Chromosome
Wilhelm Gottfried Waldeyer
(1836 – 1921)
Basics of chromosome structure
Eukaryotic chromosomes
• Usually linear
• Variable in number
• DNA interacts with proteins to form chromatin
• Centromeres ensure segregation
• Telomeres cap ends
• Must be compacted to fit in nucleus
• highly coiled DNA
• histones
• non-histone chromosomal proteins (DNA & RNA polymerase,
transcription factors, topoisomerases, histone modifying proteins)
chromatin
(DNA & proteins)
Chromatin helps to fit the long DNA molecules into
small cells or nuclei
Escherichia coli
4.6  106 bp = 1.5 mm (a 1000-fold compression)
1.5 m
Histones and nucleosomes
10 nm
10-nm fibre
30-nm fibre
Chromosome packing
(loop domains)
nucleosome
not a regular structure ?
Chromosome organisation:
Strings & Binders Switch (SBS) model
Barbieri M et al. (2012) Complexity of chromatin folding is captured by the strings and binders switch model. PNAS
109:16173-16178.
Interphase chromosomes – chromosome territories
The distribution of chromosomes and genes is nonrandom with some chromosomes
preferentially occupying internal positions and others occupying peripheral positions.
Chromosome territories
Chromosome territories
T Nagano et al. Nature (2013) doi:10.1038/nature12593
Chromosome territories
Structural modelling of X chromosomes
X
Y
Chromosome Conformation Capture (3C) and 3C-Derived Methods
A Hi-C map of chromatin interaction frequencies
high levels of intra-chromosomal interactions
less frequent inter-chromosomal interactions
Chromosome territories - separate, yet interacting nuclear domains; important
long-range chromatin interactions
Territories partitioned in (i) megabasepair-long domains with frequent internal
contacts = topological associated domains (TADs), and (ii) the lamina-associated
domains (LADs) interacting with the nuclear lamina, and with other functional
compartments
Specialized transcription factories = genes come together; proximity between
different transcription units
Splicing factors (splicing nascent transcripts into messenger RNA) accumulated
in splicing speckles - often associated with active genes
Repressed chromatin associates with heterochromatic regions
Chromosome territories
Topological associated domains (TADs)
• TADs show high levels of chromatin interaction and coincide with the presence of
tissue-specific genes and their associated enhancers (the interactions of which with their cognate
promoters are facilitated by the presence of cohesin and CCCTC-binding factor (CTCF))
• the border regions between TADs are enriched for housekeeping genes, which are
often clustered together; show high levels of CTCF and cohesin binding, although
only CTCF seems to prevent interactions between TADs.
Model of nuclear organization (at different resolutions)
described for animal models
a particular locus can be surrounded by an active (A compartment) or repressive environment (B compartment)
The nonrandom organization of
genes and chromosomes contributes
to the formation of translocations.
Proximity of chromosome territories and
chromosome translocations
Relative interphase positions of chromosomes in NHBE
cells. Panel A shows chromosomes 1 (red) and 13 (green),
Panel B shows chromosomes 9 (green) and 17 (red) while
Panel C shows chromosomes 16 (red) and 21 (green). Panel
D outlines a ‘map’ of the relative positioning of
chromosome territories in NHBE cells.
Foster et al. (2013): Relative proximity of chromosome territories
influences chromosome exchange partners in radiation-induced
chromosome rearrangements in primary human bronchial epithelial cells
Chromosome organization at interphase (in plants)
Rabl configuration
Radial loop model
more organisation models ?
Cowan C R et al. Plant Physiol. 2001;125:532-538
Rabl configuration
Pecinka et al. (2004) Chromosoma
Chromosome territories - Arabidopsis
nucleolus
NADs : genomic regions with heterochromatic
signatures and include transposable elements (TEs),
sub-telomeric regions, and mostly inactive protein coding
genes. However, NADs also include active
rRNA genes and the entire short arm of chromosome 4.
Hypothesis: telomeres, NORs and NADs anchor chromatin
loops to nucleolus
TELs clustered around
nucleolus
nu
Pontvianne et al. (2016) Cell Reports
radial loop model
NADs: nucleolus-associated domains
Chromatin and chromosomes
Heterochromatin and euchromatin
DAPI-stained chromosomes of
Fritillaria spp. (B/W, inverted)
Chromosomes and nuclei stained by fluorescent dyes
4',6-Diamidino-2-phenylindole (DAPI)
Caenorhabditis elegans
AT-specific fluorescent dye
Chromatin structure: eu- and heterochromatin
heterochromatic bands
• Traditional view: chromatin compaction limits or enhances access to transcription
factors
• Accessible chromatin is referred to as euchromatin and is active (Emil Heitz, 1928)
(transcription facilitated)
• Inaccessible chromatin is called heterochromatin and is generally inactive (thought
that regulatory proteins, e.g. transcription factors, cannot access DNA templates)
• Today - restriction of DNA accessibility is a local property of chromatin and not
necessarily a consequence of microscopically visible compaction
Histones
10 nm
10-nm fibre
30-nm fibre
Histone modifications (marks)
10-nm fibers: mechanisms of signal
spreading in chromatin
3D spreading of a signal in all directions
from a nucleation center resulting in
modification of multiple chromatin
regions both in cis and in trans
A classical view of the linear
spreading of a signal in two
directions along the chromatin
fiber
Histone modifications (marks)
 acetylation
 methylation
 phosphorylation
Histone acetylation (ac) – usually higher gene expression
Histone methylation (m) – activation or repression of gene expression (often depending
on the number of methyl groups – for example, H3K4m1, H3K4m2, H3K4m3)
Histone phosphorylation (ph) – most commonly during cellular responses to DNA
damage (phosphorylated histone H2A separates large chromatin domains around the site of DNA breakage)
lysine (K) residues, arginine (R) residues
serine (S) and threonine (T) residues
lysine (K) residues [1, 2 or 3 methyl groups]
Methylation of X chromosome in mammals (Barr body)
Methylation of X chromosome in mammals (Barr body)
DNA methylation in plants
Methylation at cytosines on the carbon
no. 5 (within the pyrimidine ring) – m5C
Arabidopsis (157 Mb) – c. 6% of the cytosine
residues methylated
Maize (2 300 Mb) – c. 25%
Zhang et al. (2018) Dynamics and function of DNA methylation in
plants. Nat Rev Mol Cell Biol 19: 489-506.
Heterochromatin in plant species
Arabidopsisbarley
chromocenters
heterochromatin
density of inaccessible region
protein-coding genes
TEs along chromosomes
Heterochromatin in plant species: Arabidopsis
Shu et al. (2012), Nature Comm
heterochromatin
Genomic features in inaccessible
and hyper-accessible regions
heterochromatin:
5-cytosine methylation,
dimethylation of H3 (H3K9me2)
euchromatin:
trimethylation of H3 (H3K9me3)
Scheme of plant chromosome (after Haslop-Harrison)
centromere
Arabidopsis chromosomes
centromere
Arabidopsis Genome Initiative, Nature 408, 2000
The frequency of features was given pseudo-colour assignments, from red (high density)
to deep blue (low density).
Gene density (`Genes') ranged from 38 per 100 kb to 1 gene per 100 kb; Transposable
element densities (`TEs') ranged from 33 per 100 kb to 1 per 100 kb.
ChTeAtEuSyCh ChTeAn BrHuAn
(a) (b) (c) (d)
DoMiAt
HeSyAt
MaInAt
BuOrAt
DoMiAn
HeSyAn
MaInAn
BuOrAn
(e) (f) (g) (h)
(i) (j) (k) (l)
Chromosome structure
small genomes
(c. 150 – 600 Mb)
large(er) genomes
(> c. 1400 Mb)
(a)
(b)
(c)
ChTeAt
MaInAt
BuOrAt
(d) HeSyAt
HeS
y1
Chromosome structure – different interphase organization
small genomes
(c. 150 – 600 Mb)
large(er) genomes
(> c. 1400 Mb)
Mitotic and meiotic chromosomes
meiotic (pachytene) chromosomes of Antirrhinum
mitotic chromosomes of Pinus
1 chromosome = 2 chromatids 1 bivalent = 2 chromosomes = 4 chromatids
Cell cycle, chromosomes and chromatids
interphase
Mitosis
Figure 3.16 Genomes 3 (© Garland Science 2007)
Meiosis
Chromosomes and chromatids during mitosis and meiosis
1 chromatid2 chromatids
2 chromatids
1 chromatid
1 chromatid
1 chromatid
Mitosis
Meiosis
2 chromatids
Chromosome morphology
Centromere structure,
function & evolution
Centromere function
• chromosomes can be monocentric or holocentric (Luzula, Eleocharis, some insects)
• dicentric chromosomes usuallly unstable (anaphase bridges >> breakage), one centromere
has to be inactivated epigenetically (cf. dicentric Robertsonian fusions)
• acentric chromosome fragments are unstable at mitosis/meiosis and lost
• sister chromatid cohesion throughout cell cycle until sister chromatid segregation at
mitosis/meiosis II (centromeres enriched with cohesin)
• sites of kinetochore formation ensuring correct chromosome position on mitotic/meiotic
spindle: chromosome congression (kinetochore: spindle microtubules attached)
Centromere function: mitotic chromatid segregation
Accurate chromosome segregation requires that kinetochores from each
sister chromatid bind microtubules that emanate from opposing spindle
poles (amphitelic attachment). This is achieved by a process called
chromosome bi-orientation. Incorrect attachments can lead to improper
chromosome segregation and aneuploidy.
Chromosomal bi-orientation on a bipolar mitotic spindle
Centromeres and microtubules (monocentric chromosomes)
Wanner et al. (2015) Chromosoma
Kinetochore
inner kinetochore - associated with the
centromere DNA; specialized form of chromatin
persistent throughout the cell cycle
outer kinetochore - interacting with
microtubules; functional only during cell division.
Even the simplest kinetochores consist of more
than 45 different proteins!
Many conserved between eukaryotic species,
including a specialized histone H3 variant (called
CENP-A or CenH3) which helps the kinetochore
associate with DNA.
Kinetochore
Zhang and Dawe (2011) Mechanisms of plant spindle formation
Microtubules (tubulin)
CENH3 (an inner kinetochore protein)
Chromosomes
Microtubules interact with kinetochores even in the earliest stages of
prometaphase (immediately following nuclear envelope breakdown).
Mitosis in barley (immunofluorescence)
(methylation of H3 on lysine 9)
(di-methylation of H3 on lysine 4)
protein
otr : outer repeat
imr : innermost repeat
cnt : central sequence
Drosophila
H. sapiens
fission yeast
S. pombe
The overall chromatin structure of the centromere
is conserved among different species
Structure of plant centromeres
CENH3 (CENP-A)-associated and H3associated
nucleosomes
The CENH3-binding domain contains
active genes (red bars), but with a
lower density than the flanking
domains.
centromere of rice chromosome 3
Rice centromeres contain a satellite repeat CentO and centromere-specific
retrotransposon CRR.
In monocentric chromosomes, the centromere is
characterized by a single CenH3-containing region within a
morphologically distinct primary constriction. This region
usually spans up to a few Mbp composed mainly of
centromere-specific satellite DNA.
• long primary constrictions that contain 3–5 explicit
CenH3-containing regions
• the size of the chromosome segment delimited by two
outermost domains varies between 69 Mbp and 107 Mbp
(several factors larger than any known centromere
length)
• 13 distinct families of satellite DNA and one family of
centromeric retrotransposons (unevenly distributed
among pea chromosomes)
Neumann et al. (2012) PLoS Genet
Pea: monocentric chromosomes with multiple centromere domains
Holokinetic Chromosomes Do Not Possess a Localized Centromere
Chromosomes with more than one centromere:
consequences and solution
Neocentromeres
•a de novo centromere formation occurring after chromosome
breakage or endogenous centromere inactivation
•kinetic motility of terminal or subterminal heterochromatin,
which is pulled to the cell poles during meiosis in plants
(heterochromatic knobs)
Two meanings in literature:
Formation and behavior of de novo centromeres
PNAS 2013
The small chromosome has no detectable
canonical centromeric sequences, but contains
a site with protein features of functional
centromeres such as CENH3, the centromere
specific H3 histone variant, and
CENP-C, a foundational kinetochore protein,
suggesting the de novo formation of a
centromere on the chromatin fragment.
A Model of Centromere Evolution
Gong Z et al. Plant Cell (2012)
The satellite repeat may be
derived from other centromeres,
such as rice Cen8, or a new
repeat, such as potato Cen9.
Centromeres may survive for
several million years without
satellite repeat invasion (slow
evolution through DNA
mutations and accumulation of
transposable elements).
A de novo DNA amplification of a
satellite repeat, possibly based
on an eccDNA-mediated
mechanism, and insertion of the
repeat (yellow) in the CENH3
domain can turn an
evolutionarily new centromere
into a repeat-based centromere.
new satellite
repeat emerges in
the neocentromere
centromeric
satellite repeats
neocentromere
emergence
original
centromere
inactivation
satellite array in
the inactivated
centromere
degrades or
eliminated during
evolution
new satellite
repeats array
expands
A model of neocentromere-mediated centromere
evolution in plants (rice)
Centromere repositioning in curbit species
• centromere repositioning (CR) extensively
documented in mammalian species (e.g. 5 CRs
in the donkey after its divergence from zebra)
• scarce reports on CR in other eukaryots including plants
Cucumis sativus
2n = 14
Cucumis melo
2n = 24
• centromeres of cucumber and melon chromosomes are
associated with distinct pericentromeric heterochromatin
• centromere activation or inactivation were associated
with a gain or loss of a large amount of pericentromeric
heterochromatin
Centromere repositioning in curbit species
Han et al. 2009, PNAS 106
Cross-species fosmid FISH in cucumber and melon (Cucurbitaceae)
Fosmids (40 kb) are based
on the bacterial F-plasmid.
The cloning vector is
limited, as a host (usually
E. coli) can only contain
one fosmid molecule. Low
copy number offers higher
stability than comparable
high copy number cosmids.
66
Arabis alpina – centromere repositioning
5 reciprocal translocations
4 pericentric inversions
3 centromere repositions
1 centromere loss
1 new centromere emergence (?)
D
E
2
I
J
4
O
P
Q
R
6AK1
A
B
C
5
K
L
M
N
7
S
T
U
8
V
W
X
3
F
G
H
2
D
E
AA1
Ab
B
Aa
4
T
Jb
C
5
M
Na
K
L
Nb
7
Ua
Ub
8
Xb
W
Qb
R
Xa
Qa
3
H
Fa
G
Fb
6
I
S
O
V
Ja
Pa
Pb
Willing et al., Nature Plants 2015
Telomeres
The Nobel Prize in Physiology or Medicine 2009 was awarded jointly
to Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak
"for the discovery of how chromosomes are protected by telomeres
and the enzyme telomerase".
Carol W. Greider Jack W. SzostakElizabeth H. Blackburn
Telomeres
Telomeres
Nicotiana tabacum
 T. Mandáková
Keywords on telomeres
• solving chromosome shortening (loss of DNA sequences)
• protects against DNA repair (repair of double-strands)
• evolutionary conserved telomeric repeats
• telomere-binding proteins (shelterin complex)
• synthesis by the telomerase enzyme
• ribonucleoprotein, enzyme
• adds telomeric repeats (e.g. TTAGGG in all
vertebrates) to the 3‘ end of DNA strands at the
ends of eukaryotic chromosomes
• preventing constant loss of DNA sequences
from chromosome ends
• composed of own RNA and reverse
transcriptase (TERT)
Telomeres are made by telomerase
Telomeres of plants
human TTAGGG
Tetrahymena TTGGGG
Arabidopsis TTTAGGG
Sequences of telomere repeats
mutation altering the RNA template subunit
of telomerase, c. 80 million years ago
Telomeres – when something goes wrong
telomere dysfunction  ring chromosomes
Wikipedia:
Human genetic disorders can be caused by spontaneous ring
chromosome formation; although ring chromosomes are very
rare, they have been found in nearly all human
chromosomes.
Disorders arising from the formation of a ring
chromosome include ring chromosome 20 syndrome where
a ring formed by one copy of chromosome 20 is associated
with epilepsy; ring chromosome 14 and ring chromosome 13
syndrome are associated with mental retardation and
dysmorphic facial features; ring chromosome 15 is
associated with mental retardation, dwarfism and
microcephaly. Ring formation of an X-chromosome causes
Turner syndrome.
Symptoms seen in patients carrying ring
chromosomes are more likely to be caused by the deletion of
genes in the telomeric regions of affected chromosomes,
rather than by the formation of a ring structure itself.
In the absence of a protein protecting telomeres, chromosomes fuse abnormally
data from the T. De Lange lab
Telomeres – when something goes wrong
Murnane JP (2012) Telomere dysfunction and chromosome instability
1. telomere dysfunction
2. sister chromatid fusion (2 centromeres)
3. bridge during anaphase
4. breakage
(breakage occurs at locations other than the site of
fusion, resulting in large inverted repeats on the end of
the chromosome in one daughter cell and a terminal
deletion on the end of the chromosome in the other
daughter cell)
5. fusion, bridge, breakage,…
… the B/F/B cycles will continue until the
chromosome acquires a new telomere, most
often by translocation
Telomeres – when something goes wrong
Breakage-fusion-bridge cycle
The telomeres (gray squares), centromeres (circles),
subtelomeric sequences (horizontal arrows)
rDNA loci on chromosomes
- routinely detected by FISH
- diagnostic value, position and the number usually species-specific
- 45S rDNA usually in different position on chromosome(s) than 5S rDNA
- 45S formed at nucleolar organizing regions (NORs) associated with nucleolus
rDNA = ribosomal DNA = genes coding ribosomal RNAs
Physical mapping of 45S rDNA (red) and 5S rDNA (green) to
metaphase chromosomes of Larix leptolepis. Chromosomes
counterstained with DAPI (blue) (Zhang et al. 2010)
Satellites (different from satellite repeats), satellite
chromosomes: chromosomes with nucleolar organizing region
(NOR) = secondary constriction. Short chromosome part beyond
the NOR is called a satellite (trabant).
SAT chromosome: Sine Acid thymonucleinico (without
thymonucliec acid or DNA). Because of relative deficiency of
DNA in the nucleolar organizing region, NORs show less intense
staining.
Satellite (SAT) chromosomes, secondary constrictions
CEN
NOR
NOR
NOR
Nucleolus
- ribosomal DNA (rDNA = rRNA genes) is transcribed
and ribosomes are assembled within the nucleolus
- ribosomes are exported to the cytoplasm. They
remain free or associate with the endoplasmic
reticulum (rough endoplasmic retictulum).
- one or several nucleoli in a nucleus
- after a cell division, a nucleolus is formed around
nucleolar organizing region (NOR) on some
chromosomes (chromosomes are brought together by
nucleolar organizing regions)
- cell division: nucleolus disappears
Pecinka et al. (2004) Chromosoma
Chromosome territories in Arabidopsis: NOR-bearing chromosomes
associated more frequently than all other chromosomes
18S, 5.8S, and 28S - genes coding 18S, 5.8S, and 28S RNA molecules
NTS - nontranscribed spacer
ETS - external transcribed spacer
ITS - internal transcribed spacers 1 and 2
transcription of rDNA 45S pre-rRNA processing 18S RNA, 5.8S and 28S RNA molecules
45S and 5S ribosomal DNA (rDNA)
Structure of the 45S rDNA tandem repeat
Ribosomes – proteins and RNA molecules. In eukaryotes, small (40S) and large (60S)
subunit. The 18S rRNA in the small subunit, large subunit contains
3 rRNA types (5S, 5.8S, and 28S rRNA).
In eukaryotes, the 5S rRNA gene is separated from the 45S rRNA genes. But together in
Artemisia, gymnosperms, and some other plants.
Heterochromatin
and heterochromatic knobs
Het knobs are located on chromosomes:
a) terminally
b) insterstitially
c) at pericentromeres
meiotic (pachytene) chromosomes of Antirrhinum
Het knobs in Brassicaceae species
Myagrum perfoliatum
Thellungiella halophila
Mandáková & Lysak (2008), Plant Cell
Het knobs in rice
CEN
het knobs
rice chromosome 4
Jiao et al. 2005, Plant Cell 17
Heterochromatic segment 1 found in
Brachycome dichromosomatica (Asteraceae)
Houben et al. 2000, Chromosoma 109
The terminal knob contains the Bds1 tandem repeat.
174-bp satellite repeat
Large Heterochromatin Knobs (Segments) in Ballantinia antipoda
Het knobs
? origin
? composition
? function (if any)
Het knob hk4S in Arabidopsis
The hk4S originated by an inversion event that
relocated pericentromeric sequence to an
interstitial position.
Fransz et al. 2000, Cell 100
Het knobs were discovered by McClintock in maize
McClintock B (1929) Chromosome morphology in Zea mays. Science 69
Barbara McClintock
(1902-1992)
America’s most distinguished
cytogeneticist, was initially denied
acceptance to Cornell University’s
Dept. of Plant Breeding because she
was a woman. Eventually allowed to
study plant genetics, McClintock
received her Ph.D. from Cornell in
1927, and later formulated one of
the most important genetic theories
of the 20th century.
• knobless and knobb-bearing accessions
• the number, size and position of knobs are variable and they are found in
23 locations on the ten maize chromosomes
Het knobs in maize
CENs
abnormal chromosome 10
Ab10
Rhoades 1952
(discovered by Albert Longley; 1937, 1938)
the 180-bp and TR-1 (350-bp) tandem repeats are the major components of
knob heterochromatin (Peacock et al. 1981, Ananiev et al. 1998) + different
retrotransposons
Het knobs in maize
Wang et al. 2006, Plant Cell 18
mFISHed pachytene chromosomes of the
Kansas Yellow Saline (KYS) inbred line
180-bp repeat (green)
TR-1 element (pink)
CEN 10
Kanizay et al. (2013) Heredity
TR-1 repeat
knob 180 repeat
Structural variants of maize chromosome 10 (Ab10)
Meiotic drive (transmission distortion)
described by Marcus Morton Rhoades
Rhoades MM (1942) Preferential segregation in maize. Genetics 27: 395–407.
Birchler et al. 2003, Genetics 164
Meiotic drive
the 1:1 segregation
(normal chromosome 10)
preferential transmission
of the Ab 10 chromosome
The ability of one homolog to enhance its probability of transmission at
the expense of its partner (e.g. in Aa heterozygote, A-bearing gametes
are produced more frequently than a-bearing gametes).
Meiotic drive in maize
Birchler et al. 2003, Genetics 164
Preferential transmission of the knob-bearing
chromosomes during female meiosis. But only if
the Ab 10 chromosome is present.
heterozygote for Ab 10
crossing-over located between
the knob and centromere
cross-over products that carry the knob
on only one of its two chromatids
(heteromorphic dyad)
pseudokinetochore activity of the knob direct the
knob-bearing chromatides to two of the four products
of meiosis II
Megasporogenesis and meiotic drive in maize
Birchler et al. 2003, Genetics 164
Female meiosis (megasporogenesis) is asymmetric:
-out of 4 haploid products only one will become the
egg; other three degenerate
- the outermost (basal) megaspore differentiates
into the megagametophyte via a few mitoses to
produce the egg, polar nuclei, and associated cells
Knob-bearing chromatids are pulled towards the
outermost megaspores during meiosis II ahead of the
centromeres.
Consequently, instead of a 50% expected ratio of
transmission in a heterozygote, knob transmission in
female meiosis varies from 59 to 82%.
Meiotic drive in maize