CG920 Genomics
Lesson 7
Protein Interactions in Gene Regulations
Jan Hejátko
Functional Genomics and Proteomics of Plants,
CEITEC - Central European Institute of Technology
And
National Centre for Bimolecular Research,
Faculty of Science,
Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.eu
2
2
 Literature sources for Chapter 06:
 Wilt, F.H., and Hake, S. (2004). Principles of Developmental Biology. (New York ;
London: W. W. Norton).
 Ainger, K., Avossa, D., Morgan, F., Hill, S.J., Barry, C., Barbarese, E., and Carson, J.H.
(1993). Transport and localization of exogenous myelin basic protein mRNA microinjected
into oligodendrocytes. J Cell Biol 123, 431-441.
 Alberts, B. (1998). The cell as a collection of protein machines: preparing the next
generation of molecular biologists. Cell 92, 291-294.
 Grefen, C., Stadele, K., Ruzicka, K., Obrdlik, P., Harter, K., and Horak, J. (2008).
Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene
receptor family members. Molecular Plant 1, 308-320.
 Hu, C.D., and Kerppola, T.K. (2003). Simultaneous visualization of multiple protein
interactions in living cells using multicolor fluorescence complementation analysis. Nat.
Biotechnol. 21, 539-545.
 Shahbabian, K., and Chartrand, P. (2012). Control of cytoplasmic mRNA localization.
Cellular and molecular life sciences : CMLS 69, 535-552.
 Van Leene, J., Witters, E., Inze, D., and De Jaeger, G. (2008). Boosting tandem affinity
purification of plant protein complexes. Trends Plant Sci 13, 517-520.
 Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D.,
Grefen, C., Schumacher, K., Oecking, C., Harter, K., and Kudla, J. (2004). Visualization of
protein interactions in living plant cells using bimolecular fluorescence complementation.
Plant J 40, 428-438.
Literature
3
3
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
 Practical use of methods for in vivo studies of protein
interactions
Outline
4
4
 Functional importance of specific protein
interactions
 Most of the proteins in the cell exist in the
form of complexes which may further
interact with each other
 Proteasome
 protein complex responsible for the
degradation of obsolete proteins in
the cell
Importance of Protein
Interactions
Once obsolete proteins are tagged with at least four ubiquitin molecules, they are
destroyed by proteasomes. Proteasomes are voracious protein shredders, but
the destructive machinery is carefully protected so that it can't attack all of the
normal proteins in the cell. The proteasome, shown here from PDB entry 1fnt, is
shaped like a cylinder, with its active sites sheltered inside the tube. The caps on
the ends regulate entry into the destructive chamber, where the protein is
chopped into pieces 3 to 23 amino acids long.
Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is performed
by a nonspecific and abundant barrel-shaped complex called the 20S
proteasome. Substrates access the active sites, which are sequestered in an
internal chamber, by traversing a narrow opening (alpha-annulus) that is blocked
in the unliganded 20S proteasome by amino-terminal sequences of alphasubunits.
Peptide products probably exit the 20S proteasome through the same
opening. 11S regulators (also called PA26 (ref. 4), PA28 (ref. 5) and REG) are
heptamers that stimulate 20S proteasome peptidase activity in vitro and may
facilitate product release in vivo. Here we report the co-crystal structure of yeast
20S proteasome with the 11S regulator from Trypanosoma brucei (PA26). PA26
carboxy-terminal tails provide binding affinity by inserting into pockets on the 20S
proteasome, and PA26 activation loops induce conformational changes in alphasubunits
that open the gate separating the proteasome interior from the
intracellular environment. The reduction in processivity expected for an open
conformation of the exit gate may explain the role of 11S regulators in the
production of ligands for major histocompatibility complex class I molecules.
(PDB, Whitby et al., (2000) Nature 408: 115-120,
http://www.rcsb.org/pdb/explore/explore.do?structureId=1fnt).
5
Proteasome
 Consisting of a core, also being
designated as 20S and regulatory
portions (19 or 11S)
 Allows targeted degradation of
proteins labelled by a specific marker small
polyppetide (76 aa) called
ubiquitin
The importance of
protein interactions
6
Proteasome –targeted proteolysis
7
7
 Functional importance of specific protein interactions
 Chromatin structure
Importance of Protein
Interactions
8
Regulation by histone
acetyl transferases or
histone deacteylases
Regulation of the chromatin structure represents one of the very basal gene
expression regulatory levels. Chromatin is a substrate for DNA-dependent RNA
polymerases that transcript the DNA encoded information into the “words and
sentences” of RNA.
Regulation of chromatin structure and its accessibility to DNA-dependent RNA
polymerases depends on many factors, one of the most important is the
regulation of chromatin binding to nucleosomes and chromatin methylation.
Regulation of chromatin interaction with histones, the positively charged proteins
forming the core of nucleosomes, is performed via modification of acetylation
status of the N-terminal portion of histones, especially histones H3 and H4. This
occurs via action of histone acetyl transferases or histone deacteylases.
8
9
CpG or CpNpG
CpNpNp
CpG
DNA methylation in animals vs. in plants
methylation status methylation status
Cell-specific methylation allows maintain of
tissue-specific gene expression profiles
Mechanism of transcriptional regulation by
DNA methylation mostly unknownImprinting and “cell memory”
Modification of the chromatin methylation is performed via DNA methyltransferases.
Interestingly, there is difference in the methylation in animals and in plants.
In animals, the methylation takes place mostly on the cytosine that occurs next to
guanosine (the sequence is denoted as CpG). In mammals, 60-90% of all CpGs are
methylated.
In plants, cytosines are methylated both symmetrically (CpG or CpNpG) and
asymmetrically (CpNpNp), where N is any nucleotide.
Methylation status is usually “reset” in the zygote and is reconstituted during
development again. E.g. the methylation is very low in the mouse embryo at the blastula
stage, however, DNA derived from later stages when organogenesis is initiated is
substantially more modified by methylation.
DNA methylation also stably alters the gene expression pattern in cells such that cells
can "remember where they have been"; in other words, cells programmed to be
pancreatic islets during embryonic development remain pancreatic islets throughout the
life of the organism without continuing signals telling them that they need to remain
islets.
DNA methylation is involved in the genomic imprinting, i.e. the genes originating from
both parents are often diversely methylated, which results into differential expression of
parental genomes (for the importance of the imprinting in the parental conflict and
epigenetics, see the lecture “Bi0580 Developmental genetics” by prof. Vyskot).
Up to know it is not clear how methylation regulates transcription. Possibly, methylation
status affects chromatin configuration or binding general repressor factors.
9
10
10
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
Importance of Protein
Interactions
11
Initiation of Transcription
Regulation of transcription occurs via specific interaction of both general and
tissue specific transcription factors (TFs) with promoter and/or enhancer
sequences.
The scheme above shows simplified subsequent formation of the complex of TFs
involved in the regulation of transcription. Interaction of general TFIID with the
TATA box induces distortion of the DNA structure (see the next slide).
11
12
Induction of structural changes upon interaction of TFIID with DNA. This may be
important for the assembly of other TFs involved in the formation of transcription
initiation complex.
This change of confirmation provides a kind of “signature” that is recognized by
other proteins and NA polymerase to recognize the proper binding site. However,
there are also TATA box-less promoter, where probably other types of
“signatures” occur.
12
13
Positive TFs
Negative TFs
Initiation of Transcription
The scheme showing the formation of the transcription initiation complex and the
interaction of both positive (open symbols) and negative (solid symbols) factors.
These proteins bind to the regulatory sequences that might be hundreds or even
thousands of base pairs away from the promoter. These protein interact with
each other and with the RNA polymerase, integrating thus many signals into a
“yes” or “no” response of the basal promoter, i.e. the region adjacent to the TATA
box and recognized by the RNA polymerase.
The individual positive or negative factors are complex and their activity might be
regulated by their phosphorylation status or via their interaction with other
proteins (i.e. momomeric or dimeric) etc..
13
14
Transcriptional Regulation by
TAFs
Signal recognition
Dimerization
DNA binding
and
transcription
activation every 7th aa
There is a whole family of transcription activating factors (TAFs) that interact with
signalling molecules, e.g. steroid hormones, thyroid hormones or retinoic acid
and in a response to the signal transfer to the nucleus where they regulate
transcription.
One of the type of TAF are leucine zipper or bZIP type TAFs. These TAFs are
dimeric, with leucine-rich hydrophobic face formed by the Leu that occurs every
7th aa.
That allows the factor to take the proper configuration, which provides the dimer
with the ability to bind DNA via charged aa.
14
15
Multifactorial Promoters Control
ProENDO16:REPORTER (sea urchin)
Deletion
mutagenesis Positive, interaction with
TAFs
Upregulation in the
presence of A and B
Developmental specificity
Combinatorial control
Midgut
Primary or skeletogenic
mesenchyme cells
Vegetal plate
An example of the “microprocessor”-like acting promoter is a promoter of the endo16
gene from the sea urchin.
There have been identified several gene regulatory modules in the endo16 gene that
have positive or negative regulatory role. These modules were identified via formation of
deletion mutants of the transcriptional fusions with reporter gene.
The analysis has revealed that the module A has a positive function and must interact
with its cognate TAFs for transcription to occur.
Module G enhances the expression when the A and B are active.
C, D, E and F are responsible for the specificity of the expression of endo16 during sea
urchin development.
Each of the modules has several protein interaction sites, some of them general, other
unique. Site for the protein SpGCF1 is present in many modules and is probably
responsible for looping of chromatin, allowing thus bringing of distal regulatory modules
close to the basal promoter.
This type of regulation, i.e. based on the different activities of diverse regulatory
sequences is sometimes called combinatorial and is common for development of many
living creatures.
In the combinatorial type of regulations, some modules may act synergistically, some of
them antagonistically, some may have both positive and negative roles (e.g. the module
B, see the figure). This variability allows very precise and responsive regulations towards
changing environmental conditions.
15
16
Regulation of β-globin type of
hemoglobin chains expression
Locus control region
Development-dependent
activation by LCR
•Acetylation of H3?
•Involvement of other
genes?
Cca 50 kbp
Multifactorial Promoters Control
An example of the combinatorial gene regulation is the regulation of β-globin type of
hemoglobin chains of humans.
As discussed in the Lesson 5 (course Bi8940 Developmental biology), the type of
hemoglobin produced by the fetus changes during development. The hemoglobin
present in the erythrocytes is composed of two α- and two β-type chains. The β-type
hemoglobin chains are of several developmental types, produced by ε, γ1, γ2 and β (in
this order). In addition, there is minor adult type of β-type hemoglobin, called δ globin.
The genes for the β-type chains are aligned on the chromosome in the order, in which
they are expressed during development (see the figure).
For the expression of individual cell types is distinctive an upstream regulatory sequence
called locus control region (LCR). LCR is located about 50 kbp away from the most
proximal ε gene.
The LCR structure is different in erytrocyte precursor cells in comparison to other cells
that could be demonstrated by the changes in the sensitivity to low concentrations of
DNase, suggesting low amount of nucleosomes bound.
For the expression of the particular genes, the interaction of their regulatory sequences
with LCRs is necessary. Because of LCR can interact only with one regulatory sequence
at a time, only one type of genes for the particular β-type chain is activated (the first
interaction of LCR with ε gene, which is later in development replaced by the other one,
is shown by the double-headed arrow).
The underlying molecular mechanisms of the specific pattern of the LCR movement from
the most proximal towards the most distal gene cannot be satisfactory explained.
Probably, acetylation of H3 histones might play a role and possibly, other genes outside
of the β-type chain family are involved in the regulation of LCR activity. That seems to
be confirmed by the identification of other human genes with similar structure,
suggesting common regulatory mechanisms via LCRs.
16
17
17
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
Importance of Protein
Interactions
18
BICOID mRNA NANOS mRNA
 Importance of mRNA localization
 Control over spatiotemporal
localization of gene product (protein)
 Asymmetric cell division during
development
 Embryo polarization
mRNA localization
Shahbabian and Chartrand, 2012
ASH1 mRNA
18
19
 Role of mRNA localization
 Attenuating the expression of
potentially toxic proteins
 Localization of
expression of MYELIN
BASIC PROTEIN (MBP)
into myelination regions
of nerve cells
mRNA localization
Ainger et al., 1993
MBP mRNA
Myelin basic protein (MBP) is a protein believed to be important in the process of
myelination of nerves in the nervous system.
The images show localization of mRNA for MBP. Digoxigenin-labeled MBP RNA
was microinjected into mouse oligodendrocytes growing in primary culture. The
injected RNA appeared as small granules which were present throughout the
cytoplasm and processes, and was also found dispersed in the peripheral
membranes of the cell.
To analyze the three dimensional distribution of microinjected labeled MBP mRNA
throughout the cell, consecutive optical sections through a single oligodendrocyte
were collected, reconstructed, and visualized using volume rendering (Fig. A) or
isosurface rendering (Fig. B) techniques. An oligodendrocyte microinjected with
MBP mRNA, visualized by volume rendering is shown in Fig. A. RNA granules
were observed throughout the perikaryon and in some, but not all, processes.
The granules in the perikaryon and in the processes appeared to be equivalent in
size. In some regions the granules in the processes were aligned in tracks.
Although not apparent from this image, the nucleus was devoid of granules.
19
20
Shahbabian and Chartrand, 2012
 Diffusion and entrapment of mRNA
 During the early stages of Xenopus
oogenesis, Xcat-2 mRNA is restricted to
a specific structure in the cytoplasm
called the mitochondrial cloud (MC,
Balbiani body)
 MC movement is partly dependent on
the depolymerization of microtubuls (socalled
„molecular motor“)
 Entrapment on the vegetal pole via
interaction of MC and ER
mRNA localization
Mechanisms
Another well studied example of the diffusion-entrapment mechanism is the
Xenopus Xcat-2 mRNA, which encodes a Nos related zinc-finger RNA-binding
protein.
During the early stages of Xenopus oogenesis, Xcat-2 mRNA is restricted to a
specific structure in the cytoplasm called the mitochondrial cloud (MC). The
mitochondrial cloud, also called Balbiani body, consists mostly of mitochondria
and small vesicles, and is the source of germinal granule material [32]. The
movement of the MC in the cytoplasm results in the localization of the Xcat-2
mRNA at the vegetal cortex (Shahbabian and Chartrand, 2012).
20
21
 Localized mRNA degradation
 During embryogenesis in
Drosophila m. Hsp83 mRNA is
localized at the posterior pole of
embryo, similarly to NANOS mRNA
 Hsp83 mRNA is localized in the
whole embryo, however, it is
destabilized by cis elements both
in 3’UTR (HDE) and in coding
region (HIE).
 HIE elements are recognized by SMAUG protein, which
mediates binding of degradation complex CCR4/POP2/NOT
 In the posterior pole the Hsp83 mRNA is protected from the
effects of SMAUG by the so-called HPE element in 3’UTR;
mechanism of this protection is still unknown
Shahbabian and Chartrand, 2012
mRNA localization
Mechanisms
Localized stabilization of a transcript is another mechanism by which an mRNA can be
subcellularly targeted. In this case, an mRNA is rapidly degraded in most parts of the cell, but it is
protected from degradation at a specific location. The hsp83 mRNA, which encodes a heat shock
protein in Drosophila, is a well-characterized example of this kind of localization (Fig. 2b). This
transcript is localized at the posterior pole of the early Drosophila embryo by the selective
stabilization of the mRNA at the posterior pole and degradation of the transcript elsewhere in the
cytoplasm.
The level of hsp83 mRNA, which is a maternally encoded transcript, decreases more rapidly in
embryos than in unfertilized eggs, which suggests that two separate mechanisms control the
stability of this transcript [38]. These two independent pathways, which are called ‘‘maternal’’ and
‘‘zygotic’’ pathways, use maternally and embryonic encoded proteins, respectively, to degrade the
hsp83 transcript [38]. By analyzing the 3’UTR of hsp83 mRNA, a region from nucleotides 253–349
was identified as the Hsp83 degradation element (HDE), which directs the destabilization of this
mRNA in unfertilized eggs. However, this region has no effect in the zygotic degradation pathway,
and transcripts without the HDE domain are subject to degradation by the embryonic degradation
machinery [38]. The hsp83 ORF has also been shown to affect the stability of the transcript. A
region at the 3’ end of the ORF, which comprises 615 nucleotides, has been found to be
responsible for this destabilization, and was consequently called Hsp38 instability element (HIE)
[39]. This region, which has the major effect in the destabilization of the transcript, functions
together with the HDE for complete degradation. The HIE domain contains six stem-loop
structures that are recognized by the maternally encoded RNA-binding protein Smaug [39, 40]. It
was shown that in Smaug mutants, degradation and thus localization of hsp83 mRNA are
impaired. Smaug recruits the CCR4/POP2/NOT deadenylase complex, triggering deadenylation
and thus degradation of the hsp83 transcript [40]. Although Smaug is present throughout the pole
plasm, the hsp83 mRNA is protected from Smaug action at the posterior pole. This protection is
related to a 57 nt region in the 3’UTR (nucleotides 351–407) downstream of HDE, which is called
HPE (Hsp83 protection element). HPE is sufficient to confer stability to an unstable transcript at
the pole plasm [40]. The mechanism by which this domain functions is not clear, and may include
interaction of trans-acting factors that block the availability of the transcript to Smaug (Shahbabian
and Chartrand, 2012).
21
22
Shahbabian and Chartrand, 2012
 Active transport of mRNA
 Asymmetric Synthesis of HO1
(ASH1) is represor of the HO
endonuclease in S. cereviseae;
inhibition of HO results in inhibition
of mating-type switching in daughter
cells
 ASH1 mRNA is actively transported
by „molecular motors“ associated
with actin
 ASH1 mRNA contains 4 cis
elements (3 in the coding
sequence and 1 in the
3’UTR), which are recognized
by RNA-binding protein SHE2
 SHE2 interacts with SHE3, an
adaptor protein, which links
SHE2 to the molecular motor
MYO4, which then binds to
actin and allows transport of
ASH1 mRNA into the
daughter cell
Shahbabian and Chartrand, 2012
ASH1 mRNA
mRNA localization
Mechanisms
Localization of ASH1 mRNA is essential for the asymmetric distribution of Ash1, which acts as a
transcriptional repressor of the HO endonuclease and results in inhibition of mating-type switching in
daughter cells [88, 89]. The ASH1 mRNA contains four localization elements, three in the coding sequence
(E1, E2A, and E2B) and one overlapping the end of the coding sequence and the 3’UTR (E3) [25, 90]. While
the presence of these four elements leads to an optimal localization, deletion analysis revealed that each
element is sufficient for localization of a reporter mRNA to the bud.
When each of these elements was inserted in multiple copies in the 30UTR, the new constructs showed
nearly normal localization. However, for these mRNAs, the asymmetric distribution of Ash1 was impaired,
suggesting that the position of these elements is important for Ash1 sorting but not for ASH1 mRNA
localization [91]. Although the primary sequences of the four ASH1 localization elements are different, they
all fold into a stem-loop structure that contains a few conserved nucleotides [92, 93]. All four elements
interact with the same RNA binding protein called She2, which is involved in the localization of bud-localized
mRNAs in S. cerevisiae.
She2 forms a tetramer under physiological conditions, and mutations that disrupt this tetrameric state
abolish its RNA-binding capacity and impair She2-dependent localization to the bud tip [94]. She2 interacts
directly with the C-terminal domain of She3, an adaptor protein that links the She2–mRNA complex to the
molecular motor Myo4 (Fig. 2c) [55, 95, 96]. Recent evidence also suggests that She3, besides its role in
connecting the She2–RNA complex to Myo4, is itself able to bind RNA and acts synergistically with She2 to
increase the affinity and specificity of RNA binding [97].
Recent studies on Myo4 helped to explain why multiple localization elements are required for proper ASH1
mRNA localization. Myo4 is a class V myosin whose main function is the transport of mRNAs to the bud tip
using actin filaments [98–100]. Myo4, unlike other type V myosins, is a nonprocessive monomer in vivo, but
it becomes processive when present in the form of oligomers [101, 102]. Purification of the localization
complex associated with a single localization element revealed that multiple copies of Myo4 are associated
with this RNA [103]. Moreover, increasing the number of Myo4 attached to the ASH1 mRNA increased the
efficiency of localization of this transcript. These results suggest that each localization element interacts with
higher order protein complexes in which a She2 tetramer may recruit multiple copies of Myo4, thus ensuring
a continuous and processive movement of the mRNP complex into the bud. Moreover, it is possible that a
She2 tetramer binds simultaneously to the localization elements of a single transcript or, alternatively, to
those of different mRNAs. This would bring multiple mRNAs together within a single complex in which
several Myo4 molecules modulate their transport to the bud tip (Shahbabian and Chartrand, 2012).
22
23
23
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
Importance of Protein
Interactions
24
24
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
 Protein stability
Importance of Protein
Interactions
25
Jing and Strader, Plant Structural Biology, Hormonal Regulations (2018)
Auxin Signalling
Scheme of the auxin signaling pathway as an example of the role of protein
stabilization leading to regulation of gene expression.
Under low intracellulr auxin concentrations, the transcription activators of auxinregulated
genes, which are called auxin responsive factors (ARFs), are in a
complex with negative regulators of transcription, so called AUX/|IAA proteins. In
the complex, Arfs can not activate transcription.
After auxin is imported into the cell, it binds to the TIR1 protein, that allows
interaction with AUX/IAA-ARF complex and targets AUX/IAA protein for the
degradation via proteosome.
That allows ARFs to enter nucleus and activate trancription of auxin-induced
genes.
25
26
26
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
 Protein stability
 Signal transduction
Importance of Protein
Interactions
27
27
 PI and signal transduction
 through G protein and
phospholipase C
 Signalling cascades using cAMP
Signal transduction
28
28
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
Outline
29
29
PI in vivo
Co-immunoprecipitation
 Isolation of protein complexes using
antibodies recognizing one of the interacting
proteins
CKI1
MY
C
CKI1
HA
αMYC
αHA
CKI1
MY
C
AHK3
HA
αMYC
CKI1-MYC
CKI1-HA
AHK3/4-HA
CKI1-HA
30
30
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
Outline
31
31
PI in vivo
Tandem affinity purification (TAP-tag)
 Isolation of protein complexes using recombinant
proteins fused with two different binding domains -
tags
 Isolated protein complexes are separated using
1D ELFO and then identified by MS
 calmodulin-binding protein (CBP)
 IgG binding domains of protein A (ProtA)
 TEV (tobacco etch virus) protease
recognition site
POI
 Advantage: using two independent protein
domains for affinity purification -> therefore high
specifity
32
32
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
Outline
33
33
PI in vivo
Yeast two-hybrid assay (Y2H)
 Isolation of protein complexes using recombinant
proteins, each fused to a part of Gal4 transcription
factor
 One of the proteins (bait) fused to DNAbinding
domain of Gal4 (Gal4-BD)
 The other protein (prey) fused to
activation domain of Gal4 (Gal4-AD)
 Protein interactions enable reconstitution of
binding domains with activation domain and
triggers the expression of a reporter gene
 Visual detection (blue color, LacZ)
 Auxotrophic selection (growth on
medium lacking histidine, His)
 Method used for searching for interaction partners
in expression libraries of individual organisms
34
34
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
Outline
35
35
 Protein interaction is detected by
reassociation of the fluorescent protein
 Each of the potential interaction
partners is fused to one of the
subunits of the fluorescent protein,
e.g. YFP
 In case of interaction, the fluorescence
appears
 Apart from identification of the interaction,
this method allows you to localize the
interaction within the cell
PI in vivo
Bimolecular fluorescence
complementation (BiFC)
36
36
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
Outline
37
37
PI in vivo
Membrane Recruitment Assay (MeRA)
 Method for identification of interactions of
cytoplasmic proteins with the membrane proteins
 Membrane protein is fused with a
fluorescecnt protein
 Potential interaction partner is fused
with another fluorescent protein with
different emission spectra
 In case of interaction the localization
of the cytoplasmic protein is changed
– it is colocalized on the membrane
with the membrane protein
38
38

PI in vivo
Membrane Recruitment Assay (MeRA)
39
39
GFP
GFPGFPGFP
P35S::ERS1:RFP + P35S::ΔTM-ETR2:GFP
PI in vivo
Membrane Recruitment Assay (MeRA)
40
40
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
 Practical use of methods for in vivo studies of protein
interactions
Outline
41
D’Agostino et al., Plant Phys, 2000
Signal Transduction via MSP
NUCLEUS
CYTOKININ
PM
AHK sensor histidine kinases
• AHK2
• AHK3
• CRE1/AHK4/WOL
REGULATION OF TRANSCRIPTION
INTERACTION WITH EFFECTOR PROTEINS
HPt Proteins
• AHP1-6
Response Regulators
• ARR1-24
CK primary response genes
- Type-A ARRs expression
Recent Model of the CK Signaling via Multistep Phosphorelay (MSP)
Pathway
41
42
AHP1
AtHK1
AHP2
AHP3
AHP4
AHP5
AHK2
AHK3
AHK4
CKI1
CKI2
ETR1
ETR2/EIN4
Is there any specificity in plant
MSP?
□ Is there a signalling specificity of MSP in plants?
42
43
Specificity of CKI1 signalling
□ CKI1 interacts in vivo with only subset of AHPs
BiFC Y2H
AHP1
AHP2
AHP3
AHP4
AHP5
AHP6
CKI1 CKI1RD
Pekárová et al., Plant Journal (2011)
CYTOKININ
43
44
Specificity of CKI1 Signalling
□ Specificity of CKI1 interaction was confirmed in vitro
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 50 100 150 200 250
AHP concentration [nM]
A450
AHP2 AHP3 AHP5
AHP3: Kd ~ 10,5 nM
AHP2: Kd ~ 9,17 nM
AHP5: Kd ~ 108 nM
Pekárová et al., Plant Journal (2011)
44
45
Structure of CKI1RD
□ X-ray crystallography revealed conserved (α/β)5 structural fold
of CKI1RD
Pekárová et al., Plant Journal (2011)
45
46
Dynamics of CKI1RD
□ Mg2+binding leads to remodelling of active centre of
CKI1RD
Pekárová et al., Plant Journal (2011)
46
47
CKI1RD structural changes are
associated with its binding specificity
□ Mg2+- and BeF3
--induced structural changes fine-tune
binding specificity of CKI1RD
Pekárová et al., Plant Journal (2011)
47
48
AHP1
AtHK1
AHP2
AHP3
AHP4
AHP5
AHK2
AHK3
AHK4
CKI1
CKI2
ETR1
ETR2/EIN4
Model Suggestion
□ YES, there is signalling specificity of MSP in plants.
P
P
P
P
P
P
P
48
49
49
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
 Practical use of methods for in vivo studies of protein
interactions
Summary
50
50
Discussion
51
51
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
Outline
52
 Identification and cloning of the promoter region of the gene
 Preparation of recombinant DNA carrying the promoter and
the reporter gene (uidA, GFP)
TATA box
Iniciation of transcription
promoter
5’ UTR
ATG…ORF of reporter gene
Transcriptional Fusion
52
53
 Identification and cloning of the promoter region of the gene
 Preparation of recombinant DNA carrying the promoter and
the reporter gene (uidA, GFP)
 Preparation of transgenic organisms carrying this
recombinant DNA and their histological analysis
Transcriptional Fusion
53
54
GUS Reporter in Mouse
Embryos
54
55
55
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
Outline
56
 Identification and cloning of the promoter and coding region of
the analyzed gene
 Preparation of a recombinant DNA carrying the promoter and
the coding sequence of the studied gene in a fusion with the
reporter gene (uidA, GFP)
TATA box
promoter
5’ UTR
ATG…ORF of analysed gene…..….ATG…ORF of reporter gene….….....STOP
Translational Fusion
56
57
 Preparation of transgenic organisms carrying the
recombinant DNA and their histological analysis
 Compared to transcriptional fusion, translation fusion allows
analysis of intercellular localization of gene product (protein)
or its dynamics
Histone 2A-GFP in Drosophila embryo by PAMPIN1-GFP in Arabidopsis
Translational Fusion
57
58
Translational Fusion
58
59
59
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
Outline
60
Databases
□ Analysis of expression using Genevestigator (AHP1 and
AHP2, Arabidopsis, Affymetrix ATH 22K Array)
60
61
Databases
□ Analysis of expression using Genevestigator (AHP1 and
AHP2, Arabidopsis, Affymetrix ATH 22K Array)
61
62
Databases
□ Analysis of expression using ePlant
62
63
Databases
□ Analysis of expression using ePlant
63
64
□ Analysis of expression using Genevestigator (AHP1 and
AHP2, Arabidopsis, Affymetrix ATH 22K Array)
Databases
64
65
65
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
Outline
66
Fluorescence-Activated Cell Sorting (FACS)
□ High-Resolution Expression Map in Arabidopsis Root
Expression Maps - RNA
Brady et al., Science, 2007
Microarray expression profiles of 19 fluorescently sorted GFP-marked lines were
analyzed (3–9, 23, 24). The colors associated with each marker line reflect the
developmental stage and cell types sampled. Thirteen transverse sections were
sampled along the root's longitudinal axis (red lines) (10). CC, companion cells.
66
67
□ High-Resolution Expression Map in Arabidopsis Root
Expression Maps - RNA
Brady et al., Science, 2007
(A) The majority of enriched GO terms (hierarchically clustered) are associated
with individual cell types (blue bar). A smaller number are present across multiple
cell types (green bar). (fig. S2) (B) GO category enrichment for hair cells
confirms a previous report (15). Enriched cis-elements and an enriched TF family
were also identified. (C) From the top 50% of varying probe sets, 51 dominant
radial patterns were identified. Pattern expression values were mean-normalized
(rows) and log2 transformed to yield relative expression indices for each marker
line (columns). Marker line order is the same for all figures; see table S1 for
marker line abbreviations. (D) Pattern expression peaks were found across one
to five cell types. (E to G) Patterns where expression is enriched in single and
multiple cell types support transcriptional regulation of auxin flux and synthesis. In
all heat maps with probe sets, expression values were mean-normalized and log2
transformed. Expression is false-colored in representations of a root transverse
section, a cut-away of a root tip, and in a lateral root primordium. (E) Auxin
biosynthetic genes (CYP79B2, CYP79B3, SUPERROOT1, and SUPERROOT2)
are transcriptionally enriched in the QC, lateral root primordia, pericycle, and
phloem-pole pericycle (P = 1.99E–11, pattern 5). All AGI identifiers and TAIR
descriptions are found in table S14. (F) Auxin amido-synthases GH3.6 and
GH3.17 that play a role in auxin homeostasis show enriched expression in the
columella, just below the predicted auxin biosynthetic center of the QC (P =
8.82E–4, pattern 13). (G) The expression of the auxin transporter, PINFORMED2,
and auxin transport regulators (PINOID, WAG1) are enriched in the
columella, hair cells, and cortex (P = 1.03E–4, pattern 31).
67
68
□ High-Resolution Expression Map in Drosophilla
Expression Maps - RNA
Nikos Karaiskos et al. Science 2017;science.aan3235
Deconstructing and reconstructing the embryo by single-cell
transcriptomics combined with spatial mapping.
(A) Single-cell sequencing of the Drosophila embryo: ~1000 handpicked stage 6
fly embryos are dissociated per Drop-seq replicate, cells are fixed and counted,
single cells are combined with barcoded capture beads, and libraries are
prepared and sequenced. Finally, single-cell transcriptomes are deconvolved,
resulting in a digital gene expression matrix for further analysis.
(B) Mapping cells back to the embryo: Single-cell transcriptomes are correlated
with high-resolution gene expression patterns across 84 marker genes, cells are
mapped to positions within a virtual embryo, and expression patterns are
computed by combining the mapping probabilities with the expression levels
(virtual in situ hybridization).
68
69
Expression Maps - Proteins
Ponten et al., J Int Med, 2011
□ Human Protein Atlas
Schematic flowchart of the Human Protein Atlas. For each gene, a signature
sequence (PrEST) is defined from the human genome sequence, and following
RT-PCR, cloning and production of recombinant protein fragments, subsequent
immunization and affinity purification of antisera results inmunospecific
antibodies. The produced antibodies are tested and validated in various
immunoassays. Approved antibodies are used for protein profiling in cells
(immunofluorescence) and tissues (immunohistochemistry) to generate the
images and protein expression data that are presented in the Human Protein
Atlas (Ponten et al., J Int Med, 2011).
69
70
□ Human Protein Atlas
(http://www.proteinatlas.org/)
Expression Maps - Proteins
70
71
□ Human Protein Atlas
(http://www.proteinatlas.org/)
Expression Maps - Proteins
71
72
72
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
Outline
73
73
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
Outline
74
 Method, which provides quick comparison of a large
number of genes/proteins between the test sample and
control
 Oligo DNA chips are used the most
 There are commercialy available kits for the whole genome
 company Operon (Qiagen), 29.110 of 70-mer oligonucleotides
representing 26.173 genes coding proteins, 28.964 transcripts and 87
microRNA genes of Arabidopsis thaliana
 Possibility of use for the preparation of photolithography chips – facilitation
of oligonucletide synthesis e.g. for the whole human genome (about 3,1 x
109 bp) jit is possible to prepare 25-mers in only 100 steps, by this
technique
Affymetrix ATH1 Arabidopsis genome array
 Chips not only for the analysis of
gene expression, but also for e.g.
Genotyping (SNPs, sequencing
with chips, …)
DNA Chips
74
75
 For the correct interpretation of the results, good knowledge of
advanced statistical methods is required
 Control of accuracy of the measurement (repeated
measurements on several chips with the same
sample, comparing the same samples analysed on
different chips with each other)
 It is necessary to include a sufficient number of controls and
repeats
 Control of reproducibility of measurements
(repeated measurements with different samples
isolated under the same conditions on the same
chip – comparing with each other)
Che et al., 2002
 Identification of reliable measurement treshold
nespolehlivé
spolehlivé
 Finally comparing the experiment with the control
or comparing different conditions with each other >
the result
 Currently there‘s been a great number of results of various
experiments in publicly accessible databases
DNA Chips
75
76
 Protein chips
 Chips with high density containing 104 proteins
 Analysis of protein-protein interactions, kinase substrates
and interactions with small molecules
 Possibility of using antibodies – more stable than proteins
Protein Chips
76
77
 Identification of proteins interacting with integrin αIIbβ3
cytoplasmic domain of platelets
 Expression of cytoplasmic part as a
fusion peptide biotin-KVGFFKR
 Analysis of binding to the protein
chip containing 37.000 clones of E.
coli expressing human recombinant
proteins
 Confirmation of interaction by pulldown
analysis of peptides and by
coprecipitation of whole proteins as
well (e.g. chloride channel Icln)
 Other use: e.g. in the identification of
kinase substrates, when substrates
are bound to the chip and exposed
to kinases in the presense of
radiolabeled ATP (786 purified
proteins of barely, of which 21 were
identified as CK2α kinase
substrates; Kramer et al., 2004)
Lueking et al., 2005
Protein Chips
77
78
78
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
Outline
79
WT hormonal mutant
Next Gen Transcriptional
Profiling
□ Transcriptional profiling via RNA sequencing
mRNA
Sequencing by Illumina and
number of transcripts determination
mRNA
cDNA cDNA
79
80
Results of –omics Studies vs
Biologically Relevant Conclusions
□ Transcriptional profiling yielded more then 7K differentially
regulated genes…
gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significant
AT1G07795 1:2414285-2414967 WT MT OK 0 1,1804 1.79769e+308
1.79769e+3
08 6.88885e-05
0,00039180
1 yes
HRS1 1:4556891-4558708 WT MT OK 0 0,696583 1.79769e+308
1.79769e+3
08 6.61994e-06
4.67708e-
05 yes
ATMLO14 1:9227472-9232296 WT MT OK 0 0,514609 1.79769e+308
1.79769e+3
08 9.74219e-05
0,00053505
5 yes
NRT1.6 1:9400663-9403789 WT MT OK 0 0,877865 1.79769e+308
1.79769e+3
08 3.2692e-08
3.50131e-
07 yes
AT1G27570 1:9575425-9582376 WT MT OK 0 2,0829 1.79769e+308
1.79769e+3
08 9.76039e-06 6.647e-05 yes
AT1G60095 1:22159735-22162419 WT MT OK 0 0,688588 1.79769e+308
1.79769e+3
08 9.95901e-08
9.84992e-
07 yes
AT1G03020 1:698206-698515 WT MT OK 0 1,78859 1.79769e+308
1.79769e+3
08 0,00913915 0,0277958 yes
AT1G13609 1:4662720-4663471 WT MT OK 0 3,55814 1.79769e+308
1.79769e+3
08 0,00021683 0,00108079 yes
AT1G21550 1:7553100-7553876 WT MT OK 0 0,562868 1.79769e+308
1.79769e+3
08 0,00115582 0,00471497 yes
AT1G22120 1:7806308-7809632 WT MT OK 0 0,617354 1.79769e+308
1.79769e+3
08 2.48392e-06
1.91089e-
05 yes
AT1G31370 1:11238297-11239363 WT MT OK 0 1,46254 1.79769e+308
1.79769e+3
08 4.83523e-05
0,00028514
3 yes
APUM10 1:13253397-13255570 WT MT OK 0 0,581031 1.79769e+308
1.79769e+3
08 7.87855e-06
5.46603e-
05 yes
AT1G48700 1:18010728-18012871 WT MT OK 0 0,556525 1.79769e+308
1.79769e+3
08 6.53917e-05
0,00037473
6 yes
AT1G59077 1:21746209-21833195 WT MT OK 0 138,886 1.79769e+308
1.79769e+3
08 0,00122789 0,00496816 yes
AT1G60050 1:22121549-22123702 WT MT OK 0 0,370087 1.79769e+308
1.79769e+3
08 0,00117953 0,0048001 yes
Ddii et al., unpublished
AT4G15242 4:8705786-8706997 WT MT OK 0,00930712 17,9056 10,9098 -4,405231.05673e-05 7.13983e-05 yes
AT5G33251 5:12499071-12500433 WT MT OK 0,0498375 52,2837 10,0349 -9,8119 0 0 yes
AT4G12520 4:7421055-7421738 WT MT OK 0,0195111 15,8516 9,66612 -3,900439.60217e-05 0,000528904 yes
AT1G60020 1:22100651-22105276 WT MT OK 0,0118377 7,18823 9,24611 -7,503826.19504e-14 1.4988e-12 yes
AT5G15360 5:4987235-4989182 WT MT OK 0,0988273 56,4834 9,1587 -10,4392 0 0 yes
Excample of an output of transcriptional profiling study using Illumina sequencing
performed in our lab. Shown is just a tiny fragment of the complete list,
copmprising about 7K genes revealing differential expression in the studied
mutant.
80
81
81
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
 Regulation of gene expression in the identification of
gene function by gain-of-function approaches
 T-DNA activation mutagenesis
Outline
82
 Methods for identification of gene function using gain-of-function
approaches
 T-DNA activation mutagenesis
 Method enabling isolation of dominant mutants by random
insertion of constitutive promoter, resulting in
overexpression of the gene and therefore in corresponding
phenotypic changes
 First step: preparation of mutant library prepared by
tansformation of a strong constitutive promoter or enhancer
 Next step: search of interesting phenotypes
 Identification of the affected gene, e.g. by plasmid-rescue
Gain-of-Function Approaches
82
83
TF TF
TF
40S
60S
TF TF TF TF
40S
60S
40S
60S
40S
60S
40S
60S
TF TF
TF
Activation Mutagenesis
83
84
Isolation of CKI1 Gene
- Isolation of the gene using activation mutagenesis
- Mutant phenotype is a phenocopy of
exogenous application of cytokinins (CKI1,
CYTOKININ INDEPENDENT 1)
*- Tatsuo Kakimoto, Science 274 (1996), 982-985 *
*
no hormones
t-zeatin
K1 K2plasmid
rescue
35S::CK
1 cDNA
84
85
85
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
 Regulation of gene expression in the identification of
gene function by gain-of-function approaches
 T-DNA activation mutagenesis
 Ectopic expression and regulated gene expression systems
Outline
86
35S
LhG4
pOP
TATA
CKI1
activator
reporter
activator x reporter
x
Regulated Expression
Systems
86
87
35S LhGR
pOP
TATA
CKI1
activator
reporter
activator x reporter
DEX DEX
+DEX DEX
DEX
DEX
x
Regulated Expression
Systems
87
88
35S LhGR
pOP
TATA
CKI1
activator
reporter
activator x reporter
DEX DEX
+DEX DEX
DEX
DEX
x
pOP
TATA
GUS
DEX DEX
wt Col-
0
4C
Regulated Expression
Systems
88
89
89
 UAS system
http://www.plantsci.cam.ac.uk/Haseloff/
 Regulatable gene expression systems
 Time- or site-specific regulation of gene expression, leading
to a change in phenotype and thereby identification of the
natural function of the gene
 pOP system
Regulated Expression
Systems
90
90
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
 Regulation of gene expression in the identification of
gene function by gain-of-function approaches
 T-DNA activation mutagenesis
 Ectopic expression and regulated gene expression systems
 Chemical Genetics
Outline
91
91
 New trends
 „chemical genetics“ – more than 50.000/120.417 records in PubMed
database (16.10. 2008/15.11. 2018, an increase of >240 %)
Chemical Genetics
92
92
 New trends
 „chemical genetics“ – more than 50.000/130.437 records in PubMed
database (16.10. 2008/24.10. 2019, an increase of >260 %)
Chemical Genetics
 Like in the case of genetics, there are also „forward“ and
„reverse“ genetics approaches
 Unlike in „classical“ genetics approaches, the subject of
study is not a gene, but a protein
 Chemical genetics tries to identify either the target protein
after a chemical treatment and after following phenotypic
changes („forward“ chemical genetics) or chemicals able
to interact with protein of interest („reverse“ chemical
genetics)
 For that purpose there are carried out searches in the
libraries of various chemicals (thousands of entries,
comercially available)
 example: analysis of endomembrane transport in plants
93
 Analysis of mechanisms of endomembrane transport by
chemical genetics approaches
 In plants cells there occurr very dynamic processes mediated mainly
by endomembrane transport
Chemical Genetics
GFP targeted to the ER
93
94
 Endomembrane transport is an important regulatory mechanism in
signal transduction and regulation of cellular processes
 Analysis of mechanisms of endomembrane transport by
chemical genetics approaches
 In plants cells there occurr very dynamic processes mediated mainly
by endomembrane transport (see film, GFP targeting to the ER)
Chemical Genetics
94
95
Richter et al., E J Cell Biol (2010)
Anterograde
transport
Retrograde
transport
Trans-Golgi
network
Multivesicular
bodies-late
endosome
(prevacuolar
compartment)
Recycling
endosome
Huang et al., 2010
PIN1-GFP
In the figure, there is simplified scheme of vesicle trafficking pathways, regulated
by GNOM and its closest relative, GNOM-LIKE1 (GNL1).
Secretory and membrane proteins are synthesised at the ER (blue) and passed
onto the Golgi apparatus (green) by anterograde trafficking in COPII-coated
vesicles.
The retrograde route from the Golgi apparatus to the ER is regulated by the ARFGEFs
GNOM (GN) and GNL1, which regulate the recruitment of COPI coats to
the Golgi membrane. On the secretory route, proteins are transported to the
sorting station, the trans-Golgi network (TGN; lilac).
From there, proteins are either transported to the vacuole (grey) via
multivesicular bodies (MVB, also called prevacuolar compartment, PVC, which
corresponds to the late endosome; deep blue) or trafficked to the plasma
membrane (PM).
Plasma membrane proteins like the auxin efflux carrier PIN1 (red), which
accumulates at the basal PM at steady state, are continually internalised and
trafficked to the TGN, which resembles the early endosome (EE) in plants.
From the TGN, PIN1 is recycled to the plasma membrane via the recycling
endosome (RE; light blue). This pathway is regulated by the ARF-GEF GNOM.
95
96
 Analysis of mechanisms of endomembrane transport by
chemical genetics approaches
 By searching in the „library“ of chemicals there were
identified those, that lead to the secretion of enzyme
(carboxypeptidase Y) in yeast (S. cerevisiae) – this
enzyme is normally transported to the vacuole via the
endomembrane transport
 Analysis of changes in secretion using dotblot
and immunodetection of
carboxypeptidase Y in the culture medium
with monoclonal antibodies
Zouhar et al., 2004
Chemical structure of sortins
Detection of vacuole phenotype (tonoplast shape) of
yeast by staining with a specific color (MDY-64)
Immunodetection of carboxypeptidase
Chemical Genetics
96
97
 Analysis of mechanisms of endomembrane transport by
chemical genetics approaches
 Identified compounds („sortins“) were able to induce
similar changes in Arabidopsis as well – transport
mechanisms are conserved in yeast and in plants
 For detailed identification of the molecular proces
affected by one of the identified „sortins“, the analysis
of its influence on a secretion of a marker protein
(AtCPY) was performed – sortin 1 specifically inhibits
only this secretory pathway
 Identifcation of mutants with altered sensitivity to
sortin 1 (hyper- or hypo-sensitive mutants) by EMS
mutagenesis
Zouhar et al., 2004
Shape of plant vacuoles using EGFP:-TIP
Phenotype of seedlings in the presence of sortins
Sortin 1
Sortin 2
 By searching in the „library“ of chemicals there were
identified those, that lead to the secretion of enzyme
(carboxypeptidase Y) in yeast (S. cerevisiae) – this
enzyme is normally transported to the vacuole via the
endomembrane transport
 Analysis of changes in secretion using dotblot
and immunodetection of
carboxypeptidase Y in the culture medium
with monoclonal antibodies
Chemical Genetics
97
98
 Analysis of mechanisms of endomembrane
transport by chemical genetics approaches –
summary
 GFP::d-TIP vacuole membrane
(tonoplast) labelling and identification
of mutations leading to altered
tonoplast morphology
 Chemical genetics in combination
with classical genetics – identification
of proteins participating in regulation
of endomembrane transport
 Proteomics approaches –
identification and analysis of vacuole
proteome
98
99
99
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
 Regulation of gene expression in the identification of
gene function by gain-of-function approaches
 T-DNA activation mutagenesis
 Ectopic expression and regulated gene expression systems
 Chemical Genetics
Summary
100
100
Discussion