Centromere Chromatine

The centromere fulfils multiple

functions during cell division:

1- governs kinetochore assembly

2- ensures equal chromosome

segregation to daughter cells
during mitosis and meiosis

Alteration in centromere function leads to aneuplody (gain or loss of

chromosomes) that most frequently results in lethality. Aneuploidy
is also a common event in various diseases, both congenital and
acquired, including tumour progression, infertility and birth
defects.
During mitosis, chromosomes are segregated
to daughter cells through interactions
between centromeres and microtubules in
the mitotic spindle.

Centromere domains
have evolved to nucleate formation
of the kinetochore, which is essential
for establishing connections between
chromosomal DNA and microtubules
during mitosis.

constitutive centromere associated network (CCAN)

Centromeres are typically formed on highly repetitive DNA that is
not conserved in sequence or size among organisms and can differ
substantially between individuals within the same organism.

The genomic organization of human centromeres.

The primary sequence at
human centromeres is alpha
satellite DNA that is based on
171 bp monomers (colored
arrows) organized in a tandem
head-to tail fashion.

The monomeric sequences

differ by as much as 40%. A
set number of monomers
give rise to a higher order
repeat (colored bars with
black arrowhead) and confer
chromosome-specificity.
Structural organization of human centromeric and pericentromeric
regions

Each small arrow represents a single satellite monomer of n bp, with n being
characteristic of a given satellite family (e.g., 171 for alpha satellite). In the
pericentromeric regions, blocks of tandem satellite monomers from a single
family (indicated by pink versus gray) occasionally contain embedded
interspersed repetitive elements (e.g., LINEs and short interspersed
nucleotide elements).
Centromeric DNA has certain sequence
characteristics, such as an AT-rich composition,
and is repetitive in nature.
These characteristics may be a prerequisite for
the formation of higher order structures at
centromeres.

The existence and conservation of higher order structures may be

necessary for centromere function but the sequence itself may
vary.

Interestingly the length of one repeat unite of satellite DNA might

be important for the formation of higher order structures
Constitutive Heterochromatine contain repetitive DNA
sequences

1) Satellite DNA

2) Transposable
elements
PHC (pericentric heterochromatin) is replicated in mid S phase and centromeric
chromatin in late S phase. Bromodeoxyuridine (BrdU) shows distinct patterns in
mid and late S phase, and replication timing was determined by colocalization of
BrdU with fluorescent major or minor satellite probes in mouse 3T3 cells

Pericentric HC

Centromeric HC
Pericentric heterochromatin is a major repressive chromatin domain
in the nucleus.

It is mainly composed of major

and minor satellite repeats that
can easily by visualized as DAPI-
dense spots in interphase nuclei.

Pericentric heterochromatin is
enriched for epigenetic
modifications which correlate
with gene repression and
therefore it represents a valuable
model to study mechanisms of
gene silencing.
 Centromeric DNA
In multicellular organisms, the centromere is embedded within constitutive
centric heterochromatine

Different classes of
eukaryotic centromeres.
In holocentric organisms (C.
elegans), centromeres form
along the entire
chromosome.
Most eukaryotes contain
monocentric chromosomes.
In S. cerevisiae, centromeric
function resides in a small
125 bp long conserved DNA
sequence.
Comparative analysis of centromere DNA organization in S. cerevisiae,
S. pombe, D. melanogaster and H.sapiens

most eukaryotic centromeres are

organized on short tandem repeats
assembled into higher order
repeats which differ dramatically
among species and exhibit
significant variation between
individuals of the same species
Lack of conservation of centromeric DNAs
suggests that DNA sequence is not the main
determinant of centromere identity and
function.
Centromere identity and function is
regulated epigenetically through the
formation of specialised chromatin structure
(CenpA, HP1)
CenH3 chromatine is mainly responsable for kinetochore
assembly
the surrounding heterochromatine domain seems to have
a determinant function in sister-chromatid cohesion.
Posttranslational modifications on histone H3 (H3K4me2 and
H3K36me2) at centromere nucleosomes resemble transcriptionally
active domains found in housekeeping genes.

Depletion of these marks causes:

1- a rapid loss of transcription at centromere,
2- inefficient CENP-A loading and
3- kinetochore inactivation
linking centromere transcription to centromere identity.
S. cerevisiae centromere
The three centromeric regions CDEI, CDEII and CDEIII serve as binding
sites for different centromere binding proteins, e. g. Cse4. Cse4
replaces histone H3.

By contrast, the regional centromeres of the fission yeast Schizosaccharomyces

pombe are longer, have multiple microtubule attachment sites per centromere
and do not contain a conserved DNA sequence common to all chromosome
Vertebrate kinetochore:
Structures that mediate attachment between chromosome and
microtubule assemble on each sister chromatid at a region called the
centromere.

Yeast kinetochore are attached by a single microtubule to their spindle pole.

Human kinetochores are attached by about 30.
Plant chromosome by hundreads.
Kinetochore assemble on regions of chromosomal DNA marked by
a centromeric specific H3 histone variant CENP-A

A. Vertebrate kinetochore
based on electron
micrographs.

B. Models of metaphase
vertebrate and budding
yeast kinetochores
bipolarly attached to
spindle microtubules
Chromosomal segregation during mitosis as well as meiosis is
regulated by kinases and phosphatases.

Fine tuning between

the mitotic protein
kinases and protein
phosphatases
regulates mitotic
progression

The relative activities of major mitotic protein kinases including Cdk1,

AURKA, AURKB, and Plk1, indicated in blue spectrum, increase as the
cells enter mitosis. This increase is accompanied by a relative decrease in
the activities of major mitotic phosphatases including.
Aurora B kinase is a protein that functions in the attachment of the mitotic
spindle to the centromere. During prometaphase Aurora kinase B localizes to
microtubules near kinetochores

Localization of Aurora B
to the centromere
during prometaphase
requires
phosphorylation of
CENP-A.
Phosphorylation of
CENP-A by Aurora A
recruits Aurora B to the
centromere. Aurora B
itself can also
The stability of the attachments might depend on phosphorylate CENP_A
tension across the centromere. Aurora B might be at the same residue
involved in sensing tension across the centromere. once it is recruited.
Centromeric DNA is
constitute of tandem
repeats which recruit the
centromeric histone H3
variant, CENH3 (CENP-A in
mammals), and bind tightly
to a kinetochore protein
known as CENP-B.
In human tandem repeat are
known as α satellites (171
pb)
CENH3 and CENP-C are conserved in plants and animals and
are the only two kinetochore proteins known to bind DNA in
both kingdoms

Several other inner and outer kinetochore proteins have clear homologs in
plants

CENP-B is an example of an important inner kinetochore protein in animals

that is absent in plants
Upon entry in mitosis,
chromosome condense and
the primary constriction
forms at the centromere, the
region of the chromosome
defined by the incorporation
of a histone variant CenpA
Human centromeric proteins

All centromeres
are associated
with CENP-A, and
it is required to
recruit many
other centromere
and kinetochore
proteins,
Inactivation of CENP-A family proteins leads to severe
chromosome missegregation events or even to cell death

CENP-A overexpression and mislocalization are observed

in several cancers and reported to be associated with
increased invasiveness and poor prognosis.

CENP-A overexpressing cells showed altered localization

of centromere and kinetochore associated proteins
leading to weakened native kinetochores as shown by
reduced interkinetochore distance and CIN
(chromosomal instability), changes in chromosome
structure and number that lead to chromosomal gain or
loss. CENP-A
overexpression
induces
mislocalization in
non-transformed
diploid RPE1 cell
line
The kinetochore forms on the microtubule plus end as a
‘basket’ of elongated molecules (namely Ndc80) that recruit
the outer-kinetochore components of the KMN network
(which comprises the Knl1 complex, the Mis12 complex and
the Ndc80 complex).
These outer-kinetochore proteins dangle from the expanded
basket surface generated by Ndc80 and can interact with the
chromatin and proteins of the constitutively centromere-
associated network (CCAN).
CENP A-C : components of the inner centromere.
CENP-A: H3 histon variant;
CENP-B. binds 17 bp in alpha satellite sequence (CENP-B box)
CENP-C: binds chromatin , may act as a bridge between internal and external
kinetochores
Arrangement of constitutive proteins on higher order α satellite DNA

CENP-B contains a
dimerization domain so that 2
CENP-B boxes are brought
into proximity.

CENP-B is located more

extensively along α-satellite
DNA in an arrays of 4 Mbs

CENP-A and core histones are assembled into nucleosomes

between the CENP-B binding region, and the combination of the
two proteins at different regions of the α-satellite DNA establishes
centromeric specific higher order structure that visually appears as
a primary constriction.
Pericentric chromatin modifications serve to recruit proteins such
as cohesin and to maintain the structure of pericentric chromatin.

Heterochromatin surrounding the centromere is known to

contribute to sister chromatid cohesion and condensation.
At regional centromeres in organisms such as humans, the
nucleosomes containing the canonical H3 histone are dimethylated
at lys 4 (H3K4me2) a modification associated with euchromatin.

Modification
associated with
euchromatin

H2AZ containing
nucleosomes are more
resistent to
condensation and form
boudary between
heterochromatin and
euchromatin
H3K4me2 is thought to be important for the physical organization
of the centromere, indeed depletion of H3K4me2 has been
shown to result in a lack of recruitment of HJURP leading to failed
incorporation of CENPA.

The acidic region on the surface of the H2AZ containing

nucleosomes allows interactions with non histone proteins and
serves as a signpost to direct chromatin-remodelling factors.
MODIFICATIONS OF CORE ISTONES CONTRIBUTE TO CENTROMERE
SPECIFICITY
(a) Subdomains of nucleosomes containing histone
(b) At metaphase, when
CENP-A (red) are interspersed with H3K4me2
mitotic chromosomes
(green) to form a domain of CEN chromatin on
condense, the
a fraction of the megabase regions of human α-
interspersed domains
satellite DNA (purple) .
promote coiling of the
DNA so that stacks of
CENP-A nucleosomes
are presented to the
poleward face of the
chromosome where
they can interact with
other kinetochore
proteins. H3-K4diMe
containing nucleosomes
are oriented between
sister kinetochores.
Summary of physical differences between CENP-A- and H3-containing
nucleosomal arrays.

CENP-A-containing arrays are generally more condensed than canonical arrays

upon folding (indicated by closer spacing of adjacent CENP-A-containing
nucleosomes). At the same time, the DNA at the entry/exit sites is less constrained
(indicated by uncrossed internucleosomal DNA for CENP-A-containing arrays).
Interestingly, the flexible ends of CenH3 (CENP-A)
nucleosomes prevent binding of histone H1 , which is a
crucial structural component binding to H3.1 and H3.3
nucleosomes.

This contributes to the formation of a chromatin structure

that is distinct to other regions.
Both characteristics are important for centromeric chromatin:

- The condence nature of the array may reflect strong self-self interactions
that culminate in the coalescence of discontinuous CENP-A nucleosomes
on each centromere into a discrete focus on the surface of the
chromosome. In addition the condensed nature of the array may be
important to help maintain structural integrity of centromeric
chromatin under mitotic spindle stretching forces.

- The increased flexibility at the DNA entry/exit site may accomodate

internucleosomal DNA paths unique to the centromere and/or
nucleosomal binding proteins that require increased local access to the
DNA and /or histone core.
How is the CENP-A mark maintained through cell divisions
during which core histones are typically dispersed?

Unlike H3, which is incorporated into chromatin in early S-phase

during replication, existing CENP-A nucleosomes are not removed
and replaced, but are inherited semiconservatively, such that “old”
CENPA is divided between daughter strands. Newly synthesized
CENP-A is loaded onto centromeres in late telophase–early G1 by a
replication independent mechanism.
Model for inheritance of human CENP-A octamers during the cell cycle.

Blocks of CENP-A nucleosomes (pink circles) at centromeres are interspersed with

blocks of histone H3 (H3.1 and H3.3) nucleosomes (blue circles) From late G1 until
late S phase, CENP-A octamers undergo an unknown structural change that may
open the nucleosome more or may decrease its stability. During replication of
centromeric DNA in late S phase, parental CENP-A octamers are redistributed to
daughter DNA strands, and this results in CENP-A dilution by 50% (gray arrow,
direction of replication-fork movement). Histone H3.3 nucleosomes are deposited
as placeholders to offset this dilution.
CENP-A, synthesized in G2 phase, is bound to its chaperone HJURP as CENP-
A–histone H4 dimers in the cytosol. Cells proceed through mitosis and form a
kinetochore with 50% of CENP-A at centromeres. In late telophase–early G1
phase, two CENP-A–histone H4 dimers are deposited by HJURP to replace the
histone H3.3 placeholders, and this produces a CENP-A octamer and restores
CENP-A occupancy to 100%.
The uncoupling of CenH3 CENP-A deposition from S phase may
give the cell the opportunity to avoid adverse interference during
replication and regulate the spatial deposition of CenH3 (CENP-A)
along with other components, which may help to prevent ectopic
localization.
The centromere-kinetochore region

Many multiprotein complexes

are recruited to the outer
kinetochore specifically in
mitosis. They provide a landing
platform for the spindle-
assembly checkpoint (SAC)
proteins.

Inner kinetochore components

associate with kinetochore
throughout the cell cycle

BOR, SUR AurB INCENP and

MCAK populate the centromere
region and regulate the stability
of microtubule-kinetochore
attachments