REVIEW / SYNTHE`SE
Epigenetic regulation of centromere formation
and kinetochore function1
Ryan Heit, D. Alan Underhill, Gordon Chan, and Michael J. Hendzel
Abstract: In the midst of an increasingly detailed understanding of the molecular basis of genome regulation, we still
only vaguely understand the relationship between molecular biochemistry and the structure of the chromatin inside of cells.
The centromere is a structurally and functionally unique region of each chromosome and provides an example in which
the molecular understanding far exceeds the understanding of the structure and function relationships that emerge on the
chromosomal scale. The centromere is located at the primary constriction of the chromosome. During entry into mitosis,
the centromere specifies the assembly site of the kinetochore, the structure that binds to microtubules to enable transport
of the chromosomes into daughter cells. The epigenetic contributions to the molecular organization and function of the
centromere are reviewed in the context of structural mechanisms of chromatin function.
Key words: centromere, kinetochore, chromatin, CENP-A, intrinsic disorder, pericentromeric heterochromatin, histone H3
lysine 9 methylation.
Re ´sume ´ : Malgre ´ la compre ´hension de plus en plus de ´taille ´e que l’on a des bases mole ´culaires de la re ´gulation ge ´no-
mique, on ne connaı ˆt encore que vaguement la relation qui existe entre la biochimie mole ´culaire et la structure de la chro-
matine a ` l’inte ´rieur des cellules. Le centrome `re est une re ´gion structurellement et fonctionnellement unique a ` chaque
chromosome qui fournit un exemple ou ` la compre ´hension mole ´culaire de ´passe de beaucoup la compre ´hension de la rela-
tion qui existe entre la structure et la fonction a ` l’e ´chelle chromosomique. Le centrome `re est localise ´ a ` la constriction pri-
maire du chromosome. Lors de l’entre ´e en mitose, le centrome `re spe ´cifie le site d’assemblage du kine ´tochore, la structure
qui se lie aux microtubules et permet le transport des chromosomes dans les cellules filles. La contribution e ´pige ´ne ´tique a `
l’organisation mole ´culaire et a ` la fonction du centrome `re est passe ´e en revue dans le contexte des me ´canismes structuraux
de la fonction de la chromatine.
Mots cle ´s : centrome `re, kine ´tochore, chromatine, CENP-A, disordre intrinse `que, he ´te ´rochromatine pe ´ricentrome ´rique, his-
tone H3 me ´thylation de lysine 9.
[Traduit par la Re ´daction]
The centromere is essential for the correct segregation of
sister chromatids during cell division. During entry into mi-
tosis, the centromeric (CEN) chromatin specifies the initia-
tion of the assembly of the kinetochore (Chan et al. 2005),
which is a massive multiprotein assembly occupying a sur-
face area of approximately 0.2 ?m2/kinetochore (Cherry et
al. 1989). The kinetochore mediates microtubule attachment
at the centromeres during mitosis. Remarkably, as CEN se-
quences have been characterized across species, the centro-
mere was found to be one of the fastest evolving regions of
the genome (Malik et al. 2002). Although this may account
for the lack of primary sequence conservation of the centro-
mere, certain elements, such as alphoid satellite DNA and
GGAAT repeats, are common (Grady et al. 1992; Nakano
et al. 2003) and are known to contribute to the assembly of
the human centromere. For instance, the centromere protein
(CENP) CENP-B is a DNA binding protein that binds spe-
cifically to a 17 bp sequence repeated in a-satellite DNA
(Masumoto et al. 1989; Muro et al. 1992). Ectopic alphoid
satellite sequences will preferentially recruit kinetochore
proteins and form active centromeres when placed under se-
lective pressure (Nakano et al. 2003).
Although the DNA binding specificity of CENP-B is con-
sistent with a model in which DNA sequence dictates cen-
tromere formation, this can be ruled out because CENP-B is
not required for the formation of a functional centromere
(Kipling and Warburton 1997) and under normal conditions,
Received 20 March 2006. Revision received 24 May 2006.
Accepted 29 May 2006. Published on the NRC Research Press
Web site at http://bcb.nrc.ca on 17 August 2006.
R. Heit, G. Chan, and M.J. Hendzel.2Department of
Oncology, University of Alberta, Edmonton, AB T6G 1Z2,
D.A. Underhill. Department of Medical Genetics, Faculty of
Medicine, University of Alberta, Edmonton, AB, Canada.
1This paper is one of a selection of papers published in this
Special Issue, entitled 27th International West Coast Chromatin
and Chromosome Conference, and has undergone the Journal’s
usual peer review process.
2Corresponding author (e-mail: email@example.com).
Biochem. Cell Biol. 84: 605–618 (2006) doi:10.1139/O06-080
##2006 NRC Canada
alphoid DNA is not sufficient for the recruitment of many
essential kinetochore proteins, such as CENP-A, CENP-C,
and CENP-E (Nakano et al. 2003; Sullivan and Schwartz
1995; Warburton 2001; Warburton et al. 1997) (see the sec-
tion of this review entitled The biochemistry of chromatin at
the centromere). The migration of centromeres within other-
wise conserved arrangements of genes and the existence of
neocentromeres in humans provide convincing evidence that
centromere specification is determined by an epigenetic
rather than a sequence-specific mechanism (Warburton
Warburton (2001) did a comprehensive cataloguing of
protein recruitment to normal, neo-, and inactive centro-
meres. Neocentromeres are fully functional centromeres that
do not contain a-satellite DNA, while the term inactive re-
fers to the inactive centromere in a dicentric chromosome.
Certain characteristics, such as intense 4’,6-diamidino-2-
phenylindole (DAPI) staining (denoting highly condensed
AT-rich DNA) and a-satellite DNA, were shared among the
normal and inactive centromeres but were absent from neo-
centromeres. Since neocentromeres are fully functional cen-
tromeres, yet lack a-satellite DNA, it can be concluded that
these sequences are neither sufficient nor required for the
formation of a centromere. This result was corroborated by
a recent study showing that chromatin containing CENP-A
is able to spread over non-a-satellite DNA when non-a-
satellite DNA and a-satellite DNA are used to construct hu-
man artificial chromosomes (Lam et al. 2006). Thus, it is
the composition of the chromatin, and not the underlying
DNA sequence, which is important for specifying a func-
Chromatin structure and function
Epigenetic mechanisms are mediated through chromatin
structure. This relationship has been evident since the ear-
liest light microscopy studies on chromosomes and nuclei.
The DNA present in the eukaryotic cell has been character-
ized by early light microscopists as comprising both euchro-
matin and heterochromatin. Heterochromatin is defined as
the chromatin that remains compact and visible throughout
the cell cycle. In contrast, euchromatin decondenses in inter-
phase to the extent that it is no longer directly visible in the
light microscope. Heterochromatin can be further catego-
rized as constitutive or facultative, based upon whether or
not there is a consistent relationship between the DNA se-
quence involved and a compact organization across cell
types and differentiation states. The former is exemplified
by CEN and pericentromeric heterochromatin, which are vir-
tually always highly condensed, while the latter contains se-
quences that are either heterochromatic or euchromatic,
depending on the cell type and state of differentiation. The
inactivation of 1 of the 2 human X chromosomes in females
is an example of facultative heterochromatin.
Beginning in the 1980s, biochemical properties that dis-
tinguish these different morphological classes of interphase
chromatin have been identified and a mechanistic under-
standing of how heterochromatin is established and main-
tained began to emerge. In this regard, DNase I digestion
kinetics have proven to be one of the most revealing fea-
tures of chromatin and reflect the close relationship between
structure and function, where differences in function are re-
flected in the accessibility of the underlying DNA sequence
to the nuclease probe. At the coarsest level, sequences that
are transcribed or in a chromatin conformation compatible
with transcription are digested about 3 times more rapidly
than sequences that are never transcribed (Weintraub and
Groudine 1976). Increased rates of digestion are correlated
with an enrichment in histone acetylation and reduced his-
tone H1 content (Iovcheva et al. 1984; Krajewski and
Becker 1998; Perry and Annunziato 1989; Ridsdale et al.
1988). At higher resolution, small regions of sequences in-
volved in transcriptional regulation are digested at rates that
are at least 10 times faster than the surrounding sequences.
These sites are consistently associated with the binding of
proteins directly involved in the regulation of transcriptional
activation (Keene et al. 1981; Lu and Richardson 2004).
The basic unit of chromosome structure, the nucleosome,
partitions the DNA into units of approximately 200 bp in
length. At the molecular level, each chromosome is a repeat
of nucleosomes and shorter segments of DNA that link the
individual nucleosomes. The nucleosome core particle com-
prises 147 bp of DNA that make 1.75 turns around the outer
surface of a protein octamer assembled from a tetramer of
histones H3 and H4 and 2 dimers of histones H2A and H2B
(Luger et al. 1997). The linking DNA is associated with a
fifth histone, histone H1, which binds DNA as it enters and
exits the nucleosome to stabilize 2 complete turns of the
DNA around the histone octamer (Thomas 1999). Histone
proteins are the substrate for post-translational modifica-
tions. It is these post-translational modifications, largely lo-
cated in the N-terminal domains of the histone proteins,
which encode most of the epigenetic information specifying
chromatin structure and function (Bradbury 1992; Davie
1996; Shilatifard 2006). For example, reduced acetylation
of the N-terminal lysines of histone H4 (Braunstein et al.
1993; Kristjuhan et al. 2003; Richards and Elgin 2002), tri-
methylation of lysine 20 on histone H4 (Biron et al. 2004;
Kourmouli et al. 2004; Schotta et al. 2004), trimethylation
of lysine 9 on histone H3 (Fischle et al. 2003; Gonzalo et
al. 2005; Peters et al. 2001; Rice et al. 2003), and trimethy-
lation of lysine 27 on histone H3 (Chadwick and Willard
2004) have all been correlated with heterochromatin struc-
tures. The N-terminal domains of the histones are not re-
quired for the formation of the nucleosome (Hayes et al.
1991), but do regulate the folding of the nucleosome poly-
mer into more compact conformations (Fletcher and Hansen
1995; Garcia-Ramirez et al. 1992; Krajewski and Ausio
The biochemistry of chromatin at the
Chromatin in the centromere differs biochemically from
the remainder of the genome in some very fundamental
ways. Early studies indicated that 3 proteins, CENP-A,
CENP-B, and CENP-C are specific to functional centro-
meres (Earnshaw and Migeon 1985). Following this discov-
ery, sequencing of CENP-A revealed it to be a homolog of
histone H3 (Palmer et al. 1987, 1989, 1991). These results
were corroborated by the discovery that CENP-A substitutes
for H3 in active CEN and neocentromeric nucleosomes, but
606Biochem. Cell Biol. Vol. 84, 2006
##2006 NRC Canada
is not present at inactive centromeres (Palmer et al. 1987;
Warburton 2001; Warburton et al. 1997). In addition to the
obvious functional differences between CEN and nonCEN
chromatin, CEN heterochromatin is much less effective than
the surrounding pericentromeric heterochromatin at repres-
sing transcription (Allshire et al. 1994; Lam et al. 2006; Pi-
doux and Allshire 2005).
An important advance in our understanding of the epige-
netic status of CEN heterochromatin came with the analysis
of linearized chromatin fibers. By stretching chromatin on
glass slides, Sullivan and Karpen (2004) mapped histone H3
and CENP-A distribution in HeLa and Drosophila CEN
chromatin. This study determined that the Drosophila
CENP-A homologue CID is incorporated as 10–40 kb long
clusters of nucleosomes interspersed with H3-containing nu-
cleosomes. Sullivan and Karpen (2004) were also able to de-
fine the post-translational modification status of CEN
heterochromatin. They showed that the H3-containing nucle-
osomes within CEN chromatin were hypoacetylated, which
is typical of heterochromatin, and enriched in dimethylated
lysine 4, a modification typically associated with potentiated
regions of chromatin (see also Lam et al. 2006). This result
has been validated by chromatin immunoprecipitation ex-
periments (Cam et al. 2005; Lam et al. 2006).
Using a human artificial chromosome model, Lam et al.
(2006) used this same linearization of chromatin technique,
combined with chromatin immunoprecipitation, to show that
CEN and pericentromeric heterochromatin exist in a dy-
namic equilibrium. Under normal conditions, CEN hetero-
chromatin that contains CENP-A is flanked by chromatin
enriched in dimethylated lysine 9, which separates the CEN
heterochromatin from the pericentromeric constitutive heter-
ochromatin. Constitutive heterochromatin is demarked by
enrichment in trimethylated lysine 9. When CENP-A is
overexpressed, the CEN heterochromatin expands and dis-
places dimethylated lysine 9-enriched chromatin. The trime-
thylation of lysine 9 in the flanking pericentromeric
heterochromatin also increases, which Lam et al. (2006)
suggest is a compensatory mechanism in response to ex-
panding CEN chromatin.
The centromere-specific histone H3
Collectively, these results imply that CENP-A is the crit-
ical feature that specifies the formation of a functional cen-
tromere. CENP-A association, however, is insufficient for
centromere formation. This has been shown by the incorpo-
ration of CENP-A into regions of the genome outside of
centromeres as a result of CENP-A overexpression. These
experiments have demonstrated that CENP-A is not suffi-
cient to generate a functional centromere (Van Hooser et al.
2001). Nonetheless, it is equally clear that CENP-A incorpo-
ration is a feature of all functional centromeres.
The greatest divergence in CENP-A and histone H3 is in
the N-terminal sequence. Interestingly, the N-terminus of
CENP-A also differs significantly among species (Fig. 1). It
has been suggested that this divergence of the N-terminal
tail of CENP-A homologs reflects the divergence in the
underlying DNA sequences that function as centromeres
among different species (Malik et al. 2002) . In this context,
the N-terminal domain of CENP-A is thought to interact
with linker DNA connecting individual nucleosomes. More-
over, in Saccharomyces cerevisiae, the CENP-A homolog
requires the N-terminal tail for proper function. CENP-A is
required for the assembly of at least 1 protein complex at
the yeast centromere (Chen et al. 2000), and is able to re-
cruit CENP-C, hSMC1, and hZW10 in human cells under
conditions in which functional centromeres are not formed
(Van Hooser et al. 2001).
It is noteworthy that the N-terminal domain of CENP-A
proteins contain a sequence repeat that is similar to the
SPKK DNA binding motif found in the C-terminal domain
of H1 histones (Churchill and Suzuki 1989; Malik et al.
2002). (Fig. 1). The N-terminal domain of CENP-A may be
similar to the C-terminus of histone H1 in another important
way. The C-terminus of histone H1 is unstructured in solu-
tion but is now thought to adopt secondary and tertiary
structure upon binding to DNA at the surface of the nucleo-
some (Hendzel et al. 2004; Roque et al. 2005; Vila et al.
2000, 2001). The C-terminus is also engaged in a number
of different but functionally important interactions within
chromatin (Hansen et al. 2006). The ability of a discrete pol-
ypeptide sequence to engage in multiple specific interactions
is a property that is common to proteins with unusually high
proportions of disorder-producing amino acids (see Table 1
for the composition of CENP-A). Such proteins can adopt
remarkably different structures for the same polypeptide se-
quence, thereby allowing very different yet specific protein–
protein interactions (Dunker et al. 2005). We therefore pro-
pose that the CENP-A N-terminal domain, similar to what
has been suggested for the histone tail domains (Hansen et
al. 2006), is intrinsically disordered and adopts unique sec-
ondary and tertiary structures specific to the interacting li-
gand. This would provide versatility to the domain despite
being coded for by a short stretch of sequence.
Although the N-terminal domain represents the site of
greatest divergence with the major histone H3 subtypes, the
N-terminal domain does not specify CENP-A incorporation
into the centromere. Rather, the changes that dictate incor-
poration specifically into the centromere are contained
within the globular domain of histone H3. Divergence from
histone H3 in the L1 loop and the a2-helix are necessary
and sufficient to target CENP-A to the centromere (Black et
al. 2004). When this domain is placed into the histone H3
sequence, the synthetic histone H3 targets to the centromere
(Black et al. 2004). Using deuterium exchange coupled with
mass spectrometry, Black et al. (2004) further demonstrated
that the CENP-A/H4 tetramer is more compact and structur-
ally rigid than the H3/H4 tetramer. Although speculative, an
altered nucleosome structure may contribute to the loss of
micrococcal nuclease nucleosomal repeat pattern that has
been observed at centromeres (Saitoh et al. 1997). This al-
tered micrococcal nuclease digestion pattern has been shown
to require CENP-A incorporation (Takahashi et al. 2000).
The assembly of CENP-A into centromeric
Since CENP-A replaces histone H3 in nucleosomes within
CEN chromatin, it might be expected that CENP-A incorpo-
ration occurs during S phase. It has long been known that
Heit et al.607
##2006 NRC Canada
euchromatin and heterochromatin replicate at different
points during S phase, and this could be a mechanism by
which uncommon histone variants are incorporated. By ex-
amining the replication timing of several chromosomes in
Drosophila, the timing of centromere replication was found
to vary among chromosomes from early to late S phase
(Sullivan and Karpen 2001). The replication of H3-contain-
ing nucleosomes and CID-containing nucleosomes, both
within the centromere, were also shown to occur at the
same time (Blower et al. 2002). Similar results were ob-
served in mammalian systems. When dicentric CHO cells
were examined, CENP-A was incorporated into active, but
not inactive, centromeres. Both the active and inactive cen-
tromeres were then compared, and no difference in replica-
tion timing was found (Ouspenski et al. 2003). Similarly,
HeLa cells were shown to replicate CENP-A-associated se-
quences in mid- to late-S phase, overlapping with the repli-
cation timing of other regions in the chromosome (Blower et
al. 2002; Shelby et al. 2000; Sullivan and Karpen 2001).
Although histone proteins incorporated into the nucleo-
some are among the most stable proteins in the nucleus, it
is also well established that histones can be incorporated
into chromatin outside of S-phase (Kimura 2005). CENP-A
is able to be incorporated into CEN DNA in the presence of
aphidicolin, a DNA replication inhibitor (Shelby et al.
2000), indicating that CENP-A is similarly capable of incor-
poration into chromatin outside of S phase. This implies that
replication-independent nucleosome assembly is the mecha-
nism of specifying CENP-A incorporation into the centro-
mere. Consistent with this hypothesis, the analysis of
CENP-A gene expression in HeLa cells revealed that
CENP-A mRNA and protein expression were found to be
most abundant in G2 (Shelby et al. 2000). When CENP-A
expression is forced throughout S phase, its incorporation is
not centromere specific (Shelby et al. 1997). Thus, replica-
tion-independent nucleosome assembly may be essential to
maintain the specificity of CENP-A incorporation.
The H3/H4 tetramer, which forms the core of the nucleo-
some, is vastly more stable in vivo than most protein–DNA
complexes (Kimura and Cook 2001), which turn over in sec-
onds to minutes (Phair et al. 2004). Despite this, there is a
small amount of incorporation of new H3 and H4 outside of
S phase (Hendzel and Davie 1990; Jackson 1990). During
G1 and G2, a unique histone H3 variant, histone H3.3, is in-
corporated into chromatin and serves as an example of repli-
Henikoff 2002; Hendzel and Davie 1990). This is accom-
plished through a chromatin assembly factor, HIRA, which
specifically recognizes histone H3.3 (Ray-Gallet et al. 2002;
Tagami et al. 2004). The existing data support the hypothe-
sis that CENP-A, like histone H3.3, is incorporated into nu-
cleosomes in G2 (Shelby et al. 1997, 2000). It is therefore
expected that a unique chromatin assembly factor that is
specific for CENP-A and responsible for the deposition of
CENP-A specifically at centromeres will be identified. The
CENP-A protein itself has been postulated to serve as the
epigenetic mark that directs CENP-A incorporation after S
phase. The equal segregation of nucleosomes that contain
CENP-A to daughter strands would result in the presence of
a CENP-A mark interspersed in H3.2 and (or) H3.1. Alter-
natively, other epigenetic marks that are reported to be
present at centromeres, such as unacetylated histone H3 that
is dimethylated at lysine 4, could provide the target for
chromatin remodeling machinery and the incorporation of
additional CENP-A into chromatin by a unique chromatin
In Schizosaccharomyces pombe, the genetics of CENP-A
incorporation are relatively well defined. In this organism,
there are 2 separate pathways for CENP-A incorporation and
the protein is incorporated in both S phase and in G2. S-phase
incorporation requires a GATA family member, Ams2 (Taka-
hashi et al. 2005). The G2 pathway, however, is of particular
relevance to the replication-independent assembly that ap-
pears to operate in mammalian cells. This pathway is depend-
ent upon the Schizosaccharomyces pombe homologues of
RbAp46 and RbAp48 (Hayashi et al. 2004; Takahashi et al.
2005). These proteins have a number of functions in chroma-
tin and are associated with chromatin-remodeling complexes,
(Loyola and Almouzni 2004; Zhang et al. 1999). In Schizo-
saccharomyces pombe, these proteins are responsible for re-
cruiting a complex of Mis6, Mis 15, and Mis 17 (Hayashi et
al. 2004). Depletion of RbAp46 and RbAp48, but not either
one alone, was sufficient to deplete CENP-A from the centro-
meres of HeLa cells and led to defects in chromosome segre-
gation (Hayashi et al. 2004) Thus, a complex containing both
proteins or complexes involving RbAp46 and (or) RbAp48
appear to specify a replication-independent pathway for
CENP-A assembly that is conserved from Schizosaccharomy-
ces pombe to mammals.
and histone chaperones
Fig. 1. Alignment of the N-terminal domain of vertebrate CENP-A. The N-terminal sequences from human histone H3.2 (H3) and H3.3 are
shown in comparison with CENP-A from several vertebrates. The regions highlighted in grey indicate the positions of motifs that may be
homologous to the SPKK (serine-proline-lysine-lysine)-type repeats found in the C-terminal domain of H1 histones.
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Constitutive and cell-cycle dependent
proteins associated with the centromere
In addition to CENP-A, other proteins have been found to
localize to the centromere throughout the cell cycle. Since
most kinetochore proteins are recruited to the centromere
only at mitosis, proteins that are constitutively present at
the centromere may play an important role in the specifica-
tion of the centromere. To date, it is evident that at least 6
proteins are constitutively centromeric, CENP-A, CENP-B,
CENP-C, CENP-H, CENP-I (hMis6), and hMis12 (Chan et
al. 2005), and thus each could function within the specifica-
tion of the centromere. As mentioned previously, CENP-B is
not required for a functional centromere (Perez-Castro et al.
1998; Warburton 2001) and so must not be a crucial protein
for centromere formation. Recruitment of each of CENP-C,
CENP-H, and CENP-I appear to be downstream of CENP-A
localization. CEN localization of CENP-C requires both
CENP-H and CENP-I, while CENP-H and CENP-I are de-
pendant on each other for proper localization (Fukagawa et
al. 2001; Nishihashi et al. 2002). Additionally, although
CENP-A and hMis12 are both needed for CENP-I localiza-
tion, the knockdown of any one of CENP-C, CENP-H, or
CENP-I has no effect on CENP-A localization (Fukagawa
et al. 2001; Goshima et al. 2003; Liu et al. 2003; Nishihashi
et al. 2002). This contradicts the expectation, based on
Schizosaccharomyces pombe experiments, that CENP-I, the
human Mis12 homologue, is required for replication inde-
pendent assembly of CENP-A into CEN nucleosomes (Hay-
ashi et al. 2004). However, a more recent study indicates
that a complex containing CENP-H and CENP-I, when de-
pleted from DT40 cells, results in a failure to incorporate
newly synthesized CENP-A (Okada et al. 2006). In all cases,
however, these studies demonstrate that CENP-A incorpora-
tion is central to the assembly of a functional centromere.
Epigenetic regulation of the centromere in
Schizosaccharomyces pombe has proven to be a very in-
formative genetic model system for the study of centromere
function and the epigenetic regulation of the centromere.
The fission yeast system has clearly demonstrated the im-
portance of pericentromeric heterochromatin in the function
of the centromere.
In fission yeast, the pericentromeric heterochromatin is in-
itiated through at least 2 independent pathways, the RNA in-
terference (RNAi) pathway and a clr3 pathway (Hansen et
al. 2005; Yamada et al. 2005). Clr3 is a fission yeast homo-
logue of mammalian class II histone deacetylases (HDACs)
and is required for the efficient deacetylation of lysine 14 of
histone H3 in fission yeast centromeres (Bjerling et al. 2002;
Wiren et al. 2005). Either pathway is capable of specifying
the recruitment of clr4 (Nakayama et al. 2001), which is the
fission yeast homologue of human SUV39H1 and H2, 2 en-
zymes responsible for the trimethylation of lysine 9 on his-
tone H3 (Nakayama et al. 2001). Clr3 and clr4 were
originally identified as genes required for the stable repres-
sion of the silent mating type loci in Schizosaccharomyces
pombe and are part of a complementation group that in-
cludes swi6 (Ekwall and Ruusala 1994). The trimethylated
form of histone H3 lysine 9 generates a binding site that is
recognized by the chromodomain of swi6 (Hall et al. 2002).
Swi6 is the fission yeast homologue of heterochromatin
protein 1 (HP1) (Lorentz et al. 1994), both of which con-
tain a chromodomain that binds to histone H3 trimethylated
on lysine 9 (Hall et al. 2002). Mutations in the histone H3
N-terminus at lysine 9 or 14, as well as at serine 10, are
sufficient to delocalize swi6 and result in defects in chro-
mosome segregation (Mellone et al. 2003).
Unlike the Schizosaccharomyces pombe proteins associ-
ated with the centromere, those associated with the pericen-
tromeric heterochromatin are not essential for viability. Loss
of genes involved in the dicer (Provost et al. 2002; Volpe et
al. 2003) and clr3 pathways of heterochromatinization
(Grewal et al. 1998; Nakayama et al. 2001; Yamada et al.
2005) consistently generate a mitotic defect characterized
by lagging chromosomes in anaphase. Both pathways con-
verge on the clr4 histone methyltransferase, which estab-
lishes the swi6 binding site (Yamada et al. 2005). The swi6
protein has been shown to be directly involved in the re-
cruitment of the cohesin complex, which is enriched at the
inner centromere (Bernard and Allshire 2002; Bernard et al.
2001; Nonaka et al. 2002). When cohesin is disrupted, a
similar phenotype is observed (Toyoda et al. 2002). Addi-
tional cohesion may be necessary to resist the force that is
being directly applied to the kinetochore and to mechani-
cally couple the chromatids.
During mitosis, the inner centromere binding protein (IN-
CENP) targets the Aurora B kinase to the pericentromeric
heterochromatin of the inner centromere (Adams et al.
2001a). Aurora B phosphorylates histone H3 at serine 10 at
the onset of entry into mitosis. This correlates with the dis-
placement of HP1 in both Schizosaccharomyces pombe and
human cells (Fischle et al. 2005; Hirota et al. 2005). The
purpose or function of HP1 displacement during mitosis is
not known. Site-directed mutagenesis of lysine 9, serine 10,
and lysine 14 revealed that each of these amino acids were
important in recruiting HP1 to pericentromeric heterochro-
matin in Schizosaccharomyces pombe (Mellone et al. 2003).
Moreover, mutations at each of these sites lead to chromo-
some segregation defects. The targeting of cohesin to peri-
centromeric heterochromatin has not been determined in
these mutant backgrounds, but the requirement for HP1 in
the assembly of cohesin into pericentromeric heterochroma-
tin (Nonaka et al. 2002) predicts that cohesin assembly
should also be impaired.
There may be contributions of protein methylation in ki-
netochore function that are independent of histones. In Sac-
charomyces cerevisiae, a histone methyltransferase, Set1,
and an Aurora kinase, IpI1, have been shown to have similar
antagonizing effects independent of histone modification
(Zhang et al. 2005). The common substrate that seems to
mediate this effect in Saccharomyces cerevisiae is a kineto-
chore protein, Dam1. Dam1 forms a collar around the end of
microtubules (Westermann et al. 2005) and may couple mi-
crotubule depolymerization with chromosome movement by
interacting with the kinetochore-associated Ndc80 complex
(Salmon 2005). Although it is expected that a homologue of
Dam1 exists in higher eukaryotes, none has been found to
date. In addition, Set1p catalyzes the dimethylation of lysine
4 on histone H3, and mutation of this lysine to arginine par-
tially suppresses the effects of loss of Ipl1 kinase function in
Heit et al. 609
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Saccharomyces cerevisiae (although not as completely as
the loss of Set1 function) (Zhang et al. 2005). Thus, in
higher eukaryotes, the function of lysine methylation, and
even of specific lysine methyltransferases, in kinetochore
function may be complex and involve both histone and non-
Epigenetic regulation of centromere function
and genomic stability in human cells
Human centromeres, like those of Schizosaccharomyces
pombe, are also associated with pericentromeric heterochro-
matin domains that recruit HP1 through a mechanism that
requires the methylation of histone H3 at lysine 9 (Fischle
et al. 2005). Thus, we might expect that deletions of Suv39,
the mammalian homologue of clr4, would compromise the
function of the centromere. Knockout studies of the
Suv39h1 and Suv39h2 homologs in mouse results in wide-
spread genomic instability and increased incidence of lym-
phomas (Peters et al. 2001). However, the reported
segregation defects, which are more consistent with in-
creased cohesion and an impaired ability to dissociate cohe-
sins, are not what would be predicted if the results from
Schizosaccharomyces pombe directly applied to humans.
More recently, we characterized chromosome segregation
defects in immortalized mouse embryonic fibroblasts iso-
lated from Suv39h1/h2 double null mice. We found an ap-
proximately 4-fold increase in the types of chromosome
alignment and chromosome segregation defects observed in
Schizosaccharomyces pombe (McManus et al. 2006). These
same defects are observed in wild-type cells following as lit-
tle as 2 h of treatment with a protein methylation inhibitor
(Fig. 2). These results are consistent with a requirement for
histone lysine methylation in the recruitment of cohesin to
HDAC inhibitors are being developed as potential chemo-
therapeutic agents for the treatment of a spectrum of human
cancers. Based upon the important roles of clr3 and clr6
HDACs in Schizosaccharomyces pombe, which deacetylate
lysine 14 and lysine 9 of histone H3 (Bjerling et al. 2002),
we would expect that inhibition of these proteins would
cause segregation defects due to acetylation-dependent in-
hibition of lysine 9 trimethylation. This would be expected
to result in a loss of cohesins at the inner centromere.
HDAC inhibitors have been found to alter the formation of
kinetochores on CEN DNA and cause a failure to properly
capture microtubules and an accumulation of cells at prome-
taphase (Shin et al. 2003). In our hands, however, there are
qualitative differences between the mitotic defects observed
in the absence of trimethylation of lysine 9 and those seen
with deacetylase inhibitor treatments (G.K. Chan and M.J.
Hendzel, unpublished observations). Thus, it is not entirely
clear that the mitotic defects observed with histone deacety-
lase inhibitors can be attributed to reduced recruitment of
The function of pericentromeric
heterochromatin in genomic stability and
Although the basic mechanisms of heterochromatin as-
sembly are conserved in mammals and Schizosaccharomyces
pombe, mammalian systems have additional machinery
specifying the initiation of heterochromatin formation. In
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Table 1. Amino acid composition of the N-terminus of CENP-A.
Homo sapiens (human)
Mus musculus (mouse)
Rattus norvegicus (rat)
Bos taurus (cow)
Canis familiaris (dog)
Cricetulus griseus (hamster)
Gallus gallus (chicken)
Danio renio (zebrafish)
Note: D, disorder; N, order; O, neither; pI, isoelectric point.
mammals, this machinery includes the following: pericentro-
meric heterochromatin that is characteristically enriched in
HP1 (Minc et al. 1999), trimethylation of lysine 9 on histone
H3 (Peters et al. 2003), the trimethylation of histone H4 at
lysine 20 (Biron et al. 2004; Schotta et al. 2004), hypoacety-
lated histones H3 and H4 (Johnson et al. 1998), and cytosine
methylation of DNA (Henikoff 2000). The trimethylation of
histone H4 at lysine 20 in heterochromatin is not conserved
in Schizosaccharomyces pombe (Sanders et al. 2004). The
enzymatic machinery that carries out these modifications is
regulated by the Rb family of proteins and includes HDACs,
histone methyltransferases, HP1, and DNA methylation
(Brehm et al. 1998; Luo et al. 1998; Magnaghi-Jaulin et al.
1998; Pradhan and Kim 2002; Robertson et al. 2000; Vaute
et al. 2002; Zhang et al. 2000). Not surprisingly, each of
these pathways is linked to genomic instability and is asso-
ciated with human cancers. The basis of this genomic insta-
bility, however, is not clear.
The study of mouse embryonic fibroblasts from triple-null
mice for the 3 Rb family member genes revealed that the
trimethylation of lysine 20 on histone H4, but not lysine 9
on histone H3, was lost (Gonzalo et al. 2005). Unfortu-
nately, the analysis of the mitotic defects in these cells was
restricted to karyotype analysis and ploidy determination.
Fig. 2. Treatment with a protein methylation inhibitor induces chromosome alignment defects. Three-dimensional maximum point projec-
tion deconvolution images are shown of a HeLa cell treated for 2 h with adenosine dialdehyde prior to fixation. Microtubules are shown in
blue, chromosomes are shown in blue, and anticentromeric antigen staining is shown in green.
Heit et al.611
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The phenotype of these cells includes elongated telomeres,
an apparent defect in the release of cohesion at the centro-
meres, and genomic instability with a tendency to increase
chromosome number with increased passage (Gonzalo et al.
2005). This phenotype parallels what has been reported for
the Suv39 double null mice (Peters et al. 2001). Importantly,
cells lacking all Rb family members, unlike those from
Suv39 double-null mice, maintained histone H3 lysine 9 tri-
methylation in pericentromeric heterochromatin (Gonzalo et
al. 2005). Although it has not been directly tested, it is
likely that the retention of this modification would allow
proper localization of HP1 proteins, even in the absence of
trimethyl K20. This implies the existence of a lysine 20
methylation-dependent regulation of cohesin binding or deg-
radation that operates independently of HP1. In this case, the
absence of lysine 20 trimethylation contributes to increased
stability of cohesins that are associated with pericentromeric
heterochromatin. It is important to note, however, that the
loss of lysine 9 trimethylation in pericentromeric heterochro-
matin results in the failure to properly target histone H4 ly-
sine 20 trimethylation (Schotta et al. 2004). Thus, cells
deficient in Suv39 lack both lysine 9 trimethylation and ly-
sine 20 trimethylation in pericentromeric heterochromatin.
The dynamics of pericentromeric
heterochromatin during mitosis
In early G2, the pericentromeric heterochromatin is phos-
phorylated by the Aurora B kinase (Adams et al. 2001b;
Fischle et al. 2005; Zeitlin et al. 2001). This targeting occurs
as part of the INCENP–survivin–Aurora B complex (Adams
et al. 2001a). The resulting phosphorylation of serine 10
correlates with the displacement of HP1a, HP1b, and HP1g,
all reaching a maximum in metaphase, although a small
amount of HP1a is retained near the centromeres (Bartova
et al. 2005; Fischle et al. 2005; Hirota et al. 2005). A ques-
tion that then arises is whether HP1 displacement is neces-
sary to enable cohesin dissociation following cleavage by
the anaphase promoting complex. Although the displace-
ment of HP1 by serine 10 phosphorylation implies impor-
Fig. 3. Cell-cycle associated changes in lysine 9 trimethylation. The neural tube of a 9.5-day-old mouse embryo stained with antitrimethy-
lated lysine 9 (histone H3) and 4’,6-diamidino-2-phenylindole (DNA). A portion of the neural tube is shown. The cells nearest the lumen
(left) are in various stages of mitosis, whereas the cells distant from the lumen are in interphase. The methylation is largely confined to the
Fig. 4. Aurora B localization in G2 mouse fibroblasts that are wild-type or Suv39h1/Suv39h2 double null. Two Suv39h1/Suv39h2 double-
null mouse embryonic fibroblast cell lines and 1 wild-type mouse embryo fibroblast cell line were stained with antitrimethylated K9 and
anti-Aurora B. An example of a G2 cell where, in wild-type cells, phosphorylation of serine 10 initiates in pericentromeric heterochromatin
and Aurora B is recruited to these sites. The first composite shows the staining of Aurora B (green) and trimethylated lysine 9 (red). The
second composite also includes 4’,6-diamidino-2-phenylindole stained chromatin (blue).
612 Biochem. Cell Biol. Vol. 84, 2006
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tance, mutation of this residue to alanine causes segregation
defects in Schizosaccharomyces pombe that are more consis-
tent with an inability to load condensin onto pericentromeric
heterochromatin (Mellone et al. 2003). Moreover, restricting
the function of lysine 9 trimethylation to the recruitment of
HP1 and, thus indirectly cohesin, is difficult to reconcile
with the cell-cycle-dependent changes in histone H3 lysine
9 methylation in pericentromeric heterochromatin (Fig. 3).
This post-translational modification increases 4-fold from
early G2, when the phosphorylation of serine 10 is initiated,
to metaphase, when the phosphorylation of serine 10 is
widespread throughout the genome (McManus et al. 2006).
Although there is some retention of HP1a at these sites, the
binding of HP1 is reduced relative to the levels observed
during interphase (Bartova et al. 2005).
A possible explanation for these observations is the be-
havior of the Aurora B kinase in the absence of lysine 9 tri-
methylation. Specifically, Aurora B is not properly targeted
to pericentromeric heterochromatin during G2 (Fig. 4) and
the initiation of serine 10 phosphorylation is spatially and
temporally altered relative to wild-type cells. This raises the
possibility that some of the mitotic defects observed in the
absence of histone lysine 9 trimethylation are a result of a
reduction in Aurora B kinase activity in this domain. In the
absence of Aurora kinase activity in Drosophila, histone H3
is not phosphorylated during mitosis (Adams et al. 2001b),
and this correlates with an increase in the number of lagging
chromosomes observed in anaphase figures (Adams et al.
2001b; Giet and Glover 2001). It also correlates with an in-
ability to recruit condensin, which compacts the metaphase
chromosome (Giet and Glover 2001). Thus, we must con-
sider the possibility that some of the mitotic defects ob-
served in the absence of histone H3 lysine 9 trimethylation
are a result of a reduction in Aurora B kinase activity in
From molecular biochemistry to three-
The principal determinant of centromere formation is the
replication-independent incorporation of CENP-A, a histone
H3 homolog, into the sequences that assemble the centro-
mere. Centromeres contain clusters of nucleosomes that con-
tain CENP-A, which are interspersed with clusters of
nucleosomes that contain histone H3 (Sullivan and Karpen
2004). Nonetheless, in 3 dimensions, these domains remain
segregated into a discrete chromatin structure throughout
the cell cycle (Sullivan and Karpen 2004). The properties to
specify the formation of such a structure, it would seem, re-
side in the N-terminal domain of CENP-A. While this do-
main shows the greatest divergence with the major histone
H3 subtypes, it also shows considerable length and sequence
variability among species. This remarkable conservation of
function, exemplified by the functional replacement of the
CENP-A homologue in human cells that were knocked
down using RNAi with Cse4 from Saccharomyces cerevi-
siae (Wieland et al. 2004), can be explained by the intrinsic
disorder hypothesis of histone tail function recently pro-
posed by Hansen and colleagues (Hansen et al. 2006). Like
the C-terminal domain of histone H1 and the N-terminal do-
mains of the core histones, the N-terminus of CENP-A con-
tains a very high proportion of amino acids that favor a
disordered state in solution. By coupling the adoption of a
specific three-dimensional structure with the binding of an
interacting partner, proteins with this property are able to
generate considerable structural diversity through a short se-
quence of amino acids. In this context, the N-terminus of
CENP-A may have diverged to accommodate different
DNA sequence environments, as proposed by Henikoff and
colleagues (Ahmad and Henikoff 2001; Malik et al. 2002),
while maintaining the capacity to engage in multiple spe-
cific interactions with kinetochore proteins, as well as poten-
tial interactions with DNA. One mechanism to specify the
segregation of nucleosomes that contain CENP-A from those
that contain H3 would be the self-association of CENP-A
tails. The diversity of structure and associations enabled by
an intrinsically disordered domain could allow all of these
interactions to occur despite the small size of the CENP-A
tails in many species.
Our understanding of the relationships between centro-
mere structure and function, in contrast with the biochemical
regulation of centromere function, is still in its infancy. The
centromere is consistently evident as a distinct chromatin
structure with a decreased diameter and the site of interac-
tion between chromatids when visualized in situ or in chro-
mosome spreads. The precise structure–function relationship
within the centromere and the contribution of histone post-
translational modifications to the formation of centromeric
and pericentromeric heterochromatin is largely undeter-
mined. Given that biophysical experiments have clearly es-
tablished that the sensing of tension is critical to the control
of mitosis, it might be expected that any significant change
in centromeric or pericentromeric heterochromatin structure
would compromise the function of the centromere and (or)
kinetochore during mitosis. We may be nearing the time
when all of the direct biochemical players in this process
have been identified. Nonetheless, a list of the parts and the
study of small complexes in vitro are a long way from a
complete understanding of how the massive three-dimen-
sional structures, which make up the centromere and kineto-
chore of higher eukaryotes, are assembled from these parts.
For this, we will need a much better understanding of the
quantitative relationships among post-translational modifica-
tions of histones, the recruitment of HP1 and cohesins, the
mechanical properties of chromatin during mitosis, and the
composition of the kinetochore. This level of understanding
will require a very different approach from what has been
used to assign molecules in a binary fashion as either re-
quired or not.
This work was supported by operating grants from the
Canadian Institute of Health Research (CIHR) and the Al-
berta Cancer Board. G.C. is a CIHR new investigator. M.H.
is an Alberta Heritage Foundation for Medical Research se-
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