ends of linear chromosomes, called telomeres. Chromosomal termini thus14
become shortened in each cell cycle. The maintenance of telomeres requires telomerase - a specific15
RNA-dependent DNA polymerase enzyme complex that carries its own RNA template and adds16
telomeric repeats to the ends of chromosomes using a reverse transcription mechanism. Both core17
subunits of telomerase - its catalytic telomerase reverse transcriptase (TERT) subunit and telomerase18
RNA (TR) component – were identified in quick succession in Tetrahymena more than 30 years ago.19
Since then both telomerase subunits have been described in various organisms including yeasts,20
mammals, birds, reptiles and fish. Despite the fact that telomerase activity in plants was described21
25 years ago and the TERT subunit four years later, a genuine plant TR has only recently been22
identified by our group. In this review, we focus on the structure, composition and function of23
telomerases. In addition, we discuss the origin and phylogenetic divergence of this unique RNA-24
dependent DNA polymerase as a witness of early eukaryotic evolution. Specifically, we discuss the25
latest information regarding the recently discovered TR component in plants, its conservation and26
structural features.27
Keywords: telomerase; evolution; telomerase RNA (TR); telomerase reverse transcriptase (TERT);28
plant TERT; plant TR.29
30
1. Telomerase Activity31
Telomerase reverse transcriptase is a specific nucleoprotein enzyme complex that solves the32
problem that conventional DNA replication machinery cannot - to fill the gap after removal of the33
RNA primer of a most distal Okazaki fragment at the 5’ - terminus of the lagging strand. This results34
in a loss of a small portion of chromosomal DNA. This phenomenon is called the end-replication35
problem (Figure 1), first defined by Olovnikov [1]. Moreover, the ends of eukaryotic chromosomes -36
telomeres - must be long enough to assemble a protective nucleoprotein “capping” structure that can37
distinguish a natural terminus from an unrepaired chromosomal break. Dysfunctional telomeres may38
trigger genome instability, cell cycle arrest, and – at least in humans- replicative cell senescence and39
apoptosis (reviewed in [2,3]).40
41
42
Biomolecules 2020, 10, x FOR PEER REVIEW 2 of 23
Figure 1. The replicating DNA in eukaryotes: DNA polymerases involved in replication. During43
semiconservative DNA replication, each strand serves as a template for DNA polymerases to44
synthesize a new complementary strand. A specialized RNA polymerase (primase), that is a part of45
DNA Pol α, synthesizes the RNA primer. A single RNA primer aids DNA replication on the leading46
strand and multiple primers initiate Okazaki fragment synthesis on the lagging strand. Further DNA47
synthesis is carried out by DNA Pol ε and DNA Pol δ (reviewed in [4]). The newly replicated telomere48
resulting from the lagging strand synthesis (Lagging telomere) retains the terminal RNA primer,49
which is subsequently removed. Attachment of the last RNA primer more proximally on the DNA50
strand, together with RNA-primer removal, creates an overhang on the G-rich strand. The initial51
product of the leading strand DNA synthesis (Leading telomere) is a blunt terminus whose C-strand52
is then resected by an exonuclease to create the mature G-rich overhang. In cells with an active RNA-53
dependent DNA polymerase (Telomerase), the G-rich overhangs originating from Lagging or54
Leading telomeres, can undergo elongation (reviewed in [5]). Telomerase carries its own RNA55
molecule, which is used as a template, and can anneal through the first few nucleotides of its template56
region to the distal-most nucleotides of the G-rich overhang of the telomere DNA, add a new telomere57
repeat (GGTTAG) sequence, translocate and then repeat the process. The complementary C-strand is58
then in-filled by DNA Pol α-primase [6].59
60
Biomolecules 2020, 10, x FOR PEER REVIEW 3 of 23
61
In humans, telomerase activity is detected in all early developmental stages and increases62
progressively with advancing embryonic stages. After completion of organogenesis in the human63
fetus, telomerase is expressed only in proliferating tissue-specific stem cells (e.g., bone marrow64
progenitor cells and neural stem cells), while telomerase activity in somatic cells is downregulated65
(reviewed in [7]. However, a tendency to repress telomerase in mammalian somatic tissues was66
described only for mammalian species of weight greater than 1 kg; e.g., laboratory mice have a67
constitutive telomerase. It was proposed that in long-lived species, telomerase downregulation may68
have evolved to limit cell proliferation and reduce the risk of cancer. Correspondingly, ca. 90% of all69
human tumors display telomerase reactivation to achieve cellular immortality [8].70
Telomerase, as a primary mechanism for telomere maintenance, is also conserved in plants.71
Analogous to mammals, telomerase activity is suppressed in terminally differentiated tissues, e.g.,72
mature leaves or stems. Active telomerase is detected in organs and tissues such as seedlings, shoot73
and root tips, young and middle-age leaves, flowers, and floral buds with proliferating meristematic74
cells [7,9–13].75
Telomerase carries, in addition to its protein catalytic subunit (telomerase reverse transcriptase;76
TERT), its own RNA templating subunit (telomerase RNA; TR) (reviewed in [14]). The expression of77
human TERT is strictly controlled at the transcript level and closely associated with telomerase78
activity, which suggests that hTERT is the primary determinant of enzyme activity [15]. In most79
human tissues, TR is ubiquitously expressed regardless of telomerase activity, and therefore, it has80
been considered by some authors as a non-limiting factor for telomerase activity [16]. However,81
telomerase activity in human T lymphocytes has been reported to relate to hTR levels but not hTERT82
protein levels [17,18].83
In plants, contrary to most human cells, expression of the TR subunit, recently characterized by84
Fajkus et al. (2019) [19], follows a tissue-specific pattern similar to that which is typical for expression85
of TERT. In thale cress (Arabidopsis thaliana), the highest TERT mRNA levels were detected in flower86
buds, lower transcript levels were detected in seedlings and young leaves, and the lowest levels were87
observed in aged leaves [11]. Similarly, TR transcripts were most abundant in flower buds and 7-day-88
old seedlings. Markedly lower, yet detectable levels, were observed using RT-qPCR in 3-week-old89
seedlings and young leaves. Absolute levels of TR transcripts were 60-70 times higher than TERT90
mRNA levels [19]. Whole-mount in situ hybridization detected TR transcripts in primary root and91
lateral root apices of 3-week-old seedlings and in cultured cells, but in other tissue samples, using92
northern hybridization, no TR signal was found [20]. Levels of TR or TERT transcripts correlate93
strongly with telomerase activities observed in various plant tissues [7,11].94
95
2. The Origin of Telomerase96
The telomerase RNA-dependent DNA polymerase arose specifically within the eukaryotic97
lineage and was able to successfully solve the end-replication problem of linear chromosomes that98
leads to telomere shortening [21]. Telomeres are composed of short non-coding tandem repeat units,99
the length of which can significantly vary among diverse taxons. The lengths of telomere arrays can100
also vary at the level of the species or ecotypes (reviewed in [22,23]). The human-type (TTAGGG)n101
telomeric sequence is conserved across several eukaryotic ‘supergroups’ [24] including Amorphea102
supergroup with metazoan and fungal species (reviewed in [25,26]. At the same time, several103
exceptions are known across the Amorphea supergroup, e.g., in insects (TTAGG)n [27,28], in104
Nematodes (TTAGGC)n [29] or in several fungal genera, where very complex or irregular telomeric105
runs were described [26,30] (Figure 2). Additionally, telomeres of some insects are constituted with106
unusual telomeric motifs of even with telomeric repeats that consist of arrays of non-long-terminal-107
repeat (non-LTR) retrotransposons [31–33]. The TERT gene disappeared from the genomes of some108
insects with non-LTR retrotransposons, as in the vinegar fly (e.g. Drosophila melanogaster). In silkworm109
(Bombyx mori), TERT is very weakly expressed in various tissues, telomerase activity is barely110
detectable and retrotransposition is required to maintain the length of chromosome ends [33–36].111
Biomolecules 2020, 10, x FOR PEER REVIEW 4 of 23
Figure 2. Telomeres and Telomerase in the Evolutionary Tree. A simplified phylogenetic tree is112
shown, where telomeres and telomerase evolved upon linearization of chromosomes by the insertion113
of Group II self-splicing introns [37]. In the Eukaryote branch, the groupings correspond to the114
current ‘supergroups’ according to the recent eukaryotic Tree of Life (eToL) [24]. Unresolved115
branching orders among lineages are shown as multifurcations. Broken lines reflect lesser116
uncertainties about the monophyly of certain groups. Examples of known telomeric repeat variants117
are listed next to respective supergroups (see also Table S1). The major known telomeric repeat118
variants in the supergroups are marked with a larger font [22,36,38] (see text for details). Last119
eukaryote common ancestor (LECA); last universal common ancestor (LUCA). The living species120
icons are partly adopted from Adl et al., 2012 [39].121
122
123
In the Archaeplastida supergroup that includes the land plants, mosses, red algae and green124
algae, the telomere is mostly composed of (TTTAGGG)n repeats, first described in A. thaliana125
(Arabidopsis-type repeats [40]. Despite this, the human-type telomere repeat is shared by several126
plant taxa from the order Asparagales [41], including species of the Allioideae subfamily, except for127
the Allium genus [42], where a more complex telomeric sequence (CTCGGTTATGGG)n was128
described [43]. An unusual telomeric motif (TTTTTTAGGG)n was found in the genus Cestrum129
(Solanaceae) [44], and in some species from the carnivorous genus Genlisea (TTCAGG and130
TTTCAGG) [45]. Outside of land plants, repeats other than the Arabidopsis-type were characterized131
in some algae and glaucophyte species ((AATGGGGGG)n , (TTTTAGGG)n, (TTTTAGG)n etc.)132
[22,38,46]. Unlike insects, even the unusual plant telomeric sequences characterized so far are133
synthesized by telomerases using TRs with corresponding template regions [19,43,44,47].134
Biomolecules 2020, 10, x FOR PEER REVIEW 5 of 23
Before the linear chromosomes of eukaryotes emerged, ∼1 Gy ago, circular chromosomes had135
been successfully used for 2 Gy in eubacteria and archaea, and they still predominate in most bacterial136
forms [48]. In a concept elaborated by E. V. Koonin [37], the origin of linear chromosomes, telomeres137
and telomerase is associated with invasion of archaeal hosts by an alpha-proteobacterial progenitor138
that resulted in mitochondrial endosymbiosis and invasion of Group II self-splicing introns, the most139
ancient genetic entities (Figure 2). Group II introns were suggested as eukaryotic evolutionary140
ancestors of retrotransposons and spliceosomal introns. They consist of a catalytically active intron141
RNA and an intron-encoded reverse transcriptase (RT), which is related to non-LTR-retrotransposon142
RTs and assists splicing by stabilizing the catalytically active RNA structure (reviewed in [49].143
Invasion of Group II introns resulted in the evolution of spliceosomal introns, compartmentalization144
of the majority of genetic information in the nucleus and linearization of chromosomes.At the same145
time, solution of the end-replication problem was hinted at by a mechanism pre-existing in a146
primordial pool of 'virus-like' genetic elements in the earliest stages of life's evolution [37]. It seems147
that incipient eukaryotes, due to the presence of Group II sequences, could have had stable linear148
chromosomes without the need for telomerase or telomere specific proteins [48].149
The TERT component of telomerase is highly conserved, having a centrally positioned reverse150
transcriptase motif (RT domain) [50–52]. The presence of TERT was detected in early branching151
eukaryotes [53,54]. It was proposed that telomerase originated as an ancient reverse transcriptase152
(RT) that internalized a primitive template-bearing RNA during early eukaryotic evolution and later153
evolved into modern telomerase RNPs with various indispensable and stably TR-associated154
components [55,56]. In accordance with this notion, the TERT subunit has the ability to bind the RNA155
molecule that provides the template sequence for DNA synthesis (TR) and various non-telomeric156
sequences [57,58]. The conserved RT motifs between TERT and other RTs indicates that TERT protein157
is closely related to RTs from the group of Penelope-like Elements (PLEs) and non-long-terminal-158
repeat (non-LTR) retrotransposons [55]. PLEs have an RT that lacks endonuclease activity and it is159
plausible that ancient retrotransposons similar to these terminal PLEs might be the progenitors of160
TERT proteins [56].161
Interestingly, besides the telomerase-dependent mechanism of telomere elongation, yeast,162
mammalian, as well as plant cells can use alternative mechanisms of lengthening of telomeres (ALT)163
based on homologous recombination (HR). ALT usually results in telomeres that are highly164
heterogeneous in length and sequence [59–61]. In plants, the ALT mechanism is activated in mutants165
with telomerase dysfunction and possibly also during the earliest stages of normal plant166
development [59–62].167
It is suggested that the canonical telomeric repeats have been changed or lost independently168
several times during evolution [36,46] and telomerase may have even occasionally been lost [36]. It169
would be interesting to further investigate the occurrence of unusual telomeric motifs, the co-170
evolution of genes encoding core telomerase subunits (TERT and TR), and replacement of telomerase171
by telomerase independent ALT systems (probably the ancestral telomere maintenance tools) in cases172
where there is evolutionary loss of telomerase.173
174
3. RNA subunit of Telomerase175
The non-coding RNA serving as telomerase RNA (TR), also known as TER or TERC, contains176
the template region for addition of telomeric repeats. TR is highly divergent, compared to TERT,177
ranging in size from ∼150 nucleotides (nt) in ciliates to over 3000-nt in yeasts [63]. TR has a number178
of conserved structural domains, consisting of the template/pseudoknot (t/PK) domain, template179
boundary element (TBE) and stem-loop region. In humans, the stem-loop region contains conserved180
structural domains: A conserved region 4/5 (CR4/5); the 3' H/ACA (H-box (consensus ANANNA); an181
ACA-box (ACA) domain; and a Cajal body box (CAB-box) motif (reviewed in [64,65]) (Figure 3a).182
Although the gene coding telomerase RNA had already been identified in Tetrahymena in 1989, and183
in humans in 1995 [14,66], it took another three decades for the first bona fide plant TR genes to be184
characterized, in 2019 [19]. Interestingly, the unusual length of the Allium telomere repeat unit (12-185
Biomolecules 2020, 10, x FOR PEER REVIEW 6 of 23
nt) [43] was used to identify candidate TRs not only in Allium species but subsequently across the186
phylogeny of land plants with either canonical or unusual types of telomere repeats [19]. Previously187
characterized TER1 and TER2 in A. thaliana were shown not to act as telomerase RNAs [19,67] and188
the original paper describing them has been retracted [68,69].189
190
191
Figure 3. Conservation of functional domains of two core telomerase subunits – TERT and TR.192
a) Models of secondary structures of human, Tetrahymena and Arabidospis TRs suggest193
conservation of several structural motives including pseudoknot in the vicinity of the template (t/PK194
domain) and stem-loop regions [70,71]. In humans the stem-loop region contains the conserved 4/5195
(CR4/5) region, the H (AnAnnA) and ACA-boxes (H/ACA) domains and the Cajal body box (CAB-196
box) motif that serve as binding sites for other protein components of the telomerase holoenzyme197
complex (dyskerin, NOP10, NHP2, and GAR1). In Tetrahymena the stem-loop 4 (SL4) is directly198
bound by p65 protein [72]. To date, particular interactors and their binding sites have not been199
demonstrated directly in Arabidopsis (see also Table 1). b) Domain arrangement of human (Animals),200
Tetrahymena (Ciliates) and Arabidopsis (Plants) TERTs. The supergroup for each species is given. N-201
terminus: telomerase essential N-terminal (TEN) domain and RNA‐binding domain (TRBD domain)202
are separated by Linker that contains a nucleus localization‐like signal (NLS). The central RT domain:203
catalytical part of the enzyme that contains seven evolutionary-conserved RT motifs (1, 2, A, B′, C, D204
and E motifs) and also telomerase specific 3 motif [73–75]. C-terminus: C‐terminal extension (CTE)205
domain.206
207
208
In mammals, telomerase RNA belongs to the family of small nucleolar (snoRNAs) and small209
Cajal body (scaRNAs) RNAs [76,77]. Both snoRNAs and scaRNAs are encoded in introns and210
transcribed by RNA polymerase II (RNA Pol II) along with their host structural genes [78–80]. The211
human TR primary transcript is synthesized by RNA Pol II, capped on its 5′ end with a212
monomethylguanosine (MMG) cap that is further methylated to N2, 2, 7 trimethylguanosine (TMG)213
cap [81–83], internally modified, and processed at its 3′ end to generate the mature, functional TR214
(reviewed in [65,84]. Several structural motifs and formation of the overall tertiary structure of TR215
are needed for a proper interaction with the TERT subunit (reviewed in [85]. Although TERT can216
bind to TR through the t/PK domain alone, additional binding with the CR4/5 domain is required217
[86,87].218
Biomolecules 2020, 10, x FOR PEER REVIEW 7 of 23
Like many other polymerases, telomerase catalyzes nucleotide addition to the 3′ hydroxyl group219
of a primer, forming a product–template duplex. Accurate telomeric repeat synthesis depends on220
strict boundaries of a template region within TR, which functions as a STOP signal in the telomerase221
extension step (reviewed in [70]). In humans, to synthesize 6-nt telomeric repeats of the human-type222
telomeric motif, telomerase anneals five nucleotides of its 11-nt long template region with terminal223
nucleotides of the telomere DNA and extends it with 6-nts complementary to the rest of the template224
region [88].225
Telomerase processivity requires repeated cycles of annealing, synthesis, translocation and re-226
annealing of substrate DNA–TR base-pairing [70]. Telomerase remains associated with substrate227
DNA even when DNA–RNA base-pairing is disrupted, however the exact mechanism was unknown228
[89–91]. Recently, details of processive telomerase catalysis were revealed using high-resolution229
optical tweezers. The authors demonstrated that a stable substrate DNA binding at an anchor site230
within telomerase facilitates the processive synthesis of telomeric repeats, which results in231
synthesizing multiple telomeric repeats before releasing them in a single step. The product DNA232
synthesized by telomerase can be recaptured by the anchor site or folded into G-quadruplex233
structures [92].234
It remains controversial whether active telomerase enzyme in humans functions as a dimer (TR235
and TERT) or only as a monomer of each subunit [93–96]. In contrast to the human telomerase236
complex, the affinity-purified telomerase from Tetrahymena is monomeric [97]. The possible237
dimerization of telomerase was also suggested in plants. Dimerization, modulated by a conserved238
TRBD domain from A. thaliana TERT that is able to interact separately with the N-terminal fragments239
and itself, was observed using yeast two-hybrid analysis of interactions [98].240
While the mechanism of the telomerase catalytic cycle may be similar among telomerases from241
different kingdoms, recent characterization of plant TRs across the whole land plant phylogeny242
revealed some features distinct from TRs described in mammals or fungi. First, plant TRs are243
transcribed with RNA Pol III [19], similarly to TRs in Ciliates, while mammalian or yeast TRs are244
RNA Pol II products [55]. The closer relationship between TR biogenesis in Ciliates (supergroup245
TSAR) and Plants (supergroup Archaeplastida) on the one hand, and fungi and animals (supergroup246
Amorphea) on the other hand, corresponds to current versions of the phylogenetic tree of eukaryotes,247
(which is supported by phylogenomic studies), and respective ancestral supergroups [24]. Plant TRs248
show relatively conserved structures of their RNA Pol III promoters (so called Type 3 RNA Pol III249
promoter [99] with a typical Upstream Sequence Element (USE) and TATA box. Further, TRs in land250
plants have a monophyletic origin [19]. This contradicts the previous paradigm, according to which,251
relatively conserved TERT subunits associate with very diverse - and unrelated – RNAs [57,58].252
Interestingly, template regions of plant TRs are of relatively diverse lengths. They are mostly of253
the length corresponding to one and one half of the telomere repeat, which allows for substrate DNA254
annealing. The template regions may, however, also be shorter (e.g., in A. thaliana TR, whose template255
region is only 9-nt long) or longer - as long as two complete telomere repeat units, e.g., as in wild256
carrot (Daucus carota) [19]. However, it is important to note that the authentic, functional part of the257
template region may be shorter than the putative predicted template (the region complementary to258
the synthesized telomere repeat, e.g., Cestrum elegans), as it is delimited by secondary structural259
elements in TRs. Some other secondary structural motifs of TRs - e.g., the pseudoknot structure260
downstream of the template region - seem conserved among animal, plant and fungal TRs [55,64,71].261
Whether a primary transcript of plant telomerase RNA is generated similarly as in Ciliates262
(synthesized by RNA Pol III, not spliced, leaving a 3′ polyuridine tail [64,100] or which motifs,263
domains or stems of TR are involved in TR-TERT interaction need to be clarified. Moreover, the264
functions of TR expand far beyond its templating function as it forms a flexible scaffold that functions265
in correct telomerase RNP assembly.266
267
268
4. TERT Subunit of Telomerase269
Biomolecules 2020, 10, x FOR PEER REVIEW 8 of 23
Most of the catalytic subunits of telomerase, TERTs, including the human and plant TERTs, can270
be classified into three major parts. At the N-terminus are positioned telomerase-specific motifs (N-271
terminal domain), reverse transcriptase motifs (RT domain) are positioned centrally, and at the C-272
terminus of the TERT protein are localized conserved motifs - these are more or less specific for273
particular groups of organisms (C‐terminal extension, CTE) (Figure 3b).274
The central RT domain is the catalytical part of the enzyme and contains seven evolutionarily-275
conserved RT motifs (1, 2, A, B′, C, D and E motifs). This domain is organized into two subdomains,276
the “fingers” involved in nucleotide binding and processivity, and the “palm” providing the277
polymerase catalytic residues and DNA primer grip [50,51,94,101].278
There are two main domains recognized within the N-terminal part: the TEN domain279
(telomerase essential N-terminal, also known as the RNA interaction domain 1 (RID1)) [102,103] and280
the RNA‐binding domain (TRBD) (reviewed in [75]. Moreover, a variable linker physically and281
functionally separates these two domains and has been shown to be biologically essential for the282
function of TERT (reviewed in [103]. The nucleus localization‐like signal (NLS), placed between TEN283
and TRBD domains, is responsible for nuclear import of TERTs [104].284
The TEN domain has both DNA‐binding and nonspecific RNA‐binding properties and may also285
stabilize short RNA–DNA duplexes during telomere extension: i.e., repeated cycles of telomerase286
annealing, synthesis, translocation, and re-annealing.287
Despite poor sequence homology, the CTE-part is almost universally conserved, although288
several roundworm species appear to lack this structure entirely [102]. The crystal structure of the289
human CTE domain identified three highly conserved regions within the CTE-region [105]. It is290
proposed that CTE is involved in promoting telomerase processivity, in regulating telomerase291
localization and is involved in differential binding of DNA, but not in essential catalytic functions, as292
reviewed in [106].293
It was proposed that TERT in metazoan ancestors possesses all three major parts and 11294
canonical motifs: GQ, CP, QFP, T motifs within the N-terminal part, and 1, 2, A, B′, C, D and E motifs295
within the RT part. However, GQ and CP motifs might be missing in some beetles (e.g., Tribolium296
castaneum) [103] or unicellular relatives of metazoans (e.g., Trypanosoma sp.). Similarly, the plant297
TERTs possess three major parts, 11 canonical motifs and CTE-part [75].298
Although the TERT protein is highly conserved, the gene structure differs among the group of299
organisms as they differ in exon/intron organization. In Ciliates, species with 1 exon (e.g., Euplotes300
aediculatus) to 19 exons (Tetrahymena thermophila) were identified [75]. Contrary to Ciliates, the TERT301
exon-intron structure is conserved across the Vertebrata. Mammalian TERT has 16 exons whereas302
TERTs in non-mammalian vertebrates have anywhere between 14 to 17 exons [50,103,107–109].303
Among plants, TERT genes with 12 exons are highly conserved [75]. Although most eukaryotes,304
including humans, harbor a single TERT gene, in polyploid plant species, as in allotetraploid305
Nicotiana tabacum (tobacco), multiple TERT paralogs exist that are differentially regulated [10,110].306
307
5. Telomerase Regulation308
As described above, the primary determinant for telomerase enzyme activity in humans seems309
to be a strictly controlled transcription level of the TERT subunit [15] rather than expression of the310
TR subunit, which is ubiquitously expressed in almost all tissues regardless of telomerase activity311
[16].312
Many studies have dissected the mammalian TERT promoter and identified cis-elements (E-313
boxes, GC motifs, ETS domain) bound by general transcription factors (TFs) such as c-MYC, NF-κB,314
STAT3, SP1 or ETS2 (Figure 4a) (reviewed in [101,111–113]). Moreover, these general TFs can be315
regulated by a number of other proteins; e.g., human RuvBL2 (reptin) regulates c-MYC‐dependent316
transcription of TERT [114]. The core functional promoter essential for transcriptional activation of317
human TERT in cancer cells was suggested in the 181-bp [115] or 208-bp fragment [116] respectively,318
upstream of the transcription start site. In plants, the 336 bp long promoter region of the TERT319
promoter seems to be essential for successful complementation of telomerase and reversion of the320
Biomolecules 2020, 10, x FOR PEER REVIEW 9 of 23
short telomere phenotype in tert -/- A. thaliana plants (Table 1a) [13,117]. Crhák et al. showed efficient321
and tissue-specific control of telomerase reconstitution. At the same time, the results have shown that322
the level of AtTERT transcript is not the sole determinant for the successful restoration of telomeric323
function of telomerase, which suggests posttranscriptional control of telomerase expression [117].324
Moreover, restoration of telomerase activity, as evaluated in complemented plant extracts in vitro,325
not always correlated with the ability to restore telomere maintenance in planta.326
327
328
329
Figure 4. Regulation of human telomerase biogenesis.330
Biomolecules 2020, 10, x FOR PEER REVIEW 10 of 23
a) Transcription of the human telomerase reverse transcriptase gene (hTERT) by RNA331
polymerase II (RNA Pol II) is regulated by several activators and repressors acting at the promoter332
level (e.g. c-MYC, Nuclear Factor κB (NF- κB), Signal Transducer and Activator of Transcription 3333
(STAT3), Specificity Protein 1/3 (SP1/3), MAD1). Histone modification H3K27me3 often silences334
hTERT, however the mutated hTERT allele is marked by the active histone marks H3K4me2,335
H3K4me3 and H3K9ac. hTERT pre-mRNA with a 5′ mono-methylguanosine (MMG) cap and poly(A)336
3’ tail, can be spliced into full-length (FL) or multiple alternative isoforms (Alternative splicing) that337
are catalytically inactive or even inhibit telomerase activity (e.g. minus alpha TERT (-α TERT) due to338
their competition for hTR with FL hTERT mRNA). Binding of heat shock protein 90 (Hsp90) with its339
co-chaperone (p23) in the cytoplasm enables hTERT phosphorylation (P). hTERT is further imported340
back to the nucleus by Importin α or β1 (Imp) via nuclear pores (n.p.), while export of hTERT may be341
mediated by the chromosome region maintenance 1 protein homolog (CRM1, also known as342
exportin-1). The ubiquitin (Ubq) -proteasomal degradation of TERT is driven by E3 ubiquitin-protein343
ligase makorin-1 (MKRN1), heat shock protein 70 (Hsp70) and carboxyl-terminus of Hsp70344
Interacting Protein (CHIP).345
b) Histone modifications H3K4me2/3 or H3K9Ac help to regulate read-through of the human346
telomerase RNA (hTR) gene by RNA Pol II in telomerase-positive cell lines. SHQ1 chaperone and347
RuvB-like proteins (RuvBLs) facilitate assembly of nascent RNA with RNA scaffold proteins348
(dyskerin, NOP10, NHP2, and NAF1). Mature TR is capped with a tri-methylguanosine (TMG) cap349
at the 5′ end, polyadenylated at the 3′ end and co-transcriptionally associated with scaffold proteins.350
The hTR variants with shorter or longer 3′ ends or associated with variant proteins may lead to351
degradation of hTR. NAF1 is replaced by GAR1 before the hTR ribonucleoprotein complex reaches352
the nucleolus.353
c) RuvBLs (pontin and reptin) enable telomerase assembly and allow hTERT recruitment to the354
nucleolus to form a mature telomerase complex while bound by nucleolin (NCL). PIN2/TERF1—355
interacting telomerase inhibitor 1 (PINX1), together with nucleophosmin (NPM) and microspherule356
protein 2 (MCRS2), regulate hTERT availability in a cell cycle-dependent manner. Telomere Cajal357
body protein 1 (TCAB1, also known as WRAP53) recognizes the Cajal body box (CAB-box) of the358
hTR in the mature telomerase complex and recruits it to the Cajal bodies (CBs). In CBs, hTR interacts359
with local proteins such as coilin while survival motor neuron protein (SMN) binds hTERT.360
d) In S-phase, the CBs colocalize with telomeres and facilitate the recruitment of the mature361
telomerase complex to the telomeres via interaction with TPP1 protein, which is one of the subunits362
of a protein complex localized at telomeres, termed as Shelterin. The presence of Shelterin proteins363
(telomeric-repeat binding factor 1/2 (TRF1/2), protection of telomeres protein 1 (POT1), TRF1-364
interacting nuclear factor 2 (TIN2), repressor/activator site binding protein (RAP1) and TPP1 helps365
distinguish chromosomal ends (telomeres) from DNA breaks. (For references see Text or Table 1.)366
367
368
The wild-type promoter of the human TERT gene is often silenced by the repressive369
trimethylation of Lys27 in histone H3 (H3K27me3) modification. Consistent with this finding, the370
mutated human TERT allele is marked by the active histone marks H3K4me2, H3K4me3 and371
acetylated histone H3 Lys9 (H3K9ac) [118,119]. Analysis of the epigenetic states of the TERT gene in372
Arabidopsis telomerase-positive and telomerase-negative tissues revealed differential levels of373
H3K27me3, the mark of developmentally silenced heterochromatin regions in plants, whereas374
euchromatin-specific marks (H3K4me3 and H3K9Ac) were approximately at the same levels in all375
tissues [11]. The striking stability of the epigenetic status of the TERT promoter in Arabidopsis may376
reflect a unique attribute of plants – their totipotency – which is in accordance with the reversible and377
dynamic character of telomerase silencing [120].378
379
380
Table 1. Human and Arabidopsis telomerase assembly - a comparative overview (a-d381
classification corresponds to Figure 4).382
Biomolecules 2020, 10, x FOR PEER REVIEW 11 of 23
Mammals (human) Reference(s) Plants (Arabidopsis
thaliana)
Reference(s)
a) TERT Minimal
promoter
330 bp upstream of the
translation start site to 228
bp downstream.
[115,116,121]
336 bp long promoter
region of the translation
start site with plausible
regulatory intron 1.
[13,117]
RNA Polymerase RNA Pol II [115] RNA Pol II [122]
Histone
modifications of
promoter
Telomerase-negative
tissues: H3K27me3;
telomerase-positive
tissues (mutated TERT
allele): H3K4me2,
H3K4me3 and H3K9ac.
[118,119,123] Telomerase-negative
tissues: H3K27me3,
H3K4me3, H3K9Ac;
telomerase-positive
tissues: H3K4me3,
H3K9Ac.
[11]
TERT expression
in organism
TERT expression is
strictly controlled at the
transcript level.
[15,124] The dynamics of TERT
transcripts correlates with
telomerase activity
observed in plant tissues.
[7,11]
Number of exons 16 exons [75,121] 12 exons [75]
Alternative
splicing of
mRNA
hTERT pre-mRNA can be
spliced into at least 22
isoforms.
[125] AtTERT pre-mRNA can
be spliced into 3 isoforms.
[75]
Post-translational
modifications
Phosphorylation or
ubiquitination.
[126,127] No putative
phosphorylation site in A.
thaliana TERT (but
predicted in rice or
tabacum TERT ).
[128,129]
Import to the cell
nucleus
Importin α promotes
nuclear import of the
TERT.
[130] Importin subunit alpha-4
is associated with TERT.
[98]
Protein domains TEN, TRBD, RT, CTE. [75,108] TEN, TRBD, RT, CTE. [75]
Protein length 1132 aa [108] 1123 aa [131]
b) TR Histone
modifications
hTR expression in
telomerase-positive cell
lines is associated with
H3K4me2/3, H3K9Ac and
hyperacetylation of H4.
[132,133] Not known yet.
RNA Polymerase RNA Pol II [66] RNA Pol III [19,20]
Modifications 5′ end cap, internally
modified, poly (A) tail
[83] Not known yet.
Template region 11-nt long template
region (synthesizes 6-nt
telomeric repeats
GGTTAG).
[66,88] 9-nt long template region
(synthesizes 7-nt
telomeric repeat
GGTTAG).
[19]
TR gene length 451-nt long transcript [66] 268-nt long transcript [19,20,71]
TR expression in
organism
In most tissues TR is
ubiquitously expressed
regardless of telomerase
activity.
[16,17] The dynamics of TR
transcripts correlates with
telomerase activity
observed in plant tissues.
[7,11]
c) Nucleolus
and CBs
TR scaffold
proteins
Dyskerin, NOP10, NHP2,
NAF1/GAR1.
[96] [84] Not known yet. Dyskerin
(CBF5), NOP10, NHP2,
NAF1, and GAR1 are
[19,134–136]
Biomolecules 2020, 10, x FOR PEER REVIEW 12 of 23
Additionally, TERT transcripts in many animal species, including vertebrates, insects or383
nematodes, are alternatively spliced [75,103,146,147]. Specific patterns of TERT mRNA variants384
expressed in humans and rodents during development indicate that splicing events are not random385
and could have a physiological function (Figure 4a) [75,148–151]. Human TERT pre-mRNAs in early386
development can be spliced into 22 isoforms, while telomerase activity is associated only with the387
full‐length hTERT (reviewed in [152]. Some of the alternate hTERT mRNA forms (e.g., the minus388
alpha-variant) may not only be catalytically inactive, but even show a dominant negative inhibition389
of telomerase activity [153]. Correspondingly, differential patterns of hTERT mRNA splicing were390
observed between normal (fetal human colon, FHC) and adenocarcinoma colon (HT-29) cells during391
their sodium butyrate-induced differentiation. The higher abundance of the minus alpha-variant of392
hTERT mRNA was observed in FHC cells, which may be involved in the more rapid loss of393
telomerase activity in these cells during differentiation [154]. Spliced variants may also have non-394
canonical roles, for example in cell proliferation [125,155]. Alternative splicing was also described in395
many plant species (e.g., A. thaliana, Oryza sativa (rice), Iris tectorum (roof iris)) (reviewed in [75].396
Short isoforms of TERT protein originating from the alternate splicing events could be functionally397
important, as suggested for the A. thaliana variant AtTERT V(I8)) (TERT variant in intron 8). This398
isoform of AtTERT is able to bind Protection of telomeres protein 1a (AtPOT1a), (one of the399
Arabidopsis orthologues of the human or fission yeast single‐stranded telomeric sequence binding400
protein POT1), more efficiently than full-length AtTERT [156,157].401
It has been proposed that human telomerase is subjected to posttranslational regulation such as402
phosphorylation or ubiquitination [126,127], reviewed in [112]. Putative phosphorylation sites were403
identified in TERT amino acid sequences from O. sativa [128] or N. tabacum [129] but not in AtTERT404
from A. thaliana [128].405
In plants, indirect regulation of telomerase by various proteins or hormones has also been406
described. In tobacco cell culture, phytohormones such as abscisic acid or auxin regulate407
phosphorylation of telomerase protein, which is required for the generation of a functional408
telomerase complex [129,158]. In A. thaliana, reduced endogenous concentrations of auxin in409
telomerase activator 1 (AtTAC1) mutant plants block the ability of this zinc-finger protein to induce410
AtTERT. However, AtTAC1 does not directly bind the AtTERT promoter [159,160]. Similar to411
humans, AtRuvBL2a protein may be involved in regulation of TERT transcription in plants because412
localized in the nucleolus.
Telomerase activity can
be immunoprecipitated
with dyskerin (CBF5) in
plants. Dyskerin
associates with TRB
proteins.
Nucleolin NCL involves nucleolar
localization of TERT.
[137] NUC-L1 has a role in
telomere maintenance
and telomere clustering.
[138,139]
RuvBLs RuvBLs (pontin and
reptin) interact with
TERT and dyskerin.
[140] Interactions between
TERT and RuvBL
proteins is mediated by
TRB proteins.
[134]
coilin Interacts with TR. [141,142] Colocalizes with TRB1 in
the CBs adjacent to the
nucleolus.
[143]
d) Association
with telomere
The TPP1 protein
interacts with TERT and
facilitates the recruitment
of the mature telomerase
complex to the telomeres.
[144] The TRB proteins interact
with TERT and may help
to recruit telomerase to
the plant telomeres.
[145]
Biomolecules 2020, 10, x FOR PEER REVIEW 13 of 23
in AtRuvBL2a heterozygous mutants, a moderate but significant increase in AtTERT transcripts was413
observed. Interestingly, telomerase activity in these plants was reduced to ca. 5% compared to WT414
plants [134].415
The regulation of telomerase activity may also be driven by modulation of TR maturation. As416
described previously, biogenesis of the human TR involves a complex series of posttranscriptional417
modifications (Figure 4b, Table 1b) (reviewed in [84]. In humans, the set of TR transcripts with418
heterogenous 3′-ends may be trimmed by various exonucleases [161]. Similarly, various 5′ cap-419
binding complexes can be recruited to a mono- or tri-methylguanosine cap [82,162].420
There is also evidence that in addition to its canonical role in telomere maintenance, both421
telomerase subunits - TERT and TR - can function independently of telomerase [163]. It was422
demonstrated that, e.g., the TR subunit was upregulated at very early stages of tumorigenesis,423
whereas telomerase activity was detected in end-stage tumors [164], and that the RNA component424
seems to be capable of DNA damage response (DDR regulation) [165]. The TERT subunit,425
independently of its action on telomeres, regulates the cell-cycle, inhibits apoptosis, regulates gene426
expression, modulates cell signaling (e.g., Wnt/β-catenin, NF-κB pathways) and DDR, or binds to and427
protects mitochondrial DNA (mtDNA) (reviewed in [85,163,166].428
In plants, the armadillo/β-catenin-like repeat-containing protein (ARM) or Chromatin429
remodeling 19 (CHR19) proteins associated with TERT may reflect possible non-telomeric functions430
of telomerase [167]. ARM proteins play a role in the Wnt/β-catenin signaling pathway in humans431
[168], but non-telomeric functions of plant TERT or TR remain elusive.432
6. Composition of Enzymatically Active Telomerase433
The active human telomerase is composed not only of a core complex of TR encircled by TERT434
but is assembled as a functional complex in a stepwise regulated process governed by multiple stably-435
or transiently-associated proteins.436
Human telomerase RNPs, as well as other box snoRNPs or scaRNPs, are associated with two437
conserved H/ACA boxes, (H box (consensus ANANNA) and the ACA box (ACA)) binding protein438
complexes: dyskerin, NOP10, NHP2 and NAF1 in the nucleoplasm, where NAF1 is replaced by GAR1439
before the hTR RNP complex reaches the nucleolus (Figure 4c) (reviewed in [84,169]). Assembly of440
TR and TERT into catalytically active telomerase is aided by RUVBL1 (pontin) and RUVBL2 (reptin)441
AAA+ ATPases, due to their direct interaction with TERT and dyskerin [140]. In mammals, the442
telomerase RNP is retained in nucleoli through the interaction between TERT and nucleolin in the443
dense fibrillar component [137,170]. Telomerase activity is negatively regulated by the nucleolar444
protein PIN2/TERF1—interacting telomerase inhibitor 1 (PINX1) [171]. Nucleophosmin (NPM) and445
microspherule protein 2 (MCRS2) may be S phase specific co-effectors of PINX1, working against446
each other to modulate the human TERT pool (reviewed in [172]. Telomerase is then recruited to447
Cajal bodies (CBs). CBs are spherical sub-nuclear organelles that reside at the nucleolar periphery448
and are implicated in RNA-related metabolic processes. TCAB1 (also known as WDR79, WRAP53),449
bound to the CAB-box motif of TR, promotes the translocation to CBs [96]. CB-related proteins, such450
as coilin and survival motor neuron (SMN), interact with telomerase and may regulate the formation451
of an active telomerase complex [141,173–175]. The CBs colocalize with telomeres and facilitate the452
recruitment of the mature telomerase complex to the telomeres via the telomere-associated protein453
TPP1, a subunit of the Shelterin complex localized at telomeres (Figure 4d) [144,176].454
A broad conservation of a dyskerin-TR association was proposed among diverse organisms,455
including plants. For example, telomerase activity and TR were immunoprecipitated with the anti-456
dyskerin antibody in onion (Allium cepa) (Table 1c) [19]. In A. thaliana, null mutants for the nucleolar457
protein NUCLEOLIN 1 caused telomere shortening on all chromosomal arms although a direct458
interaction between NUCLEOLIN 1 and TERT in Arabidopsis was not observed [138]. Similarly, we459
demonstrated that the plant RuvBL1 and RuvBL2a proteins interacted with TERT only indirectly in460
the nucleolus in vivo. In contrast to mammals, interactions between TERT and RuvBL proteins in A.461
thaliana were not direct but rather they were mediated by one of the Telomere Repeat Binding (TRB)462
proteins [134,177]. It was also shown that in A. thaliana, dyskerin directly interacted with NAF1. Plant463
Biomolecules 2020, 10, x FOR PEER REVIEW 14 of 23
dyskerin was localized not only in nucleoli, but was also detected in CBs [136]. The main abundant464
signature protein of CBs in plants, as well as in mammals, is coilin (reviewed in [178]). Dvořáčková465
detected significant colocalization of TRB1 with coilin present in the CBs adjacent to the nucleolus466
(Dvořáčková- thesis). TRB proteins, which are the only proteins with confirmed in vivo plant telomere467
localization and function [145,179–181], may help to recruit telomerase to telomeres as they directly468
interact with TERT (Table 1d) [145]. Moreover, TRB proteins associate with the dyskerin orthologue469
CBF5 in the nucleolus, and they directly interact with POT1b (one of the plant paralogues of the470
Shelterin POT1 subunit). For a recent list of proteins associated with human and plant telomerase or471
with telomeric sequences see Procházková Schrumpfová 2019 [7].472
Thus, while telomerase-interacting proteins (reviewed in [172] show relatively extensive473
conservation, individual interactions remain to be elucidated and carefully classified into direct and474
indirect ones. Due to the recently described differences in TR biogenesis pathways between plants475
and Ciliates on one hand, and mammals and yeasts on the other hand, orthologs of known TR476
interactors in human (dyskerin, NOP10, NHP2, NAF1 or GAR1), as well as e.g., orthologues of the477
La-family protein (p65) from Ciliates, or Sm proteins from yeasts (reviewed in [64] should also be478
examined in plants. Ideally, a new independent screen and subsequent analyses should identify plant479
TR direct interactors de novo.480
7. Conclusions481
Here we have provided an updated overview on telomerase – its origin, biogenesis, regulation482
and function. Despite the extensive conservation of telomerase as a tool to overcome the end-483
replication problem of linear eukaryotic chromosomes, subsequent evolution of this ancient484
molecular machine resulted in alternative solutions for particular aspects of its biogenesis and485
composition, as exemplified by the recent description of telomerase RNAs or telomerase interacting486
proteins in land plants. Further investigation of telomerase diversity across the width of eukaryotic487
phylogeny is needed for a deeper understanding of truly fundamental principles of telomere and488
telomerase regulation, and potential application of this knowledge in medicine, plant breeding or489
protection of biodiversity.490
491
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: Telomere492
repeats in representative species.493
Author Contributions: P.P.S. contributed to this paper with a literature review, drafting the paper, drawing the494
figures and creating the tables. J.F. contributed with a literature review and manuscript editing. Both authors495
approved the final version.496
Funding: This work was supported by the Czech Science Foundation (project 20-01331X), and the institutional497
support by the Ministry of Education, Youth and Sports of the Czech Republic [project CEITEC 2020 (LQ1601)]498
and European Regional Development Fund [project SYMBIT, reg. no.CZ.02.1.01/0.0/0.0/15_003/0000477].499
Acknowledgments: We thank to Lucie Bozděchová for discussing the MS prior to submission.500
Conflicts of Interest: The authors declare no conflict of interest501
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Table S1. Telomere repeats in representative species.
Telomere repeat Representative species Reference
TSAR
TTAGGG Ectocarpus siliculosus (Stramenopiles) [1] and references herein
TTTAGGG Phytophthora infestans (Oomycetes) [1] and references herein
TTTTGGGG Euplotes aediculatus (Ciliata) [1] and references herein
TTGGGG Tetrahymena (Ciliata) [1] and references herein
TTTGGG Chilodonella uncinata (Ciliata) [1] and references herein
TTTTAGGG Theileria annulata (Apicomplexa) [1] and references herein
TTTAGG Cryptosporidium parvum Iowa II (Apicomplexa) [1] and references herein
TTAGG Aurantiochytrium limacinum (Stramenopiles) [1] and references herein
Haptista
TTAGGG Emiliania huxleyi (Haptophyta) [1] and references herein
Cryptista
TTTAGGG Guillardia theta (Cryptophyceae) [1] and references herein
Archaeplastida
TTTAGGG Arabidopsis thaliana (Brassicaceae) [2]
TTAGGG Asparagus officinalis (Asparagales) [3]
TTTTAGGG Chlamydomonas reinhardtii (Chlorophyceae) [4]
TTTTAGG Klebsormidium subtilissimum (Charophyta) [1]
TTCAGG/TTTCAGG Genlisea hispidula (Lentibulariaceae) [5]
CTCGGTTATGGG Allium cepa (Amaryllidaceae) [6]
AATGGGGGG Cyanidioschyzon merolae (Rhodophyta) [7,8]
TTTTTTAGGG Cestrum elegans (Solanaceae) [9]
Amorphea
TTAGGG Homo sapiens (Animalia) [10]
TTAGG Bombyx mori (Insects) [11]
TTAGGC Ascaris lumbricoides (Nematodes) [12]
TCAGG Tribolium castaneum (Anthropoda) [13]
TTGCA Parascaris univalens (Nematoda) [1] and references herein
TGTGGG Bdelloidea (Rotifera) [1] and references herein
TAAGGG Polysphodylium pallidum (Amoebozoa) [1] and references herein
Discoba
TTAGGG Andalucia godoyi (Jakobida) [1] and references herein
Metamonada
TAGGG Giardia lamblia (Fornicata) [1] and references herein
Malawimonadida
TTAGGG Malawimonas californiana (Malawimonadida) [1] and references herein
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12. Müller, F.; Wicky, C.; Spicher, A.; Tobler, H. New telomere formation after developmentally regulated chromosomal breakage during the process of
chromatin diminution in ascaris lumbricoides. Cell 1991, 67, 815–822, doi:10.1016/0092-8674(91)90076-B.
13. Mravinac, B.; Meštrović, N.; Čavrak, V.V.; Plohl, M. TCAGG, an alternative telomeric sequence in insects. Chromosoma 2011, 120, 367–376,
doi:10.1007/s00412-011-0317-x.
Supplement P
Pecinka A, Schrumpfová, P.P., Fischer L., Dvořák Tomaštíková E., and Mozgová I., 2022.
The Czech Plant Nucleus Workshop 2021. Biologia Plantarum, 66: 39-45
P.P.S. was significantly involved in the ms writing and editing
39
biologia plantarum
an international journal for experimental botany
BIOLOGIA PLANTARUM (2022) 66: 39-45 DOI: 10.32615/bp.2022.003
CONFERENCE REPORT
The Czech Plant Nucleus Workshop 2021
A. PECINKA1,*
, P. PROCHÁZKOVÁ SCHRUMPFOVÁ2,3
, L. FISCHER4
,
E. DVOŘÁK TOMAŠTÍKOVÁ1
, and I. MOZGOVÁ5,6
1
Institute of Experimental Botany of the Czech Academy of Sciences, Centre of the Region Haná for
Biotechnological and Agricultural Research, CZ-77900 Olomouc, Czech Republic
2
Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of
Science, Masaryk University, CZ-61137, Brno, Czech Republic
3
Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University,
CZ-62500, Brno, Czech Republic
4
Department of Experimental Plant Biology, Charles University, Faculty of Science, CZ-12844, Prague, Czech
Republic
5
Biology Centre, Czech Acad Sci, Institute of Plant Molecular Biology, CZ-37005, České Budějovice, Czech
Republic
6
University of South Bohemia, Faculty of Science, CZ-37005 České Budějovice, Czech Republic
*
Corresponding author: E-mail: pecinka@ueb.cas.cz
Introduction
The cell nucleus is a fascinating organelle. It contains
chromosomes that periodically condense in preparation
for cell divisions in proliferating cells, separate sister
chromatids into daughter nuclei, and de-condense for the
interphase during which the chromosomes replicate and the
whole cycle repeats. Chromosomal DNA serves as a basic
template for transcription, which is orchestrated at many
levels by complex regulatory machinery and takes place
in specific nuclear compartments. In parallel to all these
functions, the cell nucleus is under constant surveillance
for the mitigation of DNA lesions.
Academic institutions within the Czech Republic have
a very long and fruitful history of plant cell nuclei and
chromosome research. To share the latest achievements in
the field and to provide a platform for establishing new
collaborations,weorganizedacommunity-focusedmeeting
Received 14 December 2021, accepted 8 February 2022.
Acknowledgements: We thank all participants at the CPNW2021 for their contributions, sharing unpublished data, and exciting and
fruitful discussions during the meeting. We thank T. Mozga for extensive help during the meeting organization and M. Kovačik for
developing and maintaining the CPNW2021 website. We acknowledge the financial support for the meeting from commercial and
public sponsors.
Conflict of interest: The authors declare that they have no conflict of interest.
Abstract
The Czech Plant Nucleus Workshop 2021 (CPNW2021) took place during mid-September 2021 in Olomouc, Czech
Republic. About 80 researchers and students working in the field of plant nuclear and chromosome biology in the Czech
Republic gathered together to present and discuss their current research. The meeting revealed many plant models
that are used to study plant genomes and their organization, and also a great diversity of topics including epigenetic
regulation of gene expression, genome stability, telomere biology, or sex chromosomes. CPNW2021 provided a broad
platform for establishing new research contacts and collaborations. Here, we summarize the main research directions
and findings presented at the CPNW2021 meeting.
Keywords: chromatin, chromosome, DNA damage repair, DNA methylation, environmental responses, nucleus, sex- and
B-chromosomes.
40
PECINKA et al.
“The Czech Plant Nucleus Workshop 2021” (CPNW2021).
The meeting took place at Fort Science, an interactive
science centre of Palacký University in Olomouc on 14th
and 15th
September 2021 and was attended by more than
80 participants from the majority of national plant research
institutions. This included the Institute of Experimental
Botany of the Czech Academy of Sciences (IEB), Biology
Centre of the Czech Academy of Sciences (BC), Institute
of Biophysics of the Czech Academy of Sciences (IBP),
the Central European Institute of Technology (CEITEC),
Charles University (CUNI), Masaryk University (MUNI),
and Palacký University (UP).
Nuclear biology research benefits from the
technological advancements
The keynote opening lecture was presented by Prof.
J. Doležel (IEB), who gave a historical overview of
the progress in nuclear and chromosome research and
emphasized that many of the fundamental findings were
based on a combination of excellent research ideas and
the use of the new technologies. Several examples of
new approaches were presented at the meeting. P. Cápal
(IEB) used advanced environmental scanning electron
microscopy (A-ESEM), which allows observing samples
in high resolution in their native state, with the aim
to investigate the surface structure of barley mitotic
chromosomes. This revealed a topologically complex
surface with numerous protrusions and regularly spaced
inter-chromatid bridges. A complex study of the higherorder
3D structure of both metaphase and interphase
chromosomes was presented by H. Šimková (IEB). By a
combination of Hi-C and chromosome painting techniques,
shedemonstratedthatsisterchromatidsofbarleymetaphase
chromosomes have a helical structure, where one turn
contains 20 - 38 Mbp chromatin-packed DNA, depending
on the position on the chromosome arm (Kubalová et al.
2021a). Microscopy is a classical technique to study cell
nuclei and chromosomes. However, understanding their
3D organization using classical microscopic techniques is
hampered by the diffraction limit. E. Hřibová (IEB) and
M. Franek (CEITEC/MUNI) introduced super-resolution
microscopy techniques including structural illumination
microscopy (SIM), stimulated emission depletion (STED)
microscopy, and direct stochastic optical reconstruction
microscopy (dSTORM), and discussed challenges in the
selection of fluorochromes and preparation of the plant
samples (Kubalová et al. 2021b).
Chromatin regulates plant development and
environmental responses
Very high developmental plasticity represents a unique
and integral component of plant environmental responses.
Well-controlled dynamics of chromatin structure that
ensures stable but responsive gene expression is therefore
of utmost importance in orchestrating developmental and
environmental cues.Among the crucial developmental and
cell identity modulators in plants as well as animals are the
PolycombRepressiveComplexes1and2(PRC1andPRC2)
that establish two key repressive histone post-translational
modifications H2Aub and H3K27me3, respectively
(Bieluszewski et al. 2021). A. Sharaf and V. Mallika (BC)
presented the identification of the components of PRC2
complexes in the basal eukaryotes (Sharaf et al. 2021)
and algae of the green lineage. M.G. Trejo Arellano (BC)
presented an evolutionary study of H3K27me3 distribution
in unicellular and multicellular eukaryotes. Several
contributions also tackled the recently emerging view of
PRC2 involvement in environmental sensing and response
in plants (Shen et al. 2021, Kim et al. 2021). I. Mozgová
and M. Zhou presented recent findings on the role of PRC2
in ambient light response and photoautotrophic growth
in Arabidopsis and T. Konečný (BC) showed enhanced
heterochromatin formation in PRC2 mutant plants during
de-etiolation. Using Physcomitrium patens, K. Sobotková
(BC) demonstrated that the function of PRC2 in finetuning
primary metabolism during photoautotrophic
growth might be evolutionarily conserved.
Long non-coding RNAs (lncRNAs) are emerging as
important players in chromatin modulation. For instance,
the introduction of H3K27me3 by PRC2 in the FLC locus
is aided by a lncRNA called COLDAIR (Xu and Chong
2018), and lncRNA APOLO has been implemented
in PRC2-associated repressive chromatin looping at
the PINOID locus, that encodes a major regulator of
polar auxin transport (Ariel et al. 2014). J. Hajný (IEB)
introduced a newly identified lncRNA that is expressed in
the root protophloem and regulates the transcription of a
xylem-expressed leucine-rich receptor-like kinase. This
gene in turn controls the relationship between longitudinal
anticlinal divisions in the endodermis and the stele area.
The presented findings uncover an intriguing mechanism
of cell division plane specification by long-distance
coordination of lncRNA production and associated target
gene expression. A.J. Wiese (IEB) demonstrated that
upon heat stress, two bZIP transcription factors, bZIP18
and bZIP52, undergo dephosphorylation and relocate
into the nucleus. Here, bZIP18 and bZIP52 regulate the
transcription of several common genes, including lncRNA
genes that are elevated in response to heat treatment
(Wiese et al. 2021). These results demonstrate that
phosphorylation can mediate extra-nuclear sequestration
of transcription factors that orchestrate heat stress
response, and perhaps suggest a more general mechanism
of stress response attenuation under optimal conditions but
rapid transcriptional response upon exposure to adverse
environmental conditions.
Vernalization is a key trait regulating flowering time
in plants relative to the winter or extended period of
cold conditions. Among the well-described mechanisms
is the vernalization response in Brassicaceae governed
by PRC2, whereby the flowering inhibitor locus FLC
is subjected to H3K27me3-mediated stable repression
upon extended periods of cold (Xu and Chong 2018).
Interestingly, the mechanism of vernalization response
differs in monocot grasses including crops, where the
release of transcriptional repression of the flowering
41
THE CZECH PLANT NUCLEUS WORKSHOP 2021
MADS-box activator VERNALIZATION 1 (VRN1) is
required for the induction of flowering. Analysis of over
100 hexaploid bread wheat cultivars by B. Strejčková
(IEB) revealed several known as well as new vernalization
insensitive VRN1 alleles, supporting earlier evidence that
VRN1 is the major breeding locus in cereals (Strejčková
et al. 2021). J. Šafář (IEB) showed that genetic and
epigenetic regulation of VRN1, including a putative role
of the recently identified bread wheat PRC2 components
(Strejčková et al. 2020), remains unknown. Therefore,
future plans towards the development of modifier and
reporter VRN1 lines were presented.
Cereal grains are complex structures harbouring
diploid embryo, triploid endosperm, and diploid seed
coats of maternal origin. Cereal grain development starts
with fertilization and consists of several phases including
syncytium, cellularization, maturation, and desiccation
(Nowicka et al. 2021). A. Pečinka (IEB) presented a
transcriptomic meta-analysis of the embryo, endosperm,
and seed maternal tissues from developing barley grains
(Kovacik et al. 2020). This atlas of barley seed expression
provides ample marker genes, indicates local and temporal
specificity of biological pathways, and points to the
dynamic role of epigenetic pathways.
DNA methylation - the guardian of the
heterochromatic genome fraction
Methylation of cytosines is an important epigenetic
mark used by plant cells to label chromatin for distinct
functions. It is mostly present in the transcriptionally
inactive chromatin where it occurs together with specific
histone marks. De novo DNA methylation of native loci
is driven by small RNAs (sRNAs) (Zhang et al. 2018).
L. Fischer (CUNI) showed that the potential of sRNAs to
induce DNA methylation depends not only on the level
of sRNAs but also on their origin and likely also on the
epigenetic state of the target locus (Čermák et al. 2020).
Analysis of the dynamics of the initial phases of DNA
methylation, using an experimental system for inducible
production of sRNAs in a homogeneously responding
tobacco BY-2 cell line, demonstrated that de novo cytosine
methylation can occur already 12 h after the exposure to
sRNAs (Přibylová et al. 2019). Furthermore, A. Přibylová
(CUNI) showed that changing the chromatin state by de
novo DNA methylation could affect the activity of the
CRISPR/Cas9 editing tool and the subsequent repair of
double-strand DNA breaks.
The analysis of epigenetic marks including DNA
methylation is challenging in the repetitive genomic
regions. Mapping DNA methylation in repeats is
problematic when averaging cell populations or analyzing
clusters of repeats in single-cell analysis. This problem
can be overcome by analyzing individual DNA/chromatin
fibres by an optimized method introduced by A. Kilar
(CEITEC/MUNI). This DNA fibre extension technique
combines immunofluorescence and fluorescence in-situ
hybridization signals detected using super-resolution
microscopy followed by the quantitative evaluation of
DNA methylation levels using an image analysis approach
(Franek et al. 2021).
Chromosome organization and regulation in
large and polyploid genomes
Large grass genomes are generally thought to display
the Rabl chromosome organization with centromeres
and telomeres clustered at opposite nuclei poles (Rabl
1885). A. Doležalová (IEB) investigated the relationship
between DNA replication, chromosome organization, and
genome size in Poaceae. While there was a Rabl genome
organization in Brachypodium distachyon, Hordeum
vulgare, and Triticum aestivum, the non-Rabl organization
was found in Oryza sativa and Zea mays. Prevailing
replication of telomeric sequences was observed in the
early and middle S phase, in contrast to centromeric
sequences which underwent replication during the middle
and late S phase (Němečková et al. 2020). Using FISH
against major repetitive DNA sequences on isolated
embryos and endosperm barley nuclei, A. Nowicka (IEB)
showed striking differences in chromosome organization.
While embryo nuclei showed typical Rabl configuration
at all times, endosperm nuclei progressively lost Rabl
organization in age and nuclear DNA content-dependent
manner.
D. Kopecký (IEB) provided an overview of the
research questions connected to interspecific hybridization
and polyploidy with a particular focus on the allopolyploid
genome evolution and stability and the phenomenon of
genome dominance (Glombik et al. 2020). J. Majka (IEB)
further corroborated this topic on the examples of Allium
roylei × Allium cepa and Festuca pratensis × Lolium
multiflorum hybrids and suggested that the shifts in genome
composition towards one parent could be due to uneven
behaviour of parental chromosomes during meiosis in
these hybrids. There is a continuous debate on the effects of
hybridization and polyploidization on gene transcription.
M. Glombik (IEB) showed that in allopolyploids of Festuca
pratensis × Lolium multiflorum, the overall transcript
profile is much closer to that of Lolium parent, suggesting
it as a transcriptionally dominant genome, presumably via
trans-acting regulatory factors (Glombik et al. 2021). K.
Perničková (IEB) analyzed the 3D nuclear positioning of
a pair of rye chromosomes introgressed to the hexaploid
wheat genome. She reported an occasional lack of contact
between telomeres of the additional chromosomes and
nuclear envelope, which could be responsible for the
observed less efficient chromosome pairing in meiosis
and transmission into subsequent generations (Perničková
et al. 2019).
Unravelling the mysteries of plant sex- and
B-chromosomes
R. Hobza (IBP) showed that separated sexes evolved
independently and repeatedly in about 5 % of plant
species. In many species, dioecy has evolved recently, so
42
PECINKA et al.
these plants provide an excellent model for studying the
early stages of sex chromosome divergence, which later in
evolution leads to gradual sex chromosome degeneration
(Hobza et al. 2018). Z. Kubát (IBP) showed that some
TEs proliferate preferentially either in the male or in the
female germline which can be connected with specific
circumstances affecting TE management and activity in
male and female plants and during gametophyte formation
(Jesionek et al. 2021). The process of evolutionary
diversification of sex chromosomes is also connected
with specific chromatin modifications of both histones
and cytosines in Silene latifolia as demonstrated by
M. Hubinský (IBP) (Rodríguez Lorenzo et al. 2020).
In addition to the pivotal role of S. latifolia in studying
plant sex chromosome evolution, V. Hudzieczek (IBP)
with colleagues aims to establish the genus Silene as
a common model to study also other aspects of plant
ecology, evolution, and development. For this purpose,
they established a set of methods for S. latifolia, including
Agrobacterium-mediated gene delivery, genome editing by
TALENs and CRISPR/Cas9 or gene silencing via RNAi.
Certain genomes contain specific supernumerary
chromosomes that carry a generally low number of proteincoding
genes but often harbour molecular toolkits for their
preferential inheritance. J. Bartoš (IEB) introduced plant
B chromosomes and presented maize and Sorghum as
promising models to study molecular mechanisms of nondisjunction,
preferential fertilization, and chromosome
elimination (Blavet et al. 2021, Karafiátová et al. 2021).
Plant nuclei functions and chromatin
organization during DNA damage repair
DNAis constantly exposed to a variety of genotoxic factors
that may alter its chemical and/or physical structure and
result in DNA lesions. DNA damage response (DDR) is
thereforeakeymechanismcontributingtogenomestability.
DDR is a very complex process, ultimately leading to DNA
damage repair or cell death. After DNA damage is sensed,
the local chromatin environment must be reorganized
and histones are displaced. In accordance, FASCIATA1
(FAS1), a subunit of the H3-H4 histone chaperone
complex CHROMATINASSEMBLY FACTOR 1 (CAF1),
is important for genome stability and DNA damage repair
(Kolářová et al. 2021). In her talk, M. Nešpor Dadejová
(CEITEC/MUNI) introduced a newly developed in vivo
method employing laser microirradiation-induced DNA
damage. Using the PCNA1-GFP marker line (Yokoyama
et al. 2016), this technique allows observing immediate
relocation of PCNA1 during DNA damage response in
wild type cells.
The Structural Maintenance of Chromosomes (SMC)
complexes are key components of higher-order chromatin
structure. The SMC5/6 complex is involved in homologous
recombination, replication fork stability, and DNAdamage
repair and its basic structure is evolutionarily conserved
(Díaz and Pecinka 2018). J. Paleček (MUNI) presented a
detailed architectural analysis of the SMC5/6 complex in
humans and yeast (Adamus et al. 2020), highlighted this
SMC complex as the most ancestral in eukaryotes, and
showed various models of its possible DNA processing
activity. His laboratory identified the SMC5/6 complex
subunits in the moss Physcomitrium patens and is planning
to reveal the SMC5/6 complex architecture in plants.
M. Holá (IEB) presented functional characterization of
SMC5/6 in P. patens. Sensitivity assays revealed a critical
role of SMC5/6 in double-strand-break (DSB) repair and
proposed that the circularization of SMC5/6 complex by
the kleisin subunit NSE4 is indispensable for the P. patens
SMC5/6 function (Holá et al. 2021).
DNA-protein crosslinks (DPC) represent a specific
type of DNA damage, caused by the covalent trapping
of virtually any protein to DNA (Stingele and Jentsch
2015). E. Dvořák Tomaštíková (IEB) presented a forwarddirected
genetic screen that aimed to identify factors
involved in the repair of zebularine-induced DPCs in
Arabidopsis (Procházková et al. 2022). Besides several
unknown factors undergoing characterization, the SMC5/6
complex was identified as an important DPC repair factor,
representing a new DPC repair pathway.
Maintaining genome stability over generations is a key
issue for all living organisms. F. Yang (IEB) explained
how SMC5/6 complex functions contribute to normal
male meiosis in Arabidopsis (Yang et al. 2021). Loss-offunction
mutants in SMC5/6 subunits generate unreduced
microspores. The diploid pollen leads to an unbalanced
maternal and paternal genome dosage in endosperm, which
is responsible for a frequent seed abortion but in about 10 -
15 % of seeds leads to the production of triploid offspring.
Thus, SMC5/6 has an important role in the maintenance of
gametophytic ploidy in Arabidopsis.
Maintenance of the chromosome ends
Telomeres consist predominantly of non-coding repetitive
tandem repeats and protect the ends of linear eukaryotic
chromosomes from progressive shortening and erroneous
recognition as unrepaired chromosome breaks. A rosettelike
organization of chromosomes, where telomeres
show persistent clustering at the nucleolus in interphase
nuclei while centromeres associate with nuclear envelope
was observed in A. thaliana (Fransz et al. 2002). M.
Kubová (CEITEC/MUNI) showed that the rosette-like
configuration is not a universal model for interphase
genome organization in Brassicaceae. In species with
large-genome (≥ 1 Gb), centromeres and telomeres
adopt either the Rabl-like configuration or a dispersed
distribution in the nuclear interior, with telomeres being
only rarely anchored to the nucleolus (Shan et al. 2021).
TelomericrepeatsacrosstheEukaryotestypicallyfollow
the formula (TxAyGz)n (Schrumpfová and Fajkus 2020). In
plants, telomeres are mostly composed of the Arabidopsistype
TTTAGGGn repeats (Richards and Ausubel 1988).
However, recent studies revealed significant variability in
telomere sequences in lower and also higher plants (Peska
and Garcia 2020). Moreover, telomeric or telomeric-like
repeats are found also at multiple intra-chromosomal
regions (Uchida et al. 2002, Majerová et al. 2014). This
43
THE CZECH PLANT NUCLEUS WORKSHOP 2021
phenomenon observed in many plant families is most
likely caused by genome rearrangements during evolution.
Interestingly, larger genomes in gymnosperm species than
in angiosperms were previously reported to be associated
with a larger proportion of repetitive sequences (Novák
et al. 2020). A wide survey in gymnosperms, namely in the
Cycadaceae family by R. Vozárová (IBP), has shown that
canonical Arabidopsis-type telomeric repeats are located
predominantly at chromosome ends, while pericentromeric
blocks comprise other telomeric variants. Telomeric
repeats are a natural target of epigenetic regulation and
are traditionally considered heterochromatic regions.
Recent studies show that telomeres in A. thaliana possess
both euchromatic (H3.3, H3K4me3) and heterochromatic
(H3K9me2, H3K27me1, H3K27me3) marks (Procházková
Schrumpfová et al. 2019). Histone H3 deposition is
maintained by histone chaperone proteins. A. Machelová
(CEITEC/MUNI) focused on H3 chaperones and showed
that HISTONE CELL CYCLE REGULATOR (HIRA) and
ANTI-SILENCING FUNCTION 1 (ASF1) are required for
telomere maintenance in A. thaliana, as their simultaneous
depletion causes a lethal phenotype.
TELOMERE REPEAT BINDING (TRB) family
represents a rare example of proteins with confirmed in
vivo telomere localization in plants (Schrumpfová et al.
2014). These proteins bind telomeric DNA through the
MYB-like domain and they possess plant-specific proteindomain
organization (Peska et al. 2011). Conserved
features of TRB proteins in P. patens and A. thaliana were
discussed by A. Kusová (MUNI). Apart from binding
telomeric sequences, TRB proteins directly interact with
the protein catalytic subunit of telomerase (Schrumpfová
et al. 2014). Telomerase adds telomeric repeats to the ends
of chromosomes and consists of telomerase RNA (TR)
and Telomerase reverse transcriptase (TERT) subunits.
In humans, the expression of TERT is strictly controlled
at the transcript level, but TR is ubiquitously expressed.
Interestingly, the expression of the TR subunit follows a
tissue-specific pattern similar to that of TERT expression
in plants, and the plant TR gene is transcribed by RNA
Polymerase III (Pol III), but not by Pol II, as in mammals or
yeasts (Fajkus et al. 2019). All these features as well as the
evolution of both subunits of telomerase were presented by
P. Procházková Schrumpfová (SCI, MUNI) (Schrumpfová
and Fajkus 2020; Fajkus et al. 2021). K. Konečná (IBP,
CEITEC, MUNI) presented characterization of the
Armadillo (ARM) repeat type plant-specific protein found
as an interactor with TERT (Dokládal et al. 2018). The
results suggested thatARM cellular activity is independent
of the telomerase canonical function and implied the
involvement of ARM protein in response to a drug, biotic,
and abiotic stimuli.
Summarizing remarks
TheCPNW2021showedaremarkablediversityofthetopics
broadly linked to plant nuclear and chromosome biology
that were studied in the Czech Republic. Particularly
prominent were the topics of 3D genome organization,
chromatin modifications, epigenetic regulation of gene
expression, sex chromosomes, and genome stability.
A variety of plant model species was also introduced (only
selected species are listed here) Arabidopsis thaliana,
Silene latifolia, Nicotiana tabacum, Allium sp., temperate
cereals (Triticum aestivum, Hordeum vulgare), Zea
mays, or Physcomitrium patens. This provided unique
opportunities to discuss research questions and to establish
new research connections. At the end of the meeting,
the young researchers (under 35 years) were awarded
in several categories. The awards for the best poster
presentations were given to K. Kaduchová (IEB) and M.G.
Trejo Arellano (BC). M. Glombik (IEB) and A. Přibylová
(CUNI) were awarded for the best Ph.D. student talks and
Mingxi Zhou (BC) for the best post-doc talk. Finally, it
was announced that the next CPNW meeting will be held
in 2023 in Brno.
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THE CZECH PLANT NUCLEUS WORKSHOP 2021
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© The authors. This is an open access article distributed under the terms of the Creative Commons BY-NC-ND Licence.
Supplement Q
Dvořák Tomaštíková E., Yang F., Mlynárová K., Hafid S., Schořová Š., Kusová A., Pernisová
M., Přerovská M., Klodová B., Honys D., Fajkus J., Pecinka A., and Schrumpfova P.P.*, 2023
RUVBL proteins are involved in plant gametophyte development, The Plant Journal 114, 325–
337
P.P.S. participated in the design of experiments, data evaluation and wrote the ms
RUVBL proteins are involved in plant gametophyte
development
Eva Dvorak Tomastıkova1
, Fen Yang1,2
, Kristına Mlynarova3
, Said Hafidh4
, Sarka Schorova3
, Alzbeta Kusova3,5
,
Marketa Pernisova3,5
, Tereza Prerovska3
, Bozena Klodova4,6
, David Honys4,6
, Jirı Fajkus3,5,7
, Ales Pecinka1,2
and Petra Prochazkova Schrumpfova3,5,*
1
Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany, Czech Academy of Sciences,
Slechtitelu 31, 77900 Olomouc, Czech Republic,
2
Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Slechtitelu 27, 77900 Olomouc, Czech
Republic,
3
Laboratory of Functional Genomics and Proteomics, Faculty of Science, National Centre for Biomolecular Research,
Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic,
4
Laboratory of Pollen Biology, Institute of Experimental Botany of the Czech Academy of Sciences, Rozvojova 263, CZ-165 02
Prague, Czech Republic,
5
Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Kamenice
5, CZ-62500 Brno, Czech Republic,
6
Department of Experimental Plant Biology, Faculty of Science, Charles University, Vinicna 5, 128 00 Praha 2, Czech
Republic, and
7
Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, CZ-61265 Brno, Czech Republic
Received 12 May 2022; revised 25 January 2023; accepted 1 February 2023; published online 8 February 2023.
*For correspondence (e-mail schpetra@sci.muni.cz).
SUMMARY
The proper development of male and female gametophytes is critical for successful sexual reproduction and
requires a carefully regulated series of events orchestrated by a suite of various proteins. RUVBL1 and
RUVBL2, plant orthologues of human Pontin and Reptin, respectively, belong to the evolutionarily highly
conserved AAA+
family linked to a wide range of cellular processes. Previously, we found that RUVBL1 and
RUVBL2A mutations are homozygous lethal in Arabidopsis. Here, we report that RUVBL1 and RUVBL2A
play roles in reproductive development. We show that mutant plants produce embryo sacs with an abnormal
structure or with various numbers of nuclei. Although pollen grains of heterozygous mutant plants
exhibit reduced viability and reduced pollen tube growth in vitro, some of the ruvbl pollen tubes are capable
of targeting ovules in vivo. Similarly, some ruvbl ovules retain the ability to attract wild-type pollen tubes
but fail to develop further. The activity of the RUVBL1 and RUVBL2A promoters was observed in the embryo
sac, pollen grains, and tapetum cells and, for RUVBL2A, also in developing ovules. In summary, we show
that the RUVBL proteins are essential for the proper development of both male and particularly female
gametophytes in Arabidopsis.
INTRODUCTION
Successful plant sexual reproduction requires the formation
of male and female gametophytes that contain male sperm
cells and female egg cells. In angiosperms, microgametogenesis
and megagametogenesis produce male and female
gametophytes, respectively. During microgametogenesis,
the meiosis of a microspore mother cell gives rise to a tetrad
of haploid male spores (microspores). After being
released from a tetrad, each microspore undergoes mitotic
division (pollen mitosis I) to develop into a binucleate
microspore containing a large vegetative cell and a small
generative cell. The generative cell then migrates into the
vegetative cell to form a unique cell-within-a-cell structure.
The generative cell undergoes pollen mitosis II to produce
two sperm cells enclosed in the vegetative cell (Hafidh &
Honys, 2021). During female gametophyte development,
typically three of the female spores generated by meiosis
degenerate, and the remaining megaspore undergoes three
rounds of mitotic divisions followed by cellularization. This
results in eight nuclei within seven cells organized in a precise
manner within the embryo sac. The mature embryo sac
contains an egg cell flanked by two synergid cells at the
Ó 2023 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited and
is not used for commercial purposes.
325
The Plant Journal (2023) 114, 325–337 doi: 10.1111/tpj.16136
micropylar region, a homodiploid central cell in the middle
region, and three antipodal cells in the chalazal region. The
mature embryo sac develops within a specialized structure,
the ovule, and is surrounded by diploid integuments of
maternal origin (Drews & Yadegari, 2002; Kawashima &
Berger, 2014).
Male and female gametogenesis are highly coordinated
developmental processes. Many proteins regulate
the various metabolic pathways or genetic and epigenetic
processes that mediate the specification of cell identity
(Hafidh & Honys, 2021; Sundaresan & Skinner, 2018).
Defective female gametogenesis usually causes abnormal
embryo sac development, presenting various arrest phenotypes,
thus reducing the seed set (Yang, Fernandez
Jimenez, Majka, et al., 2021). Male gametophyte mutants
in non-essential genes do not generally result in reduced
seed sets because the abundance of pollen is not usually a
limiting factor (Drews & Koltunow, 2011).
RUVB proteins belong to the evolutionarily highly conserved
superfamily of AAA+ ATPases (i.e. ATPases associated
with various cellular activities) (Neuwald et al., 1999).
This class of ATPases contains conserved motifs for ATPbinding
and hydrolysis, such as Walker boxes (Walker A
and B) (Walker et al., 1982), Sensor residues (Sensor I and
II), and Arg-fingers. AAA+ proteins use the hydrolysis of
ATP to exert mechanical forces, and this is essential for
their biological activity (Matias et al., 2006). Despite human
RUVBL proteins sharing a high degree of homology with
the bacterial RuvB helicase, the helicase activity of human
RUVBL is surprisingly low (Matias et al., 2006).
Human RUVBL1 and RUVBL2 (also known as Pontin/
Pontin 52/TIP49/TIP49a/INO80H/TIH1/Rvb1 and Reptin/Reptin
52/TIP48/TIP49b/INO80J/TIH2/Rvb2, respectively) are
essential for many cellular activities and take part in various
cellular complexes. Mammalian RUVBLs are members
of chromatin remodeling complexes INOsitol auxotrophy
80 (INO80) and Swi2/Snf2-related (SWR1) (Jin et al., 2005;
Willhoft & Wigley, 2020) or the Fanconi anemia core complex
involved in DNA damage repair signaling (Rajendra
et al., 2014). RUVBLs are important for the assembly of telomerase
holoenzyme via interaction with telomerase catalytical
subunit (TERT) (Venteicher et al., 2008) or assembly
of the mammalian target of rapamycin complex (Kim et al.,
2013; Shin et al., 2020). They are also involved in the biogenesis
of small nucleolar ribonucleoproteins (Cloutier
et al., 2017). In addition, RUVBLs were shown to be important
transcriptional regulators, regulators of the cell cycle,
and mitotic progressiondue to their interaction with MYC,
E2F1, and other transcription factors. They are also
involved in promoting mitotic spindle assembly (Mao &
Houry, 2017). RUVBL1 assists actin-based cellular motility
(mediate actin polymerization) (Taniuchi et al., 2014).
These processes underline the central role of RUVBLs in
promoting cell proliferation and survival.
The Arabidopsis thaliana (Arabidopsis) genome
encodes one homolog of RUVBL1 and two homologs of
RUVBL2–RUVBL2A and RUVBL2B, where only RUVBL1 and
RUVBL2A appear to be functional (Brunkard et al., 2020;
Julca et al., 2021). Importantly, RUVBL1 and RUVBL2A
were suggested as essential for gametophyte development
(Brunkard et al., 2020; Holt et al., 2002; Schorova et al.,
2019).
Similar to their homologs in diverse organisms, Arabidopsis
RUVBL1 and RUVBL2A can form both homo- and
heteromers (Majerska et al., 2017; Schorova et al., 2019).
Similarly, RUVBL proteins are not associated with just one
cellular process but rather have roles in the regulation
of various cellular mechanisms. We have previously
described the association of RUVBL1 and RUVBL2A, but
not RUVBL2B, with TERT from Arabidopsis (Majerska
et al., 2017). Additionally, Arabidopsis RUVBL proteins contribute
to the TOR signaling network by providing chaperone
activity for the correct assembly of the TORC1
complex (Brunkard et al., 2020; Van Leene et al., 2019).
Similarly, RUVBL1 and RUVBL2 are components of the
plant chromatin remodeling complexes SWR1 and INO80,
where they might have a regulatory role (Bieluszewski
et al., 2015; Luo et al., 2020; Zander et al., 2019). RUVBL1
was suggested as a regulator of specific disease resistance
(R) genes (Holt et al., 2002).
To shed light on the roles of RUVBL proteins during
sexual reproduction, we analyzed the gametophyte development
of ruvbl mutants. We performed reciprocal crosses
and identified major problems in the development and fertilization
of the female gametophyte. Microscopic analyses
showed that ruvbl1 and ruvbl2a failed to develop viable
ovules. Moreover, RUVBL1 and RUVBL2A are involved in
the development of viable pollen and pollen tube, although
this role is less critical. These findings are in agreement
with the reduced transmission of the mutant alleles also
from the paternal side. Our observation revealed the
essential role of RUVBL proteins in plant haploid stages in
Arabidopsis because the RUVBL proteins are needed for
the proper development of both the male and especially
female gametophytes.
RESULTS
Mutations in RUVBL1 or RUVBL2A result in seed-setting
defects
Our previous study showed that homozygous T-DNA insertion
mutants of Arabidopsis RUVBL1 and RUVBL2A are not
viable and, from several T-DNA insertion lines, only ruvbl1-
1 and ruvbl2a-1 alleles (Figure S1) showed heterozygous
progeny. Their offspring genotypic ratios did not follow the
expected Mendelian frequencies (Schorova et al., 2019).
To gain insight into the function of RUVBL1 and RUVBL2A
in gametophyte and early sporophyte development,
Ó 2023 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2023), 114, 325–337
326 Eva Dvorak Tomastıkova et al.
1365313x,2023,2,Downloadedfromhttps://onlinelibrary.wiley.com/doi/10.1111/tpj.16136byCzechs-MasarykUniversity,WileyOnlineLibraryon[28/04/2023].SeetheTermsandConditions(https://onlinelibrary.wiley.com/terms-and-conditions)onWileyOnlineLibraryforrulesofuse;OAarticlesaregovernedbytheapplicableCreativeCommonsLicense
we examined the seed production of the self-pollinated
heterozygous ruvbl1-1 and ruvbl2a-1 mutant plants (hereafter
ruvbl1-1/+ and ruvbl2a-1/+, respectively) using the
fully elongated siliques of the main inflorescence stem.
Our microscopic analyses revealed up to 41.7% (Æ 3.4%)
and 51.1% (Æ 5.9%) aborted ovules in ruvbl1-1/+ and
ruvbl2a-1/+ mutant siliques, respectively, whereas 0.5%
(Æ 0.2%) of aborted ovules were observed in selfpollinated
wild-type (WT) plants (Figure 1a,b). Next to the
aborted ovules were seeds that appeared to have developed
normally, and these were indistinguishable from
those in WT plants. To validate our initial findings, we
screened several additional available T-DNA insertional
mutants and isolated more RUVBL1 and RUVBL2A T-DNA
insertional mutants ruvbl1-6+/À and ruvbl2a-2+/À (ruvbl1-
6/+ and ruvbl2a-2/+; Figure S1). Microscopic analyses
revealed up to 55.4% (Æ 5.6%) and 49.7% (Æ 5.2%) aborted
ovules in ruvbl1-6/+ and ruvbl2a-2/+ mutant siliques
(Figure S2a,b). This suggested that the absence of homozygous
ruvbl1 and ruvbl2a plants is not a result of embryo
lethality, but might be the effect of almost complete lethality
from the female gametophyte, which would correspond
to 50% of aborted ovules according to Mendelian rules.
RUVBL genes are necessary for both male and female
fertility
To assess the maternal and/or paternal effects on the frequency
of aborted ovules, we performed reciprocal crosses
between the ruvbl1/+ or ruvbl2a/+ and WT plants. We
analyzed at least five fully elongated siliques of the main
inflorescence stem from two to three plants for each combination
specified in Figure 1. The percentages of aborted
ovules in fully developed siliques of ruvbl1-1/+ and ruvbl1-
6/+ mother plants pollinated by WT plants were 51.4%
(Æ 2.3%) and 47.4% (Æ 2.3%), respectively. The percentages
of aborted ovules of ruvbl2a-1/+ and ruvbl2a-2/+
mother plants pollinated by WT plants were 62.9%
(Æ 6.7%) and 46.8% (Æ 3.1%), respectively. The control
manual cross between two WT parents produced only
1.1% (Æ 0.4%) of such aborted ovules. Pollination of WT
mother plant with ruvbl1-1/+ and ruvbl1-6/+ pollen resulted
in 17.7% (Æ 10.6%) and 4.2% (Æ 4.5%) aborted ovules. Similarly,
pollination of WT mother plant with ruvbl2a-1/+ and
ruvbl2a-2/+ pollen resulted in 19.5% (Æ 1.4%) and 2.9%
(Æ 3.4%), of aborted ovules, respectively (Figure 1c,d;
Figure S2c,d). The number of the normally developed and
aborted seeds was not affected by an error in plant handling
during reciprocal crosses, as observable in Figure 1
(b,d).
Although reciprocal crosses between plants heterozygous
for RUVBL1 and RUVBL2A with WT revealed both
male-related and female-related reduced fertility, female
gametophyte development was more severely impaired
by RUVBL1 and RUVBL2A loss-of-function than male
Figure 1. Analysis of seed setting in RUVBL1 and RUVBL2A mutant plants
and transmission ratio distortion in reciprocal crosses.
(a) Representative content of siliques in self-pollinated WT, ruvbl1-1/+, and
ruvbl2a-1/+ plants. White asterisks indicate aborted ovules. Scale
bar = 1 mm.
(b) Percentage of aborted ovules of self-fertilized WT and mutant plants.
Numbers on top of bars correspond to the number of analyzed seeds.
***P < 0.00001 via Fisher’s exact test (chi-squared). Error bars indicate the
SD of the means of individually analyzed siliques.
(c) Representative content of siliques from reciprocal crosses between
mutants and WT. The first genotype indicates the mother plants. Scale
bar = 1 mm.
(d) Percentage of aborted ovules of manual reciprocal crosses between WT
and mutant plants. Numbers on top of the bars correspond to the number
of analyzed seeds. ***P < 0.00001 via Fisher’s exact test (chi-squared). Error
bars indicate the SD of the means of individual siliques analyzed.
(e) Transmission ratio distortion in reciprocal crosses between each of the
ruvbl1-1/+ or ruvbl2a-1/+ and WT plants. Flowers were emasculated and fertilized
manually with pollen. Numbers on top of the bars correspond to the
number of analyzed plants. Error bars indicate the SD of the means of the
individual analyzed siliques. WT, wild-type.
Ó 2023 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2023), 114, 325–337
RUVBL proteins in gametophyte development 327
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gametophyte development. The phenotypic analysis of
seed abortion of self-pollinated or reciprocally crossed
plants, described above, indicates the essential role of
RUVBLs during the haploid gametophyte phase.
Reduced transmission of ruvbl mutant alleles
Our observation indicates that RUVBLs are required for the
normal development of Arabidopsis gametophyte. To test
whether male or female gametophytes are affected by dysfunctional
RUVBL proteins, we analyzed the transmission
efficiency of the ruvbl1 and ruvbl2a mutant alleles through
the female and the male gametophytes in the F1 progeny
of the reciprocally crossed plants described above.
Assuming that RUVBLs are essential for the development
of both micro- and megagametophytes, the expected
number of heterozygous offspring would be 0%. Pollination
of ruvbl1-1/+ or ruvbl2a-1/+ maternal plants with WT
yielded 0% (0.0 Æ 0.0%) or 0.3% (0.3 Æ 0.6%), respectively,
of heterozygous offspring. When pollen grains from
either ruvbl1-1/+ or ruvbl2a-1/+ plants were used to pollinate
WT mother plants, we identified 27.2% (Æ 2.8%) and
4.6% (Æ 3.9%), respectively, of heterozygous offspring
(Figure 1e). This indicates that male gametophyte development
is also affected by the ruvbl1-1/+ or ruvbl2a-1/+
mutations.
To investigate whether RUVBLs are also required for
the development of other plant organs, we performed an
extensive analysis of the heterozygous RUVBL1 and RUVBL2A
mutants using a semi-automated plant phenotyping
system. However, the heterozygous plants for both genes
did not reveal any significant differences concerning leaf
morphology (shape, structure or diameter), growth dynamics,
growth weight or shoot growth rate compared to WT
Arabidopsis plants (Figure S3). This indicates that the
RUVBL1 and RUBL2A mutations used are recessive.
Our results suggest that the RUVBL1 and RUVBL2A
proteins have a very important role in plant gametophyte
development.
RUVBL1 and RUVBL2A are essential for ovule
development
To examine the defects in female gametophyte development,
we took a closer look at the development of the
embryo sac in ruvbl1-1/+ and ruvbl2a-1/+ plants at stage 6
of female gametophyte development using confocal
microscopy (Christensen et al., 1997). We observed a typical
configuration in the WT embryo sac with one large central
cell nucleus, one small egg cell nucleus, two synergid
cell nuclei, and three antipodal cells. By contrast, ruvbl1-1/
+ plants produced 63.2% (86/136) WT-like embryo sacs,
2.2% (3/136) with zero nuclei, 8.8% (12/136) with one
nucleus, 14.7% (20/136) with two large nuclei, and 11.0%
(15/136) with three or four nuclei. In ruvbl2a-1/+ plants, we
found 53.3% (49/96) WT-like embryo sacs and 2.2% (2/92)
with zero nuclei, 6.5% (6/92) with one nucleus, 26.1% (24/
92) with two large nuclei, and 12.0% (11/92) with three or
four nuclei (Figure 2). Similar numbers were obtained with
differential interference contrast of cleared ovules (64.4%
and 51.6% of WT-like embryo sacs) in ruvbl1-1/+ and
ruvbl2a-1/+, respectively (Figure S4).
To test whether the ruvbl ovules with abnormal
embryo sacs could still be viable to attract pollen tubes for
fertilization, we pollinated ruvbl1-1/+ and ruvbl2a-1/+ pistils
with WT pollen and monitored pollen tube attraction by
aniline blue callose staining (Figure 3a,b) (Billey et al.,
2021). Mutant ovules can be easily distinguished by callose
accumulation in the embryo sac (Sun et al., 2004). In
ruvbl1-1/+, 36.7% of the ovules failed to attract pollen
tubes, and only 6% correctly received the pollen tube but
failed to develop further (Figure 3c–f). In ruvbl2a-1/+, 47.1%
of the ovules were not targeted by WT pollen tubes that
failed to enter the micropyle, suggesting a more severe
phenotype (Figure 3c–f).
The severe defects of the embryo sac with zero nuclei
indicate either a failure to enter female meiosis or an
abnormal female meiosis process that leads to megagametophyte
nuclei degradation in ruvbl1-1/+ and ruvbl2a-1/+
plants. The severe defects of the embryo sacs from heterozygous
plants indicate that there are also problems during
mitosis. Interestingly, some of the ruvbl targeted ovules
can still receive the WT pollen tube correctly, but then
apparently fail to develop further.
RUVBL1 and RUVBL2A are required for pollen fertility
Microgametogenesis is a post-meiotic process that initially
leads to the development of unicellular microspores and
later tricellular mature pollen. In the mature Arabidopsis
pollen grain, two sperm cells, enclosed within the vegetative
cell, are assembled with the vegetative nucleus into
the male germ unit.
To screen for the putative phenotypic defects, we used
pollen grains and performed 40
,6-diamidino-2-phenylindole
(DAPI) staining for the position and shape of vegetative
and generative nuclei, as well as fluorescein diacetate
(FDA) staining to distinguish intact and defective pollen
grains. We restricted the phenotype screen to mature pollen
because disorders, even in early developmental stages,
are likely to be reflected at the mature pollen stage. We
also paid special attention to structural disorders and
nuclei organization (Renak et al., 2012).
Pollen grains of ruvbl1-1/+ and ruvbl2a-1/+ plants
showed standard phenotypes with no changes in the
shape, size or cell wall structure compared to WT
(Figure S5a; Table S1a). DAPI staining revealed that almost
all mature pollen grains of WT, ruvbl1-1/+, and ruvbl2a-1/+
plants have a centrally positioned vegetative nucleus and
two nearby located generative nuclei. The aberrant positioning
of the nuclei and predominantly eccentric position
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of individual sperm nuclei was found only in 1.9%
(Æ 1.9%), 2.4% (Æ 1.5%) or 3.7% (Æ 1.7%) pollen grains
from WT, ruvbl1-1/+, and ruvbl2a-1/+ plants, respectively.
These data indicate that the position of nuclei in the
mature pollen grains differs in ruvbl2a-1/+, but not in
ruvbl1-1/+, compared to WT (Figure S5b; Table S1b). Analysis
of intact pollen grains with FDA staining revealed significant
reductions from 93.7% (Æ 2.8%) in WT to 56.8%
Figure 2. Confocal fluorescence micrographs of ovules from ruvbl1-1/+ and ruvbl2a-1/+ plants. Scale bar = 10 lm.
(a) Representative WT-like embryo sac with two synergid cell nuclei (sn), one egg cell nucleus (ecn), one central cell nucleus (ccn), and three antipodal cells (an).
(b–e) Examples of defective embryo sacs from ruvbl1-1/+ plants. Embryo sac with three nuclei (b, c). Embryo sac with one nucleus (d). Ovule with a degraded
embryo sac (e). Black squares indicate aberrant ecn and ccn.
(f–h) Abnormal embryo sacs from ruvbl2a-1/+ plants. Embryo sac with three nuclei (f). Embryo sac with two large nuclei (g). Embryo sac with one nucleus (h).
Black squares indicate aberrant ecn and ccn.
(i) Percentage of normal and abnormal ovules from analyzed genotypes. Ovules were analyzed from at least three different plants. Numbers on top of the bars
correspond to the number of analyzed ovules.
(j) Schematic of female gametophyte stages (FG1–6) showing the sequence of syncytial mitotic divisions that lead to cellularization and formation of the embryo
sac with fused central cell nucleus. an, antipodal cells; ccn, central cell nucleus; ecn, egg cell nucleus; mn, megaspore nucleus; sn, the synergid cell nucleus.
WT, wild-type.
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Figure 3. ruvbl1 and ruvbl2a control ovule competence for pollen tube attraction.
(a) Surgically dissected pistil 24 h after pollination (hap) with wild-type pollen stained with aniline blue. Yellow arrows indicate the position of successful pollen
tube entry and reception at the micropyle. Asterisks point out untargeted ruvbl ovules. Scale bar = 50 lm.
(b) Seed size differences of successfully targeted WT and ruvbl ovule 48 hap. Scale bar = 50 lm.
(c–e) Three phenotypic classes scored for panel (f). WT ovule, correct pollen tube attraction and reception (c); ruvbl ovule, correct pollen tube attraction and
reception (d); ruvbl ovule, attraction (e). pt, pollen tube. Yellow arrows point to the micropyle pollen tube entry and reception site. Scale bars = 50 lm.
(f, g) Quantification of pollen tube ovule-targeting competence. Numbers on top of the bars correspond to the number of analyzed ovules. ***P < 0.001. Twosided
Student’s t-test was used to calculate P values between the groups.
Ó 2023 The Authors.
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330 Eva Dvorak Tomastıkova et al.
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(Æ 12.4%) in ruvbl1-1/+ and 51.3% (Æ 10.2%) in ruvbl2a-1/+
(Figure 4a–g; Table S1c).
Our observations suggest that the paternally induced
abortion ratio is partially caused by decreased viability of
pollen grains in ruvbl1/+ and ruvbl2a/+ plants, but is
accompaniedby only minor changes in mature pollen phenotypes
relating to nucleus positioning in ruvbl2a/+ plants.
RUVBL1 and RUVBL2A are involved in pollen tube growth
To investigate whether ruvbl pollen can germinate and
grow pollen tubes, we analyzed pollen tube growth in vitro
for both heterozygous ruvbl1-1/+ and ruvbl2a-1/+ lines. In
vitro germination assays showed a reduced germination
rate of 54.1% in ruvbl2a-1/+ (n = 196) and 61.8% in ruvbl1-
1/+ (n = 236), whereas the germination rate of WT was
75.8% (n = 182) (Table S2). Germinated pollen tubes from
ruvbl1-1/+ and ruvbl2a-1/+ plants grew significantly shorter
(447.4 lm in WT to 302.6 lm in ruvbl1-1/+ and 215.3 lm in
ruvbl2a-1/+) relative to WT, suggesting an additional defect
in pollen tube growth (Figure 4h–k).
We took advantage of the fact that the ruvbl1-1/+ SAIL
T-DNA insertion line contains an uidA gene encoding
b-glucuronidase under the control of pollen-specific LAT52
promoter (proLAT52::GUS) (Sessions et al., 2002). Histochemical
GUS activity staining of tissues from ruvbl1-1/+
allowed us to specifically follow ruvbl1 mutant pollen tube
growth within the pistil. This staining revealed very limited
and shorter ruvbl1-1 pollen tube growth in the pistil, consistent
with our in vitro observation (Figure S6a).
Because ruvbl1-1/+ and ruvbl2a-1/+ germinate poorly
and grow significantly shorter pollen tubes, we considered
whether pollen tubes from ruvbl1-1/+ and ruvbl2a-1/
+ plants are sufficiently competent to target WT ovules
for fertilization. To assess their competence, we pollinated
WT pistils with ruvbl1-1/+ and ruvbl2a-1/+ pollen,
followed by pollen tube callose staining with aniline blue
(Billey et al., 2021). Twenty-four hours after pollination
(24 hap), aniline blue staining revealed that both ruvbl1-1
and ruvbl2a-1 pollen tubes are partially capable of targeting
WT ovules. In WT plants pollinated with ruvbl1-1/+
and ruvbl2a/+ pollen, we observed 11.5% (n = 663) and
17.6% (n = 682) of non-canonically targeted ovules,
respectively. However, only 2% (n = 619) of aberrant,
non-canonically targeted ovules were observed in control
WT plants pollinated with WT pollen (Figure 3g;
Figure S7). Additionally, we used the proLAT52::GUS
marker of ruvbl1-1 SAIL T-DNA line to observe ruvbl1
mutant pollen targeting the WT ovules using the blue-dot
GUS assay. We pollinated emasculated WT pistils with
ruvbl1-1/+ pollen grains. As a control, we pollinated WT
pistil with proLAT52::GUS/+ pollen. Blue-dot assays
revealed that 11.67% (n = 411) of the WT ovules were targeted
by ruvbl1 pollen tubes, whereas 47.68% (n = 409)
of the WT ovules were targeted by control proLAT52::
GUS pollen tubes (Figure S6b,c).
Overall, our results show that ruvbl1-1/+ and ruvbl2a-
1/+ lines have a reduced germination rate and defects in
pollen tube growth. In addition, we observed that pollen
Figure 4. ruvbl1-1/+ and ruvbl2a-1/+ plants produce defective pollen grains.
(a–f) Fluorescein diacetate (FDA) epifluorescence micrographs of stained
pollen grains. Asterisks mark defective pollen grains. Scale bars = 50 lm.
The pollen in ruvbl1-1/+ remains attached to a tetrad due to homozygous
qrt1 mutation.
(g) The proportion of live pollen grains determined by FDA staining. Mean
values from three independent experiments with three different plants are
plotted. Statistical significance was calculated using Fisher’s exact test.
***P < 0.00001.
(h–j) In vitro WT, ruvbl1-1/+, and ruvbl2a-1/+ pollen tube growth. Green lines
indicate representative short pollen tubes in ruvbl mutants.
(k) Box-and-whisker plot showing the pollen tube length distribution of the
WT, ruvbl1-1/+, and ruvbl2a-1/+. Middle bars are median values; boxes indicate
the first to third interquartile ranges, whiskers indicate 1.5 of minimum
and maximum interquartile, and dots outside interquartile boxes indicate
outlier values. Numbers on top of the bars correspond to the total number
of measured pollen tubes. WT, wild-type.
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tubes from ruvbl1-1/+ and ruvbl2a-1/+ plants are partially
capable of targeting WT ovules.
Tissue-specific expression of the RUVBL genes in
Arabidopsis
To obtain further insight into the RUVBL expression pattern,
we generated stable reporter lines where RUVBL1
and RUVBL2A promoters were fused to the GUS. In 7-dayold
seedlings, high activity of the RUVBL2A promoter was
detected in the root apical meristems and at the positions
of forming lateral roots (Figure S8). The activity of the
RUVBL1 promoter was found only in lateral root primordia.
The activity of both promoters was not detected in other
seedling tissues or in some other plant tissues (e.g. true
leaves, stems, sepals, petals). Consistent with gametophytic
lethality, we observed RUVBL1 and RUVBL2A promoter
activity in inflorescences in different stages of
flower development (Alvarez-Buylla et al., 2010) (Figure 5a,
f), which prompted us to analyze individual tissues. Activity
of the RUVBL1 and RUVBL2A promoters was observed
in pollen as well as in the adjacent tapetum layer
(Figure 5b–d,g–i). Furthermore, we detected RUVBL2A promoter
activity in developing ovules and strong promoter
activity of both RUVBL1 and RUVBL2A genes in fertilized
ovules, but not in ovule integuments (Figure 5e,j).
To consider the broader context of developmental
processes and conditions in which RUVBL proteins might
play a role, we compared our observations with global
transcriptome data from various developmental stages and
tissues from Arabidopsis (Julca et al., 2021) (Figure S9).
These data supported our observation that RUVBL1 and
RUVBL2A genes are transcribed during pollen development
in ovules and meristems. Taken together, the data
relating to the expression of RUVBL1 and RUVBL2A are
consistent with the genetically determined role of RUVBL
proteins during gametophyte development.
DISCUSSION
RUVBL proteins are highly conserved ATPases from the
AAA+ superfamily (Neuwald et al., 1999). They are related
to bacterial RUVB helicase, which participates in Holliday
junction resolution (Putnam et al., 2001). Despite their
importance in transcriptional regulation, chromatin remodeling,
DNA damage signaling and repair, and their extensive
characterization in animals and yeasts (Dauden et al.,
2021), only limited information is available regarding their
functions in plants.
The human genome encodes two homologs of RUVB
proteins, RUVBL1 (Pontin) and RUVBL2 (Reptin), whereas
the Arabidopsis genome contains three RUVBL genes. Arabidopsis
RUVBL1 protein has a high sequence similarity
(88%) to human RUVBL1/Pontin (Figure S10). Two RUVBL2
proteins in Arabidopsis, namely RUVBL2A and RUVBL2B,
show high mutual sequence similarity (91%), suggesting
that they are paralogs, and also a high similarity with
human RUVBL2 (87 and 82%, respectively) (Figure S11).
Although the overall sequence similarity between Arabidopsis
RUVBL1 and RUVBL2A or RUVBL2B proteins is
lower (63 or 60%, respectively), all three proteins share
conserved Domains (DI, DII, and DIII) with several highly
conserved motifs. Domain II (DII) is an insertion unique to
RUVBL compared to other AAA+ family members (SilvaMartin
et al., 2016). Walker A/B motifs are proposed to
coordinate ATP binding and hydrolysis, sensor I/II motifs
sense whether the protein is bound to di- or triphosphates,
and the Arg finger allows the efficient hydrolysis of ATP
(Matias et al., 2006) (Figure S12).
Phylogenetic studies showed that the RUVBL1 gene
diverged from an archaeal RUVBL2 ancestor when eukaryotes
evolved from archaea (Kurokawa et al., 1999; Schorova
et al., 2019). This corresponds to our observation that plant
RUVBL proteins are not functionally redundant because
loss-of-function mutations in either RUVBL1 or RUVBL2A
lead to gametophyte lethality. Moreover, RUVBL2B is
considered a pseudogene because RUVBL2B is not
expressed and no phenotypes in a line carrying a T-DNA
insertion in the RUVBL2B coding sequence were observed
(Brunkard et al., 2020).
The present study aimed to investigate the involvement
of RUVBL proteins in plant sporophyte and male/
female gametophyte development. Our extensive semiautomated
phenotyping analysis did not reveal any significant
differences in ruvbl1-1/+ and ruvbl2a-1/+ T-DNA
insertion mutant plants during the development of sporophytes
(seedlings, roots, stem, leaves). However, future
experiments with weak alleles of ruvbl1 and ruvbl2a are
needed to better understand the function of RUVBL1 and
RUVBL2A in the sporophyte development of Arabidopsis.
Mammalian RUVBLs interact with many molecular
complexes with vastly different downstream effectors,
which is also true for their plant homologs. It was
observed that Arabidopsis RUVBL proteins associate with
the TERT subunit of telomerase via interaction with TELOMERE
REPEAT BINDING (TRB) proteins that colocalize with
Figure 5. Detection of RUVBL1 and RUVBL2A promoter activity by GUS histochemical staining.
(a, f) Inflorescence. Scale bars = 2 mm.
(b, g) Pollen grains from flower stage (from left) 9, 10/11, 12, 15/16. Scale bars = 10 lm.
(c, h) Anthers isolated from flower stages 10 (left) and 16 (right).
(d, i) Flowers at different stages of development; flower stages (from left) 8/9, 9, 10/11, 12, 14, 15/16. Scale bars = 500 lm.
(e, j) An unfertilized ovule from flower stages 9, 10/11, 11/12, 12, and 12. A fertilized ovule from floral stage 15 (bottom right corner). Scale bars = 50 lm.
Ó 2023 The Authors.
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(a) (b)
(c)
(d)
(e)
(f) (g)
(h)
(i)
(j)
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RUVBL proteins in gametophyte development 333
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telomeric sequences in situ and in vivo (Dvorackova et al.,
2010; Mozgova et al., 2008; Schrumpfova et al., 2004;
Schrumpfova et al., 2016). Telomere- and telomeraserelated
functions of RUVBL proteins (Schrumpfova &
Fajkus, 2020) correspond well to the RUVBL expression
pattern observed in the present study (Figure 4). RUVBL
proteins interact with ACTIN-RELATED PROTEIN 4 (ARP4).
ARP4, in addition to being a component of the SWR1 chromatin
remodeling complex (Bieluszewski et al., 2015), also
interacts directly with TERT (Fulneckova et al., 2021), thus
representing another link between RUVBL and telomerase,
in addition to that of RUVBL-TRB-TERT (Schorova et al.,
2019). RUVBL1 was reported to be a regulator of disease
resistance (R) genes (Holt et al., 2002). Arabidopsis
RUVBL1 and RUVBL2A were also reported as members of
the plant TOR signaling network, similar to that of yeast or
mammals (Brunkard et al., 2020; Van Leene et al., 2019).
However, further studies are needed to understand the full
complexity of the involvement of RUVBL proteins in various
signaling pathways.
We analyzed the transcription of RUVBL1 and RUVBL2A
using promoter-reporter lines and found high expression
of both genes in both male and female reproductive
structures and meristem tissues. We did not observe transcription
in the root differentiation zone in seedlings, nor
in cotyledons, true leaves, stems, sepals or petals. We
detected RUVBL1 and RUVBL2A promoter activity in fertilized
ovules and for RUVBL2A also strong activity in developing
ovulesbut not in ovule integuments. In male
gametophytes, we observed that RUVBL promoters are
active not just in pollen grains, but also in the adjacent
tapetum. These observations are not surprising because
tapetum cells form the innermost of the four anther
somatic layers, surrounding the developing reproductive
cells to provide materials for early pollen development,
after which they undergo programmed cell death (Ischebeck,
2016). These data agree very well with the available
transcriptomic data (Julca et al., 2021; Susaki et al., 2021).
Recent analyses of female gametophyte cells using
markers of cell fate to distinguish different cell types
showed increased expression of both RUVBL genes in central
and egg cells, but not in synergids (Susaki et al., 2021),
which further supports our observations. The involvement
of RUVBL proteins in the regulation of early developmental
processes is consistent with high transcription of RUVBL1
and RUVBL2A, but not RUVBL2B, detected in eggs, ovules,
endosperm, pollen (Julca et al., 2021) and the tapeta (Figure
5; Figure S9).
The loss-of-function mutants of ruvbl1-1 or ruvbl2a-1
strongly impacted female gametogenesis producing an
aberrant embryo sac. Similarly, male gametogenesis was
compromised by reduced pollen germination and pollen
tube guidance. Our results imply that the failure to
transmit the ruvbl mutant allele through the female is most
likely attributable to the presence of an aberrant embryo
sac in ruvbl1-1 and ruvbl2a-1 ovules. This is further aggravated
by the lack of ruvbl ovule competence to attract pollen
tubes for fertilization, resulting in collapsed ovules in
ruvbl1-1/+ and ruvbl2a-1/+ siliques. The genetic data correspond
with the GUS signals in the reproductive tissues of
the mutants, showing that the major problem arises from
embryo sac development and microspores. These are the
exact same tissues where we detected transcriptional activities
of RUVBL1 and RUVBL2A using promoter GUS
reporter lines (Figure 5).
In summary, the RUVBL1/2 complex has been implicated
in many cellular and physiological processes, ranging
from DNA repair, chromatin remodeling, small
nucleolar ribonucleoprotein, telomerase assembly, TOR
complex assembly, cell cycle, transcription regulation and
others (Dauden et al., 2021; Mao & Houry, 2017). Here, we
have uncovered a function of RUVBL proteins in gametophyte
development in plants, and we have described the
crucial dominant role of RUVBL proteins in megagametogenesis,
as well as in microgametogenesis.
EXPERIMENTAL PROCEDURES
Plant material
Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Col-0) was
used as WT control. Previously described T-DNA insertion lines
used in the present study were: ruvbl1-1 (SAIL_397_C11), ruvbl1-6
(SALK_133101), ruvbl2a-1 (GK-543F01), and ruvbl2a-2 (SALK_
071103) in the Col-0 background were used. For genotyping of TDNA
mutants, genomic DNA was isolated from small leaves
according to Edwards et al. (1991) and used for PCR analysis using
DreamTaq polymerase (Thermo Fisher Scientific, Waltham, MA,
USA) with primers listed in Schorova et al. (2019). The conditions
used were in accordance with the manufacturer’s instructions. For
promoter-reporter constructs, regions 1000 bp upstream of the
RUVBL1 or RUVBL2A transcription start sites were PCR-amplified,
cloned into pDONR207, and recombined into the binary Gateway
vector pKGWFS7.0 containing the uidA gene encoding GUS. The
final plasmids were transformed into Agrobacterium tumefaciens
strain GV3101 and then into Arabidopsis Col-0 using the floral dip
method (Clough & Bent, 1998). T1 generation seeds were screened
on 1/2 MS plates containing 50 lg mlÀ1
kanamycin (Duchefa, Haarlem,
The Netherlands) and resistant plants were transferred to soil.
T2 populations with approximately 75% resistant seedlings, indicating
single locus T-DNA insertions, were considered for further
analyses. Primers used for cloning are listed in Table S3.
Semi-automated phenotyping
Plants were grown in soil under a 12:12 h light/dark photocycle,
with a light intensity of 120 lmol mÀ2
secÀ1
white light and
4.5 lmol mÀ2
secÀ1
far-red light at 21°C and 60% relative humidity.
Twenty plants per line were used for phenotyping. Data acquisition
and image analysis were performed using Photon Systems
Instruments, spol. s r.o. (Drasov, Czech Republic). The relative leaf
growth rate, roundness, compactness or growth weight were
measured every third day.
Ó 2023 The Authors.
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Plant growth conditions
Plants were grown in soil for 16 h, with a light intensity of approximately
200 lmol mÀ2
secÀ1
and 21°C during the day, and for 8 h
at 19°C during the night, and 70% relative humidity. For seedling
growth, seeds were surface-sterilized for 5 min in 70% ethanol
and 5 min in 10% sodium hypochlorite, followed by three washes
with sterile water. Sterilized seeds were kept in the dark at 4°C for
2 days for stratification and germinated on vertical plates with 1/2
MS medium (Duchefa) containing 0.05% MES, 0.8% agar at 22°C
under a 16:8 h light/dark photocycle.
Genetic transmission through male and female gametes
To determine the gametophytic transmission of T-DNA, reciprocal
test crosses were performed between the WT and each heterozygous
mutant. Plants were emasculated approximately 48 h before
pollination in crossing experiments. Harvested seeds from individual
siliques were sown on 1/2 MS containing plates containing
50 lg mlÀ1
Phosphinothricin for ruvbl1-1/+ and 75 lg mlÀ1
sulfadiazine
for ruvbl2a-1/+, DNA was extracted by the standard Edwards
protocol (Edwards et al., 1991), and plants were genotyped as
described in the section above on ‘Plant material’.
GUS histochemical staining
GUS staining was performed as described in Nowicka et al. (2020)
and Vladejic et al. (2023) with minor modifications. Briefly, 7-dayold
seedlings of proRUVBL1::GUS and proRUVBL2A::GUS were
stained in GUS staining buffer [0.1 M sodium phosphate buffer
(pH 7.0), 0.05% Triton X-100, 0.1% X-Glc sodium salt (Duchefa)]
containing 1 mM K3[Fe(CN)6] and 1 mM K4[Fe(CN)6] for 7 h, at 37°C.
The inflorescences from 40-day-old plants were stained in GUS
staining buffer containing 2 mM K3[Fe(CN)6] and 2 mM K4[Fe(CN)6]
for 48 or 72 h for pollen or ovules stained in inflorescence, respectively,
at 37°C. Subsequently, the samples were incubated overnight
in 80% ethanol (v/v) at room temperature shaking at 50 rpm,
as described previously (Malamy & Benfey, 1997). Ovules and pollen
grains were extracted from inflorescences before microscopic
analysis, which was performed using an AxioZoom.V16Apotome2
microscope or AxioImager.Z2-ZEN in a bright field
(Carl Zeiss GmbH, Jena, Germany).
Blue dot GUS assay
Young buds of WT-plants before anther dehiscence were emasculated
and left to mature for 24 h until the stigma papillae fully
developed. Pistils were then pollinated with ruvbl1-1/+ pollen or
control WT pollen grains as described by Khouider et al. (2021)
expressing the proLAT52::GUS. Pistils were collected at 24 hap.
Fertilized pistils were dissected under a binocular dissection
microscope (Leica Microsystems, Wetzlar, Germany) and strips of
ovules attached to the septum were transferred to GUS staining
solution [0.2 M Na2HPO4, 0.2 M NaH2PO4, 10 mM EDTA, 0.1% Triton
X-100, 1 mM K3Fe(CN)6, 2 mM X-Gluc]. Samples were stained for
24 h. Stained tissues were cleared of chlorophyll by bleaching in
an ethanol series of 90, 70, 50, and 30% (v/v) before imaging.
Stained pistils were imaged with a stereomicroscope SteREO Discovery
V8 (Carl Zeiss GmbH) equipped with an AxioCam HRc camera
and Zeiss Axioimager Apotome2 using ZEN software.
Aniline blue callose staining
Aniline blue pollen tube stain was performed as described by
Billey et al. (2021) and Liu and Meinke (1998).
FDA staining
FDA staining was performed as described by Li (2011) with modifications
(Yang, Fernandez Jimenez, Majka, et al., 2021; Yang,
Fernandez-Jimenez, Tuckova, et al., 2021). Fluorescence was
observed after 20 min of staining using an inverted microscope
(IX 83; Olympus, Tokyo, Japan) at 543/620 nm excitation/emission
wavelengths and the same region was photographed with differential
interference contrast optics to determine the quantities of
pollen grains.
DAPI staining buffer
For phenotype analysis, three fully open flowers in developmental
stage 14 (Smyth et al., 1990) were collected. Fifty microlitres of
DAPI was added to each sample as previously described in Renak
et al. (2012) and shaken briefly to release pollen grains from
anthers. Pollen was observed using an inverted microscope Zeiss
Axio Imager.Z1 in bright field and UV epifluorescence. The percentage
of phenotypic defects was calculated from observations
of at least 200 pollen grains in each sample.
Ovule clearing
Clearing of ovules was performed as described by Liu and
Meinke (1998).
Confocal microscopy of ovules
The confocal observation of ovules was performed according to
the method described by Christensen et al. (1997) with the modifications.
Flowers at stage 13 were emasculated approximately 48 h
before the ovules were examined. Sepals, petals, and stigmas
were removed from the isolated flowers. Then, the remaining pistils
were exposed in fixation buffer [4% glutaraldehyde and
12.5 mM cacodylate (pH 6.9)] for 2 h at room temperature. The initial
15 min of fixation was performed under in-house vacuum. Following
fixation, the tissue was dehydrated using a graded ethanol
series (20, 40, 60, 80, and 96% for 20 min each). Following dehydration,
the tissue was cleared in a 2:1 mixture of benzyl benzoate:benzyl
alcohol. Ovules were mounted in immersion oil under
coverslips (25 9 4024 9 40 mm). The autofluorescence of ovules
was observed using a confocal microscope (TCS SP8; Leica Microsystems)
and HC PL PAO CS2 639/1.4 OIL objective equipped with
LAS-X software (Leica Microsystems) with 488 nm laser lines for
excitation and 550 nm emission filters.
In vitro pollen tube growth
In vitro pollen tube growth was performed according to the protocol
of Boavida and McCormick (2007). Approximately 300 ll of
freshly prepared pollen tube growth medium [0.01% boric acid,
5 mM CaCl2, 5 mM KCl, 1 mM MgSO4, 10% sucrose, pH 7.5 (KOH),
1.5% Phytagel (Sigma-Aldrich, St Louis, MO, USA)] was directly
poured onto a microscope slide to create a pollen tube germination
pad. Mature pollen was deposited on the solid medium and
slides incubated for 4 h in a humid chamber at 22°C in the dark.
Pollen tube lengths were measured after 4 h of in vitro growth
using the ZEN BLUE software (Carl Zeiss GmbH).
Multiple sequence alignment
Multiple sequence alignment was performed using CLUSTAL OMEGA
(Madeira et al., 2019). This alignment was then edited using JALVIEW
2 (Waterhouse et al., 2009). Regions with conserved physicochemical
properties (Conservation) were colored using JALVIEW 2 according
Ó 2023 The Authors.
The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.,
The Plant Journal, (2023), 114, 325–337
RUVBL proteins in gametophyte development 335
1365313x,2023,2,Downloadedfromhttps://onlinelibrary.wiley.com/doi/10.1111/tpj.16136byCzechs-MasarykUniversity,WileyOnlineLibraryon[28/04/2023].SeetheTermsandConditions(https://onlinelibrary.wiley.com/terms-and-conditions)onWileyOnlineLibraryforrulesofuse;OAarticlesaregovernedbytheapplicableCreativeCommonsLicense
to the CLUSTAL X color scheme (Jeanmougin et al., 1998; Livingstone
& Barton, 1993). Consensus prediction of the secondary structure
(JNetPRED) of the first sequence in each alignment was performed
using two independent prediction algorithms: the hidden Markov
model and position-specific scoring matrices using JPRED (Drozdetskiy
et al., 2015). The confidence of secondary structure prediction
was estimated using JPRED (JNetCONF) (Cole et al., 2008).
ACCESSION NUMBERS
RUVBL1 (AT5G22330); RUVBL2A (AT5G67630); RUVBL2B
(AT3G49830).
ACKNOWLEDGEMENTS
This work was supported by the Czech Science Foundation projects
21-15841S (PPS and MK), 20-01331X (AK and JF), 22-00871S
(EDT, FY, and AP), and 22-29717S (SH). AP and FY also received
funding from the Czech Academy of Sciences (Purkyne fellowship
and PPPLZ, respectively). We acknowledge the core facility CELLIM
supported by MEYS CR (LM2018129 Czech-BioImaging). The
Plant Sciences Core Facility of CEITEC Masaryk University is
acknowledged for its technical support. We acknowledge Leon
Jenner for editing and proofreading the manuscript submitted for
publication.
AUTHOR CONTRIBUTIONS
PPS and EDT designed the study, supervised the project
and wrote the manuscript with support from AP, JF, and
DH. EDT, FY, KM, SH, SS, AK, MP, and TP conducted
experiments and analyzed data. BK analyzed the transcriptomic
data.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version
of this article.
Table S1. Percentage of aberrant pollen grains determined in
bright field (BF), 40
,6-diamidino-2-phenylindole (DAPI) and fluorescein
diacetate (FDA) staining and pollen grain germination.
Table S2. Quantification of pollen tube germination determined by
an in vitro test.
Table S3. List of the primers used in the present study.
Figure S1. Schematic illustration of T-DNA insertions within the
RUVBL1 and RUVBL2A genes.
Figure S2. Analysis of seed setting in the identified RUVBL1 and
RUVBL2A T-DNA insertional mutants and transmission ratio distortion
in reciprocal crosses.
Figure S3. Phenotypic analysis of plants heterozygous for RUVBL1
and RUVBL2A mutations.
Figure S4. Differential interference contrast micrographs of cleared
ovules from ruvbl1-1/+ and ruvbl2a-1/+ plants.
Figure S5. Phenotypic analysis of mature pollen grains.
Figure S6. ruvbl1-1 pollen tubes lack the competence to target
ovules for fertilization.
Figure S7. Phenotypic classes observed after pollination of WT
ovules with mutant pollen.
Figure S8. Detection of RUVBL1 and RUVBL2A promoter activity
in seedlings by GUS histochemical staining.
Figure S9. Graphical summary of RUVBL mutations on plant
viability.
Figure S10. Multiple sequence alignment of RUVBL1 from human
and A. thaliana.
Figure S11. Multiple sequence alignment of RUVBL2 from human
and A. thaliana.
Figure S12. Multiple sequence alignment of RUVBLs from A.
thaliana.
OPEN RESEARCH BADGES
This article has earned Open Data and Open Materials badges.
Data and materials are available.
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RUVBL proteins in gametophyte development 337
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Supplemental Figures:
Figure S1. Schematic illustration of T-DNA insertions within the RUVBL1 and RUVBL2A
genes. Blue boxes indicate exons and black lines introns. Locations of various T-DNA
insertions in the coding sequences of the RUVBL1 and RUVBL2A genes available from several
plant databases are marked with triangles. Dark grey triangles mark T-DNA insertion lines with
a limited number of heterozygous progeny (ruvbl1-1 (SAIL_397_C11), ruvbl1-6
(SALK_133101), ruvbl2a-1 (GABI-Kat_543F01), and ruvbl2a-2 (SALK_071103)). Light grey
triangles mark lines that did not provide any homozygote or heterozygote plants.
Figure S2. Analysis of seed setting in newly identified RUVBL1 and RUVBL2A T-DNA
insertional mutants and transmission ratio distortion in reciprocal crosses.
(a) Representative content of siliques in self-pollinated WT, ruvbl1-6/+, and ruvbl2a-2/+ plants.
The white asterisks indicate aborted ovules.
(b) Percentage of aborted ovules of self-fertilized WT and mutant plants. The numbers on top
of the bars correspond to the number of seeds analyzed. ***P < 0.00001 in Fisher´s exact test
(X2). Error bars indicate the standard deviation of individually analyzed siliques.
(c) Representative content of siliques from reciprocal crosses between mutants and WT. The
first genotype indicates the mother plants. Scale bar, 1 mm.
(d) Percentage of aborted ovules of manual reciprocal crosses between WT and mutant plants.
The numbers on top of the bars correspond to the number of analyzed seeds. ***P < 0.00001
in X2. Error bars indicate the standard deviation of the means of individual siliques analyzed.
Figure S3. Phenotypic analysis of plants heterozygous for RUVBL1 and RUVBL2A mutations.
The WT and the PCR-validated ruvbl1-1/+, and ruvbl2a-1/+ plants were analyzed using a semiautomated
phenotyping pipeline (Photon System Instruments). (a), Top view imaging using
RGB cameras. (b), Side view imaging using RGB cameras 50 days after sowing. Scale bars,
6.5 cm.
Figure S4. Differential interference contrast micrographs of cleared ovules from ruvbl1-1/+ and
ruvbl2a-1/+ plants. Scale bar, 20 µm.
(a) Representative WT-like embryo sac with one egg cell nucleus (ecn) and one central cell
nucleus (ccn).
(b-d) Examples of defective embryo sacs from ruvbl1-1/+ plants. Ovule with a degraded
embryo sac (b). Embryo sac with two large nuclei (c). Embryo sac with three nuclei (d).
(e-g) Abnormal embryo sacs from ruvbl2a-1/+ plants. Ovule with a degraded embryo sac (e).
Embryo sac with one nucleus (f). Embryo sac with three nuclei (g).
(h) Percentage of normal and abnormal ovules from analyzed genotypes. Ovules were
analyzed from at least three independent plants. The numbers on top of the bars correspond
to the number of analyzed ovules.
Figure S5. Phenotypic analysis of mature pollen grains.
(a) Bright field (DIC), and corresponding 4′,6-diamidino-2-phenylindole (DAPI) epifluorescence
micrographs. Scale bars, 50 µm.
(b) Examples of ruvbl2a-1/+ pollen grains with aberrant positioning of the nuclei stained with
DAPI. Scale bars, 20 µm.
Figure S6. ruvbl1-1 pollen tubes lack competence to target ovules for fertilization.
(a) In vivo pollen tube growth 24 hap of control WT expressing proLAT52::GUS and ruvbl1-1/+
containing proLAT52::GUS associated with the SAIL T-DNA allele. Red arrows mark the
approximate pollen tube length detected within the pistil.
(b) Representative classes of WT ovules targeted with WT pollen tubes (no GUS stain) or with
ruvbl1-1 pollen tubes (with GUS blue dot). Scale bar, 50 µm.
(c) Quantification of the ovule classes from b. Statistics were performed with two-sided
Student’s t test.
Figure S7. Phenotypic classes observed after pollination of WT ovules with mutant pollen.
Scale bars, 20 µm.
Figure S8. Detection of RUVBL1 and RUVBL2A promoter activity in seedlings by GUS
histochemical staining.
(a, d) 7 days old seedlings. Scale bars, 1 mm. (b, e) Primary roots. Scale bars, 200 µm. (c, f)
Lateral roots. Scale bars, 100 µm.
Figure S9. Graphical summary of RUVBL mutations on plant viability.
(a) The analysis of global transcriptome data from various developmental stages. Data were
generated using the CoNekT Database and adapted (Julca et al., 2021). Pictograms were
created with BioRender.com. TPM, transcript per million.
(b) Graphical summary of the role of RUVBL1 and RUVBL2A in securing the viability of male
and female gametophytes. Heterozygous mutant plants produce 50% of gametes containing
WT allele (green) and 50% containing mutant allele (red). All megagametes carrying mutant
alleles die (50% lethality) and the remaining are mix of pollen carrying WT and mutant alleles.
The frequency of the pollen with the mutant allele participating in fertilization is again very
much reduced resulting in a low number of heterozygous offspring.
Figure S10. Multiple sequence alignment of RUVBL1 from human and A. thaliana. Regions
with conserved physicochemical properties were coloured according to the Clustal X colour
scheme (Jeanmougin et al., 1998). Domains and highly conserved motifs (Motifs) were marked
according to similarity with human RUVBL1 protein (Matias et al., 2006; Gorynia et al., 2011).
The secondary structure (Secondary Structure) of the first sequence in the alignment was
predicted, helices are marked as red tubes, beta sheets are marked as dark green arrows.
Confidence values for secondary structure prediction were estimated (JNetCONF) - high
values indicate high confidence. Regions with high conserved physicochemical properties
(Conservation) are marked in light yellow. The alignment consensus (Consensus) is given in
the percentage of the modal residue per column and the sequence logo indicates the relative
number of residues per column.
Figure S11. Multiple sequence alignment of RUVBL2 from human and A. thaliana. The
analyses were performed as described in Figure S10.
Figure S12. Multiple sequence alignment of RUVBLs from A. thaliana. The analyses were
performed as described in Figure S10.
Gorynia, S., Bandeiras, T.M., Pinho, F.G., et al. (2011) Structural and functional insights into
a dodecameric molecular machine – The RuvBL1/RuvBL2 complex. J. Struct. Biol., 176,
279–291.
Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G. and Gibson, T.J. (1998) Multiple
sequence alignment with Clustal X. Trends Biochem. Sci., 23, 403–405.
Julca, I., Ferrari, C., Flores-Tornero, M., et al. (2021) Comparative transcriptomic analysis
reveals conserved programmes underpinning organogenesis and reproduction in land
plants. Nat. Plants, 7, 1143–1159.
Matias, P.M., Gorynia, S., Donner, P. and Carrondo, M.A. (2006) Crystal Structure of the
Human AAA+ Protein RuvBL1. J. Biol. Chem., 281, 38918–38929.
Supplement R
Kusova A., Steinbachova L., Přerovská T., Záveská Drábková L., Paleček J., Khan A., Rigóová
G., Gadoiu Z., Jourdain C., Stricker T., Schubert D., Honys D., and Schrumpfová P.P.*, 2023.
Completing the TRB family: newly characterized members show ancient evolutionary origins
and distinct localization, yet similar interactions Plant Molecular Biology 112:61–83.
P.P.S. participated in the design of experiments, data evaluation and wrote the ms
Vol.:(0123456789)1 3
Plant Molecular Biology (2023) 112:61–83
https://doi.org/10.1007/s11103-023-01348-2
Completing the TRB family: newly characterized members show
ancient evolutionary origins and distinct localization, yet similar
interactions
Alžbeta Kusová1,2
   · Lenka Steinbachová3
   · Tereza Přerovská1
   · Lenka Záveská Drábková3
   ·
Jan Paleček1,2
   · Ahamed Khan4
   · Gabriela Rigóová1
   · Zuzana Gadiou3
   · Claire Jourdain5
   · Tino Stricker5
   ·
Daniel Schubert5
   · David Honys3,6
   · Petra Procházková Schrumpfová1,2
 
Received: 9 December 2022 / Accepted: 2 March 2023 / Published online: 28 April 2023
© The Author(s) 2023
Abstract 
Telomere repeat binding proteins (TRBs) belong to a family of proteins possessing a Myb-like domain which binds to telomeric
repeats. Three members of this family (TRB1, TRB2, TRB3) from Arabidopsis thaliana have already been described as
associated with terminal telomeric repeats (telomeres) or short interstitial telomeric repeats in gene promoters (telo-boxes).
They are also known to interact with several protein complexes: telomerase, Polycomb repressive complex 2 (PRC2) E(z)
subunits and the PEAT complex (PWOs-EPCRs-ARIDs-TRBs). Here we characterize two novel members of the TRB family
(TRB4 and TRB5). Our wide phylogenetic analyses have shown that TRB proteins evolved in the plant kingdom after the
transition to a terrestrial habitat in Streptophyta, and consequently TRBs diversified in seed plants. TRB4-5 share common
TRB motifs while differing in several others and seem to have an earlier phylogenetic origin than TRB1-3. Their common
Myb-like domains bind long arrays of telomeric repeats in vitro, and we have determined the minimal recognition motif of
all TRBs as one telo-box. Our data indicate that despite the distinct localization patterns of TRB1-3 and TRB4-5 in situ,
all members of TRB family mutually interact and also bind to telomerase/PRC2/PEAT complexes. Additionally, we have
detected novel interactions between TRB4-5 and EMF2 and VRN2, which are Su(z)12 subunits of PRC2.
Key message 
TRB proteins bind short/long telomeric repeats and attract telomerase, PRC2 or PEAT complexes. Here we show the unique
features of novel members of TRB family that have earlier phylogenetic origin.
Keywords  Telomere repeat binding · TRB · PRC2 · PEAT · TERT · Telomeric
Introduction
Telomere repeat binding proteins (TRBs) were originally
characterized as proteins in Arabidopsis thaliana with a
binding affinity to telomeric DNA sequences proportional
to the number of telomeric repeats (Schrumpfová et al.
2004). They belong to the plant-specific Single myb histone
1 (SMH) family with an N-terminal Myb-like domain
(Myb-like), a central histone-like (H1/5-like) domain, and
a coiled-coil domain near the C-terminus (Marian et al.
2003). Three members of the TRB family (TRB1, TRB2
and TRB3) in A. thaliana exhibit self-interactions and
mutual interactions in the yeast-two hybrid (Y2H) system
(Kuchař and Fajkus 2004; Schrumpfová et al. 2004).
Alžbeta Kusová and Lenka Steinbachová shared co-first authorship.
Accession numbers  TRB1 (AT1G49950); TRB2, formerly
TBP3 (AT5G67580); TRB3, formerly TBP2 (AT3G49850);
TRB4 AT1G17520; TRB5 AT1G72740; RUVBL1
(AT5G22330); RUVBL2A (AT5G67630); TERT (AT5G16850);
POT1a (AT2G05210); POT1b (AT5G06310); Fibrillarin1
(AT5G52470); SRp34 (AT1G02840); EMF2 (AT5G51230);
VRN2 (AT4G16845); CLF (AT2G23380); SWN (AT4G02020);
PWO1 also named as PWWP1 (AT3G03140); PWO2 also named
as PWWP2 (AT1G51745); PWO3 also named as PWWP3
(AT3G21295).
*	 Petra Procházková Schrumpfová
	schpetra@sci.muni.cz
Extended author information available on the last page of the article
62	 Plant Molecular Biology (2023) 112:61–83
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They bind plant telomeric repeats (TTT​AGG​G)n through
the Myb-like domain (Mozgová et al. 2008), while the
H1/5-like domain is responsible for dimerization with
other TRB proteins (Schrumpfová et al. 2008).
TRB1-3 proteins are proposed to participate in telomerase
biogenesis. They interact directly with the catalytic
protein subunit of telomerase (TERT) (Schrumpfová
et al. 2014) and mediate interactions between TERT and
Recombination UV B-like (RUVBL) proteins (Schořová
et al. 2019), homologs of the essential mammalian telomerase
assembly components Pontin and Reptin (Venteicher
et al. 2008). Nuclear and predominantly nucleolar localization
of TRB1-3 interacting with TERT and RUVBLs, as
well as with the plant ortholog of dyskerin, CBF5 (Lermontova
et al. 2007), was observed using Bimolecular fluorescence
complementation (BiFC) (Sweetlove and Gutierrez
2019). Moreover, the TRB1 protein interacts via its
H1/5-like domain with Protection of telomeres 1 (POT1b)
(Schrumpfová et al. 2008), an A. thaliana homolog of the
G-overhang binding protein Pot1, a core component of
mammalian telomere cap complex, Shelterin (Tani and
Murata 2005; Surovtseva et al. 2007). Additionally, in situ
co-localization of TRB1 with telomeric DNA repeats has
been detected in plant cells (Schrumpfová et al. 2014; Dreissig
et al. 2017).
Telomere shortening was observed in trb1 mutants in the
A. thaliana ecotype Columbia, with otherwise-stable telomere
lengths (Shakirov and Shippen 2004; Schrumpfová
et al. 2014). In contrast, telomere extension was detected
in trb2 knockout mutants of the A. thaliana ecotype Wassilewskija,
which exhibits telomere length polymorphism
in wild-type plants (Shakirov and Shippen 2004; Maillet
et al. 2006; Lee and Cho 2016). Triple homozygous mutant
plants, containing the alleles from Columbia (trb1 and trb3)
and from Wassilewskija (trb2), exhibit telomere shortening
(Zhou et al. 2018).
In multicellular organisms, Polycomb repressive complex
1 (PRC1) and PRC2 repress target genes through histone
modification and chromatin compaction. In Drosophila melanogaster,
four core PRC2 subunits are present: the histone
methyltransferase Enhancer of zeste [E(z)], Suppressor of
zeste 12 [Su(z)12], Extra sex combs (Esc), and the histonebinding
nucleosome remodelling factor 55 kDa (Nurf55).
The E(z) homologs in A. thaliana, named CURLY LEAF
(CLF) and SWINGER (SWN), are implicated in sporophyte
development (reviewed in Mozgova and Hennig 2015). The
PRC2 complex primarily methylates histone H3 on lysine 27
(H3K27me3), a mark of transcriptionally silent chromatin.
TRB1–3 interact with the PRC2 proteins CLF and SWN
(Zhou et al. 2018). We have shown that TRB1 proteins are
not only associated with long arrays of telomeric repeats
but also with interstitially located short telomeric sequences
telo-box motifs, especially in the promoters of translation
machinery genes (Schrumpfová et al. 2016). It was further
shown that these telo-boxes are part of the cis-regulatory
elements that may relate to PRC2 recruitment (Zhou et al.
2016, 2018).
Besides the PRC2 complex, TRB1-3 are components of
the PEAT complex (PWOs-EPCRs-ARIDs-TRBs) mediating
histone acetylation/deacetylation and heterochromatin condensation.
They potentially regulate the RNA-directed DNA
methylation (RdDM) pathway (Tan et al. 2018; Tsuzuki and
Wierzbicki 2018). The involvement of TRB proteins in histone
deacetylation supports the previous observation that
TRB2 directly interacts with histone deacetylases (HDT4
and HDA6) (Lee and Cho 2016). Recently, it has also been
proposed that histone 1 (H1) selectively prevents H3K27me3
accumulation at telomeres and large pericentromeric interstitial
telomeric repeat (ITR) domains by restricting DNA
accessibility to TRB proteins (Teano et al. 2020).
Here we show that the plant-specific TRB proteins can
be recognized in lower plants, such as Streptophytic algae,
as well as in higher plants. In seed plants, TRB proteins
are divided into three main lineages. We speculate that due
to whole genome duplication (WGD) in A. thaliana, three
ancestral TRB proteins have multiplied to the current five
TRB members. We characterize new members of TRB family
in A. thaliana (TRB4 and TRB5) and demonstrate that
all members of the TRB family can bind long arrays of telomeric
repeats with high specificity. We defined the minimal
recognition motif for all TRBs as one telo-box. Even though
TRB4 and TRB5 share very high sequence and structural
homology with TRB1, TRB2 and TRB3, they differ in terms
of the surface of their Myb-domains and their cellular localization.
We provide evidence that TRB4-5 mutually interact
with other members of the TRB family and physically interact
with TERT, POT1a/b, SWN/CLF, and PWO1-3. Novel
interactions were also detected between TRB4-5 and EMF2/
VRN2, which represent the Su(z)12 subunits of PRC2.
Completing TRB family analysis permits further exploration
of the biological roles of these important plant-specific
proteins.
Materials and methods
Primers
The sequences of all primers and probes used in this study
are provided in Supplemental Table 1.
Plant material
For transient assays, Nicotiana benthamiana plants were
grown in soil in LD conditions up to 4 weeks and subsequently
used for Agrobacterium tumefaciens infiltration.
63Plant Molecular Biology (2023) 112:61–83	
1 3
Phylogenetic analyses
We combined two homology searches based on A. thaliana
TRB1-5. First, we searched completely sequenced genomes
using Phytozome v12 and second using BLASTP from available
databases (NCBI) and publicly available sequences.
Protein sequences were aligned using the Clustal
Omega (Sievers et al. 2011) algorithm in the Mobyle platform
(Néron et al. 2009), with homology detection by
HMM–HMM comparisons. We screened data after alignment
in the BioEdit program (Hall 1999).
Maximum likelihood (ML) analyses of the matrices were
performed in RAxML 8.2.4 (Stamatakis 2014) to examine
differences in optimality between alternative topologies.
1000 replications were run for bootstrap values. Phylogenetic
trees were constructed and modified with iTOL v3.4
(Letunic and Bork 2016). The MEME search was set to identify
domains and conserved amino acid (aa) sequence motifs
under these conditions: a maximum of 15 motifs for each
protein with a wide sequence motif from 2 to 50 and a total
number of sites from 2 to 600 MEME 4.11.2 (Bailey et al.
2009). The evolutionarily conserved aa residues were visualized
using ConSurf 2016 (Ashkenazy et al. 2016).
Analysis of protein structures
The AlphaFold (Jumper et al 2021; Varadi et al 2022) and
SwissModel (Waterhouse et al 2018) tools were used to generate
in silico protein models. Structural models were compared
as previously described (Palecek & Gruber 2015). All
structures including electrostatic potential of their molecular
surfaces were visualized using PyMOL Molecular Graphics
System, Version 2.4.1, Schrödinger, LLC.
Cloning
For yeast two-hybrid assays (Y2H), most of the Y2H constructs
in pGADT7-DEST or pGBKT7-DEST (Horák et al.
2008) were prepared previously: TERT constructs (RID1,
TEN, Fw1N, Fw3_NLS, Fw3N and RT) were reported in
Majerská et al. 2017, TRB1-3 were reported in Schrumpfová
et al. 2014, RUVBL1 and RUVBL2A were reported
in Schořová et al. 2019, POT1a and POT1b were reported
in Majerská et al. 2017, ­SWNΔSET and ­CLFΔSET were
reported in Chanvivattana et al. 2004 and Hohenstatt et al.
2018). PWO1 was reported in Hohenstatt et al. 2018, VRN2
and EMF2 were reported in Lindner et al. 2013. The coding
sequences of PWO2 and the fragment of PWO3 were cloned
in the pGBKT7 and pGADT7 vectors (Clontech), passing
through pDONR221 (Invitrogen) as described in Hohenstatt
et al. 2018.
For the TRB4 construct, the cloned cDNA sequence
of TRB4 (G60951 from Arabidopsis Biological Resource
Center; ABRC, https://​abrc.​osu.​edu/) in pENTR223 was
used as the entry clone. Site-directed mutagenesis using the
QuikChange XL Site-Directed Mutagenesis Kit (Agilent
Technologies) was performed following the manufacturer’s
instructions to remove the mutation V122A in the protein
encoded by pENTR223-TRB4 as described in Wiese et al.
2021. To generate an entry vector containing the cDNA
sequence of the TRB5 gene, the total RNA from 7-day-old A.
thaliana Col-0 seedlings isolated by TRI reagent (Molecular
Research Center) was used for cDNA preparation using
M-MuLV Reverse Transcriptase (New England Biolabs).
The cDNA sequence of TRB5 was amplified using gene specific
Gateway-compatible primers according to the manufacturer’s
instructions with primers specified in Supplemental
Table 1, and the RT-PCR products were recombined into
the Gateway donor vector pDONR207 (Invitrogen). DNA
fragments were introduced into the destination Gateway vectors
pGBKT7-GW, pGADT7-GW (Addgene) using the LR
recombinase reaction (Invitrogen).
For BiFC experiments, the Multisite Gateway® system
(Invitrogen) was used to create pBiFCt-2in1 constructs
(Grefen and Blatt 2012). The genes encoding TRB1-5 from
A. thaliana Col-0 were PCR-amplified from the constructs
used in the Y2H system described above by two-step PCR
using primers specified in Supplemental Table 1 and Phusion™
High-Fidelity DNA Polymerase (Thermo Fisher
Scientific) as described in Wiese et al. 2021. The amplicons
with TRB1,2,3,5 genes were cloned into Gateway
pDONR221 entry vectors (Thermo Fisher Scientific) carrying
either attP1-P4 or attP3-P2 recombination sites using
the BP Clonase™ II enzyme mix (Thermo Fisher Scientific).
To generate pDONR221 entry clones carrying the TRB4
gene, the In-Fusion® Snap Assembly cloning kit (Takara
Bio USA) was used, where PCR-generated TRB4 amplicons
with one half of att-sites obtained in the first Phusion PCR
step of the Gateway® cloning were fused with linearized
PCR-generated pDONR221 backbone with appropriate
attL sites by recognizing 15-base pairs (bp) overlaps at their
ends according to the manufacturer’s instructions. All entry
clones were subsequently used in LR Clonase™ II Plus
(Thermo Fisher Scientific) reactions to create pBiFCt-2in1CC
and pBiFCt-2in1-NN expression constructs harboring
two protein coding regions C- or N-terminally fused to
either the N- or C-terminal eYFP halves (e.g., TRB1-nYFP
and TRB1-cYFP). After verification by Sanger sequencing
(Macrogene), the constructs were used for transient expression
in N. benthamiana leaves.
For transient expression of TRB1-5 fused with GFP in N.
benthamiana leaf epidermal cells, the specific entry clones
described above (TRB1-3, 5 in pDONR207 and TRB4 in
pENTR223) were used in LR Clonase™ II (Thermo Fisher
Scientific) reactions to create the pGWB6 (N-terminal
GFP fusion under the 35S promoter) expression vectors
64	 Plant Molecular Biology (2023) 112:61–83
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(Nakagawa et al. 2007). To label nucleolus or nucleoplasm,
we co-transfected N. benthamiana leaf epidermal cells with
constructs expressing Fibrillarin1-mRFP (Koroleva et al.
2009) or SRp34-mRFP (Lorković et al. 2004; Koroleva et al.
2009), respectively.
For protein expression in Escherichia coli, constructs in
pDONR207 (for TRB1 and TRB5) or in pENTR223 (for
TRB4) were used as donor vectors for LR Clonase™ reactions
(Thermo Fisher Scientific), where genes coding proteins
of interest (TRB1, 4 and 5) were transferred into the
destination vector pHGWA (Busso et al. 2005).
Yeast two‑hybrid assays
Y2H was performed using the Matchmaker TM GAL4-based
two-hybrid system (Clontech) as described in Schrumpfová
et al. 2014. Successful co-transformation of each bait/prey
combination into Saccharomyces cerevisiae PJ69-4a was
confirmed on SD plates lacking Leu and Trp, and interactions
assessed on SD medium lacking Leu, Trp and His
(with or without 1 mM or 3 mM 3-aminotriazol (3-AT)) or
SD medium lacking Leu, Trp and Ade at 30 °C. Co-transformation
with an empty vector and homodimerization of
the TRB1 protein served as a negative and positive control,
respectively (Schrumpfová et al. 2014). Each combination
was co-transformed at least three times, and at least three
independent drop tests were carried out. Protein expression
was verified as described in Schořová et al. 2019.
In vitro translation and co‑immunoprecipitation
Proteins were expressed from constructs similar to those
used in the yeast two-hybrid system with a hemagglutinin
tag (pGADT7-DEST; VRN2, EMF2 and POT1a proteins)
or a myc-tag (pGBKT7-DEST; TRB proteins) using a TNT
Quick Coupled Transcription/Translation system (Promega)
in 50 µl reaction volumes according to the manufacturer’s
instructions. The VRN2, EMF2 or POT1a proteins
were radioactively labelled using 35
S-Met. The co-immunoprecipitation
procedure was performed as described by
Schrumpfová et al. 2008. Input, Unbound and Bound fractions
were separated by 10% SDS – PAGE, and analyzed
using a FLA7000 imager (Fuji-film).
Transient heterologous expression
A. tumefaciens competent cells (strain GV3101) were transformed
with selected expression clones and selected on
YEB medium supplemented with gentamycin (50 µg/mL),
rifampicin (50 µg/mL), and a vector-specific selection agent
(pBiFCt-2in1: spectinomycin 100 µg/mL, pGWB6: kanamycin
and hygromycin both 50 µg/mL, pROK2: kanamycin
50 µg/mL) at 28 °C for 48 h. Colonies were inoculated in
the same media lacking agar and grown overnight at 28 °C.
Bacterial cells of overnight cultures were pelleted by centrifugation
(5 min at 1620 g), washed twice, re-suspended,
and diluted to an ­OD600 of 0.5 with infiltration medium
(10 mM MES pH 5.6, 10 mM ­MgCl2 and 200 µM acetosyringone).
A suspension of Agrobacterium cells carrying
the p19 repressor plasmid was added in a 1:1 ratio with
Agrobacterium suspensions harboring plasmids of interest
to suppress gene silencing and enhance transient expression
(Gehl et al. 2009). Mixed suspensions were incubated with
moderate shaking for 1.5–2 h at room temperature and subsequently
injected into the abaxial side of leaves of 4-weekold
N. benthamiana plant. On the third day after infiltration,
tobacco epidermal cells were microscopically analyzed.
Microscopy images were acquired using the Zeiss LSM880
laser scanning microscope (Axio Observer Z1, inverted)
with Definite Focus 2 (excitation 488 nm for GFP/YFP and
561 nm for mRFP). Images were processed using Fiji/ImageJ
(Schindelin et al. 2012) and Adobe Photoshop CS6 (Adobe,
San Jose, CA, USA) software.
Nuclei isolation and immunofluorescence
Isolation of nuclei was performed using 10-day-old seedlings
as described in (McKeown et al. 2008). Nuclei were
centrifuged for 20 min at 350 g and 4 °C, resuspended in
1 × PBS and spotted onto slides. Nuclei were briefly dried
at 4 °C, then fixed in 4% paraformaldehyde in 1 × PBS,
0.5% Triton-X for 10 min. Nuclei were rinsed three times
in 1 × PBS, blocked in 5% goat serum with 0.05% Tween-
20 for 30 min at RT and incubated overnight with antibody
anti-TRB 5.2 (Schrumpfová et al. 2014) diluted 1:300 in 5%
BSA in 1 × PBS. Nuclei were washed three times for 5 min
in 1 × PBS supplemented with 0,05% Tween-20 (PBST),
then incubated 1 h with an anti-mouse Alexa 488 antibody,
Invitrogen (1:750 dilution). Slides were washed three times
for 5 min in 1 × PBST, then dehydrated in ethanol series as
described in Kutashev et al. 2021. Coverslips were mounted
in 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI),
2 µg/mL in Vectashield and imaged using fluorescence using
a Zeiss AxioImager Z1 epifluorescence microscope.
Expression of TRB1, 4 and 5 in E. coli
Proteins fused with His-tags were expressed in E. coli
(BL21(DE3) RIPL) from the destination vector pHGWA.
BL21(DE3) RIPL were grown in Luria–Bertani medium
supplemented with ampicillin and chloramphenicol to final
concentration 100 ug/ml and 12,5 ug/ml at 37 °C until ­OD600
reached 0.5. Cells were cultured for 4 h at 25 °C after the
addition of IPTG to a final concentration of 0.5 mM. Cells
were collected by centrifugation (8,000 g, 8 min, 4 °C). The
cell pellet was dissolved in lysis buffer containing 50 mM
65Plant Molecular Biology (2023) 112:61–83	
1 3
sodium phosphate (pH 8.0), 300 mM NaCl, 10 mM imidazole,
5 mM β-mercaptoethanol, and protease inhibitor cocktail
cOmplete tablets EDTA-free (Roche). The cell suspension
was sonicated on ice for 6 min of process time with
1 s pulse and 2 s pause (Misonix). Cell lysate supernatant
was collected after centrifugation at 20,000 g, 4 °C for 1 h.
Proteins were purified by immobilized-metal affinity chromatography
using TALON® metal affinity resin (Clontech),
where filtered supernatant (0.45 μm filter) was mixed with
TALON® beads and incubated for 1 h. The proteins of interest
were eluted with 100 mM imidazole in the same buffer.
These proteins were then concentrated, and the buffer was
exchanged for 50 mM sodium phosphate (pH 7.0), 300 mM
NaCl by ultrafiltration (Amicon 3 K/30 K, Millipore). The
concentration of purified proteins was determined using the
Bradford assay. We evaluated protein purity using SDS–polyacrylamide
gel electrophoresis, with gels stained by BioSafe
Coomassie G250 (Bio-Rad). To ensure successful
purification, the purified proteins were detected by specific
monoclonal mouse anti-polyhistidine antibody as described
previously (Schrumpfová et al. 2004) and by matrix-assisted
laser desorption-ionization tandem mass spectrometry
(MALDI-MS/MS) (CEITEC Proteomics Core Facility).
Electrophoretic mobility shift assay (EMSA)
DNA probes and competitors used are described in Supplemental
Table 1. To reduce a non-specific DNA–protein
binding, 10 pmol of purified TRB1, 4 or 5 were preincubated
with 1, 10, or 100 pmol of a specific competitor (oligodeoxynucleotides
of non-telomeric or competitor telomeric
sequence in double-stranded form, as indicated in Results)
for 20 min in EMSA buffer (10 mM Tris–HCl pH 8.0, 1 mM
EDTA, 1 mM dithiothreitol, 50 mM NaCl, and 5% w/v
glycerol). Probes were end-labelled using [γ-32
P]ATP and
polynucleotide kinase (New England Biolabs), according
to (Sambrook et al. 1989). A probe (1 pmol) was added to
the reaction mixture on ice and incubated for 20 min before
the mixture was loaded onto a 7.5% w/v non-denaturing
polyacrylamide gel (AA:BIS = 37.5:1, 0.5 × TBE, 1.5 mm
thick). Electrophoresis was at 15 °C for 3 h at 10 V/cm in a
precooled buffer. Signals of labelled oligonucleotides were
detected using a phosphorimager FLA-7000IP (Fujifilm).
Results
Sequence and structural divergences in the TRB
family
Two decades ago, in silico analysis already predicted that the
A. thaliana SMH family contains five TRB members (Marian
et al. 2003). We used a combination of recent genome
and transcriptome annotations (Lamesch et al. 2012; Cheng
et al. 2017) and predicted sequence and structural similarity
to characterize the TRB family members TRB4 and TRB5.
These were each found to contain an N-terminal Myblike
domain, a central H1/5-like domain, and a C-terminal
coiled-coil domain, similar to previously characterized
family members TRB1-3 (Fig. 1A) (Marian et al. 2003;
Schrumpfová et al. 2004; Mozgová et al. 2008).
We then performed a phylogenetic reconstruction of TRB
proteins from the Brassicaceae family based on a matrix
including 52 TRBs, with 357 aligned positions. The tree
shows that in A. thaliana, as well as in other Brassicaceae,
TRB4 and TRB5 are grouped in a monophyletic lineage that
is distant from TRB1-3 (Fig. 1B, Supplemental Table 2).
The Myb-like domain is composed of a helix-turn-helix
(HTH) motif (Ogata et al. 1992; Bilaud et al. 1996). Even
though the domain composition of SMH proteins is unique
to the plant lineage, the Myb-like domain shows high aa
sequence conservation across the plant and animal kingdoms
(Fig. 1C). Predicted three-dimensional (3D) models
of A. thaliana TRB Myb-like structural features were overlaid
with the X-ray diffraction-resolved crystal structure of
human Telomeric repeat-binding factor 2 (hTRF2) (Fig. 1D)
and it was found that the 3D structure of the Myb-like
domain is well conserved in both plant and animal kingdoms.
Interestingly, all TRBs show a slight difference in the
composition of the second helix. However, the aa residues
mediating the interaction between hTRF2 and the human telomeric
repeat sequence (TTA​GGG​) are fully conserved in A.
thaliana even though the plant telomeric sequence slightly
differs (TTT​AGG​G) from the human one (Fig. 1C, D).
The central linker histone globular domain (H1/5-like
domain) adopts a winged-helix fold including a HTH motif
and a “wing” defined by two β-loops (Ramakrishnan et al.
1993). Using the SWISS-MODEL tool, the Xenopus laevis
(Xl) H1 domain (XlH1.0) appeared to be the closest template
to A. thaliana TRBs H1/5-like domain (Bednar et al.
2017). We compared the 3D structure of the histone globular
domain from XlH1.0-B, obtained by cryo-electron microscopy
(Cryo-EM) and X-ray crystallography (Bednar et al.
2017), with the AlphaFold protein structure predictions of
the H1/5-like domain of A. thaliana TRBs (Varadi et al.
2022). Although the sequence conservation between the
XlH1 domain and the H1/5-like domain of A. thaliana TRBs
is lower than in the Myb-like domain, the 3D structure of
the H1/5-like domain in TRBs is highly similar (Fig. 1C, D).
The C-terminal coiled-coil domains usually contain a
repeated pattern of hydrophobic and charged aa residues,
referred to as a heptad repeat (Lupas and Gruber 2005). This
repeating pattern enables two helices to wrap/coil around
each other. The conservation of hydrophobic residues (Alanine
(A), Isoleucine (I), Valine (V), Phenylalanine (F) or
Methionine (M)) and an acidic residue (Glutamic acid (E))
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Fig. 1  Sequence and structural alignments of TRB family proteins.
(A) Schematic representation of the conserved domains of TRBs
from A. thaliana. Myb-like, Myb-like domain; H1/5-like, histone-like
domain; coiled-coil, C-terminal domain. (B) Unrooted Maximum
likelihood (ML) phylogenetic tree of Brassicaceae TRB proteins. The
length of the branches are proportional, and the black dots indicate
the position of TRB1-5 from A. thaliana. For a list of species, see
Supplementary Table  2. (C) Multiple alignments of the Myb-like,
H1/5-like and coiled-coil domains. The positions of α-helices or
β-sheets of the uppermost or the lowermost sequence in each alignment
are highlighted: bold, experimentally determined structures
(cryo-EM or X-ray crystallography); thin, AlphaFold prediction.
Human Telomeric repeat-binding factor 2 (hTRF2) and Xenopus laevis
histone H1.0 (Xl H1.0-B) were used to show the most conserved
amino acid (aa) residues. Amino acid shading indicates the following
conserved amino acids: dark green, hydrophobic and aromatic;
light green, polar; blue, basic; magenta, acidic; yellow, without side
chain (glycine and proline). The aa of hTRF2 that mediate intermolecular
contacts between telomeric DNA and hTRF2 are marked with
an asterisk. (D) Structural models of Myb-like, H1/5-like and coiledcoil
domains. AlphaFold protein structure predictions deposited in
the EMBL database were used (Varadi et al. 2022). The three-dimensional
model of the Myb-like domain fits best the hTRF2-DNA interaction
structure (PDB: 1WOU) (Court et al. 2005). The structure of
the histone-like domain is most similar to X. laevis histone H1 structure
(PDB: 5NL0) (Bednar et al. 2017). The positions of the aa of
hTRF2 that mediate intermolecular contacts between telomeric DNA
and hTRF2 are marked with an asterisk
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is obvious in all coiled-coil domains from A. thaliana TRBs.
Only one long α‐helix is predicted by AlphaFold in TRB4-5,
while the coiled-coil domains from TRB1 and TRB2 seem
to have an additional short α‐helix (Fig. 1C, D). However,
these predictions await experimental verification.
The most divergent of the three domains in TRBs (Myblike,
H1/5-like and coiled-coil) is the coiled-coil domain.
Our alignments suggest that although TRB4 and TRB5 are
distant from TRB1-3 members in terms of sequence, they
are folded into similar three-dimensional structures, with
only minor differences.
The evolution of TRBs within the plant lineage
We performed comprehensive phylogenetic analysis to
investigate whether TRBs are conserved across lower and
higher plants, and whether TRB4-5 form a distinct group
to TRB1-3 in all species, as observed in the higher plant
A. thaliana. We used a data set of 268 proteins and 599
aligned positions and found that TRB proteins first evolved
in Streptophyta in Klebsormidiophyceae, although TRBs are
missing in the other Streptophyte algae studied, including
Charophyceae and Zygnematophyceae. In Klebsormidium
nites only one TRB homolog was identified. Following the
evolutionary tree, an increasing number of TRB homologues
were found in Bryophyta and Tracheophyta. There
are three homologues of TRB in mosses Sphagnum fallax
and Physcomitrium patens, and these TRBs share very high
sequence similarities. Contrastingly, there is only one homologue
in the moss Ceratodon purpureus. Perhaps due to
limited sequence information available for the genomes of
hornworts or liverworts, TRB was not found in these lineages.
In seed plants, which have undergone more rounds of
whole genome duplication events (WGDs) than Bryophyta
and Lycophyta (Clark and Donoghue 2018), predominantly
three TRB proteins were recognized. Within Brassicaceae,
which has undergone an additional recent round of WGD
(Walden et al. 2020), five TRB homologs were revealed
(Fig. 2A).
Next, the MEME search (Bailey et al. 2009) was used to
identify the 15 most typical motifs in TRBs across the plant
kingdom (Supplemental Fig. 1). For ease of presentation,
a simplified tree was used with 83 representatives from 33
families and 26 orders, each family having only one member
(Fig. 2B, Supplemental Table 3). TRB proteins were
divided into three main lineages named TRB_A, TRB_B
and TRB_C, into the latter of which the TRB_D sub-lineage
is integrated. The lineage TRB_A includes Streptophyte
algae, Bryophyta and Lycophyta and diverges from seed
plant lineages TRB_B and TRB_C in terms of the length
of introns, disordered parts of proteins or additional motifs.
A clear diversification of TRBs into monocots and dicots
was revealed in both TRB_B and TRB_C lineages. In seed
plants, the TRB_B lineage seems to be less abundant than
the TRB_C lineage.
Consistent with previous findings, the canonical N-terminal
Myb-like (motif 1), a central H1/5-like (motifs 2, 4 and
7), and a C-terminal coiled-coil (motif 3) domains include
the top conserved motifs present in all TRBs (Fig. 2B).
Within the lineage TRB_A, a unique aa motif was detected
in Bryophytes (motif 15; EEREH). Interestingly, the coiledcoil
domains of the proteins from TRB_B lineage contain
the specific motif 11 (EDTDS), but most of the proteins
from the TRB_A and TRB_C lineage contain the motif 6
(DAEAA) instead. However, motif 6 is not present in the A.
thaliana protein TRB5 that has diverged from TRB4. The A.
thaliana proteins TRB4 and TRB5 (belonging to the TRB_B
lineage) lack motif 8 (DDVKI) adjacent to the coiled-coil
domain. In contrast, this motif is present within proteins of
the TRB_C lineage, including A. thaliana TRB1-3 proteins.
The TRB_D sub-lineage is embedded within TRB_C
lineage but supported by a high 99% bootstrap value.
TRB2 and TRB3 from A. thaliana are nested within the
TRB_D sub-lineage. This lineage lacks motif 5 (MSVMA),
a motif adjacent to the Myb-like domain, which is present
in the rest of the TRBs in the TRB_C lineage. Similarly
to Brassicaceae, divergence to the TRB_D sub-lineage was
detected within several other dicot families (e.g., Rham-
naceae - Ziziphus jujuba, Cucurbitaceae - Cucumis sativus,
Euphorbiaceae - Ricinus communis, Rutaceae - Citrus
sinensis, Malvaceae - Glycine max, Cleomaceae - Tarenaya
hassleriana).
In general, TRBs were detected in lower and higher
plants. TRBs in Streptophyte algae, Lycophyta and Bryophyta
(grouped in TRB_A lineage) are more closely related
to A. thaliana TRB4 and TRB5 proteins (TRB_B lineage)
than to A. thaliana TRB1-3 (TRB_C lineage). Lineage
TRB_B and TRB_C differ in several motifs accompanying
the canonical H1/5-like or coiled-coil domains. The specificity
of the sub-lineage TRB_D, embedded into TRB_C lineage,
is highlighted by its lack of a specific motif 5 following
the canonical Myb-like domain.
The TRB4‑5 from dicots differ in solution accessible
surface of the Myb‑like domain
The N-terminal Myb-like domain is the most conspicuous
structural unit in TRBs. In order to compare the structural
characteristics of this, we further examined the sequence
conservation and estimated evolutionary conservation of
predicted 3D structures. The TRB groups (TRB_A, B, C,
D), established in Fig. 2B, were subdivided into Lycophytes,
Bryophytes, Monocots and Dicots. Consensus sequences of
the Myb-like domain in each individual group are visualized
in Fig. 3A.
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The evolutionary dynamics of aa substitutions in A.
thaliana TRB4, 1 and 2, representing Dicots from lineages
TRB_B, C and D, respectively, were visualized using
ConSurf 2016 (Ashkenazy et al. 2016). Visualization has
shown a very strong conservation of the DNA-binding
surface in all lineages (Supplemental Fig. 2), consistent
with the above analysis (Figs. 1 and 2). In comparison,
opposite solution-accessible surface was less conserved;
in Dicots from the TRB_D lineage, conservation was
lower than in Dicots from TRB_B and TRB_C lineages
(Fig. 3B, Supplemental Fig. 2). Dicots in all three lineages
exhibit a variant unstructured N-terminus of the
MYB-domain which is conserved within the lineage. The
aa at the N-terminal position 3 in Dicots from the TRB_B
lineage, including TRB4-5 from Arabidopsis, show significantly
conserved substitutions of Alanine (A) residues
to the polar uncharged aa Asparagine (N) (Fig. 3,
Inverted triangle). The unstructured C-terminal tail of the
Myb-like domain in the TRB_B lineage (Fig. 3, Triangle,
Supplemental Fig. 2A) shows less conservation than in
TRB_C and TRB_D lineages, where these residues are
part of the third helix.
Surface models showing the charge of the Myb-like
domain were visualized using PyMol viewer (Fig. 3C,
Supplemental Fig. 2C, F). In the TRB_B lineage, visualizations
reveal a negatively charged aa (E/D) at position 14
(Fig. 3, Rhombus) flanking conserved EEE motif (Fig. 3,
Circle), whereas proteins in the TRB_A and TRB_C lineages
possess uncharged Alanine (A) at the position 14.
Interestingly, the aa (positively charged aa K/R at position
17) proximal to the E/D motif, are replaced to uncharged
(L) in Dicots from TRB_B lineage (Fig. 3C, Trapezoid).
These data indicate that the E/D motif at position
14 together with aa at position 17 are responsible
for the additional areas of negative charge on the
solution-accessible surface of the Myb-domain in Dicots
from TRB_B lineage.
Even one telomeric unit is sufficient for TRB binding
Our previous findings revealed that the N-terminal Myblike
domains of TRB1-3 are responsible for specific recognition
of long arrays of telomeric DNA (Schrumpfová
et al. 2004; Mozgová et al. 2008). The DNA-binding preference
for oligodeoxynucleotide (oligo) substrates was tested
using the electrophoretic mobility shift assay (EMSA). Oligo
sequences were designed to assess the effect of TRB4 and
TRB5 binding to long arrays of telomeric sequences as well
as to interstitially located telo-boxes (Fig. 4A, Supplemental
Table 1). Either a tetramer of telomeric sequences or a telobox
sequence (1.2 telomeric units) flanked with non-telomeric
DNA were used as the labelled probes. A non-telomeric
oligonucleotide, added in 1, 20 and 100-fold excess, served
as competitor DNA and vice versa. Parallel experiments
with the TRB1 protein were used for comparison.
The results obtained from these experiments clearly
show that TRB4 and TRB5 do indeed preferentially bind
long arrays of telomeric sequences or telo-boxes positioned
within a non-telomeric DNA sequence (Fig. 4B–D). TRBs
bind to telomeric dsDNA in a similar mode as was described
for TRB1-3, forming a high-molecular-weight complex
which does not migrate into the gel (Schrumpfová et al.
2004; Mozgová et al. 2008). The binding of all three TRB
proteins to the telomeric sequence is highly specific, as even
a 100-fold higher concentration of non-telomeric competitor
does not prevent the formation of protein-telomeric DNA
complex.
Our data indicate that TRB4-5, as well as other TRBs,
are capable of binding long arrays of telomeric sequences
or short motifs with as little as one telomeric repeat. This is
in contrast to human TRF1/TRF2, which bind two telomeric
repeats as preformed dimers (Court et al. 2005) and also to
previous predictions (Hofr et al. 2009). Having defined the
TRB minimal recognition motif as one telo-box, the binding
properties of TRBs are poised for further investigation.
Unlike other TRB family members, TRB5
is preferentially localized in the cytoplasm
To compare Arabidopsis TRB proteins further, we examined
the subcellular localization of native TRB proteins in Arabidopsis
cells, using a mouse monoclonal antibody developed
in our laboratory specific for the conserved section of the
Myb-like domain found in the TRB family. The anti-TRB
5.2 antibody recognizes all five members of TRB family
(Schrumpfová et al. 2014). Nuclei isolated from 10-dayold
seedlings were subjected to immunofluorescence using
Fig. 2  Phylogenetic analysis of TRB proteins. (A) Simplified evolution
of the main Viridiplantae lineages with known TRB proteins.
One TRB protein evolved initially in Streptophyta in Klebsormidiophyceae,
then diversified to three similar homologues in Mosses
and Lycophyta and several diverse homologues in seed plants, with
five members in A. thaliana from Brassicaceae. The evolutionary tree
was adopted from Rensing 2020 and Cheng et al. 2019. (B) ML phylogenetic
tree of TRB proteins. Twenty-seven species were included
in 83 sequences with 465 bp in the final data set. Only one species
from the family was selected for the final analysis. The ML likelihood
is -24,356.573420. The numbers below branches indicate bootstrap
support values > 50%. Four major groups are shown in the phylogenetic
tree: TRB_A for Streptophyte algae, Lycophyta and Bryophyta;
TRB_B including AtTRB4 and AtTRB5 for Embryophyta; TRB_C
for Embryophyta with AtTRB1 and the nested TRB_D encompassing
AtTRB2 and AtTRB3. Motifs are ranked and ordered by the highest
probability of occurrence. Fifteen most probable motifs are depicted.
For sequence and sequence conservation information, see Supplementary
Fig. 1. For a list of species, see Supplementary Table 3
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this anti-TRB antibody combined with DAPI staining. We
observed a speckled distribution of TRBs in the nucleus and
nucleolus (Fig. 5A).
As the anti-TRB 5.2 antibody does not distinguish the
localization of individual proteins, we proceeded to the
subcellular localization of individual members of TRB
family using TRBs fused with green fluorescent protein
(GFP). Confocal microscopy showed that TRB1-3 fused
with GFP expressed from pGWB6 after transient Agrobacterium-mediated
transformation in N. benthamiana leaf
epidermal cells are localized mainly in nucleoli and nucleoplasmic
fluorescence foci, as was described previously
(Schrumpfová et al. 2014; Zhou et al. 2016). Interestingly,
TRB4 is distributed not only in nucleoli and nucleoplasmic
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fluorescence foci of different sizes, but also throughout the
nucleoplasm. Conversely, TRB5 fused with GFP is localized
mainly in the cytoplasm with only minor localization
in the nucleolus and nucleoplasm (Fig. 5B, Supplemental
Figs. 3, 4).
Co-expression of GFP-TRBs with the nucleolar marker
Fibrillarin1 or nucleoplasm marker Serine-arginine-rich
proteins 34 (SRp34) fused with a monomeric red fluorescent
protein (mRFP) after transient Agrobacterium-mediated
transformation in N. benthamiana leaf epidermal cells
verified the subnuclear localizations of the TRB4 and TRB5
described above. Using Fibrillarin-mRFP we clearly identified
the stronger localization of GFP-TRB4 in the nucleolus,
but the weak nucleolar localization of GFP-TRB5. The relative
positioning of GFP-TRB and the nucleoplasm marker
SRp34-mRFP also supports our observation that the GFPTRB5
is preferentially localized in the cytoplasm (Fig. 5C).
TRB4-5 fused with GFP show a distinct localization in
the plant cell compared to TRB1-3, proteins with a later evolutionary
origin. In particular, GFP-TRB5 manifests strong
cytoplasmic localization, suggesting a possible specific
functional role or spatiotemporal regulation.
Dimerization of TRB proteins
To shed light on the conservation of mutual interactions
between TRB proteins from Arabidopsis, we analyzed
interactions between all members of TRB family. To date,
self and mutual dimerization of TRB1-3 has been investigated
by Y2H or by co-immunoprecipitation experiments
(Co-IP) (Kuchař and Fajkus 2004; Schrumpfová et al. 2004,
2008; Mozgová et al. 2008), however knowledge of the precise
subcellular localization of mutual TRB interactions is
missing.
First, the interactions of TRB4 and TRB5 with other TRB
family members were investigated using a GAL4 based Y2H
assay. Mutual interactions between TRB family members
from Arabidopsis appear to be conserved, as both TRB4
and TRB5, interact with all TRB family members. Moreover,
TRB4-5 form self-dimers in a similar way to TRB1-3
(Fig. 6A).
Next, BiFC assays with equal protein levels were used
to detect self- and mutual interactions of TRBs at the level
of cellular compartments. The TRB coding sequences were
introduced into 2in1 multiple expression cassettes within a
single vector backbone with an internal marker for transformation
and expression-mRFP1 (Grefen and Blatt 2012).
TRBs were fused with an N- and/or C-terminal half of yellow
fluorescent protein (nYFP, cYFP) as both N-terminal
and C-terminal fusions. A list of all constructs is provided
in Supplemental Table 4. Fluorescence was detected using
confocal laser scanning microscopy after transient Agrobacterium-mediated
transformation in N. benthamiana epidermal
cells. Our observations show self- and mutual interactions
of TRB1-4 in the nucleolus and/or in nucleoplasmic
fluorescence foci that were of different numbers and sizes.
Interestingly, the TRB5 homodimeric interaction was found
to be clearly cytoplasmic with a reduced nuclear speckle size
compared to other TRB interactions. Additionally, TRB1-3,
but not TRB4, interact moderately with TRB5 in the nucleolus,
as well as in more prominent nucleoplasmic fluorescence
foci (Fig. 6B, Supplemental Figs. 5, 6).
Overall, our characterization of mutual TRB interactions
revealed that all TRB members have the ability to form selfdimers
and mutually interact. However, TRB5 homodimeric
interactions are predominantly localized in the cytoplasm,
which differs from TRB1-4 homodimeric interactions, which
are nucleolar or localized in nucleoplasmic fluorescence
foci.
Novel interaction partners of TRB proteins—PRC2
subunits EMF2 and VRN2
To further elucidate the conservation of protein interaction
partners throughout the TRB family, we looked
for interactions between TRB4 and TRB5 with TERT,
RUVBLs, POT1b, PWO1-3, SWN, CLF interactors, which
have already been described as interacting with TRB1-3
(Schrumpfová et al. 2008, 2014; Hohenstatt et al. 2018;
Zhou et al. 2018; Tan et al. 2018; Schořová et al. 2019;
Fig. 3  Conserved features of MYB-like domain through TRB lineages.
(A) Consensus sequences of Myb-like domain in each evolutionary
lineage were visualised using sequence logos. Colored
squares, represent TRB1-5 proteins from A. thaliana; Inverted triangle,
the aa at position 3 (B) at N-terminus of TRBs that is replaced in
Dicots from TRB_B lineage to N; Asterisk, the aa at position 5 (K)
that mediates intermolecular contacts between telomeric DNA and
N-terminus of all TRBs (Fig. 1C); Circle, the negatively charged aa
(E/D) at position 11 conserved across all TRB lineages; Rhombus, the
aa at position 14 replaced from uncharged (A/S) to negatively charged
(E/D) in the whole TRB_B lineage; Trapezoid, the aa at position 17
that is in Dicots from TRB_B lineage partially replaced from positively
charged (K/R) to hydrophobic (L); Triangle, the aa at position
60 that shows lower conservation in Dicots from TRB_B lineage than
in other lineages. (C) The representative members of Dicots from
TRB_B, TRB_C and TRB_D lineages, namely A. thaliana TRB4,
TRB1 and TRB2, respectively, were analyzed. The three-dimensional
model of the Myb-like domains from the site opposing the DNAbinding
viewpoints are based on the hTRF2-DNA interaction model
(PDB: 1WOU) (Court et al. 2005). The evolutionary dynamics of aa
substitutions among aa residues within Myb-domain of these exemplified
were visualized using ConSurf 2016 (Ashkenazy et al. 2016).
The conservation of residues is presented in a scale, where the most
conserved residues are shown in dark magenta and non-conserved
residues as white. (D)  Surface models showing the charge on the
Myb-like domain are presented from the site opposing the DNAbinding
viewpoint for each model. Residue charges are coded as red
for negative, blue for positive, and white for neutral, visualised using
PyMol, Version 2.4.1, Schrödinger, LLC
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Mikulski et al. 2019). These interactions were investigated
using a GAL4-based Y2H assay, and TRB interactions with
newly identified protein interactors were verified by Co-IP.
Our results revealed that TRB4-5 interact with the
N-terminal domains of TERT called telomerase essential
N-terminal (TEN, 1 – 233 aa) and RNA interaction domain
1 (RID1, 1 – 271 aa) fragments, in a similar manner to that
described for TRB1-3 (Schrumpfová et al. 2014). Interactions
of TRBs with TERT seem to be mediated only via the
N-terminal domain of TERT, as the centrally positioned
Telomerase RNA-binding domain (TRBD) or reverse transcriptase
domain (RT) domain do not show interactions
(Supplemental Fig. 7). We also observed interactions of
TRB4-5 with RUVBL1, RUVBL2A and POT1b in the
same manner as was observed for TRB1-3. Moreover, Y2H
experiments detected novel interactions between TRB4-5
with a second homolog of the human POT1 protein in A.
thaliana - POT1a. These interactions were further confirmed
by the Co-IP of these proteins (Fig. 7A, Supplemental
Fig. 8A).
Furthermore, TRB4-5 interact with PWOs, members of
the PEAT complex, as was already observed for TRB1-3
(Tan et al. 2018). A strong interaction was detected between
TRB4-5 and PWO1 full-length (Supplemental Fig. 8B) or
PWO1 N-terminal sections, including PROLINE-TRYPTOPHANE-TRYPTOPHANE-PROLINE
(PWWP) domain
(1–223 aa) (Fig. 7B). PWO2 and 3 interact with TRB4 via
their N-terminal parts (1–216 and 1–204 aa, respectively).
Similarly, we detected the interactions of the N-terminal
parts of PWO1 and PWO3 with TRB5.
Fig. 4  EMSA of TRB1, TRB4 and TRB5 binding of radioactivelylabelled
oligonucleotides. (A) Schematic depiction of oligonucleotides
employed. Telomeric, four repeats of plant telomeric DNA
sequence; telo-box, 1.2 plant telomeric units flanked with non-telomeric
DNA sequence; non-telomeric, oligonucleotide with non-telomeric
DNA. (B) EMSA of TRB1, TRB4 and TRB5 proteins binding
radioactively-labelled double-strand (ds) tetramers of telomeric
sequence with unlabelled tetramers of non-telomeric oligonucleotides
as competitor DNA. The concentration of unlabelled competitor
increases from 1-, 20- to 100-fold the concentration of the labelled
probe (as depicted by the triangle). Oligo*: protein ratio is 1:10. (C)
EMSA of the same proteins with a radioactively-labelled ds telo-box
oligonucleotides with unlabelled non-telomeric oligonucleotides as
competitor DNA, performed as in B. (D) EMSA of the same proteins
with a radioactively-labelled ds of non-telomeric oligonucleotides
with unlabelled ds tetramers of telomeric sequence as competitor
DNA, performed as in B
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To identify TRB interactions with E(z) homologs,
members of the PRC2 complex, we used SWN and CLF
proteins without the SET [Su(var)3–9, E(z), Trx] domain
that confers histone methyltransferase activity ­(SWNΔSET
and ­CLFΔSET) (Chanvivattana et al. 2004; Hohenstatt
et al. 2018). The SET-domain does not seem to be involved
in interactions of SWN and CLF with TRB4-5 as Y2H
experiments identified interactions between TRB4-5 and
­SWNΔSET and ­CLFΔSET (Fig. 7C).
It was shown recently that the rice homologue of
TRB, Telomere repeat-binding factor 2 (TRBF2), and
rice Su(z)12 homologues of the PRC2 complex named
EMBRYONIC FLOWER 2b (EMF2b) interact with each
other (Xuan et al. 2022). We observed novel interactions,
not only between Arabidopsis TRB4-5 and EMF2, but also
between TRB4-5 and other Su(z)12 homologue VERNALIZATION
2 (VRN2). The observed interactions from Y2H
system were verified by Co-IP (Fig. 7C).
This broad screen of TRB4-5 protein interaction partners
suggests a role of TRBs in various protein complexes. TRBs
might contribute to the recruitment of these complexes to
telomeric DNA repeats (e.g., Telomerase complex to telomeres)
(Fig. 8A) or to telo-boxes localized in the promoter
regions of the various genes (e.g., PEAT complex or PRC2
subunits to telo-boxes) (Fig. 8C, D, F).
Fig. 5  Subcellular localization
of native TRBs and
GFP-TRB fusion proteins. (a)
Isolated nuclei from A. thaliana
seedlings were subjected to
immunofluorescence using an
anti-TRB antibody combined
with DAPI staining. All five
native members of the TRB
family are visualized. Scale
bar = 10 µm. (b) TRB1-5 were
fused with GFP (N-terminal
fusions), expressed in N.
benthamiana leaf epidermal
cells and observed by confocal
microscopy. Single images of
areas with nuclei are presented.
Scale bars = 5 µm. (c) Colocalization
of TRB4 and TRB5
(N-terminal GFP fusions) with
a nucleolar marker (FibrillarinmRFP)
and a nucleoplasm
marker (SRp34-mRFP) was
performed as described in B).
Single images of areas with
nuclei are presented. Scale
bars = 5 µm
74	 Plant Molecular Biology (2023) 112:61–83
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Discussion
Although the TRBs were originally characterized as being
associated with long arrays of telomeric repeats (Schrumpfová
et al. 2004, 2014; Mozgová et al. 2008; Dvořáčková
et al. 2010; Dreissig et al. 2017) (Fig. 8A), recent observations
indicate broad engagement of TRB proteins in
various cellular pathways. The most important TRB functions
(Fig. 8B–F) include interactions with the telomerase
complex (Schrumpfová et al. 2014; Schořová et al. 2019),
association with telo-boxes in the promoters mainly of
translation machinery genes (Schrumpfová et al. 2016),
recruitment of PRC2 and PEAT complexes to telo-boxes
(Zhou et al. 2018; Tan et al. 2018; Tsuzuki and Wierzbicki
2018; Mikulski et al. 2019) or antagonisms between TRB1
and H1 at long interstitial telomeric DNA repeats (Teano
et al. 2020).
Fig. 6  Dimerization of TRB proteins. (a) The Y2H system was used
to assess mutual protein–protein interactions of TRBs. Two sets of
plasmids carrying the indicated protein fused to either the GAL4
DNA-binding domain (BD) or the GAL4 activation domain (AD)
were constructed and introduced into yeast strain PJ69-4a carrying
reporter genes His3 and Ade2. Interactions were detected on histidine-deficient
SD medium (–His), or under stringent adenine-deficient
SD medium (–Ade) selection. Co-transformation with an empty
vector (AD, BD) served as a negative control. (b) Interactions of
TRBs fused with nYFP or cYFP part were detected using the Bimolecular
fluorescence complementation (BiFC) assay in N. benthamiana
leaf epidermal cells. Shown here are single images of merged
signals of reconstructed YFP (interaction of the tested proteins) and
signals of mRFP (internal marker for transformation and expression)
fluorescence detected by confocal microscopy. For separated fluorescent
emissions, see Supplemental Fig. 5. Scale bars = 5 µm
Fig. 7  Interaction of TRB4-5 with various partners. (A) The Y2H
system was used to assess protein–protein interactions of TRB4-5
proteins with TERT fragments, RUVBLs and POT1a/b as in Fig. 6.
Co-transformation with an empty vector (AD, BD) served as a negative
control. Asterisks, 1  mM 3-aminotriazol (3-AT); cross, 3  mM
3-AT. (B) Interactions between N-terminal domain of PWO1-3
and TRB4-5 were detected as in A). Interactions with full length
PWO1-2 proteins are in Supplementary Fig. 8B. (C) Novel interactions
between TRB4-5 and EMF2/VRN2, as well as interactions with
­SWNΔSET/CLFΔSET were tested using Y2H system as in A). Novel
interactions were verified by Co-IP. The TNT expressed VRN2 and
EMF2 (35
S-labelled*) were mixed with TRB4-5 (myc-tag) and incubated
with anti-myc antibody. In the control experiment, the VRN2
and EMF2 proteins were incubated with anti-myc antibody and Protein
G magnetic particles in the absence of partner protein. Input (I),
unbound (U), and bound (B) fractions were collected and separated in
SDS–10% PAGE gels
75Plant Molecular Biology (2023) 112:61–83	
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TRB4‑5 are evolutionarily closer to TRBs from lower
plants
Our phylogeny indicates that TRB proteins with N-terminal
Myb-domains are conserved in plants and probably arose in
Streptophyte algae within the family Klebsormidiophyceae.
This corresponds to the transition of plants to a terrestrial
habitat 800 Mya ago (Cheng et al. 2019) (Fig. 2). However,
other groups of Streptophyte algae do not have TRB
proteins, favoring a birth and death model of gene evolution
(Nei et al., 2005; Eirín- López et al., 2012) or total
divergence (Pinho and Hey 2010), such as to the TRF-like
(TRFL) genes with a C-terminal Myb-domain (Karamysheva
et al. 2004; Hwang and Cho 2007; Fulcher and Riha 2016;
Majerská et al. 2017).
Recurrent gene duplications over many generations have
created orthologs and paralogs in the plant genome, giving
rise to several new protein functions. In green algae, the
majority of gene families contain only one gene (Clark and
Donoghue 2018; Qiao et al. 2019), consistent with this, only
one copy of the TRB protein was detected in Streptophyte
algae. An increased number of TRB homologs were found
in Embryophyta, which could be a result of WGD within
these derived groups. In Mosses (taxon Bryophyta), the
genome was duplicated in several independent events. Two
WGD events in Sphagnum and Physcomitrium around 200
Fig. 8  Overview of the main Telomere repeat binding proteins
(TRBs) functions. (A) TRBs are associated with the physical ends of
chromosomes (telomeres) via their Myb-like domain (Schrumpfová
et al. 2004, 2014; Mozgová et al. 2008; Dvořáčková et al. 2010; Dreissig
et al. 2017). TRBs interact with Arabidopsis homologs of the
G-overhang binding protein Protection of telomere 1a, b (POT1a, b)
(Schrumpfová et al. 2008, this study). (B) TRBs mediate interactions
of Recombination UV B – like (RUVBL) proteins with the catalytic
subunit of telomerase (TERT), and participate in telomerase biogenesis
(Schrumpfová et al. 2014; Schořová et al. 2019). TRBs are associated
in the nucleus/nucleolus with POT1a (Schořová et al. 2019),
and also with a plant orthologue of dyskerin, named CBF5 (Lermontova
et al. 2007) that binds the RNA subunit of telomerase (TR)
(Fajkus et al. 2019; Song et al. 2021). (C) TRBs are associated with
short telomeric sequences (telo-boxes) in the promoters of various
genes in  vivo, mainly with translation machinery genes (Schrumpfová
et  al. 2016). ORF, Open reading frame. (D) Telo-box motifs
recruit Polycomb repressive complexes (PRC2) via interactions of
PRC2 subunits with TRB (Zhou et al. 2016, 2018, this study) CLF,
CURLY LEAF; SWN, SWINGER; EMF2, EMBRYONIC FLOWER
2; VRN2, VERNALIZATION 2. (E) Histone H1 prevents the invasion
of H3K27me3 and TRB1 over telomeres and long interstitial
telomeric regions (Teano et al. 2020). (F) TRB proteins, as subunits
of the PEAT (PWO-EPCR-ARID-TRB) complex, are involved in heterochromatin
formation and gene repression, but also have a locus‐
specific activating role, possibly through the promotion of histone
acetylation (Tan et al. 2018; Tsuzuki and Wierzbicki 2018; Mikulski
et al. 2019)
76	 Plant Molecular Biology (2023) 112:61–83
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Mya and 120 Mya, respectively (Lang et al. 2018; Clark and
Donoghue 2018), resulted in three almost identical TRB proteins
clustered in the lineage TRB_A. The moss Ceratodon
also underwent WGD, but unlike Sphagnum and Physcomitrium,
the TRB duplication was not detected. No TRBs were
found in hornworts and liverworts, but this may be due to the
limited data available on these genomes.
Our results suggest diversification of the TRB gene family
in seed plants which is linked with multiple subsequent or
independent events of WGD. Lineage TRB_B seems to have
evolved in the ancient past and is closely related to TRB_A
in Bryophytes. The sub-lineage TRB_D is present in dicots
in many families and embedded within the TRB_C lineage.
Within Brassicaceae, nested WGDs resulted in multiple
TRB homologs. A. thaliana has five TRBs with closely
related proteins TRB2-3 and TRB4-5. However, within
Brassicales the situation differs. The genome of Carica
papaya (Caricaceae) possesses only two TRB proteins, each
belonging to one of the lineages TRB_B or TRB_C lineages.
This observation is in agreement with an older paleoploidy
event in the Brassicaceae lineage (β) that is not shared by
C. papaya but is shared by all other analyzed Brassicaceae
species (Dassanayake et al. 2011; Wang et al. 2011). As
summarized by Rockinger et al. 2016, the ancestor of all
Caricaceae underwent only a single WGD event, in comparison
to the ancestor of A. thaliana, which underwent a more
recent, additional round of WGD (α) (Bowers et al. 2003;
Kagale et al. 2014). Similarly, in monocots, where several
independent WGDs have occurred, an increased number of
TRB proteins were found, e.g., six TRBs were detected in
Zea mays (Fig. 2B).
The complexity of plant genomes is extremely high,
and annotations of plant genomes are undergoing intensive
improvement at present (Kress et al. 2022). Further revision
and reinvestigation of the TRB evolutionary tree may help
to elucidate species-specific TRB variants such as the one
identified in Malus domestica.
Conserved structure of individual domains
Although TRB4-5 are grouped in a monophyletic lineage
that is distant to TRB1-3 proteins, our assessment of predicted
structural models implies that all TRB family members
are folded into similar three-dimensional structures.
The TRB Myb-like domain is very closely related to that
of other telomere-binding proteins, including human TRF1
and TRF2 (Chong et al. 1995; van Steensel et al. 1998;
Smogorzewska and de Lange 2004). TRF1 and TRF2 bind
to DNA as preformed homodimers (Bianchi et al. 1997,
1999; Broccoli et al. 1997). It was suggested that in vitro the
Myb-like binding domain of TRF1 binds to DNA essentially
independently of the rest of the protein (König et al. 1998;
Bianchi et al. 1999; Court et al. 2005). Specificity in DNA
recognition of TRF1 and TRF2 is achieved by several direct
contacts from aa side chains to the DNA, mainly via helix 3
and an extended N-terminal arm (Hanaoka et al. 2005; Court
et al. 2005). Predicted models of the plant Myb-like domain
(Fig. 1) show that the overall 3D structure is preserved in
both plant and animal kingdoms, and that the surface mediating
protein-DNA interactions are fully conserved. However,
the surface side and extended N-terminal arm of the
Myb-domain of proteins from the TRB_B family differ to
those of the TRB_A or TRB_C families (Fig. 3).
Consistent with the conserved features of the Myb
domain, our EMSA results showed the conserved ability of
TRB4-5 proteins to bind telomeric sequences. Notably, not
all proteins with Myb-like domains are able to bind telomeric
repeats, as Arabidopsis proteins from the TRFL family,
with this motif at the C-terminus, need an accessory Mybextension
domain for telomeric dsDNA interactions in vitro
(Karamysheva et al. 2004; Ko et al. 2008). Moreover, unlike
TRBs, only Arabidopsis plants deficient in one particular
member (Hwang and Cho 2007), but not plants deficient for
all six TRFL proteins with Myb-extension domain, exhibit
changes in telomere length (Fulcher and Riha 2016). Our
EMSA results showed the ability of TRB4-5 proteins to bind
longer telomeric tracts as well as short telo-boxes, containing
roughly one telomeric repeat flanked by non-telomeric
sequence (Fig. 4). These observations suggest that TRB4-5
proteins may be associated with cis-regulatory elements in
the promoter regions of Arabidopsis genes as was described
for TRB1-3 (Zhou et al. 2016; Schrumpfová et al. 2016).
The presence of only one telomeric repeat in the telo-box
raises the question of whether TRB proteins operate on promoter
regions as monomers/dimers or multimers. Based on
our quantitative DNA-binding study, we propose that TRB1
and TRB3 bind long telomeric DNA arrays with the stoichiometry
of one protein monomer per one telomeric repeat
(Hofr et al. 2009), but the stoichiometry of TRBs in regulatory
complexes associated with telo-boxes needs further
elucidation.
The sequence-specific interaction between telomeric
dsDNA and TRB1-3 is mediated predominantly by the Myblike
domain, although additional domains from TRBs can
also contribute to non-specific DNA interactions (Schrumpfová
et al. 2004; Mozgová et al. 2008; Hofr et al. 2009).
The H1/5-like domains of TRB proteins belong to the same
group as central globular domain of core H1 histones, the
incorporation of which directly influences the physicochemical
properties of the chromatin fiber and further modulates
nucleosome distribution, chromatin compaction and contributes
to the local variation in transcriptional activity by
affecting the accessibility of transcription factors and RNA
polymerases to chromatin (Fan and Roberts 2006; Zhou
77Plant Molecular Biology (2023) 112:61–83	
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et al. 2015; Hergeth and Schneider 2015; Bednar et al. 2017;
Fyodorov et al. 2018). The H1/5-like domain of TRB mediates
non-specific DNA interactions (Mozgová et al. 2008) as
well as interactions with the other members of TRB family
and also with the POT1b protein (Schrumpfová et al. 2008).
The globular domain of H1 adopts a winged-helix fold
with a “wing” defined by two β-sheets. 3D model predictions
suggest only minor differences between TRB4-5 and
TRB1-3 within the H1/5-like domain, as the loop between
two antiparallel β-sheets in the H1/5-like domain of TRB4-5
is longer than in TRB1-3.
Coiled-coil domains are structural motifs that consist of
two or more α-helical peptides that are wrapped around each
other in a superhelical fashion that may mediate interactions
between proteins (Lupas and Gruber 2005; Apostolovic et al.
2010). Only minor differences were predicted for the 3D
structure of the coiled coil domain of TRB4-5 and TRB1-3.
However, the motifs at the C-terminal sections of the coiledcoil
domains in TRBs from TRB_B differ from those motifs
at the C-terminal parts of TRB_C lineages, and clearly distinguish
these two evolutionarily distinct groups.
TRB4‑5 differ in subcellular localization from TRB1‑3
In previous studies, it was shown that TRB1-3 are highly
dynamic DNA-binding proteins with cell-cycle regulated
localization. During interphase, GFP-fused TRB1-3 proteins
are preferentially localized in the nucleus, with a
strong nucleolar signal and relatively strong nuclear speckles
of different sizes (Dvořáčková et al. 2010; Schrumpfová
et al. 2014; Zhou et al. 2018). A similar pattern was
observed in transiently or stably transformed Arabidopsis
cells (Dvořáčková et al. 2010) or in tobacco epidermal cells
(Schrumpfová et al. 2014) despite the fact that tobacco
telomeres are dispersed throughout the nucleus (so-called
non-Rabl chromosome configuration), while Arabidopsis
telomeres are clustered around the nucleolus (rosette-like
chromosome configuration) (Shan et al. 2021). Our results
show that even native TRBs in isolated Arabidopsis nuclei
can be detected with a specific antibody, and are localized
in the speckles. Some of these speckles in the vicinity of the
Arabidopsis nucleoli might be telomeric (Dvořáčková et al.
2010), or might be Cajal bodies (Dvořáčková 2010), as was
demonstrated for speckles detected in tobacco nuclei.
Here we show that TRB4 and TRB5 fused to GFP have,
in contrast to TRB1-3, a distinct subcellular localization pattern
(Fig. 5B, C). In addition, unlike all other members of
the TRB family, TRB5 is preferentially localized in the cytoplasm.
It remains to be clarified whether it is sequestered
there or plays a specific functional role.
Our Y2H or BiFC assays proved that TRB4-5 could form
homo- and hetero-dimers, as observed in TRB1-3 (Kuchař
and Fajkus 2004; Schrumpfová et al. 2004). In addition to
dimers, TRB1-3 are capable of forming both homo- and heterotypic
multimers via their H1/5-like domain (Schrumpfová
et al. 2004; Mozgová et al. 2008; Hofr et al. 2009). Similar
multimerization can also be assumed for TRB4 and TRB5,
as we observed the formation of high molecular weight complexes
for TRB4-5 in EMSAs, which did not migrate into the
gel (Warren et al. 2003).
Despite distinct subcellular localization of GFP-TRB4
and GFP-TRB5, we observed mutual interactions between
all TRB family members using BiFC, predominantly in the
nuclear speckles (Fig. 6B). Only the TRB5 homodimeric
interaction was found to be clearly cytoplasmic with nuclear
speckles of decreased size compared to the size of speckles
in the other homodimeric or mutual TRB interactions. Our
BiFC assays showed that TRB1-3, but not TRB4, have the
ability to drag TRB5 into the nucleolus, as TRB4 does not
interact with TRB5 in the nucleolus. We can assume that
TRB proteins form various heteromers in different subcellular
compartments that might possess different functions
related to distinct biochemical pathways.
Interconnection of TRBs with various protein
complexes
Even though TRB4-5 show slightly distinct localization patterns
compared to TRB1-3, it appears that all TRBs may
interact with similar partners in vitro (Fig. 7). TRB4-5 proteins
directly interact with the N-terminal domains of TERT
as was also shown for TRB1-3 proteins (Schrumpfová et al.
2014). Additionally, TRB4-5 interact with both RUVBL1
and RUVBL2A proteins, which may imply that they may
mediate interactions in the trimeric complex RUVBL-TRBTERT
as was proposed for TRB1-3 (Schořová et al. 2019).
Telomerase might also be modulated by POT1 proteins: A.
thaliana POT1a positively regulates telomerase activity,
POT1b is proposed to negatively regulate telomerase and
promote chromosome end protection (Beilstein et al. 2015).
We observed not only the expected interaction between
TRB4-5 and POT1b (Schrumpfová et al. 2008), but also
revealed novel interactions of TRB proteins with POT1a.
Altogether, we can assume that TRB4-5 are associated with
the telomerase complex as was proved for TRB1-3.
In addition to telomeres (Schrumpfová et al. 2014),
TRB1-3 regulate the PRC2 target genes (Zhou et al. 2016,
2018). Interestingly, H3K27me3 is present at telomeres of
Arabidopsis (Adamusová et al. 2020; Vaquero-Sedas and
Vega-Palas 2023). The observation that not only TRB1-3
(Zhou et al. 2018), but also TRB4-5, physically interact with
homologs of E(z) subunit of PRC2 complex named CLF
and SWN suggests that all TRBs can target PRC2 to Polycomb
response elements (PREs) including telo-boxes (Deng
et al. 2013; Zhou et al. 2016; Godwin and Farrona 2022).
Moreover, the novel interaction between TRBs and Su(z)12
78	 Plant Molecular Biology (2023) 112:61–83
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A. thaliana homologues EMF2 and VRN2 described here,
tightly interconnects all TRBs with the core PRC2 components.
These observations support the recently published
observation that O. sativa single Myb transcription factor
TRBF2 forms phase-separated droplets, which aggregate
with PRC2 via rice OsCLF and OsEMF2 (Xuan et al. 2022).
In Arabidopsis, the PWO1 protein interacts with all three
E(z) homologs, including CLF and SWN through its conserved
N-terminal PWWP domain (Mikulski et al. 2019).
Furthermore, PWO1-3 are associated with members of the
PEAT complex, which was recently identified as being able
to silence transposable elements (Tan et al. 2018). Our data
suggest that the interaction between TRBs and PWOs is not
restricted to only TRB1 and 2 (Tan et al. 2018), instead other
TRB members, including TRB4-5, interact with N-terminal
part of PWO1 and PWO3 from A. thaliana, containing
PWWP domains. Additionally, the N-terminal part of PWO2
is recognized by Arabidopsis TRB4. Interestingly, in tobacco
PWO1 tethers CLF to nuclear speckles (Hohenstatt et al.
2018; Mikulski et al. 2019) which have a similar distribution
to speckles of TRB proteins.
Overall, we conclude that TRB proteins, including the
newly characterized TRB4-5, are associated with several
complexes, including telomerase, PRC2 or PEAT complexes.
However, TRBs are unlikely to be permanently associated
with all of these complexes (Tan et al. 2018; Schubert
2019), and we might speculate that TRB subunits are partially
interchangeable within these complexes.
It should be noted that the number of identified interaction
partners of TRBs in Arabidopsis may increase in
the future, as these promiscuous proteins may play a role
in various additional biochemical processes that are not
yet elucidated. For example, the A. thaliana TRB1 gene
is responsive to several types of hormones, including jasmonate
(JA) (Yanhui et al. 2006); TRB homologue from
apple dynamically modulates JA-mediated accumulation of
anthocyanin and proanthocyanidin (An et al. 2021); soybean
TRB homologue was identified as candidate gene regulating
total soluble sugar in soybean seeds (Xu et al. 2022).
Conclusion
Proteins from the TRB family are plant-specific and apparently
first evolved in lower plants. We speculate, that due
to WGDs one ancestral TRB was multiplied to the current
five TRB members in A. thaliana increasing the potential of
diversification of their particular functions. Further research
is needed to confirm whether newly described TRB4-5 proteins
specifically target to telomeric sequences located terminally
(Schrumpfová et al. 2004, 2014; Mozgová et al. 2008;
Dvořáčková et al. 2010; Dreissig et al. 2017) or interstitially
(Schrumpfová et al. 2016) via their Myb-like domain like
other members of this family TRB1-3. Additionally, the versatile
interactions of all TRBs members with other proteins
contribute to the multiple functions that they adopt in the
cell nucleus, including participation in telomerase biogenesis
(Schrumpfová et al. 2014; Schořová et al. 2019), recruitment
of PRC2 or PEAT complexes (Zhou et al. 2016, 2018;
Tan et al. 2018; Tsuzuki and Wierzbicki 2018; Mikulski
et al. 2019) or competing with H1 for binding to interstitially
localized telomeric sequences (Teano et al. 2020). The cytoplasmic
localization of TRB5 and its implications deserve
further investigation. As additional functions and interaction
partners of TRBs are discovered, it can be expected that
research in plants will lead to a better understanding of the
mode of action of the different TRBs and also to the elucidation
of novel functions.
Supplementary Information  The online version contains supplementary
material available at https://​doi.​org/​10.​1007/​s11103-​023-​01348-2.
Acknowledgements  This work was supported by the Czech Science
Foundation projects 21-15841S (P.P.S., M.K., D.H.) and 20-01331X
(A.K), and Czech Ministry of Education, Youth and Sports grant
number LTAUSA18115 (L.S.). The Plant Sciences Core Facility of
CEITEC Masaryk University is acknowledged for its technical support.
We acknowledge the Imaging Facility of the Institute of Experimental
Botany AS CR supported by the MEYS CR (LM2018129 CzechBioImaging)
and IEB AS CR.
We acknowledge Sara Farrona (Plant and AgriBiosciences Centre,
National University Ireland, IR) for providing us with SWN, CLF and
EMF2 constructs. Moreover, we acknowledge Eva Sykorova (Institute
of Biophysics of the Czech Academy of Sciences, Brno, Czech
Republic) for providing us constructs with POT1a-b and TERT fragments
and Ali Pendle (John Innes Centre, Norwich, United Kingdom)
for the sub-nuclear localization markers. We also acknowledge Yvona
Stražická and Michal Franek for the help with the immunofluorescence
on Arabidopsis nuclei.
We acknowledge Leon Jenner and Iva Mozgová for proofreading
and manuscript editing.
The access to the computing and storage facilities owned by parties
and projects contributing to the National Grid Infrastructure MetaCentrum
provided under the programme Projects of Large Infrastructure
for Research, Development, and Innovations (LM2010005) was highly
appreciated, as was the access to the CERIT-SC computing and storage
facilities provided under the programme Center CERIT Scientific
Cloud, part of the Operational Program Research and Development for
Innovations, reg. no. CZ.1.05/3.2.00/08.0144.
Author contribution  Petra Procházková Schrumpfová and David
Honys designed the study, supervised the project. Petra Procházková
Schrumpfová wrote the manuscript with support from David Honys,
Jan Paleček, Daniel Schubert. Lenka Steinbachová, Zuzana Gadiou,
Alžbeta Kusová, Tereza Přerovská, Claire Jourdain and Tino Stricker
performed cloning, Lenka Steinbachová performed localization and
BiFC, Alžbeta Kusová and Ahmed Khan performed Y2H, Tereza
Přerovská performed EMSA, Gabriela Rigóová performed Co-IP, Jan
Paleček analyzed 3D structures, Lenka Záveská Drábková performed
evolution analysis. Petra Procházková Schrumpfová conducted experiments
and analyzed data.
Funding  Open access publishing supported by the National Technical
Library in Prague.
79Plant Molecular Biology (2023) 112:61–83	
1 3
Data availability  All data generated or analysed during this study are
included in this published article.
Declaration 
Competing interest  The authors report no competing interests.
Open Access  This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format,
as long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate
if changes were made. The images or other third party material in this
article are included in the article's Creative Commons licence, unless
indicated otherwise in a credit line to the material. If material is not
included in the article's Creative Commons licence and your intended
use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​
org/​licen​ses/​by/4.​0/.
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Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Authors and Affiliations
Alžbeta Kusová1,2
   · Lenka Steinbachová3
   · Tereza Přerovská1
   · Lenka Záveská Drábková3
   ·
Jan Paleček1,2
   · Ahamed Khan4
   · Gabriela Rigóová1
   · Zuzana Gadiou3
   · Claire Jourdain5
   · Tino Stricker5
   ·
Daniel Schubert5
   · David Honys3,6
   · Petra Procházková Schrumpfová1,2
 
1
	 Laboratory of Functional Genomics and Proteomics, Faculty
of Science, National Centre for Biomolecular Research,
Masaryk University, Brno, Czech Republic
2
	 Mendel Centre for Plant Genomics and Proteomics, Central
European Institute of Technology, Masaryk University, Brno,
Czech Republic
83Plant Molecular Biology (2023) 112:61–83	
1 3
3
	 Laboratory of Pollen Biology, Institute of Experimental
Botany of the Czech Academy of Sciences, Prague,
Czech Republic
4
	 Institute of Plant Molecular Biology, Biology Centre
of the Czech Academy of Sciences, České Budějovice,
Czech Republic
5
	 Institute of Biology, Freie Universität Berlin, 14195 Berlin,
Germany
6
	 Department of Experimental Plant Biology, Faculty
of Science, Charles University, Prague, Czech Republic
Completing TRB family: newly characterized members show ancient
evolutionary origins and distinct localization, yet similar interactions
Alžbeta Kusová1,2+
, Lenka Steinbachová3+
, Tereza Přerovská1
, Lenka Záveská Drábková3
, Jan Paleček1,2
,
Ahamed Khan4
, Gabriela Rigóová1
, Zuzana Gadiou3
, Claire Jourdain5
, Tino Stricker5
, Daniel Schubert5
,
David Honys3,6
, and Petra Procházková Schrumpfová1,2*
1
Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research,
Faculty of Science, Masaryk University, Brno, Czech Republic
2
Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology,
Masaryk University, Brno, Czech Republic
3
Laboratory of Pollen Biology, Institute of Experimental Botany of the Czech Academy of Sciences,
Prague, Czech Republic
4
Biology Centre of the Czech Academy of Sciences, Institute of Plant Molecular Biology, České
Budějovice, Czech Republic
5
Institute of Biology, Freie Universität, 14195 Berlin, Germany
6
Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague, Czech
Republic
* Author for correspondence: schpetra@sci.muni.cz
+ Co-authors: Shared co-first authorship.
Supplemental Fig. 1 The 15 most conserved motifs in plant TRB protein motifs
Motifs are ranked and ordered by highest probability of occurrence. Motif 1 corresponds to
the MYB-like domain. Motifs 2, 4 and 7 belong to the H1/H5-like domain. Motifs 3 and 6 or 3
and 11 create the coiled-coil domain.
Supplemental Fig. 2 Conserved residues and electrostatic charge visualization of the Myblike
domain in Arabidopsis members of Dicots from TRB_B, TRB_C and TRB_D lineages
The representative members of Dicots from TRB_B, TRB_C and TRB_D lineages, namely A.
thaliana TRB4, TRB1 and TRB2, respectively, were analyzed. The three-dimensional model of
the Myb-like domains from the site opposing the DNA-binding (A-C) and from the DNA binding
(D-F) viewpoints are based on the hTRF2-DNA interaction model (PDB: 1WOU) (Court et al.
2005).
B) and E) The evolutionary dynamics of aa substitutions among aa residues were visualized in
ConSurf 2016 (Ashkenazy et al., 2016). The conservation of residues is presented in a scale,
where the most conserved residues are shown in dark magenta and non-conserved residues
as white.
C) and F) Surface models showing the charge on the Myb-like domains. Residue charges are
coded as red for acidic, blue for basic, and white for neutral, visualized using PyMol, Version
2.4.1, Schrödinger, LLC.
Supplemental Fig. 3 Maximum intensity projections and Z-stacks of TRB1-3 GFP-fusion
proteins in nuclei of N. benthamiana leaf epidermal cells
TRB1-3 fused with GFP (N-terminal fusions), expressed in N. benthamiana leaf epidermal cells
and observed by confocal microscopy. Figures represent Maximum Intensity projections (I) of
entire Z-stack images of nuclei (II; TRB1 - 0,53 µm each; TRB2 and TRB3 – 0,47 µm each). Scale
bars = 5 μm.
Supplemental Fig. 4 Maximum intensity projections and Z-stacks of TRB4-5 GFP-fusion
proteins in N. benthamiana leaf epidermal cells
TRB4-5 fused with GFP (N-terminal fusions), expressed in N. benthamiana epidermal cells, and
observed by confocal microscopy. Figures represent Maximum Intensity projections (I) of
entire Z-stack images (II) of nuclei (TRB4 - 0,52 µm each; TRB5 - 0,82 µm each; scale bars = 5
μm) and whole epidermal cell (TRB5 - 0,78 µm each; scale bar = 20 μm).
Supplemental Fig. 5 Mutual TRB interactions detected by BiFC in N. benthamiana
Protein-protein interactions of TRB proteins fused with nYFP or cYFP part were detected in N.
benthamiana leaf epidermal cells by confocal microscopy. Shown here are single images of
fluorescence signals from individual channels (YFP, Yellow fluorescence protein; RFP, red
fluorescence protein – an internal marker for transformation and expression) and merged
signals (merge, merged YFP and RFP channels). Scale bars = 5 µm.
Supplemental Fig. 6 Maximum Intensity projections and Z-stacks of N. benthamiana
epidermal cells nuclei presenting BiFC analyses
Maximum Intensity Projections (I) of entire Z-stack images (II) of nuclei of N. benthamiana leaf
epidermal cells displaying BiFC interactions of TRB proteins. Shown here are merged images
of YFP (interaction of the tested proteins) and mRFP (internal marker for transformation and
expression) fluorescence detected by confocal microscopy. Scale bars = 5 μm.
A) Interaction of TRB1 and TRB2 proteins (nYFP-TRB1 + cYFP-TRB2 pBiFCt-2in1-NN
construct). Optical sections 0,48 µm each.
B) Interaction of TRB1 and TRB4 proteins (nYFP TRB1 + cYFP TRB4 pBiFCt-2in1-NN
construct). Optical sections 0,55 µm each.
Supplemental Fig. 7 Interactions of TRB4-5 with TERT domains
A) Schematic depiction of the plant catalytic subunit of telomerase (TERT) showing
functional motifs. The regions of structural domains TEN (telomerase essential Nterminal
domain), TRBD (Telomerase RNA-binding domain), RT (reverse transcriptase
domain) and CTE (C-terminal extension) are depicted above the conserved RT motifs
(1, 2, A, B, C, D and E), telomerase-specific motifs (T2, CP, QFP and T) and a NLS (nucleus
localization-like signal). All of the depicted TERT fragments were used for proteinprotein
interaction analysis (amino acid numbering is shown).
B) TERT fragments from Majerská et al. 2017 were fused with the GAL4 DNA-binding
domain (BD). TRB4 and TRB5 were fused with the GAL4 activation domain (AD). Both
constructs were introduced into yeast strain PJ69-4a carrying reporter genes His3 and
Ade2. Interactions were detected on histidine-deficient SD medium (-His), or under
stringent adenine-deficient SD medium (-Ade) selection. Co-transformation with an
empty vector (AD, BD) served as a negative control. Asterisks *, 3 mM 3-aminotriazol.
Supplemental Fig. 8 Protein-protein interactions of TRBs with various proteins
A) TNT expressed proteins, POT1a (35S-labelled*) and TRB4/TRB5 (myc-tag), were mixed
and incubated with an anti-myc antibody and Protein G magnetic particles. In the
control experiment, the POT1a proteins were incubated with an anti-myc antibody and
Protein G magnetic particles in the absence of partner protein. Input (I), unbound (U),
and bound (B) fractions were collected and run in SDS–10% PAGE gels. Asterisks *, 35S
labelling.
B) Interactions of TRB4-5 were evaluated using the Y2H system. Interactions between
TRB4-5 and full-length PWO1-2 were tested as in Fig. 7B. Interactions were detected
on histidine-deficient SD medium (-His), or under stringent adenine-deficient SD
medium (-Ade) selection. Co-transformation with an empty vector (AD, BD) served as
a negative control. Asterisks *, 3 mM 3-aminotriazol.
Supplement S
Teano G., Concia L., Wolff L., Carron L., Biocanin I., Adamusová K., Fojtová M., Bourge M.,
Kramdi A., Colot V., Grossniklaus U., Bowler Ch., Baroux C., Carbone A., Probst A.V.,
Schrumpfová P.P., Fajkus J., Amiard S., Grob S., Bourbousse C., and Barneche F. 2023.
Histone H1 protects telomeric repeats from H3K27me3 invasion in Arabidopsis. Cell Reports,
42(8):112894.
P.P.S. participated in the design of experiments and ms editing
Article
Histone H1 protects telomeric repeats from
H3K27me3 invasion in Arabidopsis
Graphical abstract
Highlights
d H1 promotes PRC2 activity at a majority of genes
d H1 limits PRC2 activity at telomeres and interstitial telomeric
repeats (ITRs)
d H1 orchestrates the spatial organization of telomeres and
ITRs
d PRC2 repression is achieved by restricting accessibility to
TRB proteins
Authors
Gianluca Teano, Lorenzo Concia,
Le´ a Wolff, ..., Stefan Grob,
Clara Bourbousse, Fredy Barneche
Correspondence
barneche@bio.ens.psl.eu
In brief
Teano et al. report that linker histone H1
and a group of H1-related telomeric
proteins interplay to selectively influence
the Polycomb repressive landscape at
telomeric repeats in Arabidopsis. These
findings provide a mechanistic
framework by which H1 affects the
epigenome and nuclear organization in a
sequence-specific manner.
Teano et al., 2023, Cell Reports 42, 112894
August 29, 2023 ª 2023 The Author(s).
https://doi.org/10.1016/j.celrep.2023.112894 ll
Article
Histone H1 protects telomeric repeats
from H3K27me3 invasion in Arabidopsis
Gianluca Teano,1,8,9 Lorenzo Concia,1,9 Le´ a Wolff,1 Le´ opold Carron,2 Ivona Biocanin,1,8 Katerina Adamusova´ ,3,4
Miloslava Fojtova´ ,3,4 Michael Bourge,5 Amira Kramdi,1 Vincent Colot,1 Ueli Grossniklaus,6 Chris Bowler,1 Ce´ lia Baroux,6
Alessandra Carbone,2 Aline V. Probst,7 Petra Procha´ zkova´ Schrumpfova´ ,3,4 Jirı´ Fajkus,3,4 Simon Amiard,7 Stefan Grob,6
Clara Bourbousse,1 and Fredy Barneche1,10,*
1Institut de biologie de l’E´ cole normale supe´ rieure (IBENS), E´ cole normale supe´ rieure, CNRS, INSERM, Universite´ PSL, Paris, France
2Sorbonne Universite´ , CNRS, IBPS, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative (LCQB), 75005 Paris, France
3Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Brno, Czech Republic
4Laboratory of Functional Genomics and Proteomics, NCBR, Faculty of Science, Masaryk University, Brno, Czech Republic
5Cytometry Facility, Imagerie-Gif, Universite´ Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198
Gif-sur-Yvette, France
6Department of Plant and Microbial Biology & Zurich-Basel Plant Science Center, University of Zurich, Zurich, Switzerland
7CNRS UMR6293, Universite´ Clermont Auvergne, INSERM U1103, GReD, CRBC, Clermont-Ferrand, France
8Universite´ Paris-Saclay, 91190 Orsay, France
9These authors contributed equally
10Lead contact
*Correspondence: barneche@bio.ens.psl.eu
https://doi.org/10.1016/j.celrep.2023.112894
SUMMARY
While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional
interplays with chromatin factors along the epigenome are just starting to emerge. Here we show
that, in Arabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes
where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with
its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres
and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to
Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2 cis recruitment.
This study provides a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich
domains at telomeric sequences and contributes to safeguard nucleus architecture.
INTRODUCTION
Both local and higher-order chromatin architecture rely to a large
extent on the regulation of nucleosome density and accessibility,
in which linker histone H1 and Polycomb Repressive complexes
1 and 2 (PRC1/2) play distinct roles. H1 modulates nucleosome
distribution by contacting the nucleosome dyad with its central
globular (GH1) domain and by binding linker DNA at the nucleosome
entry and exit sites with its disordered carboxy-terminal
domain. This indirectly contributes to dampen transcriptional activity
by affecting the accessibility of transcription factors and
RNA polymerases to DNA but also through interactions with histone
and DNA modifiers (reviewed elsewhere1–3
).
Polycomb group activity is another determinant of chromatin
organization that extensively regulates transcriptional activity,
cell identity, and differentiation in metazoans,4,5
plants,6
and unicellular
eukaryotes.7
While H1 incorporation directly influences
the physicochemical properties of the chromatin fiber, PRC1
and PRC2 display enzymatic activities mediating histone H2A
Lysine monoubiquitination (H2Aub) and histone H3 lysine 27 trimethylation
(H3K27me3), respectively.4,5
In metazoans, chromatin
of PRC target genes is highly compacted,8–10
a feature
thought to hinder transcription (reviewed in Schuettengruber
et al. and Illingworth5,11
). PRC2 can favor chromatin compaction
either by promoting PRC1 recruitment or through its subunit
Enhancer of Zeste Homolog 1 (Ezh1) in a mechanism not necessarily
relying on the H3K27me3 mark itself.12
Mutual interplay between H1 and PRC2 activity first emerged
in vitro. Human H1.2 preferentially binds to H3K27me3-containing
nucleosomes13
while, vice versa, human and mouse PRC2
complexes display substrate preferences for H1-enriched chromatin
fragments. The latter activity is stimulated more on di-nucleosomes
than on mono- or dispersed nucleosomes.14,15
In vivo, recent studies unveiled that H1 is a critical regulator of
H3K27me3 enrichment over hundreds of PRC2 target genes in
mouse cells.16,17
Chromosome conformation capture (Hi-C)
analysis of hematopoietic cells,16
germinal center B cells,17
and embryonic stem cells18
showed that H1 triggers distinct
genome folding during differentiation in mammals. These major
advances raise the question of the mechanisms enabling H1
sequence-specific interplays with PRC2 activity in chromatin
regulation and their evolution in distinct eukaryotes.
Cell Reports 42, 112894, August 29, 2023 ª 2023 The Author(s). 1
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
ll
OPEN ACCESS
In Arabidopsis thaliana, two canonical linker histone variants,
H1.1 and H1.2, represent the full H1 complement in most somatic
cells.19–21
These two linker histones, hereafter referred to
as H1, are enriched over heterochromatic transposable elements
(TEs) displaying high nucleosome occupancy; CG,
CHG, and CHH methylation; as well as H3K9 dimethylation.22,23
H1 also contributes to CG methylation-mediated gene
silencing24
and is less abundant over expressed genes.22,23
As
in mammals, Arabidopsis H1 incorporation is thought to dampen
RNA Pol II transcription, an effect that also applies in plants to
RNA polymerase Pol IV, which produces short interfering RNAs
(siRNAs).25
Arabidopsis H1 also restricts accessibility to DNA
methyltransferases and demethylases, which mediates gene or
TE silencing.26–30
This process is counter-balanced by incorporation
of the H2A.W histone variant, presumably competing with
H1 for DNA binding through its extended C-terminal tail.31
Besides TE silencing, recent studies suggested that H1 dynamics
may affect PRC2 activity during Arabidopsis development.
The first piece of evidence is that H1 is largely absent
from the vegetative cell nucleus of pollen grain and is degraded
during spore mother cell (SMC) differentiation at the onset of
heterochromatin loosening and H3K27me3 reduction.26,32–34
Further evidence comes from the observation that H1 loss-offunction
mutant nuclei display a $2-fold lower H3K27me3 chromatin
abundance, while a few discrete H3K27me3 sub-nuclear
foci of undetermined nature displayed increased H3K27me3 sig-
nals.35
Hence, despite evidence that variations in H1 abundance
mediate epigenome reprogramming during plant development,
there is no information on how H1 interplays with PRC2 activity
and on the consequences of this interaction on the chromatin
landscape and topology in these organisms.
Here, we profiled H3K27me3 in h1 mutant plants and found
that, while a majority of genes expectedly lost H3K27me3, telomeres
and pericentromeric interstitial telomeric repeat (ITR) regions
or interstitial telomeric sequences (ITSs) were massively
enriched in this mark. We identified that H1 prevents PRC2 activity
at these loci by hindering the binding of Telomere Repeat
Binding (TRB) proteins, a group of H1-related proteins with extra-telomeric
function in PRC2 recruitment.36,37
H1 safeguards
telomeres and ITRs against excessive H3K27me3 deposition
and preserves their topological organization. Collectively, our
findings led us to propose a mechanism by which H1 orchestrates
Arabidopsis chromosomal organization and contributes
to the control of H3K27me3 homeostasis between structurally
distinct genome domains.
RESULTS
H1 is abundant at H3K27me3-marked genes
and reduces their chromatin accessibility
To assess the relationships between H1, PRC2 activity, and
chromatin accessibility, we first compared the genomic distribution
of H3K27me3 with that of H1.2, the most abundant canonical
H1 variant in Arabidopsis seedlings.22
To maximize specificity,
we used a GFP-tagged version of H1.2 expressed under
the control of its endogenous promoter.22
In agreement with previous
studies in several eukaryotes,22,23,38,39
H1.2 covers most
of the Arabidopsis genome without displaying clear peaks. However,
a closer examination revealed that, as compared to genes
and to TEs that are not enriched in H3K27me3,40–42
H1 level was
higher at coding genes marked by H3K27me3, especially toward
their 50
regions (Figures 1A and S1A–S1C).
Having found that H1 is enriched at PRC2 marked genes, we
tested whether H1 also contributes to regulate chromatin accessibility
using assay for transposase-accessible chromatin followed
by sequencing (ATAC-seq) in nuclei of wild-type (WT)
and h1.1h1.2 double-mutant plants (hereby named 2h1 for short).
As previously reported in WT plants,44
H3K27me3-marked genes
tend to display low chromatin accessibility as compared to nonmarked
genes, which are usually expressed and typically display
a sharp ATAC peak at their transcription start sites (TSSs) corresponding
to the nucleosome-free region (Figure 1B). In 2h1
nuclei, gene body regions of H3K27me3-marked loci displayed
a significant increase in accessibility (Figures 1B and S1D).
A
B
Figure 1. H1.2-GFP is enriched at PRC2-target genes where it contributes
to restrain DNA accessibility
(A) H1.2-GFP mean read coverage at protein-coding genes and TEs.
(B) ATAC-seq analysis of chromatin accessibility of genes and TEs described
in (A) in WT (plain lines) and 2h1 (dashed lines) nuclei. Chromatin accessibility is
estimated as read coverage. TSS, transcription start site; TES, transcription
end site. In (A) and (B), H3K27me3-marked genes (n = 7,542) are compared to
all other annotated protein-coding genes. Heterochromatic versus euchromatic
TEs were defined previously.43
The plots represent the mean of three
(H1.2-GFP) or two (ATAC-seq) independent biological replicates.
2 Cell Reports 42, 112894, August 29, 2023
Article
ll
OPEN ACCESS
Hence, H1 tends to abundantly occupy PRC2 target gene bodies
where it has a minor but detectable contribution in restricting
chromatin accessibility.
H1 promotes H3K27me3 enrichment at a majority of
PRC2 target genes while protecting a few genes
displaying specific sequence signatures
To determine at which loci H1 influences PRC2 activity, we profiled
the H3K27me3 landscape in WT and 2h1 seedlings. To
enable absolute quantifications despite the general reduction
of H3K27me3 in the mutant nuclei, we employed chromatin
immunoprecipitation sequencing (ChIP-seq) with reference
exogenous genome (ChIP-Rx) by spiking in equal amounts of
Drosophila chromatin to each sample.45
Among the $7,500
genes significantly marked by H3K27me3 in WT plants, more
than 4,300 were hypomethylated in 2h1 plants (Figures 2A–2C
and S2A–S2D; Table S1). Hence, general loss of H3K27me3 in
2h1 seedlings identified by immunoblotting and cytology35
results
from a general effect at a majority of PRC2-regulated
genes. It is noteworthy that $85% of the genes marked by
H3K27me3 in WT plants were still significantly marked in 2h1
plants (Figure S2D). Hence, H1 is required for efficient
H3K27me3 maintenance or spreading rather than for PRC2
seeding. Our RNA sequencing (RNA-seq) analysis showed that
genes encoding PRC1/PRC2 subunits are not downregulated
in 2h1 plants, excluding indirect effects resulting from less-abundant
PRC2 (Table S2). Unexpectedly, we also found that $500
genes were hyper-marked or displayed de novo marking in
2h1 plants (Figures 2A–2C and S2A–S2D; Table S1).
To determine whether the hypo/hyper/unaffected gene sets
had different functional properties, we inspected their transcript
levels. Hyper-marked genes correspond to the least expressed
gene category in WT plants, whereas many hypo-marked
genes are significantly more expressed than unaffected genes
(Figure 2D). Functional categorization of hypo-marked genes
notably identified an over-representation of genes involved in
transcriptional regulation and meristem maintenance (Figure
S2H). These classifications are consistent with former reports
of PRC2 repressing these biological processes.46
In
contrast, a feature of the hyper-marked gene set is the presence
of TE or TE-gene annotations (Figure S2I; Table S1). Hence, H1mediated
PRC2 activation14–16
is most likely conserved in plants,
but, in Arabidopsis, this property is contrasted by a heretoforeunsuspected
negative effect at a minority of poorly expressed
genes sometimes displaying TE features.
In vitro, PRC2 activity was proposed to be favored by local H1
abundance and/or at densely organized nucleosome arrays.14
Instead, we found that hypo-marked genes tend to display lower
H1 level, to be more accessible and expressed than other genes
marked by H3K27me3 (Figures 2D–2F and S2E). We therefore
tested nucleosome density using ChIP-seq profiling of histone
H3, confirming that hypo-marked genes display lower nucleosome
occupancy than other marked gene categories (Figure
S2G). Collectively, analysis of the hypo-marked loci suggests
that chromatin of the corresponding genes is not sufficiently
nucleosome dense to favor PRC2 local activity when H1 is absent.
We further explored whether the specific influence of H1 on
H3K27me3 enrichment at genes could rely on a sequencedependent
mechanism, especially at hyper-marked genes,
since they do not incur H1-mediated PRC2 activation. In contrast
to the promoter sequences of the hypo-marked genes in which
no such motif is significantly over-represented, we identified
three enriched motifs in the hyper-marked gene set (Figure S2J).
A poly(A) motif is present in 84% of the gene promoters, and the
AAACCCTA telomeric motif, referred to as telobox,47,48
which
serves as Polycomb Response Elements (PREs) in plants,36,37
is found in 17% of them (Table S1). Based on these observations,
we conclude that the capacity of H1 to counteract H3K27me3
enrichment at a small gene set presumably involves specific
sequence features.
H1 contributes to define accessibility and expression
of PRC2 target genes
To get insights into the functional consequences of H1 loss at
genes where it either promotes or dampens H3K27me3 enrichment,
we compared the chromatin accessibility and transcript
levels of these gene sets in WT and 2h1 nuclei. ATAC-seq
profiling showed that hypo-marked gene bodies were significantly
more accessible in the mutant than in the WT line
(Figures 2F and S2F), thereby correlating with reduced
H3K27me3 levels. Accessibility of H3K27me3 hyper-marked
genes was increased in 2h1 plants, still remaining at a very low
level as compared to non-marked genes (Figures 2F and S2F).
Conservation of this function in both hypo- and hyperH3K27me3
gene categories indicates that H1 incorporation reduces chromatin
accessibility of Arabidopsis PRC2-target genes independently
of its influence on H3K27me3 enrichment.
Confirming previous reports,23,35
our RNA-seq analysis
showed that H1 loss of function triggers minor gene expression
changes (Table S2). However, we identified a significant tendency
for increased transcript levels of the H3K27me3 hypoand
hyper-marked genes set in the 2h1 line (Figure 2G). Taken
together, these analyses showed that, at a majority of PRC2
target genes, H1 depletion triggers H3K27me3 loss associated
with a moderate increase in DNA accessibility and expression.
H1 prevents H3K27me3 invasion over a specific family
of heterochromatic repeats
Considering the observed H3K27me3 enrichment at a few TErelated
genes in 2h1 plants, we extended our analysis to TEs,
which typically lack H3K27me3 in Arabidopsis.49,50
This revealed
that 1,066 TEs are newly marked by H3K27me3 in 2h1 plants,
most frequently over their entire length, thereby excluding a priori
the possibility that H3K27me3 TE enrichment is due to spreading
from neighboring genes (Figure 3A). We clustered H3K27me3marked
TEs into two groups, TE cluster 1 and TE cluster 2, displaying
high and low H3K27me3 enrichment, respectively (Figure
3A). While TE cluster 2 (n = 850) is composed of a large
variety of TE families, TE cluster 1 (n = 216) mostly consists of
ATREP18 (189 elements) annotated in the TAIR10 genome as
unassigned (Figure 3B). In total, TE cluster 1 and 2 comprise
60% of all annotated Arabidopsis ATREP18 elements, including
many of the longest units (Figure S3A). A second distinguishing
feature of TE cluster 1 elements is their elevated H1 and H3 occupancy
(Figures 3C and S3B–S3E). Accordingly, TE cluster 1 and,
more generally, ATREP18 elements are strongly heterochromatic
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A D
B E
F
G
C
Figure 2. H1 influences H3K27me3 marking, chromatin accessibility, and expression of PRC2-target genes
(A) Identification of differentially marked genes using spike-in normalized DESeq2 ChIP-seq analysis identifies low H3K27me3 levels over a majority of the PRC2
target genes in 2h1 plants. All genes displaying an H3K27me3-enriched domain in WT or 2h1 plants (according to MACS2 peak detection; see STAR Methods) are
individually shown as dots. Red dots, differentially marked genes (false discovery rate [FDR] <0.01).
(B) H3K27me3 profiles along all genes significantly marked in WT or 2h1 plants. Genes are grouped according to differential analysis in (A) and ranked within each
group according to mean H3K27me3 levels.
(C) H3K27me3 profile of representative genes of the three sets identified in (A) exemplifying the general tendency of PRC2-target genes to keep a weak
H3K27me3 domain in 2h1 plants.
(D) Transcript levels in WT seedlings. The values represent RNA-seq log10 transcripts per million (TPM) values. The embedded boxplots display the median, while
lower and upper hinges correspond to the first and third quartiles. *p < 10À9
and **p < 10À15
, Wilcoxon rank test.
(E) H1.2-GFP ChIP-seq profiling on the indicated gene sets (mean read coverage).
(F) ATAC-seq analysis of the indicated gene sets. ATAC-seq data are presented as in Figure 1B using mean read coverage.
(G) Transcript level variations between WT and 2h1 plants in the same three gene sets. The values represent mRNA log2 fold changes. The embedded boxplots
display the median, while lower and upper hinges correspond to the first and third quartiles. *p < 5% and **p < 1%, Student’s t test. ChIP-Rx, ATAC-seq, and RNAseq
data correspond to two biological replicates each, and H1.2-GFP ChIP-seq correspond to three biological replicates.
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with elevated nucleosome occupancy, H3K9me2, cytosine
methylation, and very low chromatin accessibility (Figures 3D
and S3C–S3H and S3K). Taken together, these observations
indicate that H1 prevents H3K27me3 accumulation over a set of
H1-rich, heterochromatic, and highly compacted repeats, which
contrasts with its positive influence on H3K27me3 marking over
thousands of PRC2-target genes.
Noteworthy, while MNase-seq analyses22,23
and our ATAC-seq
data showed that heterochromatic TEs tend to be more accessible
in 2h1 nuclei, the chromatin of TE cluster 1 and ATREP18 repeats
remained very poorly accessible despite H1 loss
(Figures 3D, S3C, and S3D). Hence, chromatin "inaccessibility"
of TE cluster 1 elements is either H1 independent or compensated
by other mechanisms, possibly a local increase in PRC2 activity.
Repeats gaining H3K27me3 in 2h1 plants are parts
of two large pericentromeric telomeric regions
Aiming at determining the features potentially leading to a selective
role of H1 at TE cluster 1 elements, we first envisaged that
H1could locally prevent conversion of the H3K27me1 heterochromatic
mark into H3K27me3. However, analysis of public data-
sets51
showed that, as compared to other TEs, H3K27me1 is not
particularly abundant at TE cluster 1 or at ATREP18 elements,
therefore ruling out this first hypothesis (Figure S3F). We then
explored the possibility that H1could favor H3K27me3 de-methylation.
Examination of the H3K27me3 profile in loss-of-function
plants for the three major histone H3K27me3 demethylases
EARLY FLOWERING 6 (ELF6), RELATIVE OF ELF 6 (REF6), and
JUMONJI 13 (JMJ13)52
showed no H3K27me3 increase at TE
cluster 1 elements (Figure S3I) or at hyper-marked genes (Figure
S3J). This led us to rule out the hypothesis that, in WT
plants, H3K27me3 could be regulated at these loci though
active erasure. Last, considering the tendency for cytosine
methylation to be mutually exclusive with H3K27me3 deposition
in Arabidopsis,53–55
we envisioned that H3K27me3 enrichment
at TE cluster 1 may indirectly result from decreased DNA
methylation induced by H1 loss. Examination of cytosine methylation
patterns of TE cluster 1 elements in 2h1 plants oppositely
showed an increase in CG, CHG, and CHH methylation (Figure
S3K). We did not ascertain whether methylated cytosines
and H3K27me3-containing nucleosomes co-occur at individual
TE cluster 1 chromatin fragments, yet this observation ruled out
that H1 indirectly hinders PRC2 activity at these loci by promoting
cytosine methylation, a possibility that would have been supported
if an opposite effect was observed.
Having not found evidence for indirect roles of H1 on
H3K27me3 marking at TE cluster 1, we concluded that H1 hinders
PRC2 recruitment or activity at these repeats, and this
A B C
D
F
E
G
Figure 3. H1 hinders H3K27me3 enrichment at two pericentromeric ITR blocks spanning more than 420 kb
(A) Hyper-marked TEs were clustered into two groups according to H3K27me3 levels after spike-in normalization defining two TE clusters of 216 and 850 TEs,
respectively. H3K27me3 profiles over all TE cluster 1 and 2 elements are ranked in each group according to H3K27me3 mean spike-in normalized coverage.
(B) Relative TE superfamily composition of H3K27me3-enriched TEs. TE cluster 1 comprises a strong over-representation of "unassigned" annotations mainly
corresponding to ATREP18 elements, while TE cluster 2 elements correspond to a wide variety of TE super-families.
(C) TE cluster 1 elements display high H1 occupancy. The plot represents H1.2-GFP mean read coverage over the indicated repertoire of TEs and repeats.
(D) Chromatin accessibility of TE cluster 1 elements remains very low in 2h1 nuclei. ATAC-seq data are presented as in Figure 1B using mean read coverage.
(E) Motif enrichment search identified an over-representation of telobox motifs in TE cluster 1 sequences. E values were calculated against all TE sequences.
(F) ATREP18 repeats display outstanding density and a distinct pattern of telobox motifs as compared to the whole set of annotated TEs. The plot represents the
density of perfect telobox sequence motifs in all ATREP18s as compared to all TEs within 50-bp bins.
(G) Chromosome distribution of H3K27me3 defects in 2h1 plants and their link to ATREP18, TE cluster 1, and TE cluster 2 elements. The sharp peaks of telobox
density in the pericentromeres of chromosome 1 and 4 correspond to ITR-1R and ITR-4L. Chromosome 1 pericentromeric region displays a sharp overlap
between 2h1-specific H3K27me3 enrichment and the telobox-rich ITR-1R. Bottom panel, shaded boxes correspond to blacklisted TAIR10 genome sequences
(see STAR Methods). Complementary profiles over ITR-4L and other interspersed elements from TE cluster 2 are shown in Figures S3L and S3M.
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despite a densely packed chromatin organization that theoretically
constitutes an excellent substrate. As previously done for
hyper-marked genes, we therefore tested whether TE cluster 1
elements are distinguishable from other TEs by specific DNA
motifs. MEME search identified a prominent sequence signature,
the telobox motif (Figure 3E), which we had already identified in
17% of the hyper-marked genes (Figure S2J). As compared to all
other TEs, teloboxes were found to be $100-fold more densely
represented in ATREP18 elements as compared to all TEs
(Figures 3E and 3F). With 7,328 telobox motifs, TE cluster 1 contains
$53% of the whole TAIR10 telobox repertoire. Hence, if not
considering proper telomeres that span 2 to 5 kb at the end of
each chromosome,56,57
TE cluster 1 repeats display the majority
of telomeric motifs of Arabidopsis genome and the strongest
propensity to attract PRC2 activity upon H1 loss.
Remarkably, these two properties can be seen at a chromosomescalebycontrasting
the genomedistributionofteloboxdensity
and of H3K27me3 differential marking, since about 95% of TE
cluster 1 elements cluster within two outstandingly telobox-rich
regions situated in the pericentromeres of chromosomes 1 and
4 (Figures 3G and S3L). Given this characteristic, we consider
these domains as two of the nine Arabidopsis genome loci proposed
to constitute ITRs,58,59
hereby referred to as ITR-1R and
ITR-4L of $355 kb and $72 kb, respectively. In agreement with
the description of ITRs in plants and vertebrates,60,61
ATREP18
elements that constitute most of these domains display a high
density in telobox motifs frequently organized as small clusters
(Figures 3G, S3L, S3N, and S3O). Further supporting their telomeric
evolutionary origin, ATREP18s encode no open reading
frame or other TE features, are mostly oriented on the same
DNA strand, and are tandemly organized (nearly 90% of them being
positioned within 1 kb of each other; Figures S3P–S3R), hence
they do not constitute stricto sensu TEs. Ectopic H3K27me3
deposition was also found at several interspersed elements of
TE cluster 2 located in all pericentromeric regions outside these
two ITR blocks (Figure S3M), but our main conclusion is that H1
abundantly occupies two large blocks of pericentromeric ITRs
where it prevents H3K27me3 marking.
H1 influences telomere chromatin composition and subnuclear
positioning
Considering that telomeres display hundreds of perfect telobox
motifs, the question arose whether, similarly to ITRs, H1 also prevents
H3K27me3 deposition at chromosome ends. Because the
perfect continuum of terminal telomeric motifs is not suited for
quantitative NGS analyses, ChIPs were analyzed through hybridization
with radioactively labeled concatenated telomeric
probes.62
H3K27me3 ChIP dot blots led to the estimation that
telomeres display an average $4-fold more H3K27me3 enrichment
in 2h1 as compared to WT plants, independently of detectable
changes in nucleosome occupancy probed by anti-H3 ChIP
dot blot (Figures 4A and S4A).
To assess whether H3K27me3 enrichment concerns a few
telomeres or affects them all, we explored its occurrence in intact
nuclei using H3K27me3 immunolabeling combined with telomere
fluorescence in situ hybridization (DNA FISH). Consistent
with our ChIP-blot analysis, most telomeric foci were enriched
with H3K27me3 in 2h1 nuclei, with 2- to 4-telomere foci
frequently presenting outstandingly strong H3K27me3 signals
(Figures 4B and 4C). We could not ascertain whether some of
these strong signals corresponded to cross-hybridizing pericentromeric
ITRs, but their frequent positioning near to the nuclear
periphery may point to the latter hypothesis. Indeed, in 2h1
nuclei, telomeric foci were frequently re-distributed toward the
nucleus periphery, thereby contrasting with the telomere rosette
model proposed by Fransz et al.,63
first establishing that telomeres
cluster around the nucleolus (Figure 4D). In addition, the
number of telomere foci was reduced in the mutant nuclei
(Figures 4B and 4C), indicating that H1 not only prevents accumulation
of H3K27me3 at ITRs and at most telomeres but is
also required for the sub-nuclear organization and proper individualization
of these domains.
H1 promotes heterochromatin packing but attenuates
ITR insulation and telomere-telomere contact
frequency
To better understand the altered telomere cytogenetic patterns
of 2h1 nuclei and to extend our analysis to ITR topology,
we employed in situ Hi-C of dissected cotyledons, composed
of 80% mesophyll cells, which enabled us to reach high
resolution. In agreement with previous reports,64–69
WT plants
displayed frequent interactions within and between pericentromeric
regions, which reflect packing of these domains within
so-called chromocenters (Figures 5A, 5B, and S5E). Loosening
of these heterochromatic structures in 2h1 mutant nuclei,
formerly observed by microscopy,23,35
was expectedly identified
here as a more steep decay with distance70
and lower longrange
interaction frequency within pericentromeric regions
(Figures 5A, 5B, and S5H–S5J). However, this tendency appears
to be a general trend in the mutant nuclei since it was also
observed for chromosome arms. As also seen in crwn and condensin
mutants,71
in a matrix of differential interaction frequency
between WT and 2h1 nuclei these prominent defects are also
visible as blue squares surrounding the centromeres, which are
mirrored by increased interaction frequency between pericentromeric
regions and their respective chromosome arms (i.e.,
red crosses along chromosome arms) (Figure 5C).
Having identified large-scale defects of chromosome organization
in 2h1 mutant nuclei, we then focused on telomere-telomere
interaction frequency. Because telomeres are not included
in the TAIR10 reference genome, we used the most sub-telomeric
100-kb sequences of each chromosome end as a proxy
to estimate telomere long-distance interactions, and these
were controlled using an internal 100-kb region of each pericentromeric
region as well as 100-kb regions randomly chosen in
distal chromosomal arms. As previously noted, in WT plants,
the telomere-proximal regions frequently interacted with each
other through long-range interactions.64–68
We further observed
that ITR-1R and ITR-4L do not particularly associate with
each other or with telomeres (Figure S5H). In 2h1 nuclei,
with the exception of the regions adjacent to the nucleolar organizer
regions (NORs) of chromosome 2 and 4 (SubNOR2
and SubNOR4), which displayed atypical patterns (detailed in
Figures S5H–S5J), interaction frequencies between all sub-telomeric
regions were increased (Figure 5D). Furthermore, ITR-1R
and 4L also showed increased ITR-ITR and ITR-telomere
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A
B
C D
Figure 4. H1 influences both H3K27me3 enrichment and sub-nuclear organization of telomeres
(A) Increased H3K27me3 level at telomeres in 2h1 plants. H3 ChIP signal is used as a proxy of nucleosome occupancy. ChIPs were followed by dot-blot hybridization
with a labeled telomeric probe. Data are the mean of two biologically and technically replicated experiments ±SE. A second biological replicate is
shown in Figure S5.
(B) Most telomeric loci are enriched in H3K27me3 and re-distributed toward the nucleus periphery in 2h1 plants. Representative collapsed z stack projections of
cotyledon nuclei subjected to H3K27me3 immunolabeling and telomere DNA FISH are shown. Blue, DAPI DNA counterstaining; green, telomere FISH signals;
red, H3K27me3 immunolabeling.
(C) Quantification of sub-nuclear telomeric signal properties. *p < 1.6eÀ07, Wilcoxon signed-rank test.
(D) Quantification of nucleus classes displaying different patterns in telomere sub-nuclear localization. Number and position of telomeric foci were determined in
two independent biological replicates (n > 20 each).
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A E
B
C
D
Figure 5. H3K27me3 accumulation at ITRs and at telomeres associates with ITR insulation and more frequent telomere-telomere
interactions
(A) Mean contact count as a function of genomic distance for all chromosome arms at a 100-kb resolution.
(B) Distribution of interaction decay exponents (IDEs) determined at a 100-kb resolution for chromosome arms and pericentromeric regions of WT and 2h1 nuclei.
Median IDE values of chromosome arms and pericentromeres were determined as À0.95/À1.16 in WT and À1.05/À1.2 in 2h1 nuclei, respectively. *p = 0.076 and
**p = 0.001, pairwise Wilcoxon rank-sum test.
(C) Relative difference of interaction frequency between WT and 2h1 plants. The log2 values of observed/expected (O/E) interaction frequency along the five
chromosomes in 2h1 versus WT are shown at a 100-kb resolution. Regions in red have more frequent contacts in 2h1 than in WT plants, while regions in blue have
less. Pericentromeric regions are depicted in dark gray on the schematic chromosomes.
(D) H1 reduces the frequency of long-distance interactions between chromosome ends. Circos plots depict variations in inter-chromosomal interaction frequencies
between telomere-proximal, pericentromeric, ITR-1R, and ITR-4L 100-kb domains. Yellow boxes, ITR regions. External green/red track, H3K27me3
variations in 2h1 versus WT plants (log2 ratio). Magenta boxes, telomere-proximal regions and SubNOR2 or SubNOR4.
(E) Reduced frequency of intra-pericentromeric O/E interactions in 2h1 mutant nuclei is contrasted by TAD re-enforcement of the H3K27me3-enriched ITR-1R
355 kb block. Top panel, location of ITR-1R in chromosome 1. Middle panel, magnification of the region surrounding chromosome 1 pericentromeres at a 10-kb
resolution. Bottom panel, magnification of the pericentromere-embedded ITR-1R at a 2-kb resolution. Strong and modest increase correspond to log2FC > 1 and
log2FC 0.35–1, respectively; modest and strong decrease correspond to log2FC À0.33 to À0.65 and log2FC < À0.65, respectively. Quantitative analyses are
shown in the complementary Figures S4H–S4J. All Hi-C analyses combine three independent biological replicates.
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interaction frequency (Figures 5D, S5G, and S5J). Consistent
with a reduced number of telomere foci in intact 2h1 nuclei
(Figure 4), this observation supports an organizational model in
which telomeres tend to coalesce more frequently in the
absence of H1.
Last, we examined chromosome topology at ITR loci. In WT
plants, both of them formed large structures resembling topologically
associating domains (TADs), which are themselves
immersed within highly self-interacting pericentromeric regions.
Interestingly, in 2h1 nuclei, intra-ITR interactions were strongly
enhanced (i.e., TAD re-enforcement), while the surrounding pericentromeric
environments expectedly showed an opposite trend
linked to heterochromatin relaxation (Figures 5E and S5G). This
observation was supported by comparing distal-to-local ratios
(DLRs) of interaction frequency that showed clear local drops
at each ITR in 2h1 nuclei, hence an increased tendency for
interacting only with itself usually interpreted as increased
domain compaction (Figure S5K).72
Altogether, these observations
show that, in contrast to its general role in heterochromatin
packing, H1 dampens the local insulation of ITRs from their
neighboring environment. Remarkably, the boundaries of these
compaction defects in 2h1 nuclei sharply correspond with
H3K27me3 enrichment (Figures 5E and S5K).
H1 antagonizes TRB-mediated PRC2 activity at ITRs
With the aim to determine the molecular mechanisms by which
H1 selectively represses PRC2 activity at telobox-rich elements,
and more particularly at ITRs, we envisioned that TRB proteins
might have a prominent role (Figure 6A). The TRB1, TRB2, and
TRB3 founding members of these plant-specific single-Myb-histone
proteins constitute part of the telomere nucleoprotein structure
required to maintain telomere length73
(Figure 6B). Their
Myb domain has strong affinity to the G-rich strand of telobox
DNA motifs73–75
and, combined with a coiled-coil domain that
can associate with the CURLY-LEAF (CLF) and SWINGER
(SWN) catalytic subunits of PRC2,36,37
TRBs act as transcriptional
regulators of protein-coding genes bearing a telobox
motif.76,77
Interestingly, despite their low protein sequence similarity
to H1 (14% ± 2%; Figures S6A and S6B), TRBs display a
typical GH1 domain.19,78
Hence, we hypothesized that antagonistic
chromatin incorporation of the GH1 domains of TRB and
H1 proteins might modulate PRC2 recruitment at ITRs.
To test this model, we first compared H1 and TRB1 genomic
distribution. Analysis of available TRB1 ChIP-seq data76
showed
that TRB1 peak summits expectedly correlate with the position of
telobox motifs located in protein-coding genes. However, despite
the presence of numerous telobox sequences, TRB1 poorly occupies
TE cluster 1 elements (Figures 6C and S6C). Reciprocally,
H1 average occupancy is low at TRB1 peaks over the genome
(Figures 6D and S6D). These observations hint at an antagonistic
cis enrichment of H1 and TRB1 at chromatin. To better resolve
these general patterns and link them to linker DNA positioning,
we examined the profiles of H1, TRB1, telobox motifs, and nucleosome
occupancy around well-positioned nucleosome (WPN)
coordinates defined using MNase-seq.28
As expected, H1.2GFP
distribution was enriched at DNA linker regions. Surprisingly,
this was also the case for telobox motif distribution, which sharply
coincided with regions serving as linker DNA. While TRB1 peaks
appeared much broader, their summits were also more pronounced
at regions corresponding to linker DNA coordinates (Figure
6E). Hence, if it exists, competitive binding between H1 and
TRB proteins likely occurs at linker DNA.
These observations are all compatible with a mechanism by
which high H1 occupancy at ITRs prevents TRB1 DNA binding.
Vice versa, increased access to ITRs in 2h1 mutant plants would
facilitate TRB1-mediated PRC2 recruitment. To functionally
assess whether this model holds true, we first examined whether
GFP-TRB1 accumulates at ITRs in 2h1 plants and then determined
the H3K27me3 profile in mutant plants lacking both H1
and TRB1, TRB2, and TRB3. To undertake the first experiment,
we crossed a TRB1::GFP-TRB1 line76,77
with 2h1 and revealed
GFP-TRB1 genome association by ChIP-seq and ChIP telomere
dot blot. Comparison of GFP-TRB1 chromatin association in WT
and 2h1 plants showed a significantly increased association at
TE cluster 1, ITR-1R and ITR-4L, and at telomeres (Figures 7A,
S4B, S7A, and S7B), thereby providing evidence that H1 restricts
TRB1 binding to these loci in vivo.
We then determined whether abolishing simultaneously
the expression of linker H1 and TRB1, TRB2, and TRB3 proteins
affects PRC2 activity at ITRs. To probe H3K27me3 profiles
in h1.1h1.2trb1trb2trb3 quintuple-mutant plants (hereinafter
referred to as htrbQ for short), we crossed 2h1 double-mutant
plants to trb1(+/À)trb2trb3 triple mutant plants propagated as
a heterozygous state to accommodate the seedling lethality
induced by TRB123 combined loss of function.36,37
Homozygous
htrbQ mutant seedlings exhibited an aggravated phenotype
as compared to the trb123 triple-mutant line (Figure 7B), a
synergistic effect presumably reflecting a convergence of H1
and TRB123 functions in the regulation of common genes.79
Despite the dwarf morphology of the quintuple-mutant line, we
conducted a ChIP-Rx profiling of H3K27me3 in homozygous
WT, 2h1, trb123, and htrbQ seedlings, all segregating from a single
crossed individual. As compared to the 2h1 co-segregant
siblings, in the quintuple-mutant seedlings, H3K27me3 enrichment
was almost completely abolished at ITR-1R and more
generally at TE cluster 1 elements (Figures 7C, 7D, S7B, and
S7C). Taken together, these analyses demonstrate that H1 occupancy
at ITRs antagonizes TRB protein recruitment, thereby
constituting a mechanism preventing invasion of these large
chromosome blocks by H3K27me3.
DISCUSSION
H1 has a dual impact on H3K27me3 deposition in
Arabidopsis
We report that Arabidopsis H1 is highly enriched at PRC2 target
genes, where it typically promotes H3K27me3 enrichment and
diminishes chromatin accessibility. Contrasting with this general
tendency, we also identified an opposite role of H1 in limiting
H3K27me3 deposition at interstitial and terminal telomeres as
well as at a few genes. This unveiled that, in plants, H1 has a differential
effect on H3K27me3 levels over thousands of proteincoding
genes on the one hand and over loci characterized by
repeated telomeric motifs on the other hand.
Considering that PRC2 activation is favored at chromatin
made of closely neighboring nucleosomes,14
we postulate that
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A B
C
D
E
Figure 6. Antagonistic chromatin association of H1 and TRB1 over the genome
(A) Working model of H1/TRB1 antagonistic chromatin association at linker DNA-localized telobox motifs and its sequence-specific influence on PRC2
recruitment at distinct chromatin regions displaying telomeric repeats.
(B) TRB family members possess an amino-terminal single-Myb domain with sequence specificity for telobox motifs, a coiled-coil domain enabling their association
with PRC2 subunits,36,37
and a central GH1 domain that may trigger competitive binding with H1.
(C) H1 and TRB1 patterns are both influenced by telobox positioning, and they display an opposite trend at TE cluster 1 telobox motifs. The plots display TRB1 and
H1 mean read coverage at all TAIR10 genome telobox motifs.
(D) H1 occupancy is reduced at genome loci corresponding to TRB1 peak summits.
(E) H1, TRB1, and telobox motifs all tend to associate with DNA linker regions. Genome-wide profiles of H1, TRB1, and telobox sequence motifs were plotted over
the coordinates of all Arabidopsis WPNs defined by Lyons and Zilberman.28
In (C) and (D), shuffled controls were produced with random permutations of genomic
position of the regions of interest.
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the repertoire of genes losing H3K27me3 upon H1 depletion are
those where H1 is required to attain a compaction level enabling
efficient PRC2 cis activity. Supporting this hypothesis, genes
sensitive to H1 for efficient H3K27me3 marking tend to (1)
display lower H1 and nucleosome occupancy, (2) be more
accessible, and (3) be more expressed than genes unaffected
by H1 depletion. In contrast, genes and TEs gaining
H3K27me3 upon H1 loss tend to have an elevated nucleosome
density and to be weakly accessible while exhibiting sequence
signatures potentially triggering different mechanisms of PRC2
regulation, such as H1/TRB protein interplay. The large scale
on which these antagonistic patterns are observed sheds light
on the existence of prominent functional links between H1 and
PRC2-based regulation, two main factors in the instruction of
DNA accessibility.
Promoting H3K27me3 enrichment at genes: An
evolutionarily conserved function of H1
We identified that H1 has a general role in H3K27me3 deposition
at genes, yet most of the H3K27me3 peaks are still detectable in
2h1 plants. Hence, in agreement with the subtle phenotypes of
h1 mutant plants, H1 is likely not mandatory for the nucleation
of PRC2 activity but rather for H3K27me3 maintenance or
spreading in Arabidopsis. In term of chromatin function, H1
depletion results in a global increase in chromatin accessibility
at gene bodies but its impact on expression was apparently
more related to variations in H3K27me3 marking. Hence, consistent
with the functional categories of the misregulated genes in
2h1 plants, part of the defects in gene expression resulting
from H1 depletion might result from indirect consequences on
PRC2 activity. The recent findings that depletion of H1 variants
in mouse cells triggers widespread H3K27me3 loss and misregulation
of PRC2-regulated genes, thereby phenocopying loss
of EZH2,16,17
suggest that favoring PRC2 activity is an evolutionarily
conserved function of H1.
H1 hinders PRC2 activity at telomeric repeats by
preventing local association of TRB proteins
We provide evidence that H1 antagonizes TRB-mediated
PRC2 activity at telomeric repeats. Waiting for an assessment
A B
C D
Figure 7. H1 antagonizes TRB1-mediated PRC2 activity at ITRs
(A) H1 restricts GFP-TRB1 protein association at TE cluster 1 elements. The plots show GFP-TRB1 mean normalized coverage in WT and 2h1 seedlings at the
indicated repeat categories. ***p <1.94eÀ05, Wilcoxon signed-rank test.
(B) Homozygous h1.1h1.2trb1trb2trb3 (htrbQ) quintuple-mutant seedlings represented 25% of the segregating progeny and displayed strongly altered seedling
phenotypes with deficient cotyledon development and slow root growth, indicating that morphogenesis is strongly affected upon combined H1 and TRB123 loss
of function. WT, 2h1, and trb123 mutant lines have been selected as null F2 segregants from the same cross as the analyzed htrbQ plant line.
(C) H1 and TRB proteins are all required for H3K27me3 enrichment at ITR-1R and ITR-4L TEs. TAIR10 annotated repeats located within ITR-1R and ITR-4L
coordinates were ranked similarly in all heatmaps. H3K27me3 levels were determined using spike-in normalized ChIP-seq analysis.
(D) Browser view showing that GFP-TRB1 and H3K27me3 enrichment at ITR-1R in 2h1 is lost in htrbQ mutant seedlings. Each ChIP series is shown as equally
scaled profiles of the indicated genotypes. ChIP-seq and ChIP-Rx data represent the mean of two biological replicates each.
Cell Reports 42, 112894, August 29, 2023 11
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of their relative affinity for telobox elements in a chromatin
context, H1/TRB1 proteins’ antagonistic association along
the genome plausibly results from competitive DNA binding
of their respective GH1 protein domains. First, chromatin
incorporation of H1 and TRB1 is negatively correlated at a
genome-wide scale. Second, analysis of nucleosome positioning
showed that telobox motifs are preferentially situated
in linker DNA where TRB1 association is also pronounced;
so that competition with H1 can occur on linker DNA. Third,
profiling of TRB1 chromatin association in 2h1 plants showed
that TRB1 ectopically invades ITRs and other telobox-rich elements
upon H1 loss. These observations reveal that, in
WT plants, elevated H1 incorporation limits TRB1 enrichment
and/or accessibility on these loci despite the presence of
repeated telobox motifs for which the TRB1 Myb domain has
strong affinity.74,76
H3K27me3 profiling in quintuple 2h1trb123 seedlings showed
that H3K27me3 enrichment at ITRs in H1-depleted plants depends
on TRB proteins, thereby demonstrating a functional
framework in which repression of H3K27me3 deposition at telomeric
repeats relies on H1 preventing local association of PRC2associated
TRB proteins. Future studies will determine whether
other chromatin modifiers influencing H3K27me3 are implicated.
The latter possibility cannot be discarded as, for example, the
PRC1 subunit LIKE-HETEROCHROMATIN 1 (LHP1), acting as
a chromatin reader of H3K27me3 in Arabidopsis,80
prevents
TRB1 enrichment at PRC2 target genes displaying telobox mo-
tifs.77
The outstanding genome-wide pattern of telobox positioning
in linker DNA also suggests a capacity of this sequence
motif to influence chromatin organization, possibly by repelling
nucleosomes.
H1 has a profound influence on the Arabidopsis 3D
genome topology
Using Hi-C, we identified a reduced frequency of chromatin interactions
within and among the pericentromeres in 2h1 nuclei.
This is a typical feature of Arabidopsis mutants affecting chromocenter
formation64,65,67
or when chromocenters get disrupted in
response to environmental stress.68
These analyses refine the
recent observation that chromocenter formation is impaired in
2h1 nuclei,23,26,35
a defect that commonly reflects the spatial
dispersion of pericentromeres within the nuclear space.63
They
also shed light on a complex picture in which ITR-1R and 4L
embedded within the pericentromeres of chromosomes 1 and
4 escape the surrounding relaxation of heterochromatin induced
by H1 depletion and organize themselves as TAD-like structures.
In 2h1 nuclei, H3K27me3 invasion at ITRs might underlie the
maintenance of compacted and poorly accessible chromatin,
while neighboring heterochromatic regions tend to become
more accessible. It is noteworthy that, in the absence of CTCF
(CCCTC-binding factor) and of obvious related 3D structures,
Arabidopsis is thought to lack proper TADs81–83
; hence, H1 regulation
of ITR insulation represents a new regulatory function of
Arabidopsis genome topology.
We also report that H1 depletion leads to a reduction in the
number of telomeric foci and of their proportion near the nucleolus.
This suggested that 2h1 mutants are impaired in telomere
spatial individualization, which is indeed supported in our Hi-C
analyses by more frequent inter-chromosomal interactions between
telomere-proximal regions. As the preferential positioning
of telomeres around the nucleolus and centromeres near the nuclear
periphery is an important organizing principle of Arabidopsis
chromosome sub-nuclear positioning63
and topology,69
H1
therefore appears to be a crucial determinant of Arabidopsis nuclear
organization.
Both PRC1 and PRC2 participate in defining Arabidopsis
genome topology,64,84
and H3K27me3 is favored among longdistance-interacting
gene promoters.66
This led to the proposal
that, as in animals, this mark could contribute to shape chromosomal
organization in Arabidopsis, possibly through the formation
of Polycomb sub-nuclear bodies.66,85
Here we mostly
focused on large structural components of the genome, such
as telomeres, pericentromeres, and ITR regions. In mammals,
H1 depletion triggers not only higher-order changes in chromatin
compartmentation16,17
but also extensive topological changes
of gene-rich and transcribed regions.18
Future studies will determine
to what extent the impact of H1 on the H3K27me3 landscape
contributes to defining Arabidopsis genome topology.
H1 as a modulator of H3K27me3 epigenome
homeostasis
In Neurospora crassa, artificial introduction of an (TTAGGG)17
telomere repeats array at interstitial sites was shown to trigger
the formation of a large H3K27me2/3-rich chromosome
domain.86
Followed by our study, this illustrates the intrinsic
attractiveness of telomeric motifs for H3K27me3 deposition in
multiple organisms. With several thousands of telomeric motifs
altogether covering $430 kb, ITRs represent at least twice the
cumulated length of all telomeres in Arabidopsis diploid nuclei,
thereby forming immense reservoirs of PRC2 targets. Our findings
led us to hypothesize that H1-mediated repression of
PRC2 activity at these scaffolding domains serves as a safeguard
to avoid the formation of gigantic H3K27me3-rich blocks
in both pericentromeric and telomeric regions, which not only
can be detrimental for chromosome folding but could also be
on a scale tethering PRC2 complexes away from protein-coding
genes. In other terms, balancing PRC2 activity between proteincoding
genes and telomeric repeats, H1 protein regulation may
represent an important modulator of epigenome homeostasis
during development.
Limitations of the study
Owing to their repetitive nature,87–92
chromatin composition
and organization of plant telomeres has long remained enig-
matic.93,94
Former studies indicated a dominance of H3K9me2
over H3K27me3 histone marks.62,89,95
Using ChIP dot blot and
in situ immunolocalization with telomeric probes, here we
showed that H1 moderates the accumulation of H3K27me3 at
telomeres by 2- to 4-fold, yet this effect could be indirect and
not homogeneous. Hence, two limitations of our study are that
we could not assess the precise distribution of HK27me3 enrichment
along each telomere and whether H1 acts on PRC2 at telomeres
in cis. In agreement with the mosaic chromatin status of
telomeres in other organisms,96
Arabidopsis telomeres are
thought to be made of segments with distinct nucleosome
repeat length (NRL) with average length of 150 bp,97
a much
12 Cell Reports 42, 112894, August 29, 2023
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shorter size than the 189 bp estimated for H1-rich TEs.23
Considering
that H1 protects about 20 bp of DNA in vitro,98
an NRL
length of 150 bp is seemingly incompatible with H1 incorporation
into telomere chromatin. For instance, H1 has been proposed to
be under-represented at telomeres in plants97,99
as it is in mam-
mals.93,100–102
This could explain the short NRL of Arabidopsis
and human telomeres97,103
that, long after being suspected,104
have recently been re-constructed as an H1-free state columnar
organization.105
In conclusion, the existence of distinct chromatin
states at Arabidopsis telomeres needs to be explored in
more detail to establish whether the H1-mediated repression
of PRC2 activity is a global property of telomeres or rather affects
a few segments through H1 cis association.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DE-
TAILS
B Arabidopsis thaliana
d METHOD DETAILS
B Immuno-FISH
B ATAC-seq
B In situ Hi-C
B RNA-seq
B H1.2-GFP, GFP-TRB1 and H3 ChIP-seq experiments
B H3K27me3 ChIP-Rx
B H3K27me3 and H3 ChIP-blot analyses
B Hi-C bioinformatics
B ChIP-seq and ChIP-Rx bioinformatics
B MNase-seq bioinformatics
B ATAC-seq bioinformatics
B DNA sequence analyses
B Gene ontology analysis
B Protein alignment
d QUANTIFICATION AND STATISTICAL ANALYSES
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
celrep.2023.112894.
ACKNOWLEDGMENTS
The authors are grateful to Erwann Cailleux (IBENS, Paris, France) and David
Latrasse (IPS2, Orsay, France) for technical guidance with ATAC-seq, Nicolas
Valentin (I2BC, Gif, France) for assistance with FACS, Magali Charvin
(IBENS, Paris, France) for technical assistance with the IBENS plant growth
facility, Fre´ de´ rique Perronet (IBPS, France) for providing Drosophila samples,
and Kinga Rutowicz (University of Zurich, Switzerland) and Ange´ lique De´ le´ ris
(IBENS, Paris and I2BC, Gif-Sur-Yvette, France) for sharing unpublished results.
This work benefitted from grants from the Agence Nationale de la Recherche
projects (ANR-18-CE13-0004, ANR-17-CE12-0026-02) to F.B.
Collaborative work between F.B. and C. Baroux was supported by a
research grant from the Velux Foundation (Switzerland) and the Ricola Foundation
(Switzerland). Collaborative work between F.B. and A.P. was supported
by CNRS EPIPLANT Action (France). G.T. benefitted from a shortterm
fellowship of the COST Action CA16212 INDEPTH (EU) for training in
Hi-C by S.G. in U.G.’s laboratory, which is supported by the University of
Zurich (Switzerland) and the Switzerland National Science Foundation (project
31003A_179553). S.A. benefitted from a CAP20-25 Emergence research
grant from Re´ gion Auvergne-Rh^one-Alpes (France). Work in J.F.’s team was
supported by the Czech Science Foundation (project 20-01331X) and Ministry
of Education, Youth, and Sports of the Czech Republic, project INTERCOST
(LTC20003).
AUTHOR CONTRIBUTIONS
G.T., L.W., I.B., and C. Bourbousse performed ChIP and ChIP-RX experiments.
G.T. and L.W. performed ATAC-seq experiments. K.A. and M.F. performed
telomere dot blots. I.B. performed phenotypic analyses. M.B. contributed
to FACS nucleus sorting. S.A. performed cytological experiments. G.T.
and S.G. generated the Hi-C datasets. A.K. and V.C. developed ATAC-seq
bioinformatics tools. L. Concia, G.T., and C. Bourbousse performed RNAseq,
MNase-seq, ChIP-seq, and ATAC-seq bioinformatics analyses. L. Concia,
L. Carron, and S.G. performed Hi-C bioinformatics. G.T., C. Bourbousse,
L. Concia, and F.B. conceived the study. F.B., C. Bourbousse, A.C., C. Baroux,
C. Bowler, A.C., S.A., A.P., U.G., P.P.S., J.F., and S.G. supervised research.
F.B. wrote the manuscript and all authors edited the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
INCLUSION AND DIVERSITY
We support inclusive, diverse, and equitable conduct of research.
Received: January 21, 2021
Revised: December 2, 2022
Accepted: July 13, 2023
Published: July 28, 2023
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127. Ramı´rez, F., Ryan, D.P., Gr€uning, B., Bhardwaj, V., Kilpert, F., Richter,
A.S., Heyne, S., D€undar, F., and Manke, T. (2016). deepTools2: a next
generation web server for deep-sequencing data analysis. Nucleic Acids
Res. 44, W160–W165. https://doi.org/10.1093/nar/gkw257.
128. Boyle, E.I., Weng, S., Gollub, J., Jin, H., Botstein, D., Cherry, J.M., and
Sherlock, G. (2004). GO::TermFinder—open source software for accessing
Gene Ontology information and finding significantly enriched Gene
Ontology terms associated with a list of genes. Bioinformatics 20,
3710–3715. https://doi.org/10.1093/bioinformatics/bth456.
Cell Reports 42, 112894, August 29, 2023 17
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Rabbit anti-H3K27me3 Merck Cat# 07-449; RRID: AB_310624
Goat biotin anti Rabbit IgG ThermoFisher Cat# 65-6140; RRID: AB_2533969
Mouse anti-digoxigenin Roche Cat#11333062910; RRID: AB_514495
Rat anti-mouse FITC Invitrogen Cat# rmg101; RRID: AB_2556582
Mouse Cy3 anti-biotin antibody Sigma Cat# C5585; RRID: AB_258901
Anti-GFP Thermo Fisher Cat# A11122; RRID: AB_221569
Mouse Anti-H3 Abcam Cat# Ab1791; RRID: AB_302613
Rabbit anti-H3K27me3 Diagenode Cat# C15410069; RRID: AB_2814977
Chemicals, peptides, and recombinant proteins
Percoll Sigma-Aldrich #P1644
Protein-A/G Dynabeads Invitrogen #10004D
Agencourtâ AMPureâ XP Beads Beckman Coulter #A63880
DpnII New England Biolabs #R0543T
DNA Polymerase I, Large (Klenow) Fragment New England Biolabs #M0210L
T4 ligase (HC) Promega #MI79A
Critical commercial assays
Nextera DNA Library Prep Kit Illumina #FC-121-1030
MinEluteâ PCR Purification Kit QIAGEN #28004
RNeasy micro kit (Qiagen) QIAGEN #74004
KAPA LTP Library Preparation Kit Roche #KR0961
the PierceTM
BCA Protein Assay Kit Thermo ScientificTM
#23225
NEBNextâ UltraTM
II DNA Library Prep Kit for Illuminaâ New England Biolabs #E7645
Deposited data
ATAC-seq of WT and 2h1 plants This study GEO: GSE160408
H3K27me3 ChIP-Rx of Wt and 2h1 plants This study GEO: GSE160410
H1-2-GFP ChIP-seq of WT plants This study GEO: GSE160411
Hi-C in WT and 2h1 plants This study GEO: GSE160412
RNA-seq of WT and 2h1 plants This study GEO: GSE160413
H3K4me3 ChIP-seq of WT plants Fiorucci et al.,106
GEO: GSE124318
H2Bub ChIP-seq of WT plants Nassrallah et al.,45
GEO: GSE112952
MNase-seq of WT and 2h1 plants Lyons & Zilberman,28
GEO: GSE96994
WGBS of WT and 2h1 plants Lyons & Zilberman.,28
GEO: GSE96994
H3K9me2 ChIP-seq of WT plants Ma et al.,51
GEO: GSE111814
H3K27me1 ChIP-seq of WT plants Ma et al.,51
GEO: GSE111814
TRB1 ChIP-seq of WT plants Schrumpfova´ et al.,76
GEO: GSE69431
H3K27me3 ChIP-seq of WT and ref. 6elf6jmj13 plants Yan et al.,52
GEO: GSE106942
Experimental models: Organisms/strains
Arabidopsis: Col 0 Arabidopsis Biological Resource Center CS22625
h1.1h1.2 Rutowicz et al., N/A
TRB1::GFP-TRB1 Schrumpfova´ , P.P73
N/A
trb1 NASC Salk_001540
trb2 FLAGdb/FST collection107
Flag_242F11
trb3 NASC Salk_134641
trb1trb2trb3 This study N/A
(Continued on next page)
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RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Fredy Barneche
(barneche@bio.ens.psl.eu).
Materials availability
Arabidopsis transgenic and mutant lines generated in this study will be made available upon request.
Data and code availability
All data reported in this paper has been deposited at Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) public repository
under the accession number GSE160414. This paper analyzes existing, publicly available datasets whose accession numbers
are listed in the key resources table. All original code is available in this paper’s supplemental information. Publicly available code
used in this paper is listed in the key resources table. Any additional information required to reanalyze the data reported in this paper
is available from the lead contact upon request.
EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
Arabidopsis thaliana
Seeds were surface-sterilized, plated on half strength Murashige and Skoog (MS) medium with 0.9% agar and 0.5% sugar, and cultivated
under long-day (16h/8h) at 23/19
C light/dark photoperiod (100 mmol mÀ2
.sÀ1) for 5 days unless otherwise stated. Cotyledons,
when used, were manually dissected under a stereomicroscope. The h1.1h1.2 (2h1) Arabidopsis mutant line and the transgenic
pH1.2::H1.2-GFP line22
were kindly provided by Dr. Kinga Rutowicz (University of Zurich, Switzerland). The TRB1::GFP-TRB1 line
described in.76
The 2h1/TRB1::GFP-TRB1 transgenic line was obtained upon manual crossing of the 2h1 and TRB1::GFP-TRB1
line described previously in.76
The trb123 triple mutant line was produced by crossing a trb1trb2 double homozygous plant (derived
from a cross between trb1 (Salk_001540) and trb2 (Flag_242F11) mutant alleles) with a double homozygous trb2trb3 mutant plant
(derived from a cross between trb2 (Flag_242F11) with trb3 (Salk_134641).
METHOD DETAILS
Immuno-FISH
After fixation in 4% paraformaldehyde in 1X PME, cotyledons of 7-day-old seedlings were chopped directly in 1% cellulase, 1% pectolyase,
and 0.5% cytohelicase in 1X PME, and incubated 15 min. Nucleus suspensions were transferred to poly-Lysine-coated
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Software and algorithms
Trim Galore version: 0.6.4_dev Cutadapt version: 2.10 https://doi.org/10.5281/zenodo.5127898 N/A
STAR (version 2.7.3a) Dobin et al.,108
N/A
DESeq2 package Love et al.,109
N/A
HTSeq suite (version 0.11.3) Anders et al.,110
N/A
R package dplyr =https://CRAN.R-project.org/package=dplyr N/A
Hi-C Pro pipeline Servant et al.,111
N/A
Boost-HiC Carron et al.,112
N/A
Juicebox toolsuite Duran et al.,113
N/A
HOMER Heinz et al.,72
N/A
Bowtie2v.2.3.2 Langmead at al.,114
N/A
MACS2 Zhang et al.,115
N/A
sambamba v0.6.8. Tarasov et al.,116
N/A
bedtools v2.29.2 Qinlan et al.,117
N/A
Genomics Viewer (IGV) version 2.8.0 Thorvaldsdo´ ttir et al.,118
N/A
ASAP ATAC-Seq data Analysis Pipeline https://zenodo.org/record/1466008 N/A
MEME version 5.1.1 Bailey et al.,119
N/A
REVIGO Supek et al.,120
N/A
T-Coffee Notredame et al.,121
N/A
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slides. One volume of 1% lipsol in 1X PME was added to the mixture and spread on the slide. Then, 1 volume of 4% PFA in 1X PME
was added and slides were dried. Immunodetection and FISH were conducted as described previously78
using the following antibodies:
rabbit H3K27me3 (#07–449 - Merck) diluted 1:200, Goat biotin anti Rabbit IgG (#65–6140 - ThermoFisher) 1:500, mouse
anti-digoxigenin (#11333062910 -ROCHE) 1:125, rat anti-mouse FITC (#rmg101 - Invitrogen) at 1:500 at 1:100, mouse Cy3 anti-biotin
antibody (#C5585 - Sigma) at 1:1000. Acquisitions were performed on a structured illumination (pseudo-confocal) imaging system
(ApoTome AxioImager M2; Zeiss) and processed using a deconvolution module (regularized inverse filter algorithm). The colocalization
was analyzed via the colocalization module of the ZEN software using the uncollapsed z stack files. To test for signal colocalization,
the range of Pearson correlation coefficient of H3K27m3 vs. telomeric FISH signals were calculated with the colocalization
module of the ZEN software using z stack files. Foci with coefficients superior to 0.5 were considered as being colocalized.
ATAC-seq
Nuclei were isolated from 200 cotyledons of 5-day-old seedlings and purified using a two-layer Percoll gradient at 3000 g before
staining with 0.5 mM DAPI and sorting by FACS according to their ploidy levels using a MoFlo Astrios EQ Cell Sorter (Beckman Culture)
in PuraFlow sheath fluid (Beckman Coulter) at 25 psi (pounds per square inch), with a 100-mmmicron nozzle. We performed sorting
with $43 kHz drop drive frequency, plates voltage of 4000–4500 V and an amplitude of 30–50 V. Sorting was performed in purity
mode. For each sample, 20000 sorted 4C nuclei were collected separately in PBS buffer and centrifuged at 3,000 g at 4
C for 5 min.
The nuclei were re-suspended in 20 mL of Tn5 transposase reaction buffer (Illumina). After tagmentation, DNA was purified using the
MinElute PCR Purification Kit (Qiagen) and amplified with Nextera DNA Library Prep index oligonucleotides (Illumina). A size selection
was performed with AMPure XP beads (Beckman Coulter) to collect library molecules longer than 150 bp. DNA libraries were
sequenced by Beijing Genomics Institute (BGI Group, Hong-Kong) using the DNA Nanoballs (DNB) DNBseq in a 65 bp pairedend
mode.
In situ Hi-C
Hi-C was performed as in Grob et al. (2014)65
with downscaling using seedlings crosslinked in 10 mM potassium phosphate pH 7.0,
50 mM NaCl, 0.1 M sucrose with 4% (v/v) formaldehyde. Crosslinking was stopped by transferring seedlings to 30mL of 0.15 M
glycine. After rinsing and dissection, 1000 cotyledons were flash-frozen in liquid nitrogen and ground using a Tissue Lyser (Qiagen).
All sample were adjusted to 4 mL using NIB buffer (20 mM HEPES pH7.8, 0.25 M sucrose, 1 mM MgCl2, 0.5 mM KCl, 40% v/v glycerol,
1% Triton X-100) and homogenized on ice using a Dounce homogenizer. Nuclei were pelleted by centrifugation and resuspended
in the DpnII digestion buffer (10 mM MgCl2, 1 mM DTT, 100 mM NaCl, 50 mM Bis-Tris-HCl, pH 6.0) before adding SDS
to a final concentration of 0.5% (v/v). SDS was quenched by adding 2% Triton X-100. DpnII (200 u) was added to each sample
for over-night digestion at 37
C dATP, dTTP, dGTP, biotinylated dCTP and 12 mL DNA Polymerase I (Large Klenow fragment)
were added before incubation for 45 min at 37
C. A total of 50 unit of T4 DNA ligase along with 7 mL of 20 ng/mL of BSA (Biolabs)
and 7 mL of 100 mM ATP were added to reach a final volume of 700mL. Samples were incubated for 4h at 16
C with constant shaking
at 300rpm. After over-night reverse crosslinking at 65
C and protein digestion with 5 mL of 10 mg/mL proteinase K, DNA was extracted
by phenol/chloroform purification and ethanol precipitation before resuspension in 100mL of 0.1X TE buffer. Biotin was removed from
the unligated fragment using T4 DNA polymerase exonuclease activity. After biotin removal, the samples were purified using AMPure
beads with a 1.6X ratio. DNA was fragmented using a Covaris M220 sonicator (peak power 75W, duty factor 20, cycles per burst 200,
duration 150 s). Hi-C libraries were prepared using KAPA LTP Library Preparation Kit (Roche)65
with 12 amplification cycles. PCR
products were purified using AMPure beads (ratio 1.85X). Libraries were analyzed using a Qubit fluorometer (ThermoFisher) and a
TAPE Station (Agilent) before sequencing in a 75 bp PE mode using a DNB-seq platform at the Beijing Genomics Institute (BGI Group;
Honk Kong).
RNA-seq
Upon growth 5 days under long days conditions (16h light at 23
C, 8h dark at 19
C), seedlings were fixed in 100% cold acetone under
vacuum for 10 min. Cotyledons from 100 plants were manually dissected and ground in 2 mL tubes using a Tissue Lyser (Qiagen) for
1 min 30 s at 30 Hz before RNA extraction using the RNeasy micro kit (Qiagen). RNA was sequenced using the DNBseq platform at the
Beijing Genomics Institute (BGI Group) in a 100 bp paired-end mode. For raw data processing, sequencing adaptors were removed
from raw reads with trim_galore! 0.6.4_dev Cutadapt version: 2.10. Reads were mapped onto TAIR10 genome using STAR version
2.7.3a108
with the following parameters ‘‘–alignIntronMin 20 –alignIntronMax 100000 –outFilterMultimapNmax 20 –outMultimapperOrder
Random –outFilterMismatchNmax 8 –outSAMtype BAM SortedByCoordinate –outSAMmultNmax 1 –alignMatesGapMax
100000’’. Gene raw counts were scored using the htseq-count tool from the HTSeq suite version 0.11.3110
and analyzed with the
DESeq2 package109
to calculate Log2-fold change and to identify differentially expressed genes (p value <0.01). TPM (Transcripts
per Million) were retrieved by dividing the counts over each gene by its length in kb and the resulting RPK was divided by the total read
counts in the sample (in millions). Mean TPM values between two biological replicates were used for subsequent analyses. To draw
metagene plots, genes were grouped into expressed or not and expressed genes split into four quantiles of expression with the function
ntile() of the R package dplyr (=https://CRAN.R-project.org/package=dplyr).
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H1.2-GFP, GFP-TRB1 and H3 ChIP-seq experiments
H1.2-GFP and parallel H3 profiling were conducted as in Fiorucci et al. (2019)106
after sonicating chromatin to mono/di-nucleosome
fragment sizes. WT Col-0 or pH1.2::H1.2-GFP seedlings were crosslinked for 15 min using 1% formaldehyde. After dissection, 400
cotyledons were ground in 2 mL tubes using a Tissue Lyser (Qiagen) for 2 3 1 min at 30 Hz. After resuspension in 100 mL Nuclei Lysis
Buffer 0.1 %SDS, the samples were flash frozen in liquid nitrogen and chromatin was sheared using an S220 Focused-Ultrasonicator
(Covaris) for 17 min at peak power 105 W, duty factor 5%, 200 cycles per burst, to get fragment sizes between 75 and 300 bp. Immunoprecipitation
was performed on 150 mg of chromatin quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific)
with 60 mL of Protein-A/G Dynabeads and 3.5 mL of anti-GFP (Thermo Fisher #A11122) for H1.2-GFP and mock (WT) sample or
anti-H3 (Abcam #Ab1791) for H3 IPs. Immunoprecipitated DNA was subjected to library preparation using the TruSeq ChIP Sample
Preparation Kit (Illumina) and sequenced using a NextSeq 500 system or DNBSEQ-G400 in a single-end 50 bp mode (Genewiz, USA;
Fasteris, Switzerland and DNBseq BGI, Hong-Kong).
H3K27me3 ChIP-Rx
ChIP-Rx of WT and 2h1 plants corresponding to Figures 1, 2, 3, 4, 5, and 6 and of WT, 2h1, trb123 and htrbQ plants corresponding to
Figure 7 were performed using anti-H3K27me3 #07–449 (Millipore) and #C15410069 (Diagenode), respectively. Both ChIP-Rx series
were conducted as in Nassrallah et al. (2018)45
using two biological replicates of 8-day-old WT and 2h1 seedlings. For each biological
replicate, two independent IPs were carried out using 120 mg of Arabidopsis chromatin mixed with 3% of Drosophila chromatin
quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). DNA samples eluted and purified from the two technical
replicates were pooled before library preparation (Illumina TruSeq ChIP) and sequencing (Illumina NextSeq 500, 1x50bp or DNBSEQG400,
1x50bp) of all input and IP samples by Fasteris (Geneva, Switzerland) and BGI (Hong-Kong), respectively.
H3K27me3 and H3 ChIP-blot analyses
Anti-H3K27me3 (Millipore, #07–449 antibody) and anti-H3 (Abcam #Ab1791 antibody) ChIPs were conducted using 2 g of tissue.
Pellets of both inputs (20%) and immunoprecipitated DNA were resuspended in 40 mL of TE, pH 8.0 and analyzed through dotblot
hybridization using a radioactively labeled telomeric probe synthesized by non-template PCR.62,122
ITRs contribution to the
hybridization signal was minimized using high stringency hybridization as detailed in.62
Hi-C bioinformatics
Hi-C reads were mapped using the Hi-C Pro pipeline111
with default pipeline parameters and merging data from three biological replicates
at the end of the pipeline. Data were in visualized using the Juicebox toolsuite113
and represented in Log10 scale after SCN
normalization123
with Boost-HiC112
setting alpha parameter to 0.2. In Figure S4, we normalized the sequencing depth in each sample
and scored the number of reads in each combination of genomic regions using HOMER.72
Read counts were further normalized for
the bin size and the median value between the three biological replicates was reported. Distal-to-Local [log2] Ratios (DLR) where
implemented as described in HOMER72
and adapted to define local interactions between a defined size window (k) and the two surrounding
windows as distal regions at 10kb and 100kb for k = 2 to k = 150 bins and selected for each ITR a windows value corresponding
of 3 ITR sizes (1050 kb for ITR-1R and 240 kb for ITR-4L).
ChIP-seq and ChIP-Rx bioinformatics
For H3K27me3 spike-in normalized ChIP-Rx, raw reads were pre-processed with Trimmomatic v0.36124
to remove leftover Illumina
sequencing adapters. 50
and 30
ends with a quality score below 5 (Phred+33) were trimmed and reads shorter than 20 bp after trimming
were discarded (trimmomatic-0.36.jar SE -phred33 INPUT.fastq TRIMMED_OUTPUT.fastq ILLUMINACLIP:TruSeq3SE.fa:2:30:10
LEADING:5 TRAILING:5 MINLEN:20). We aligned the trimmed reads against combined TAIR10 Arabidopsis thaliana
and Drosophila melanogaster (dm6) genomes with Bowtie2v.2.3.2 using the ‘‘–very-sensitive’’ setting. Duplicated reads and reads
mapping to regions with aberrant coverage or low sequence complexity defined in125
were discarded with sambamba v0.6.8.116
Peaks of H3K27me3 read density were called using MACS2115
with the command ‘‘macs2 callpeak -f BAM –nomodel -q 0.01 -g
120e6 –bw 300 –verbose 3 –broad’’. Only peaks found in both biological replicates and overlapping for at least 10% were retained
for further analyses. Annotation of genes and TEs overlapping with peaks of histone marks H3K27me3, H3K4me3, and H2Bub were
identified using bedtools v2.29.2 intersect as for H3K27me3. We scored the number of H3K27me3 reads overlapping with marked
genes using bedtools v2.29.2 multicov and analyzed them with the DESeq2 package109
in the R statistical environment v3.6.2 to
identify the genes enriched or depleted in H3K27me3 in 2h1 plants (p value <0.01). To account for differences in sequencing depth
we used the function SizeFactors in DESeq2, applying a scaling factor calculated as in Nassrallah et al. (2018).45
For GFP-TRB1,
H1.2-GFP and H3 ChIP-seq datasets, raw reads were processed as for H3K27me3. We counted the reads over genes and TEs using
bedtools v2.29.2 multicov and converted them in median counts per million, dividing the counts over each gene or TE by its length
and by the total counts in the sample and multiplying by 106 to obtain CPMs (Counts per Million reads). Mean read coverage was
used in Figure 1A, while the ratio between median value between biological replicates in IP and median value in Input was used
for violin-plot analysis of H1.2-GFP in Figures S3B and S6C. To include nucleosomes in close proximity to gene TSS, an upstream
region of 250 bp was also considered for the overlap (minimum 150 bp) for H3K27me3, TRB1 and H3K4me3 (datasets detailed in the
key resources table). H3K27me3 TE cluster 1 and TE cluster 2 were identified using Deeptools plotHeatmap using the –kmeans
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option set at 2. Tracks were visualized using Integrative Genomics Viewer (IGV) version 2.8.0.118
Meta-gene plots and heatmaps were
generated from depth-normalized read densities using Deeptools computeMatrix, plotHeatmap, and plotProfile. Violin-plots, histograms
and box-plots were drawn using the package ggplot2 v3.2.1 (https://cran.r-project.org/web/packages/ggplot2/) in the R statistical
environment. All scripts used will be made publicly available. Shuffled controls were produced with random permutations of
genomic position of the regions if interest. The permutations were generated with bedtools v2.29.2 and the command "bedtools
shuffle -chromFirst -seed 28776 -chrom’’.
MNase-seq bioinformatics
MNase read density28
was obtained from NCBI GEO under the accession GSE96994. Genomic location of WPNs shared between
WT and 2h1 plants were identified as overlapping WPN coordinates between the two genotypes calculated with bedtools v2.29.2
intersect.
ATAC-seq bioinformatics
Raw ATAC-seq data were treated using the custom-designed ASAP pipeline (ATAC-Seq data Analysis Pipeline; https://zenodo.org/
record/1466008). Mapping was performed using Bowtie2 v.2.3.2114
with parameters –very-sensitive -X 2000. Mapped reads with
MAPQ<10, duplicate pairs, and reads mapping to the mitochondrial genome as well as regions with aberrant coverage of low
sequence complexity defined in125
were filtered out. Concordant read pairs were selected and shifted as previously described by
4 bp.126
Peak calling was performed using MACS2115
using broad mode and the following parameters: –nomodel –shift À50 –extsize
100. Heatmaps and metaplots were produced from depth-normalized read coverage (read per million) using the Deeptools suite.127
DNA sequence analyses
Motifs enriched in gene promoters (À500 bp to +250 bp after the TSS) and in annotated units of TE cluster 1 elements
were identified using MEME version 5.1.1.119
The following options were used for promoters: ‘‘-dna -mod anr -revcomp -nmotifs
10 -minw 5 -maxw 9’’ and for TEs: ‘‘-dna -mod anr -nmotifs 10 -minw 5 -maxw 9 -objfun de -neg Araport11_AllTEs.fasta -revcomp
-markov_order 0 -maxsites 10000’’ where Araport11_AllTEs.fasta correspond to the fasta sequence of all TEs annotated
in Araport11.
Telobox positioning was analyzed using the TAIR10 coordinates described in Zhou et al.37
and obtained from ==+https://gbrowse.
mpipz.mpg.de/cgi-bin/gbrowse/arabidopsis10_turck_public/?l=telobox;f=save+datafile. Telobox repeat numbers were scored over
10-bp non-overlapping bins, smoothed with a 50-bp sliding window and subsequently used to plot telobox density.
Gene ontology analysis
Gene ontology analysis of H3K27me3 differentially marked genes were retrieved using the GO-TermFinder software128
via the
Princeton GO-TermFinder interface (http://go.princeton.edu/cgi-bin/GOTermFinder). The REVIGO120
platform was utilized to reduce
the number of GO terms and redundant terms were further manually filtered. The Log10 p values of these unique GO terms were then
plotted with pheatmap (https://CRAN.R-project.org/package=pheatmap) with no clustering.
Protein alignment
Protein sequences of H1.1, H1.2, H1.3, TRB1, TRB2 and TRB3 were aligned using T-Coffee121
(http://tcoffee.crg.cat/apps/tcoffee/
do:regular) with default parameters. Pairwise comparison for similarity and identity score were calculated using Ident and Sim tool
(https://www.bioinformatics.org/sms2/ident_sim.html).
QUANTIFICATION AND STATISTICAL ANALYSES
Unless stated otherwise, statistical tests were performed with the R package rstatix_0.7.1 (=https://CRAN.R-project.org/
package=rstatix) using the functions wilcox_test and wilcox_effsize. All pairwise comparisons between the read coverage in WT
and 2h1 over a given set of gene or TEs were tested with Wilcoxon signed rank test for paired samples, using the wilcox_test function
with the option "paired = TRUE’’. All other comparisons were tested with Wilcoxon rank-sum test for independent samples, setting
the option "paired = FALSE".
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Cell Reports, Volume 42
Supplemental information
Histone H1 protects telomeric repeats
from H3K27me3 invasion in Arabidopsis
Gianluca Teano, Lorenzo Concia, Léa Wolff, Léopold Carron, Ivona Biocanin, Katerina
Adamusová, Miloslava Fojtová, Michael Bourge, Amira Kramdi, Vincent Colot, Ueli
Grossniklaus, Chris Bowler, Célia Baroux, Alessandra Carbone, Aline V. Probst, Petra
Procházková Schrumpfová, Jirí Fajkus, Simon Amiard, Stefan Grob, Clara
Bourbousse, and Fredy Barneche
A
H3K27me3
H2Bub
H3K4me3
Transcript level
High
Med-High
Med-Low
Low
Absent
High
Med-High
Med-Low
Low
Absent
High
Med-High
Med-Low
Low
Absent
High
Med-High
Med-Low
Low
Absent
2h1
Chromatin accessibility (ATAC-seq)
WT
-1kb TSS +1kb -1.0 TSS +1kb
10
6
2
10
6
2
10
6
2
10
6
2
80
60
40
H1.2-GFP H3
-1kb TSS +1kb
2000
1500
1000
2000
1500
1000
2000
1500
1000
2000
1500
1000
80
60
40
80
60
40
80
60
40
-1kb TSS +1kb
Histone level (ChIP-seq mean level) B
C
*
H1.2-GFPmeanlevel
Euchromatic TEs Heterochromatic TEs
**
1
2
3
0
H3K27me3
-marked
H3K27me3
-unmarked
0
2
4
6
*
D Accessibility at gene TSS
ATAC-seq(CPM)
0
100
75
50
25
H3K27me3-marked H3K27me3-unmarked
Accessibility at gene bodies
0
100
75
50
25
Euchromatic TEs Heterochromatic TEs
WT
2h1
**
*
0
100
75
50
25
Figure S1
E
ATAC-seq(CPM)ATAC-seq(CPM)
H1 over TEs H1 over gene TSS Accessibility at TEs
Figure S1: H1.2 is enriched at PRC2-target genes and at heterochromatic TEs where it contributes to modulate DNA accessibility, related to Figure 1.
(A) H1.2 distribution and its link to gene expression. H1.2-GFP and H3 levels over the TSS (+/- 250bp) of genes marked by different histone modifications characteristic of PRC2-based
repression (H3K27me3; n=7542), of transcription initiation (H3K4me3; n=18735), transcription elongation (H2Bub; n=11357) or according to gene expression quartiles. Genes with no
detectable reads in our RNA-seq analyses of WT plants were considered as not expressed genes (n=5894) as compared to other genes (n=22103). Data represent the merge of two biological
replicates. All ChIPs have been generated in this study except H2Bub and H3K4me3. (B) DNA accessibility and its link to gene expression. Same analysis as in (A) for ATAC-seq. Normalized
read coverage is used as a proxy of DNA accessibility in WT and 2h1 plants. (C) H1.2-GFP mean level at euchromatic and heterochromatic TEs (mean read coverage). Same analysis than in
Figure S1A using TE annotation defined in Bernatavichute et al., (2008). For genes pValue < 10-16; for TEs pValue <10-308 using a Wilcoxon rank sum test; effect size, moderate. (D) ATAC-seq
read mean density (CPM) over the TSS (+/- 250bp) and full gene body of H3K27me3-marked genes (n=7542) compared to all other annotated protein-coding genes. * and ** indicate a pValue
< 10-30 and < 10-100 using a Wilcoxon rank test, with small and large effects, respectively. (E) Same analysis than (B) for the whole annotations of the corresponding TE sets. * and ** indicate a
pValue < 10-122 and < 10-308 , respectively, using a Wilcoxon rank test. ATAC-sea data correspond to the mean of two biological replicates.
**
**
*
*
Figure S2
+1kbTES-1kb
G
69
120
4
303
Hypothetical protein
Overlap with a TE annotation
Annotated as transposon
None
I
Log2FC(DEseq2normalization)
Variations in H3K27me3 gene enrichment (WT vs 2h1)
Mean of normalized counts
Traditional With spike-in
Hyper-H3K27me3
n= 496
Hypo-H3K27me3
n= 4317
Unaffected
N= 3197
Unmarked
n= 25046
A
B
WT 2h1 WT 2h1 WT 2h1 WT 2h1
TSS
C
496 hyper-H3K27me3 3197 unaffected
H3K27me3meanlevel
25046 unmarked genes4317 hypo-H3K27me3
H3K27me3meanlevel
D
2h1-onlyWT-only
1165 6377 471
H3K27me3-marked genes
Shared
F Accessibility at gene bodies
WT
2h1
*
*
*
* * *
Hyper-
H3K27me3
Unaffected Hypo-
H3K27me3
ATAC-seq(CPM)
H
-1kb TSS TES +1kb
Hypo-H3K27me3 (n=4317)
Unaffected (n=3197)
Hyper-H3K27me3 (n=496)
H3meanlevel
J
1.9e-051 1094 motifs in 418 promoters (84%)
3.2e-004 114 motifs in 83 promoters (17%)
1.8e-006 104 motifs in 87 promoters (17%)
Telobox
Motif E-value Motif number (n)
H3K27me3meanlevel
E
H1.2-GFPmeanlevel
0
2
4
6
****
****
****
Hyper-
H3K27me3
n=496
Unaffected
n=3197
Hypo-
H3K27me3
n=4317
Unmarked
n=25046
H1 level at gene bodies
Figure S2: H1 influences H3K27me3 marking, chromatin accessibility and expression of PRC2-target genes, related to Figure 2.
(A) Comparison of the repertoire and chromatin properties of genes significantly marked by H3K27me3 in WT and 2h1 plants. MA-plots show DESeq2 results using either a spike-in normalization
factor or DEseq2-based normalization. The plots were drawn using two biological replicates for each sample. (B) H3K27me3 levels at differentially marked genes (normalized mean read
coverage). (C) Detailed analysis of data presented in Figure 2B. In each cluster genes were ranked according to mean H3K27me3 level. (D) Number of H3K27me3-marked genes in WT and 2h1
seedlings. Data correspond to two biological replicates. (E) H1.2-GFP level over the TSS (+/- 250bp) of the gene sets with differential H3K27me3 enrichment in 2h1 plants defined in Figure 2A.
**** indicated a pValue <10e-11 using a Wilcoxon signed rank test on paired samples. (F) ATAC-seq mean read coverage (CPM) over the whole gene body of the corresponding gene sets. *
indicated a pValue < 10e-70 using a Wilcoxon signed rank test on paired samples. (G) H3 level over the same gene sets than in (B). (H) Gene ontology analysis of the genes differentially marked
in 2h1 plants. Association to a significantly over-represented gene function is denoted as a heatmap of false discovery rate (FDR). N=4317 hypo-marked genes; 496 hyper-marked genes. (I)
Number of genes among the 496 hyper-marked genes that either overlap an annotated TE, are annotated as TEs, or are annotated as hypothetical proteins (Key resources table). (J) Sequence
motifs over-represented in the promoters of the 496 H3K27me3 hyper-marked genes in 2h1 plants (E-value < 1e-220). E-values were calculated against random sequences. The two first motifs
could not be matched to any previously known regulatory motif while the 3rd identified motif corresponds to the previously described telobox motif (AAACCCTA).
Figure S3
Figure S3: H1 hinders H3K27me3 enrichment at pericentromeric ITRs with specific chromatin and sequence signatures, related to Figure 3.
(A) Size distribution of TEs and TE-like repeats belonging to the repertoires defined in Figure 3A. (B) TE Cluster 1 is enriched in H1.2-GFP as compared to other TEs (mean read coverage). Pvalues
of differences between the medians assessed using a Wilcoxon rank-sum test are shown. (C) Independent biological replicate of ATAC-seq data completing Figure 3D. ATAC-seq mean
read coverage of the indicated TE categories in WT and 2h1 nuclei. (D) ATAC-seq read coverage (CPM) over the whole annotated TE units of corresponding sets. P-values obtained using a
Wilcoxon signed rank test on paired samples are given. (E-H) Profiling of H3, H3K9me2, H3K27me1 and H3K27me3 over indicated TE categories. (E) Histone H3 profiling indicates that
ATREP18s and TE Cluster1-ATREP18 elements display elevated nucleosome occupancy. This is consistent with the weak accessibility of these elements determined by ATAC-seq analyses.
(F) H3K27me1 is not particularly enriched at TE Cluster 1-2 nor at ATREP18 elements as compared to other TEs. (G) H3K9me2 level is high at TE Cluster 1 as compared to other TEs. (H) All
TE types investigated in this study, including ATREP18 elements, display low H3K27me3 levels in WT plants. H3K27me3 and H3 but not H3K27me1 and H3K9me2 ChIP-seq have been
generated in this study. (I) H3K27me3 profiles of the indicated gene sets in WT and ref6 elf6 jmj13 triple mutant plants impaired in H3K27me3 demethylation. (J) Same analysis than (I) for the
indicated gene categories. (K) CG, CHG and CHH mean methylation at the indicated TE categories in WT and 2h1 mutant seedings. (L) Chromosome 4 distribution of ATREP18 elements,
telobox motif distribution and H3K27me3 profiles (normalized mean coverage). Top and bottom panels are as in Figure 3G. (M) Close-up view on interspersed H3K27me3-enriched repeats of
TE cluster 2, exemplifying a physical correlation between H3K27me3 enrichment and telobox-rich domains in 2h1 pericentromeric regions. (N) Clustered patterns of telobox motif in ATREP18
elements of TE Cluster 1. (O) Genomic distances between all perfect telobox motifs on each chromosome reflecting that ITR organizations in chromosome 1 and 4 constitute important
differences as compared to other chromosomes in which interspersed telobox motifs are largely prevalent. Except at telomeres, chromosome 5 does not display adjacent telobox motifs. (P)
Frequency of open reading regions (ORFs) identified in the indicated TE categories. TE Cluster 1, which contains many pericentromeric ATREP18 elements, rarely encodes ORFs. In contrast,
TE Cluster 2 more frequently encodes ORFs as compared to the ensemble of all TEs. (Q) Strand distribution. TE Cluster 1 consists mainly of ATREP18 elements organized in a strand-specific
manner. (R) Distribution of different groups of TEs in three classes of distances: nested (0 base pairs), closely located (under 1 kb) or distantly located (above 1 kb). Compared to the ensemble
of all TEs, TE Cluster 2 elements tend to be dispersed across the genome while, conversely, TE Cluster 1 elements tend to be located in close proximity. This peculiar distribution is largely due
to the overwhelming presence of ATREP18 elements in TE Cluster 1 elements in this group (189 out of 216, 87%).
Unaffected -marked genes
All TEs
TE cluster 1
TE cluster 2
TE Cluster 1 ATREP18s
ATREP18s
-1.0 TSS TES 1.0Kb -1.0 TSS TES 1.0Kb
I
H3K27me3meanlevel
WT ref6 elf6 jmj13
800
600
400
200
0
H3K27me3
25
5
10
15
20
0
30
TEs(n)
TE Cluster 1
TE Cluster 2
ATREP18 elements in TE Cluster 1
Other TEs in TE Cluster 1
Other ATREP18s
All TEs
TE size distribution (bp)
A
ATAC-Seq
0
25
50
75
100
All TEs TE Cluster 2 TE Cluster 1
WT
2h1
***
p < 2,45e-107
***
p < 1,45e-91
***
p < 2,26e-72
Accessibility over TEs
C
D
WT rep1 WT rep2 2h1 rep1 2h1 rep2
TSS TES +1kb-1kb
ATAC-seq
All TEs TE cluster 2TE cluster 1
B
H1 density over TEs
0
1
2
3
All TEs TE cluster 2 TE cluster 1
p < 4,44e-81
***
p < 3,45e-12
***
p < 2,07e-09
***
E
G
5000
1000
2000
3000
4000
0
50
H3K9me2
10
20
30
40
0
H3 H3K27me1
F
H
25
5
10
15
20
0
30
-1kb TSS TES +1kb
All TEs
TE cluster 1
TE cluster 2
ATREP18s in TE Cluster 1
ATREP18s
Unmarked genes
Hypo-H3K27me3 genes
Hyper-H3K27me3 genes
-1.0 TSS TES Kb1.0 -1.0 TSS TES Kb1.0
800
600
400
200
WT ref6 elf6 jmj13
H3K27me3meanlevel
J
K All TEs TE cluster 1 TE cluster 2
CG
CHG
CHH
Chr4: 2500000-4000000
ITR-4L
ATREP18s
H3K27me3 - 2h1
H3K27me3 - WT
Telobox density
H1 - WT
ATREP18s
H3K27me3 - 2h1
H3K27me3 - WT
Telobox motifs
H1 - WT
L
Chr4: 3170000-3290000
Chromosome 4 pericentomeric left region
M
H3K27me3 - WT
H1 - WT
ATREP18s
H3K27me3 - 2h1
Telobox motifs
Chr1: 15950000-16350000
N
O Genome-wide telobox distance distribution
Chr1 Chr2 Chr3 Chr4 Chr5
Chr1
Chr2
Chr3
Chr4
Chr5
Chr1 Chr2 Chr3 Chr4 Chr5
Chromosome
Genomicdistance(bp)
160
6
1
0
1000
10000
200000
P
Q
R
All Araport TEs
TE cluster 1
TE cluster 2
TE Cluster 1 ATREP18s
TE Cluster 1 - other TEs
Other ATREP18s
TE H3K27me3
Distance between elements
Above 1 kb
Under 1 kb
Nested (0 bp
0% 25% 50% 75% 100%
Percentage of the group
All Araport TEs
TE cluster 1
TE cluster 2
TE Cluster 1 ATREP18s
TE Cluster 1 - other TEs
Other ATREP18s
TE H3K27me3
Minus (-)
Plus (+)
DNA strand
TE Cluster 1 ATREP18s
TE H3K27me3
All Araport TEs
TE cluster 1
TE cluster 2
TE Cluster 1 - other TEs
Other ATREP18s
TEs encoding an ORF
0 ORF
>1 ORF
0% 25% 50% 75% 100%
Percentage of the group
ID Telobox distribution in ATREP18 elements
AT4TE14840 (-)
AT4TE14815 (-)
AT4TE14800 (-)
AT4TE14780 (-)
AT4TE14760 (-)
AT4TE14690 (-)
AT4TE14685 (-)
AT4TE14660 (-)
AT1TE50745 (-)
AT1TE50740 (-)
AT1TE50700 (-)
AT1TE50685 (-)
AT1TE50650 (-)
AT1TE50620 (-)
AT1TE50615 (-)
AT1TE50575 (-)
500bp
TSS TES +1kb-1kb TSS TES +1kb-1kb TSS TES +1kb-1kb
-1kb TSS TES +1kb
ChIP-seqmeanlevelChIP-seqmeanlevel
ChIP-seqmeanlevelChIP-seqmeanlevel
WT
2h1
other TEs in TE Cluster 1
WT 2h1
IP H3 IP H3K27me3 Input Mock
WT 2h1 WT 2h1 WT 2h1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
H3/Input
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
H3K27me3/Input
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
H3K27me3/H3
WT 2h1 WT 2h1 WT 2h1
0
1
2
3
4
rep 1 rep 2 mean
TRB1-GFP/Input
WT 2h1
Rep. 1
Rep. 2
Input
WT 2h1Mock
IP GFP-TRB1
WT 2h1Mock
A
B
Rep. 1
Rep. 2
Figure S4: H1 influences H3K27me3 and TRB1 enrichment, related to Figure 4.
(A) Independent biological replicates of H3K27me3 and H3 ChIP DNA hybridization to telomeric probes completing Figure 4A. (B) GFP-TRB1 in enriched at telomeres in 2h1 nuclei as
compared to WT. Anti-GFP ChIP-blotting was performed as in Figure, 4A and S4A using TRB1::GFP-TRB1 and 2h1/ TRB1::GFP-TRB1 plants. The results of two independent biological
replicates are shown in the upper panel, and corresponding signal quantification in the lower panel.
Figure S4
MeanRep. 1 Rep. 2
Figure S5
Figure S5: H3K27me3 accumulation at ITRs and at telomeres associates with ITR
insulation and more frequent telomere-telomere interactions, related to Figure 5.
(A) Mapping results of Hi-C libraries and comparison with re-processed Hi-C datasets from
Liu et al. (2016) and Wang et al. (2015). (B) Heatmap of similarity scores among WT and
2h1 mutant datasets calculated using Spearman rank correlation between each sample as
100 kb bins. (C) Comparison of intra-chromosomal reads over the total number of valid
interactions among our samples and published DpnII-based Hi-C datasets from Liu et al.
(2016) and Wang et al. (2015). Proportion of cis (intra-chromosomal) interactions among
valid interactions is positively correlated with library quality for in situ Hi-C as in Sun et al.
(2020). (D) Estimation of the Hi-C resolution achieved in this study. The curves show the HiC
Spector score of chromosome 1 contact map down-sampled at 10% of contacts as in
Carron et al. (2019). We computed 30 down-samples at a resolution of 2kb and computed
Hi-C Spector score similarity, using an Eigen value of 10, against each Hi-C map with a
resolution from 2kb to 150 kb. For each resolution, the maximum Hi-C Spector score of the
30 down-samplings is reported. This analysis was performed using the merge of three
independent biological replicates. Public data sources are given in key resources table. (E)
Hi-C interaction frequency heatmap showing normalized Log10 contact count at 100 kb
resolution for all Arabidopsis chromosomes in WT (below the diagonal) and 2h1 (above the
diagonal) nuclei. Lateral tracks depict the positions of centromeres (black) and
pericentromeres (gray). (F) Relative differences of interaction frequency between WT and
2h1 nuclei for the ITR regions illustrated in (B). Log2 ratios of normalized interaction
frequencies of 2h1 vs WT nuclei are shown at a 10kb resolution. All Hi-C data were
analyzed using the merge of three independent biological replicates. (G) Same analysis
than (E) illustrating interaction frequencies between the chromosomal regions spanning 1
Mb around ITR-1R (Chr1:15086191-15441067) and ITR-4L (Chr4:3192760-3265098) at a
10 kb resolution. (H-I) Pericentromere-embedded ITR-1R and 4L frequently associate with
other pericentromeric regions through inter-chromosomal contacts, but less frequently with
all other tested chromosome domains except the NOR2 region. Besides frequent longrange
interactions with NOR4, the NOR2-adjacent Chr2L region also shows frequent
interactions with several genome regions including ITR-4L and its neighboring
pericentromeric regions on Chr4L. (J) Relative differences of interaction frequency between
WT and 2h1 nuclei. With the exception of NOR-proximal Chr2L and Chr4L regions,
telomere-telomere and telomere-ITR interactions tend to increase in the absence of H1.
Similarly, interactions between ITR-1R and 4L are slightly more frequent in the mutant line.
In contrast, interactions between ITRs and all pericentromeres tend to be reduced in the
mutant line. The decrease in frequency of interaction is particularly marked between the
NOR-adjacent regions and the pericentromeres of chromosome 2 and 4. Interaction
frequencies are expressed as logarithm of the observed read pairs (H and I) or logarithm of
their ratio (J) normalized for region size. The indicated values are the median of three
biological replicates (H and I) and the ratio of the medians (J). In (H-J) we probed four
groups of regions: 1) ITR-1R and 4L coordinates, 2) sub-telomeric regions defined as the
100 kb terminal chromosomal regions adjacent to the telomeres, 3) pericentromeric regions
represented by 100-kb segments located at 1 Mb from centromeres, and 4) 100-kb
chromosome arm regions located at 5 Mb from the telomere positions. The sub-telomeric
regions of chromosome 2 and 4 left arms are separated from the telomeres by the NOR2
and NOR4, and therefore referred to as subNOR2 and SubNOR4 respectively (blue label).
Distal arm regions were used as control. (K) The upper panel displays log2 ratio of O/E
interaction frequency (2h1/WT) around ITR-1R and ITR-4L regions. Middle panel, ∆DLR
represents the variations in distal-to-Local [log2] ratios in 2h1 vs WT (2h1/WT) (see
Methods). Bottom panel, ratio of H3K27me3 mean levels between 2h1 and WT nuclei
(2h1/WT). Data combine three independent biological replicates and are processed at a
10kb resolution.
A
B C D
HiCSpectorscore
Resolution
WT
W
T
2h1
Rep1
Rep2
Rep3
Rep1
Rep2
Rep3
WT
2h1
Rep1
Rep2
Rep3
Rep1
Rep3
Rep2
Value
Liu
et al. (2016)
W
ang
et al. (2015)
70% -
60% -
50% -
40% -
30% -
20% -
10% -
0% -
Rep1
Rep2
Rep3
Rep1
Rep2
Rep3
Rep1
Rep2
Rep1
Rep2
W
T
2h1
E
2h1/WT
ITR-1R TAD-like
enhancement
ITR-4L TAD-like
enhancement
F
2h1
WT
ITR-1R TAD-like
ITR-4L TAD-like
ITR-1R TAD-like
ITR-4L TAD-like
G
Pericentromeres
∆DLR
H3K27me3 level
(2h1/WT)
ITR-1R ITR-4L
9.5 19.71Mb Pericentromeres0.8 8.3MbKnob
ITR-1R ITR-4L
K
J
Relative interaction
differences
between WT and 2h1 nuclei.
Log2 of the ratio of normalized
counts (Wt / 2h1)
Sub-
telomeric
regions
Chromosome
arms
ITR
regions
Peri-
centromeric
regions
Chr1L
Chr1R
SubNOR2
Chr2R
Chr3L
Chr3R
SubNOR4
Chr4R
Chr5L
Chr5R
Chr5R
Chr5L
Chr4L
Chr3R
Chr3L
Chr2R
Chr1R
Chr1L
ITR-4L
ITR-1R
Chr1L
Chr1R
Chr2L
Chr2R
Chr3L
Chr3R
Chr4L
Chr4R
Chr5L
Chr5R
Peri-centromeric
regions
Chr5R
Chr5L
Chr4R
Chr4L
Chr3R
Chr3L
Chr2R
Chr2L
Chr1R
Chr1L
ITR-4L
ITR-1R
Chr1L
Chr1R
Chr2R
Chr3L
Chr3R
Chr4L
Chr5L
Chr5R
Chr5R
Chr5L
Chr4R
Chr4L
Chr3R
Chr3L
Chr2R
Chr2L
Chr1R
Chr1L
Sub-telomeric
regions
Chromosome
arms
ITR
regions
H
I
Hi-C interaction in 2h1
Log2 normalized counts
Chr1L
Chr1R
SubNOR2
Chr2R
Chr3L
Chr3R
SubNOR4
Chr4R
Chr5L
Chr5R
Sub-
telomeric
regions
Chromosome
arms
ITR
regions
Peri-
centromeric
regions
Chr5R
Chr5L
Chr4L
Chr3R
Chr3L
Chr2R
Chr1R
Chr1L
ITR-4L
ITR-1R
Chr1L
Chr1R
Chr2L
Chr2R
Chr3L
Chr3R
Chr4L
Chr4R
Chr5L
Chr5R
Sub-
telomeric
regions
Chromosome
arms
ITR
regions
Peri-
centromeric
regions
Peri-centromeric
regions
Chr5R
Chr5L
Chr4R
Chr4L
Chr3R
Chr3L
Chr2R
Chr2L
Chr1R
Chr1L
ITR-4L
ITR-1R
Chr1L
Chr1R
Chr2R
Chr3L
Chr3R
Chr4L
Chr5L
Chr5R
Chr5R
Chr5L
Chr4R
Chr4L
Chr3R
Chr3L
Chr2R
Chr2L
Chr1R
Chr1L
Sub-telomeric
regions
Chromosome
arms
ITR
regions
Hi-C interaction in WT
Log2 normalized counts
Chr1L
Chr1R
SubNOR2
Chr2R
Chr3L
Chr3R
SubNOR4
Chr4R
Chr5L
Chr5R
Chr5R
Chr5L
Chr4L
Chr3R
Chr3L
Chr2R
Chr1R
Chr1L
ITR-4L
ITR-1R
Chr1L
Chr1R
Chr2L
Chr2R
Chr3L
Chr3R
Chr4L
Chr4R
Chr5L
Chr5R
2h1
A B
Identity (%) H1.1 H1.2 H1.3 TRB1 TRB2 TRB3
H1.1 56.55 23.02 16.57 15.47 16.28
H1.2 22.22 12.78 13.73 13.93
H1.3 11.39 12.26 12.14
TRB1 47.73 47.87
TRB2 65.79
TRB3
C
H1.3 1 MAEDKI------------------------------------------------------
H1.2 1 MSIEEENVP-TTVDSGAAD--TT-------------------------------------
H1.1 1 MSEVEIENAATIEGNTAADAPVT-------------------------------------
TRB1 1 MGAPKQKWT--QEEESALKSGVIKHGPGKWRTILKDPEFSGVLYLRSNVDLKDKWRNMSV
TRB2 1 MGAPKQKWT--PEEEAALKAGVLKHGTGKWRTILSDTEFSLILKSRSNVDLKDKWRNISV
TRB3 1 MGAPKLKWT--PEEETALKAGVLKHGTGKWRTILSDPVYSTILKSRSNVDLKDKWRNISV
consensus 1 *........ .............. ....... . .. .. ...............
H1.3 7 --------------LKKTPAA----------------------------KKPRK-PKT--
H1.2 21 -----------VKSPEKKPAAKGGKSKKTTTA-----KATKKPV---KAAAPTKKKTT--
H1.1 24 -----------DAAVEKKPAAKGRKTKNVKEV-----K--EKKT---VAAAPKK-RTV--
TRB1 59 MANGWGSREKSRLAVKRTF-SLPKQEENSLALTN-SLQSDEENV---DA--TSGL-QVSTRB2
59 TAL-WGSRKKAKLALKRTP-PGTKQDDNNTALTIVALTNDDERA---KP--TS-P-GGSG
TRB3 59 TAL-WGSRKKAKLALKRTPLSGSRQDDNATAITIVSLANGDVGGQQIDA--PSPP-AGSconsensus
61 . .... . .......... ..... .... . . . ... .
H1.3 22 ------------TTHPPYFQMIKEALMVLKEKNGSSPYAIAKKIEEKHKSLLPESFRKTL
H1.2 60 ------------SSHPTYEEMIKDAIVTLKERTGSSQYAIQKFIEEKHKS-LPPTFRKLL
H1.1 60 ------------SSHPTYEEMIKDAIVTLKERTGSSQYAIQKFIEEKRKE-LPPTFRKLL
TRB1 110 ------SNPPPRRPNVRLDSLIMEAIATLKEPGGCNKTTIGAYIEDQYH--APPDFKRLL
TRB2 110 GGSPRTCAS--KRSITSLDKIIFEAITNLRELRGSDRTSIFLYIEENFK--TPPNMKRHV
TRB3 114 ------CEP--PRPSTSVDKIILEAITSLKRPFGPDGKSILMYIEENFK--MQPDMKRLV
consensus 121 ....... .*..*...*... *.. ..* ..**.. . .........
H1.3 70 SLQLKNSVAKGKLVKIRASYKLSDTTKMITRQQDK----------KNKKNMKQEDKEITK
H1.2 107 LVNLKRLVASEKLVKVKASFKIPSARSAATPKPAA----------PVKKK--ATV--VAK
H1.1 107 LLNLKRLVASGKLVKVKASFKLPSASAKASSPKAA----AEKS-APAKKK--PATVAVTK
TRB1 162 STKLKYLTSCGKLVKVKRKYRIPNSTPLSSHRRKGLGVFGGKQRTSSLPSPKTDIDEVNF
TRB2 166 AVRLKHLSSNGTLVKIKHKYRFSSNFIPAGARQKAPQLFLEGNNKKDP--TKPEENGANS
TRB3 164 TSRLKYLTNVGTLVKKKHKYRISQNYMAEGEGQRSPQLLLEGN-KENT--PKPEENGVKN
consensus 181 .. **.... ..***... ... . . .. .... . . . .. .... .. .
H1.3 120 RTRSS---STRPKK-TV------------------------------SVNKQEK------
H1.2 153 -PKGKVAAAVAPAKAKAAAKGTKKPAAKVVAKAKVTAKPKAKVTAAKPKSKSVAAVSKTK
H1.1 160 -AKRKVAAASKAKK-TIAVKPKTAAAKKVTAKAKAK-----------PVPRATAAATKRK
TRB1 222 QTRSQIDTEIARMK-SMNVHEAAAVAAQAVAEAEA-------------------------
TRB2 224 LTKFRVDGELYMIK-GMTAQEAAEAAARAVAEAEF-------------------------
TRB3 221 LTKSQVGGEV-MIM-GMTEKEAAAAAARAVAEAEF-------------------------
consensus 241 ......... .. .. ............... .
H1.3 140 ---------------KRKVKKA-----RQPKSIKSSVG---KKKAM-KASA---------
H1.2 212 AVAAKPKAKERPAKASRTSTRTSPGKKVAAPAKKVAVT---KKAPA-KSVKV---KSPAK
H1.1 207 AVDAKPKAKARPAKAAKTAKVTSPAKKAVAATKKVATVATKKKTPVKKVVKPKTVKSPAK
TRB1 256 -------AMAEAEEAAKEA------EAAEAEAEAAQAF---AEEAS-KTLKGRNICKM--
TRB2 258 -------AITEAEQAAKEA------ERAEAEAEAAQIF---AKAAM-KALKFRIRNHP--
TRB3 254 -------AMAEAEEAAREA------DKAEAEAEAAHIF---AKAAM-KAVKYRMHSQT--
consensus 301 ...... ..... ........ . .. .... *... . .
H1.3 167 --------A
H1.2 265 RASTRKAKK
H1.1 267 RASSRV-KK
TRB1 297 ----MI-RA
TRB2 299 --------W
TRB3 295 --------R
consensus 361 ..
GH1
GH1
D
ChIP-seqmeanlevel -2kb TRB1 peak summit +2kb
80
50
40
20
1700
1600
1500
1400
1300
H3
TRB1
Shuffled
Figure S6: H1 and TRB1 share protein sequence features and prevalently associate to the genome in a mutually exclusive manner, related to Figure 6.
(A) ClustalW protein sequence alignment of TRB1, TRB2 and TRB3 proteins with the three H1 variants showing the relative conservation of a central globular H1 domain. (B) Amino-acids
sequence identity between TRB1, TRB2 and TRB3 proteins and the three H1 variants. (C) TE Cluster 1 displays elevated H1 level as compared to the ensemble of Arabidopsis TEs and to
other telobox-containing regions. In agreement with the proposed mutually exclusive binding of H1 and TRB1 over teloboxes, the set of teloboxes matching a known TRB1 peak displays
significantly less H1 occupancy than other teloboxes of the genome. P-values of differences between the medians assessed using a Wilcoxon rank-sum test are shown. (D) Figure 6D
complementary data. H3 occupancy (mean read coverage) and randomly shuffled peaks are shown as control of data normalization. The shuffled control was produced with random
permutations of genomic position of the regions of interest. TRB1 ChIP-seq data are from Schrumpfová et al. (2014).
n=2627n=11188
n=13815n=216
All TEs TE cluster 1 All teloboxes Teloboxes
without TRB1
TRB1-associated
teloboxes
3.40E-09
4.30E-05
0.00013
2E-16
0
1
2
H1.2-GFPmeanlevel
Figure S6
Figure S7
Figure S7: H1 prevents TRB1-mediated PRC2 activity at ITRs and at a few other repeats, related to Figure 7.
(A) GFP-TRB1 level at the indicated TE categories in WT and 2h1 nuclei (mean normalized coverage from two independent biological replicates). (B) H3K27me3 and GFP-TRB1 mean
level of different TE categories in the indicated genotypes. In each heatmap, TEs were ranked from top to bottom according to H3K27me3 or GFP-TRB1 mean level after RPGC or spikein
based normalization, respectively. While GFP-TRB1 ChIP-seq were performed using parental lines, H3K27me3 ChIP-Rx was performed on WT, 2h1 and trb123 mutant lines selected
from null F2 segregants from the same cross than the analyzed 2h1trb1trb2trb3 (htrbQ) plant line. (C) Same analysis than (B) displaying H3K27me3 mean level over whole TE
annotations. All data represent the mean of two independent biological replicates. ***, pValue < 1e-30 using a Wilcoxon rank sum test; effect size: small for All TEs comparisons, large or
moderate for TE Cluster 1 and TE Cluster 2 comparisons.
TE Cluster 2
H3K27me3B
WT 2h1 trb1/2/3 htrbQ WT 2h1
TE Cluster 1
GFP-TRB1
TSS TES
All TEs
GFP-TRB1 mean levelH3K27me3 mean level
-1Kb TSS TES 1Kb -1Kb TSS TES 1Kb
2
3
WT 2h1
All TEs
TE Cluster 1
GFP-TRB1meanlevel
0
A
TE Cluster 2
TE Cluster 1
TSS TES
H3K27me3
WT 2h1 trb1/2/3 htrbQ WT 2h1
GFP-TRB1
C
H3K27me3relativelevel(CPM)
All TEs TE Cluster 2 TE Cluster 1
2h1
WT
trb123
htrbQ
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