THEJOURNALOFCELLBIOLOGY
JCB: COMMENT
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The Journal of Cell Biology, Vol. 176, No. 6, March 12, 2007 735­736
http://www.jcb.org/cgi/doi/10.1083/jcb.200702020
JCB 735
Centromeres are the specialized chromatin domains on each
eukaryotic chromosome that direct kinetochore assembly during
mitosis. Kinetochores mediate chromosome attachment to the
mitotic spindle and are required for microtubule-dependent
chromosome movement during mitosis. Kinetochores also act
as signaling centers that activate the mitotic checkpoint, delaying
the initiation of anaphase in response to improper chromosome
attachments to the mitotic spindle (Maiato et al., 2004). CENP-A
is a histone H3 variant that is present exclusively in centromeric
nucleosomes and is required to direct kinetochore assembly
(Cleveland et al., 2003; Carroll and Straight, 2006). Centromeric
DNA is not well conserved even among closely related
species, and, in humans, no individual DNA sequence is necessary
or sufficient to encode centromere function. However, all
functional centromeres contain CENP-A.As the key universally
conserved element of centromeric nucleosomes throughout
eukaryotic organisms, CENP-A is thought to provide the epigenetic
mark that specifies centromere function. A central question,
then, is how the specialized chromatin of the centromere is
assembled and stably propagated.
Two recent studies published in this issue address this
question. Applying a novel fluorescent technology in a series of
elegant pulse-chase experiments, Jansen et al. (2007) demonstrate
that the deposition of newly synthesized CENP-A is coupled
to cell cycle progression. Somewhat surprisingly, however,
these authors show that CENP-A deposition into chromatin
occurs during telophase/early G1 phase (Fig. 1). This is in
marked contrast to conventional histone H3, which is assembled
concomitantly with DNA replication. Furthermore, by exploiting
cell fusion experiments of the type pioneered in classic cell
cycle studies by Rao and Johnson (1970), Jansen et al. (2007)
show that exit from mitosis is required for the loading of nascent
CENP-A into centromeric chromatin. Similar conclusions were
recently reached for loading of the Drosophila melanogaster
CENP-A (called Cid) during early embryonic cell cycles (Schuh
et al., 2007), indicating that the timing of eukaryotic centromeric
chromatin replication is likely conserved.
In a related study also published in this issue, Maddox et al.
(2007) used a functional genomics strategy to identify genes
required for CENP-A deposition in Caenorhabditis elegans.
Disruption of a single gene called kinetochore null 2 (KNL-2)
resulted in the dramatic loss of CENP-A from chromosomes,
resulting in a phenotype that is indistinguishable from that of
CENP-A depletion itself (Oegema et al., 2001). C. elegans
chromosomes are holocentric, an interesting evolutionary variation
in which centromeres and thus CENP-A span the entire
length of the chromosome. This raised the possibility that the
mechanism of CENP-A deposition might somehow be divergent
from other organisms with monocentric chromosomes.
However, Maddox et al. (2007) identified the human homologue
of KNL-2 (also called Mis18BP1) and demonstrated that it was
also required for CENP-A localization (Fig. 1). This suggests
that mechanisms of centromere assembly are conserved in
diverse species and that KNL-2 is a key factor in this process.
The studies highlighted here represent a fundamental advance
in our understanding of how the integrity of centromeric
chromatin is established and maintained. Nevertheless, several
questions remain to be answered. For example, how is the existing
centromere recognized as the site for incorporation of new
CENP-A? One recent model has suggested that tension applied
across the centromere as a result of bipolar spindle attachment
during mitosis could condition centromeric chromatin for
CENP-A loading (Mellone andAllshire, 2003), thereby ensuring
that only active centromeres will be targeted for the deposition
of new CENP-A. Jansen et al. (2007) addressed this question by
forcing cells to exit mitosis in the absence of microtubule­
chromosome attachments. Under these conditions, CENP-A
loading occurs normally, indicating that tension across the centromere
is not required for CENP-A loading. However, structural
components of the kinetochore have been shown to be required
for CENP-A loading, suggesting that interaction between kinetochore
assembly and CENP-A deposition contributes to stable
centromere maintenance (Kline et al., 2006; Okada et al., 2006).
Centromeric chromatin gets loaded
Christopher W. Carroll and Aaron F. Straight
Department of Biochemistry, Stanford University, Stanford, CA 94305
Centromeric nucleosomes contain a histone H3 variant
called centromere protein A (CENP-A) that is required for
kinetochore assembly and chromosome segregation. Two
new studies, Jansen et al. (see p. 795 of this issue) and
Maddox et al. (see p. 757 of this issue), address when
CENP-A is deposited at centromeres during the cell division
cycle and identify an evolutionally conserved protein
required for CENP-A deposition. Together, these studies
advance our understanding of centromeric chromatin
assembly and provide a framework for investigating the
molecular mechanisms that underlie the centromere-specific
loading of CENP-A.
Correspondence to Aaron F. Straight: astraight@stanford.edu
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Another important question regards the mechanism by
which CENP-A is loaded into centromeric chromatin and the
role of KNL-2 in this process. Conventional histone H3 and the
histone variant H3.3 copurify with distinct histone chaperone
complexes called chromatin assembly factor 1 (CAF1) and
HIRA, respectively, which are required for the deposition of
their associated histones (Tagami et al., 2004). Although no
analogous histone chaperone complex specifically required for
CENP-A loading has been characterized to date, several genes
have been identified that are necessary for the centromerespecific
localization of CENP-A. Of particular interest are the
products of the Schizosaccharomyces pombe Mis16 and Mis18
genes (Hayashi et al., 2004). Mis16 is a homologue of the
closely related human proteins RbAp46 and RbAp48, which
are also required for CENP-A localization. RbAp48, the only
common subunit of CAF1 and HIRA, was also found to associate
with CENP-A purified from Drosophila cells (Furuyama
et al., 2006). Two Mis18 homologues,  and , were recently
shown to be required for CENP-A loading in human cells and
are found in a complex with human KNL-2 (Fujita et al., 2007).
Interestingly, KNL-2 and Mis18/ localize specifically to
centromeric chromatin during telophase/early G1 phase. Thus,
KNL-2 likely functions at chromatin during CENP-A loading,
possibly as a targeting element for a CENP-A­containing
histone chaperone complex.
CENP-A is the best candidate to provide the epigenetic
mark that specifies centromere identity within eukaryotic
chromosomes. Until recently, the molecular systems that control
the centromere-specific loading of CENP-A were completely
unknown. The two studies highlighted here along with several
advances in diverse experimental systems have laid the foundation
for further studies designed to elucidate a detailed molecular
understanding of how centromeric chromatin is assembled and
maintained. The continued identification of factors required for
this process along with the development of in vitro systems to
dissect the mechanisms of CENP-A deposition are now essential
to understand the molecular underpinnings of centromeric
chromatin assembly.
Submitted: 13 February 2007
Accepted: 14 February 2007
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Figure 1. A model for the centromere replication cycle for a single human
chromosome. During DNA replication (1), CENP-A­containing nucleosomes
(blue dots) are randomly distributed to each sister chromatid, resulting
in a twofold reduction in the amount of CENP-A at each centromere.
Upon exit from mitosis (2), a change in the state of KNL-2 or within the
chromatin itself targets KNL-2 to the centromere, which, in turn, recruits
newly synthesized CENP-A for deposition (3). After centromere replication,
KNL-2 dissociates from centromeres (4). The mechanisms that control KNL-2
centromere binding and dissociation are unknown.
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