Báo cáo Y học: Beyond the ABCs of CKC and SCC Do centromeres orchestrate sister chromatid cohesion or vice versa? potx

MINIREVIEW
Beyond the ABCs of CKC and SCC
Do centromeres orchestrate sister chromatid cohesion or vice versa?
Pamela B. Meluh
1
and Alexander V. Strunnikov
2
1
Memorial Sloan-Kettering Cancer Center, Laboratory of Mechanism and Regulation of Mitosis, New York, USA;
2
Unit of
Chromosome Structure and Function, NIH, NICHD, Laboratory of Gene Regulation and Development, Bethesda, MD, USA
The centromere–kinetochore complex is a highly specialized
chromatin domain that both mediates and monitors
chromosome–spindle interactions responsible for accurate
partitioning of sister chromatids to daughter cells. Cen-
tromeres are distinguished from adjacent chromatin by
specific patterns of histone modification and the presence of
a centromere-specific histone H3 variant (e.g. CENP-A).
Centromere-proximal regions usually correspond to sites of
avid and persistent sister chromatid cohesion mediated by
the conserved cohesin complex. In budding yeast, there is a
substantial body of evidence indicating centromeres direct
formation and/or stabilization of centromere-proximal
cohesion. In other organisms, the dependency of cohesion on
centromere function is not as clear. Indeed, it appears that
pericentromeric heterochromatin recruits cohesion proteins
independent of centromere function. Nonetheless, aspects of
centromere function are impaired in the absence of sister
chromatid cohesion, suggesting the two are interdependent.
Here we review the nature of centromeric chromatin, the

dynamics and regulation of sister chromatid cohesion, and
the relationship between the two.
Keywords: centromere; kinetochore; CENP-A; histone;
methylation; heterochromatin; sister chromatid cohesion;
cohesin; chromatin immunoprecipitation.
INTRODUCTION
Chromosomes are duplicated during S phase in a process
that entails not only DNA replication, but also replication of
the chromatin itself. Thus, the distribution and modification
state of nucleosomes, as well as other DNA-associated
proteins that organize the genome and specify patterns of
gene expression must be maintained. The process of high
fidelity DNA replication is well understood at this point [1].
Much less is known about the propagation of chromatin
structure and organization. Presumably, this is accom-
plished to a large degree by chromatin assembly factors that
deposit nucleosomes concomitant with DNA replication, as
well as by ÔinstructionsÕ encoded in the DNA sequence (e.g.
sequence-specific protein binding sites, intrinsic bends, etc.).
However, there are many epigenetic phenomena that cannot
be explained in this way. Thus, various mechanisms for the
self-propagation of pre-existing chromatin states have
been proposed [2,3]. We imagine that as for the DNA,
replication of chromatin must also be a faithful process.
One specialized chromatin domain whose faithful
duplication is paramount to accurate chromosome segre-
gation is the centromere–kinetochore complex (hereafter,
centromere or CKC). The CKC plays both a mechanical
and a regulatory role during mitosis. Centromeres of
paired sister chromatids capture dynamic microtubules

(MTs) within the mitotic spindle and exert force upon
them. As mitosis proceeds, sister centromeres ultimately
interact most stably with MTs from opposite spindle poles
[4]. Such bipolar attachment ensures that each daughter
cell will receive a precise complement of chromosomes.
The fidelity of chromosome transmission is enhanced not
only by the geometry of stable centromere–MT interac-
tions, but also by the action of a centromere-based
regulatory system called the mitotic checkpoint that
monitors CKC–MT interactions and delays the onset of
anaphase until stable bipolar attachment is achieved
(reviewed in [5]).
Genomic integrity is further enhanced by the cell cycle
dependent deposition of protein complexes that mediate
association, precise alignment, and efficient packaging of
sister chromatids following replication. Chief among these
factors are the evolutionarily conserved cohesin and
condensin complexes (Table 1) [6]. These complexes con-
tribute to the structural maintenance of chromosomes and
accurate chromosome transmission during meiosis and
mitosis. Not surprisingly, both complexes are essential
and contain SMC protein pairs (structural maintenance
of chromosomes [7]) in addition to unique components
that presumably confer functional specificity.
Numerous observations suggest an intimate relationship
exists between the formation and function of the CKC and
Correspondence to P. B. Meluh, Memorial Sloan-Kettering Cancer
Center, Program in Molecular Biology, Laboratory of Mechanism
and Regulation of Mitosis, 1275 York Ave.,
New York, NY 10021, USA.

Fax: + 1 646 422 2062, Tel.: + 1 212 639 7679,
E-mail:
Abbreviations: CKC, centromere–kinetochore complex; SCC, sister
chromatid cohesion; SMC, structural maintenance of chromosomes;
CAR, cohesin-associated region; MT, microtubule; ChIP, chromatin
immunoprecipitation.
Dedication: This Minireview Series is dedicated to Dr Alan Wolffe,
deceased 26 May 2001.
(Received 28 January 2002, revised 11 March 2002,
accepted 18 March 2002)
Eur. J. Biochem. 269, 2300–2314 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02886.x
Table 1. Quick guide to key cohesion, centromeric and cell cycle proteins described in the text.
Protein in:
S. cerevisiae; S. pombe;
Human/Metazoans Structural feature(s) Function
Condensin complex
Smc2; Cut14; HCAP-E ATPase, coiled-coil domain DNA binding activity
Smc4; Cut3; HCAP-C ATPase, coiled-coil domain DNA binding activity
Ycs4; Cnd1; HCAP-D2 HEAT repeats (BIR repeats)
Ycg1/Ycs5; Cnd3; HCAP-G HEAT repeats
Brn1; Cnd2; HCAP-H/Barren Barren subunit
Cohesin complex
Smc1, Psm1; hSMC1 ATPase, coiled-coil domain DNA binding activity
Smc3, Psm3; hSMC3 ATPase, coiled-coil domain DNA binding activity
Scc1/Mcd1; Rad21;
hRAD21/hSCC1
Cleaved by Esp1 at anaphase onset; C-terminal fragment
normally degraded by Ub-dependent proteolysis via
N-end rule
Rec8; Rec8; REC8 Scc1/Mcd1-related Meiosis specific cohesin subunit related to Scc1/Mcd1;

selectively retained at paired sister centromeres until
Meiosis II anaphase, when cleaved, presumably by Esp1;
proper localization requires Spo13
Scc3/Irr1; Psc3; SA-1/STAG3 (
STromal AntiGen family)
Other factors that promote SCC
Pds5; Pds5; PDS5 HEAT repeats Cohesion and condensation, chromatin association depends
on cohesin; Pds5
S.p.
interacts with cohesin complex
Scc2; Mis4; HUMHBC4244 HEAT repeats Cohesin complex loading onto chromatin during S phase
Scc4; ??; ?? Cohesin complex loading onto chromatin during S phase
Ctf7/Eco1; Eso1; ?? C
2
H
2
Zn finger-like
domain, Gcn5-related N-acetyl-
transferase super family
Interacts with PCNA; maturation of cohesion; Ctf7/Eco1
possesses in vitro lysine acetyltransferase activity toward itself,
Scc1/Mcd1, Scc3, and Pds5
domain; Eso1 also has a
polymerase domain (Pol eta)
Trf4 & Trf5; (Cid12?);
(POLS?)
DNA polymerase sigma Cohesion establishment during S phase; Trf4 associates
physically with both Smc1 and Smc2
Ctf18; (Spbc902.02c?); ?? RFC homology Cohesion establishment during S phase
Mad2; Mad2; MAD2 Mitotic checkpoint protein Accumulates on unattached CKC’s during prophase and

prometaphase (as do other mitotic checkpoint proteins);
Mad2 (or Bub1 and BubR1) can physically associate with
APC
Cdc20
to inhibit its activity
CAR (
Cohesin
Associated Region)
Often intergenic A+T-rich region DNA sequence bound by cohesin (in some cases, can promote
cohesion when introduced at an ectopic site); centromeric
DNA has portable CAR activity; may or may not correspond
to a site of cohesion
Factors that can disrupt SCC
Esp1; Cut1; Separase CD-clan caspase-like protease Cleaves Scc1/Mcd1 to promote sister chromatid separation at
the metaphase-to-anaphase transition; regulated by Securin
Pds1; Cut2; Securin/PTTG D-box (APC substrate) Chaperone and inhibitor of Esp1/Separase; degraded via
APC-directed Ub-dependent proteolysis during mitosis
Ubr1; ??; UBR1 RING-H2 domain Ubiquitin-protein ligase (E3) for N-end rule pathway;
required for clearance of Scc1/Mcd1 cleavage products
APC/C or ‘‘
Anaphase
Promoting Complex’’
RING-H2 domain subunit,
cullin subunit
Multi-subunit ubiquitin-ligase (E3) for mitotic progression and
cyclin B destruction; Cdc20 is the specificity factor for Pds1;
or ‘‘Cyclosome’’ APC
Cdc20
cyclin B-ubiquitin ligase activity is inhibited by Mad2
Cdc5; Plo1; Polo Polo Kinase (S/T kinase) Among other things, may phosphorylate Scc1/Mcd1 to enhance

its Esp1-dependent cleavage
Cdc28; Cdc2; hCdc2 CDK Kinase Among other things, hCdc2 may phosphorylate hRad21 to
enhance its separase-dependent cleavage; high Cdc2 kinase
activity leads to inhibitory phosphorylation of separase
Ó FEBS 2002 Centromeric chromatin and sister chromatid cohesion (Eur. J. Biochem. 269) 2301
the establishment and maintenance of sister chromatid
cohesion (SCC). Cytologically, centromeres of mitotic and
meiotic chromosomes appear as sites of avid cohesion, and
may, in fact, direct the formation and maintenance of a
centromere-proximal domain of cohesin and other cohesion
promoting factors [8–12]. Consistent with this, in Drosophila
and vertebrate cells, cohesin subunits at centromeres are
specifically retained until anaphase, whereas the vast
majority of cohesin dissociates from chromosome arms
during prophase [13–16]. Similarly, during meiosis I,
meiosis-specific cohesin complexes persist at centromeres,
even while arm cohesion is dissolved to facilitate dissolution
of the chiasmata [17,18]. Retention of SCC at centromeres
during meiosis I is, of course, critical for the reductional
pattern of meiosis I chromosome segregation. However,
SCC is also essential for accurate equational chromosome
segregation during mitosis (and meiosis II), and when
compromised, leads to chromosome nondisjunction and
loss [17,19–24].
SCC at or near the centromere clearly opposes the
MT-dependent pulling forces exerted by the spindle prior to
the onset of anaphase, and therefore helps to prevent
premature dissociation of sister chromatids [25]. Perhaps
more importantly, centromere-proximal cohesion (and in
the case of larger centromeres, perhaps condensin-mediated

packaging) apparently serves to sterically constrain sister
CKCs in a Ôback-to-backÕ orientation, thereby precluding
merotelic or monopolar attachments in favor of bipolar
attachment of the paired sisters to the spindle [25–27].
Finally, the metaphase-to-anaphase transition is triggered
by the timely degradation of a particular cohesin subunit
known as Scc1/Mcd1 in S. cerevisiae, and Rad21 in other
Table 1. continued.
Protein in:
S. cerevisiae; S. pombe;
Human/Metazoans Structural feature(s) Function
Heterochromatin proteins
??; Swi6; HP-1 Chromo domain Heterochromatin protein; binds histone H3 methylated at
Lysine 9; necessary for proper chromosome segregation;
required in S. pombe for centromeric (but not arm) cohesion
and recruitment of cohesin to silent chromatin (including
centromeres)
??; Clr4; SUV39H1 Chromo domain, SET domain Heterochromatin protein; histone H3 Lysine 9 methyltransferase
Centromere proteins
Cse4; Cnp1; CENP-A Histone H3 fold domain Essential CKC determinant; may replace histone H3 in
centromere specific nucleosomes; uniquely marks centromeric
chromatin; required for proper localization of CENP-C and
INCENP to CKC; might directly or indirectly recruit cohesin
Mif2; Cnp3; CENP-C Essential CKC determinant; may have DNA binding activity;
localization depends on CENP-A
Sli15, ??, INCENP IN box Chromosomal passenger protein; interacts with Aurora B kinase;
localization to CKC in mid-mitosis is dependent on cohesin
Ipl1; Ark1; Aurora B/AIM-1 Aurora kinase Chromosomal passenger protein; interacts with INCENP;
localization to CKC in mid-mitosis is dependent on cohesin;
substrates include histone H3, CENP-A, and Ndc10

(S. cerevisiae centromere protein)
Bir1; Bir1/Cut17; Bir1/Survivin IAP-related Chromosomal passenger protein; interacts with INCENP and
Aurora B kinase; localization to CKC in mid-mitosis is
dependent on cohesin
Ndc80/Hec1; Ndc10; HEC All subunits have coiled-coil
domains
Component of conserved CKC protein complex (includes
Ndc80/Hec1, Nuf2, Spc25, Spc24); in S. cerevisiae,
Ndc80/Hec1 physically and genetically interacts with cohesin
subunit Smc1; Nuf2
S.c.
and Spc25
S.c.
also interact with Smc1
Slk19; ??; ?? Coiled-coil domain Non-essential CKC-associated protein; requires separase (Esp1)
for cleavage and localization to the spindle midzone in
anaphase; null mutants show high frequency of premature
sister chromatid separation in meiosis I
Spo12; (Wis3?); ?? Non-essential; mutants show equational division (i.e. premature
sister chromatid separation) in meiosis I
Spo13; ??; ?? Non-essential meiosis-specific protein; required for SCC during
meiosis I; mutants fail to localize Rec8 properly and show
equational division (i.e. premature sister chromatid
separation) in meiosis I
??; ??; MEI-S322 Coiled-coil domain Drosophila protein present at CKCs of both meiotic and mitotic
chromosomes; dissociates at anaphase; required for main
tenance of SCC at the CKC in mitosis and especially in meiosis
2302 P. B. Meluh and A. V. Strunnikov (Eur. J. Biochem. 269) Ó FEBS 2002
species. In so far as the CKC controls this transition
through activation of the mitotic checkpoint [5], it does so

by indirectly inhibiting the degradation of the Scc1/Mcd1
cohesin subunit (reviewed in [28–30]). Below we review in
further detail the nature of centromeric chromatin and SCC
and the relationship between them.
THENATUREOFCENTROMERIC
CHROMATIN
Centromeres are defined genetically by phenotypic or
molecular markers [31] that always segregate away from
one another during meiosis I. Centromeres can be visua-
lized cytologically as the primary constriction of metaphase
chromosomes in vertebrate cells, and correspond to the site
of kinetochore formation. Centromeric chromatin struc-
ture and composition have been studied primarily
in human and rodent cells, Drosophila, Caenorhabditis
elegans, fission yeast, and budding yeast, using a combi-
nation of genetics, cell biology and more recently, bioin-
formatics. With the apparent exception of budding yeast
and possibly C. elegans, natural centromeres occur within
the context of constitutive heterochromatin. As such, these
centromeric regions are largely nontranscribed, exert
position effects on gene expression, and show reduced
recombination. They typically contain extended arrays of
repetitive, often A+T-rich, DNA sequence elements (e.g.
alpha satellite in human cells) [31–34], and by indirect
immunofluorescence and/or chromatin immunoprecipita-
tion, are organized by nucleosomes that are hypo-acetyl-
ated and hyper-methylated (i.e. on lysine 9 of histone H3)
(Fig. 1) [35–38]. The latter features account for the
Fig. 1. Cross talk between centromere components and cohesion proteins. Diagram summarizes potential relationships compiled from findings from
several species. The centromeric chromatin is characterized hypo-acetylated, hyper-methylated (ÔMeÕ) histone H3-containing nucleosomes as well as

the CENP-A-containing variety (ÔAÕ). The degree of nucleosome interspersion is unresolved and may be species-specific. Histone H3 methylation by
ÔSETÕ domain-containing methyltransferases [e.g. Clr4
S.p.
, Su(var)3–9
D.m.
; SUV39H1] is critical for recruitment of chromo domain proteins (e.g.
HP1 or Swi6
S.p.
.) and cohesin to heterochromatic regions. Recruitment of other centromere proteins (e.g. CENP-C (ÔCÕ); INCENP; and possibly the
Ndc80/Hec1 complex) is dependent on CENP-A (ÔAÕ). Under certain conditions CENP-A can be shown to recruit cohesin, and Ndc80/Hec1
genetically and physically interacts with cohesin. Mitosis-specific centromere proteins such as the mitotic checkpoint protein Mad2 indirectly
promote SCC by inhibiting the activity of APC towards Pds1/Securin. Conversely, cohesin is important for the recruitment of some centromeric
proteins, namely the chromosomal passenger proteins aurora B/Ipl1, INCENP, and Bir1. Tension exerted across the CKC leads to disruption of
SCC (at least in budding yeast) and release of passenger proteins. Aurora B/Ipl1 kinase, which can phosphorylate (ÔPÕ) CENP-A, as well as histone
H3, has been implicated in detecting tension at the CKC and may therefore play an active role in the dissolution of SCC, perhaps by acting on
CENP-A. The mechanism(s) whereby CENP-A distribution and HMTase activity are directed to centromere-proximal regions is currently
unknown but clearly of great interest.
Ó FEBS 2002 Centromeric chromatin and sister chromatid cohesion (Eur. J. Biochem. 269) 2303
presence of chromo domain-containing heterochromatin
proteins (which bind lysine 9-methylated histone H3) at
centromeres in Schizosaccharomyces pombe (Swi6 and
Clr4), Drosophila and mammalian cells [HP1 and Su
(var)3–9]. Recently, these chromo domain proteins have
been implicated in the recruitment and/or stabilization of
centromere-proximal cohesin [25,39].
The pattern of core histone tail modification within
centromeric chromatin is a critical determinant of CKC
structure and function. Treatment of S. pombe or human
cells with trichostatin A, a histone deacetylase inhibitor,
leads to increased histone acetylation throughout the

genome. Within centromeric chromatin, increased acetyla-
tion correlates with profound effects on centromere struc-
ture and function, including decreased centromeric gene
silencing, loss of associated HP1 proteins, and pronounced
chromosome segregation defects [36,40]. Similarly, muta-
tions in S. pombe and Drosophila that affect centromeric
gene silencing or position effect also affect centromere
structure and function [25,41–43]. In several cases, the genes
defined by these mutations encode histone-modifying
enzymes such as deacetylases (Clr3) or methylases (Clr4),
strongly supporting the idea that centromeric chromatin
must ÔsportÕ a particular histone code.
Centromeric chromatin is distinct from surrounding
chromatin, not only with respect to its histone modification
pattern as discussed above, but also at an even more
fundamental level of chromosome organization: namely,
that of the nucleosome itself. While the sequence and size
of underlying centromeric DNA varies greatly among
organisms [2,34], centromeric chromatin in all eukaryotes
studied to date, including budding yeast and C. elegans,
contains a unique and essential histone H3 variant
(reviewed in [44]). The founding member of this group,
CENP-A, was originally identified as a prominent auto-
antigen in human CREST serum [45]. CENP-A is a
constitutive centromere component, and localizes to the
inner kinetochore plate of mitotic chromosomes [46]. In
mammalian cells, CENP-A is incorporated into nucleo-
some-like particles along with histones H2A, H2B, and H4
[47,48] and in vitro, CENP-A can replace histone H3 within
reconstituted nucleosomes [49]. Importantly, alpha satellite

DNA, the major DNA repetitive element present at human
centromeres, copurifies with CENP-A-containing nucleo-
somes [50]. Given that CENP-A is found only at active
centromeres [46,51], it has been proposed that centromeric
chromatin is uniquely marked by centromere-specific
nucleosomes in which CENP-A replaces histone H3.
Whether such specialized nucleosomes are present at
centromeres in other organisms remains to be determined,
but is likely to be the case. For example, in Saccharo-
myces cerevisiae or S. pombe alterations in histone dosage
can affect centromere chromatin structure and impair
chromosome segregation [52–55], as do certain point
mutations in histones H2A and H4 [56,57]. While such
phenotypes could reflect the indirect effect of altered gene
expression, allele-specific genetic interactions between his-
tone H4 and the yeast CENP-A homolog, Cse4, suggest
these two proteins physically associate, possibly in the
context of a nucleosome-like particle [57,58]. Regardless, it
is clear from genetic studies that CENP-A is required for
the assembly of a functional kinetochore, and in its
absence other essential centromere components, such as
CENP-C, are no longer recruited to the CKC [59–64].
That said, CENP-A is not sufficient for centromere
specification. When a functional Cse4–Gal4 DNA binding
domain fusion is directed to an ectopic locus, neocentro-
mere activity is not detected [65] (P. Meluh, unpublished
results). Similarly, when overexpressed, CENP-A is misdi-
rected to noncentromeric chromatin, where it recruits and/
or stabilizes some (e.g. CENP-C and hSmc1), but not all
centromere factors [147].

SISTER CHROMATID COHESION
Sister chromatid cohesion (SCC) refers to the physical, and
as we now know, protein-mediated linkage that exists
between replicated sister chromatids from the onset of DNA
replication until anaphase. Thus, SCC exists throughout a
significant portion of both the mitotic and meiotic cell
cycles. Moreover, SCC persists, at least at or near
centromeres, even when M phase is prolonged by drug
treatment or checkpoint activation. Establishment, main-
tenance and timely resolution of SCC are essential steps in
ensuring proper chromosome segregation and, accordingly,
the genetic stability of a eukaryotic cell. Mutations that
impair any of these steps would be expected to cause gross
chromosome missegregation, cell growth arrest and/or
inviability. Uncovering such relevant phenotypes as prema-
ture sister chromatid separation in prometaphase or failure
to separate sisters in anaphase has led to the genetic
identification of a number of SCC structural and regulatory
components (Table 1) [19–22,24,66–68]. These genetic find-
ings have been strikingly consistent with biochemical
approaches aimed at defining the molecular nature of
SCC (reviewed in [6,29,30]).
Thus, in a few short years, we have gone from regarding
SCC as a Ôcytological formalismÕ to a clear appreciation
of SCC as a complex cellular process that involves specific
cis-acting chromosomal loci (e.g. the centromere), dozens of
protein factors, and that is intimately associated with the cell
cycle machinery. The precise molecular mechanism whereby
sister chromatids are held together remains elusive, as
biochemical activities and dependency relationships for

assembly have been assigned to only a few SCC proteins.
On the other hand, our knowledge about SCC regulation
and the chromosomal localization of SCC activity has
accumulated at a fast-pace. Chromosomal ÔaddressesÕ for
SCC activity can be divided into two major classes:
centromeric cohesion and chromosomal arm cohesion. As
mentioned above, location is not the only distinction
between these two classes. Centromeric and arm cohesion
can be differentially regulated, such that centromere-prox-
imal cohesion is more stable and thus, presumably, the more
relevant target of cell cycle regulation [69]. The molecular
basis for the distinction is the subject of great interest.
PROTEINS INVOLVED IN
ESTABLISHMENT OF SISTER
CHROMATID COHESION
What mediates SCC? The landmark study by Holloway
et al. [70] established that at least one noncyclin protein
must be degraded via ubiquitin-dependent proteolysis to
allow for sister chromatid separation during mitosis. This
observation, combined with studies that ruled out DNA
2304 P. B. Meluh and A. V. Strunnikov (Eur. J. Biochem. 269) Ó FEBS 2002
topological constraints as the sole mediator of SCC [20,71],
led to the proposal that SCC must be protein mediated. This
view has since been validated by genetic and biochemical
studies that have identified chromosome-associated proteins
involved in SCC (reviewed in [6,29,30]).
Perhaps the best characterized of these SCC factors is
the evolutionarily conserved cohesin complex, components
of which have been genetically identified in several model
organisms. Cohesin was first biochemically identified as a

multicomponent complex in Xenopus embryonic extracts
[13], and similar complexes have since been purified from
yeast [72,73] and other vertebrate species [14,74,75]. In all
cases, the cohesin complex consists of four types of
subunit, some of which have tissue- or developmental
stage-specific paralogs (Table 1 [17,18,74,76,77]). The
cohesin core consists of a heterodimeric pair of SMC
(structural maintenance of chromosomes) proteins, Smc1
and Smc3. Like the related Smc2–Smc4 heterodimer found
in the condensin complex [78], the Smc1–Smc3 heterod-
imer forms an extended coiled-coil with two catalytic
domains possessing DNA-binding and ATPase activities
[6,79]. Smc1 and Smc3 are likely to interact through their
hinge regions [80,81] to form a clamp around the
chromatin fiber [82]. In budding yeast, Smc1 and Smc3
are constitutively bound to chromatin throughout the cell
cycle and presumably serve as platforms for assembly of
mature cohesin early in S phase. At that time, two
additional subunits, Scc3 (i.e. SA1/STAG in mammals)
and Scc1/Mcd1 (called Rad21 in other organisms), are
recruited to chromatin in an Smc1- and Smc3-dependent
fashion [10,20]. It is possible that assembly of tetrameric
cohesin also occurs in a soluble phase during S phase to
compensate for chromatin replication. Interestingly, the
S. pombe Scc3 homolog, Psc3, does not behave as a stable
component of the cohesin complex, but this could simply
reflect the method of cell lysate preparation [73]. Regard-
less, it is believed that within replicated chromosomes only
the mature tetrameric form of cohesin is competent to
bridge sister chromatids. This view is supported by the

DNA binding properties of cohesin in vitro [79], and also
by the fact that cleavage of Scc1/Mcd1 at the metaphase-
to-anaphase transition and the accompanying loss of Scc3
from chromatin coincides with, and indeed is a prerequis-
ite for, dissolution of SCC [67] (see below).
Fig. 2. Regulation of sister chromatid cohesion establishment and release during the budding yeast cell cycle. Schematic diagram summarizing factors
cited in the text that govern SCC. Left panel. Several key regulatory steps in SCC formation and resolution are shown in grey boxes. Green arrows
indicate poleward pulling forces exerted on the centromere–kinetochore complex (CKC) by the mitotic spindle. Blue arrows indicate a chromatin
recoil force that may allow for transient re-establishment of SCC at the CKC in budding yeast. Right panel. Consequences of impaired SCC.
Metaphase arrest, chromosome loss (ÔCutÕ phenotype), and/or nondisjunction can result from SCC misregulation. Ac, acetylation; P, phos-
phorylation; APC, anaphase promoting complex.
Ó FEBS 2002 Centromeric chromatin and sister chromatid cohesion (Eur. J. Biochem. 269) 2305
Other factors essential for the establishment and main-
tenance of SCC have been identified, largely through genetic
screening (Table 1; Fig. 2). Although these proteins colo-
calize with cohesin in chromatin and/or genetically interact
with cohesin subunits, they are not stably associated with
soluble cohesin complexes. One such factor is the conserved
Pds5/Spo76/BimD protein [22,75,83–86]. Mutations in Pds5
homologs confer SCC and chromosome segregation defects
similar to those seen in cohesin mutants, yet Pds5 is not an
integral part of the cohesin complex per se. For example, in
S. cerevisiae, Pds5 is significantly less abundant than
cohesin itself [85], and although Pds5 and cohesin have
been shown to physically interact in S. pombe and human
cells, only a subfraction of total cohesin is associated with
Pds5 [75,86]. Nonetheless, Pds5 homologs undergo cell-
cycle regulated localization to mitotic and meiotic chromo-
somes in their respective organisms, and at least in budding
and fission yeast, chromatin localization is dependent upon

cohesin function [22,85,86]. However, cohesin complex
localization to chromosomes is independent of Pds5 func-
tion. Thus, Pds5 either acts downstream of cohesin in an
SCC assembly pathway, or it provides an SCC fidelity or
optimization function. In this regard, S. pombe strains
lacking Pds5 are viable and establish SCC in S phase
normally. However, pds5D mutants are unable to maintain
SCC during prolonged G2-arrest [86]. In contrast, in
budding yeast, Sordaria,andA. nidulans, Pds5 is essential
for both establishment and maintenance of SCC
[22,84,85,87]. These observations suggest that Pds5 pro-
motes maturation or stabilization of the cohesin-mediated
linkage between sisters, and that different organisms require
Pds5 activity to different extents.
Although cohesin subunits can associate with chromatin
thought out the cell cycle, execution point studies indicate
that productive and faithful SCC strictly requires that
mature cohesin assembly and deposition occur in S phase
[21,72,88–90]. Several proteins promote this timely incor-
poration of cohesin into chromatin, and hence, SCC. For
example, mutations in the highly conserved Scc2 (Mis4 in
S. pombe) and Scc4 proteins, phenocopy cohesin mutants
in that loading of Scc1/Mcd1 and Scc3 onto chromatin in
S phase, and consequently SCC, fails [91,92]. However,
unlike cohesin mutants, loss of Scc2 or Scc4 function does
not preclude assembly of soluble mature cohesin complexes
[91]. Thus, Scc2 and Scc4 might function in early S phase to
chaperone Scc1/Mcd1 and Scc3 to pre-existing chromatin-
bound Smc1–Smc3 heterodimers or to make newly repli-
cated chromatin accessible for cohesin assembly.

The sequence and/or the genetic interactions of other
proteins required for cohesion suggest that productive
cohesin deposition is intimately associated with the act of
replication. Budding yeast Ctf7/Eco1 and the related
S. pombe protein Eso1 [72,89,90] are required specifically
for establishment of SCC in S phase. Cohesin binding to
chromatin per se is independent of Ctf7/Eco1/Eso1, but
SCC linkages are either not formed or break prematurely in
ctf7/eco1/eso1 mutants. Ctf7/Eco1 was recently shown to
exhibit lysine acetyltransferase activity in vitro, both towards
itself and toward cohesin subunits, suggesting that Ctf7/
Eco1 promotes establishment of SCC via post-translational
modification of cohesin [93]. Ctf7/Eco1 and Eso1 interact
with PCNA both physically and genetically [86,89] and
Eso1 itself has a DNA-polymerase-like domain that is
functionally separable from the domain involved in SCC.
These data suggest a possible mechanistic link between
replication fork movement and cohesin deposition onto
chromatin. The budding yeast Trf4 protein reinforces such a
connection. Trf4 is itself a DNA polymerase and when
inactivated, leads to a profound defect in cohesion estab-
lishment [94,95]. To date, it has not been possible to
genetically separate the polymerase activity of Trf4 from its
role in cohesion. While the DNA polymerase domains in
proteins required for SCC might be a red herring, Ctf18, an
RFC-like protein has also been recently implicated in SCC
[89,96]. Thus, an attractive hypothesis is that a polymerase
switch similar to that which occurs during Okazaki
fragment synthesis might take place at cohesion sites to
facilitate duplication and assembly of cohesion complexes

when sister chromatids are in close proximity [95,96]. This
model remains to be tested.
Obviously, what Ôduplication and assemblyÕ entails must
await structural and biochemical characterization of the
critical proteins. However, a detailed understanding of the
molecular basis of SCC will also require the development of
a comprehensive in vitro system. This will be an ambitious
undertaking given that SCC assembly seems tightly coupled
to DNA and chromatin replication. Moreover, as described
below, it is unclear what would constitute an adequate DNA
template on which to reconstitute SCC as our understanding
of the nature of cohesion sites in vivo is meager.
CIS-ACTING SITES REQUIRED
FOR SISTER CHROMATID COHESION
Given that cohesin complex deposition occurs coincident
with replication, or shortly thereafter, why invoke the
existence of specific heritable, DNA- or chromatin-encoded
cohesin binding sites? This concept may have derived in part
from the observation that centromeric regions often appear
as avid and persistent sites of cohesion. In addition, models
of higher order mitotic chromosome structure envision two
closely associated sister chromatid cores with sister DNA
loops extending in opposite directions [97,98]. Cohesin-
mediated Ôspot-weldingÕ of homologous sister chromatid
domains would be a prerequisite for such models.
To date, the most comprehensive analyses of chromo-
somal sites potentially required for SCC have been carried
out in budding yeast. Two fundamentally different approa-
ches have been used. One approach has exploited chromatin
immunoprecipitation (ChIP) [99] and makes the assumption

that sites of cohesin binding (e.g. Scc1/Mcd1 or Smc1)
correspond to sites of cohesion. A second approach has been
to functionally map the sequence(s) required for SCC using
centromere-based plasmids. Like authentic chromosomes,
these minichromosomes are replicated in S phase and persist
as paired sister minichromatids until anaphase [8,71].
The ChIP studies have revealed that cohesin complexes
do not bind uniformly along yeast chromosomes. Rather,
cohesin associated regions (CARs) are 300–1000 bp in
length and sparsely distributed on the chromosome, occur-
ring only every 8–13 kbp on average [9,10,100,101]. It is
noteworthy that such CARs correspond to only a few
nucleosomes’ worth of DNA. There is, however, one
striking exception to this rule, namely, an enormous con-
centration of cohesin binding sites occurs over a 10–20 kb
domain encompassing each centromere [9,10,73,100]. It is
2306 P. B. Meluh and A. V. Strunnikov (Eur. J. Biochem. 269) Ó FEBS 2002
important to note that functional centromeres, which in
yeast are specified by a  125 bp DNA sequence, are both
necessary and sufficient for formation of much larger
cohesin domains [8,10]. Moreover, using a recombinase
strategy, Megee et al. have shown that the centromere is a
major determinant of SCC on yeast minichromosomes [8].
Taken together these data suggest that the centromere
somehow directs cohesin recruitment (or stabilization) over
a long distance and that the resulting assemblage is directly
responsible for centromere-dependent SCC. The mechan-
ism of such localized recruitment (e.g. active spreading,
polymerization, post-translational modifications), if it
occurs at all, is unknown.

There are several observations that seem at odds with this
Ôcentromere-directed SCCÕ model for budding yeast SCC.
First, by ChIP, cohesion proteins are not enriched to the
same extent around the centromeres of minichromosomes
as they are around centromeres within the chromosome [8].
Moreover, localization of cohesion proteins within cells or
chromosome spreads as determined by microscopic tech-
niques does not agree with the ChIP data. By indirect
immunofluorescence or GFP-tagging, cohesion proteins
are broadly distributed on chromosomes, and do not appear
to be enriched at centromeres in yeast [20,73,102,103]
(A. V. Strunnikov, unpublished results) or, for that matter,
in vertebrate cells prior to prometaphase [16,74,75,104].
Finally, one of the most paradoxical observations is that
soon after their replication, pericentric regions of sister
chromatids in yeast appear to Ôsplit apartÕ owing to
MT-dependent spindle forces [10,105–107]. In other words,
despite their apparent load of cohesion proteins, centro-
meric regions may not be closely paired.
These discrepancies could in part be explained by the
in vivo and in vitro binding preference of the cohesin
complex for A+T-rich DNA [9,100,101,108]. However,
they might also reflect the limitations of ChIP, an assay that
relies on the geometry of reactive amino groups to promote
formaldehyde-dependent protein–DNA cross-linking [99].
Conceivably, the microscopic localization data accurately
reflect a broad distribution of cohesion proteins on chro-
mosomes. In this case, ChIP must be revealing a functional
or structural difference in cohesion proteins or chromatin as
a whole around the centromere, such that cohesion proteins

are more accessible to cross-linking reagents and/or anti-
bodies. This altered state could be related to the weakened
(or absence of) cohesion at the centromeres in mitosis (see
above) or to the stretching of centromere chromatin [26,109]
that could alter DNA conformation, making it more
accessible to cross-linking.
Another possibility is that we have been misled by the
pervasive concept of SCC as Ômolecular glueÕ,which
connotes SCC as inert and unchanging. Perhaps cohesion
protein binding at the centromere, or for that matter, at all
CARs, is dynamic. In this case, the apparent binding
differences revealed by ChIP would reflect localized differ-
ences in cohesin on-off rates (as influenced by DNA
sequence, chromatin structure, Smc1–Smc3 enzymatic
cycle, local kinase activities, etc.). Dynamic cohesin binding
is consistent with several observations: (a) cohesin proteins
rapidly dissociate from the chromosome when a CAR (i.e.
the centromere) is excised [8]; (b) over-expression of cohesin
subunits can expand CAR size as determined by ChIP
(P. Megee, Department of Biochemistry and Molecular
Genetics, University of Colorado Health Sciences Center,
DN, USA, personal communication); (c) CARs are Ôport-
ableÕ, but only in a qualitative sense, the range over which
a given CAR promotes cohesin binding varies with its
chromosomal (or minichromosomal) location; (d) cryptic
cohesion sites on a minichromosome are revealed when its
centromere is excised [9,10]; and (e) the transient but
persistent separation of paired sister centromeres following
replication (i.e. ÔbreathingÕ) [26,105–107] and that re-associ-
ation of sister centromeres early in mitotic prophase requires

cohesin [26]. Taking these considerations into account, it is
not presently possible to pinpoint a specific sequence within
the centromeric region that could rightly be called a
Ônucleation siteÕ for cohesin recruitment.
In higher eukaryotes, the existence of natural cis-acting
sites that mediate SCC is largely inferred, based on current
models of chromosome architecture. Assuming there are
specific cohesion sites, those on chromosome arms and at the
centromere are differentially regulated in vertebrate cells.
Thus, cohesin largely disappears from chromosome arms
during prophase, but persists at centromeres until anaphase
[13–16] (see below). Another difference appears to be that
once established in S phase, SCC on chromosome arms is
less static than that at centromeres, as sister sequences can
display dynamic association and dissociation behavior [110].
The nature of this phenomenon remains to be elucidated.
Although the nature and organization of SCC sites in
higher eukaryotes remains elusive, several repetitive DNA
sequences have been shown to promote (albeit misregulated)
SCC when integrated into an ectopic chromosomal location
[111,112]. Most notable among these, apropos this review, is
the normally centromeric human alpha-satellite repeat.
That human centromeric DNA can promote SCC in
mammalian cells is consistent with findings from budding
yeast with one important distinction. Namely, in this case,
SCC establishment does not correlate with centromere
function because integrated alpha-satellite DNA rarely
directs formation of a functional centromere. Similarly,
the repressed centromeres of stable dicentric chromosomes
often remain heterochromatic and show avid SCC [113].

This suggests that it is possible to genetically separate
centromeric cohesion and segregation functions and lends
support to studies in S. pombe indicating cohesin is
attracted to heterochromatin and possibly plays an inde-
pendent structural role in interphase chromatin [25,39].
REGULATION OF SISTER CHROMATID
COHESION
In budding yeast, positive regulation of SCC establishment
is mediated by strong transcriptional induction of SCC1/
MCD1 and SCC3 expression, accompanied by smaller
increases in the mRNA levels for other genes involved in
cohesion establishment. Indeed, cluster analysis of genome-
wide expression data from budding yeast revealed that
several cohesion protein genes, including SCC1/MCD1,are
coregulated by the MBF transcription factor along with
many DNA biosynthetic genes [114].
Once established in S phase, how is cohesion maintained?
Is it stable or dynamic? Besides an obvious requirement for
continued integrity of the cohesion proteins (and perhaps
the underlying CAR, as in the case of centromere-proximal
cohesion), there is little information about the mechanism of
Ó FEBS 2002 Centromeric chromatin and sister chromatid cohesion (Eur. J. Biochem. 269) 2307
SCC maintenance. As noted above, chromatin association
of at least some cohesion proteins may be dynamic. Indeed,
one ChIP study suggested that Scc1/Mcd1 is redistributed
from arm sites to centromeric regions during the transition
from the S phase to mitosis [100]. This has not been
rigorously tested, but if true, would be reminiscent of
cohesin dynamics in higher eukaryotes, where the bulk of
cohesin and Pds5 dissociates from chromosome arms as

cells enter mitotic prophase [15,16,75]. While cohesin may
be concentrated (or selectively retained) at centromeres
when yeast cells enter mitosis, as noted above, centromere-
proximal cohesion per se appears to be lost or weakened
prior to metaphase (Fig. 2 [26,105–107]). If true, then the
crucial regulatory step governing loss of SCC at the
metaphase-to-anaphase transition in yeast must involve
arm cohesion sites, or at least the most centromere-proximal
CARs that do not ÔbreatheÕ ( 30 kb from the centromeric
DNA) [26,106,115].
Work from several labs has revealed that the mechanism
whereby such hypothetical ÔstrongÕ cohesion sites are
dissociated or disassembled entails proteolytic destruction
of the cohesin subunit Scc1/Mcd1 (reviewed in [29,30,
116,117]). First, it was shown that at the nonpermissive
temperature, esp1–1
S.c.
. mutants are defective for sister chro-
matid separation and subsequent anaphase [118]. Second, it
was noted that a subset of Scc1/Mcd1p undergoes specific
proteolytic cleavage at the onset of anaphase that is Esp1-
dependent [67]. Importantly, a noncleavable form of Scc1/
Mcd1 behaves in a dominant fashion to retard sister
chromatid separation, thus mimicking the esp1–1 phenotype
[67,68]. Similar observations were made for S. pombe
mutants in the Esp1-related protein Cut1 [73,119]. Esp1
was subsequently shown to be a conserved caspase-like
CD-clan cysteine endopeptidase (i.e. ÔseparaseÕ) capable of
cleaving Scc1/Mcd1 in vitro [120]. In vivo, this cleavage event
is followed by the destruction of Scc1/Mcd1 fragments in a

Ubr1-dependent fashion (i.e. by the N-end rule degradation
pathway) [121]. Destruction of Scc1/Mcd1 is accompanied
by dissociation of Scc3 (and Pds5) from chromosomes,
which together presumably inactivate the cohesin complex
[85]. In this way, the critical connections that hold sisters
together from S phase through early mitosis are literally cut
by Ômolecular scissorsÕ in the form of Esp1/separase at the
onset of anaphase.
The elucidation of separase function provided great
insight into the destruction of SCC, but in itself, neither
explained the timeliness of that destruction, nor the
requirement for ubiquitin-mediated proteolysis in sister
chromatid separation (i.e. as mediated by anaphase pro-
moting complex/cyclosome or APC/C) [70]. Indeed, bud-
ding yeast Esp1 and fission yeast Cut1 are present
throughout the cell cycle and may have additional functions
in spindle morphogenesis and exit from mitosis [104,119,
122–127]. Not surprisingly, the activity of Esp1 towards
Scc1/Mcd1 is coordinated with the cell cycle by multiple
levels of regulation (Fig. 2).
Esp1 exists in a complex with a ÔsecurinÕ protein known as
Pds1 in budding yeast [118]. Unrelated proteins that are
functionally equivalent to Pds1 have been identified in many
systems and appear to regulate Esp1 in two ways (i.e.
vSecurin in Xenopus, PTTG in human cells, Cut2 in
S. pombe [72,128–130]). In each case, sequestration by its
cognate securin inhibits separase’s sister chromatid separ-
ation activity. In addition, Pds1 (and perhaps its analogs)
serves as a chaperone/activator for Esp1, in part by promo-
ting its efficient nuclear targeting [127]. Pds1 contains a

B-type cyclin destruction box and accordingly is degraded
inmitosisinanAPC
Cdc20
-dependent fashion [131,132].
The regulated degradation of Pds1 in mitosis frees Esp1
to act on Scc1/Mcd1, and explains the requirement for
ubiquitin-dependent proteolysis in the metaphase-to-ana-
phase transition. However, recent studies suggest that
similar to authentic caspases, Esp1 itself might undergo
proteolytic cleavage to become catalytically active [16]. In
addition, Xenopus separase is subject to inhibitory phos-
phorylation by Cdc2 [133].
Scc1/Mcd1 cleavage is also regulated at the level of
substrate in that phosphorylation of Scc1/Mcd1 enhances
its cleavage by Esp1-like proteins both in vitro and in vivo
[73,120,134]. Phosphorylation has been attributed to the
Cdc2 kinase in human cells [74] and to the polo-like protein
kinase Cdc5 in yeast [134]. Cdc5 is proposed to facilitate
cleavage of Scc1/Mcd1 via phosphorylation of the endo-
peptidase recognition site; however, whether Cdc5 directly
phosphorylates Scc1/Mcd1 in vivo is unknown [134].
Nonetheless, when Cdc5 is inactivated, separation of sister
chromatid arms is delayed compared to that of centromeric
regions [134]. This observation is consistent with an earlier
study showing that separation of sister centromeric regions
is unaffected by mutations that prevent Esp1-mediated
cleavage of Scc1/Mcd1 [26], and provides additional
evidence that during an unperturbed mitosis, arm cohesion
sites in yeast are ÔstrongerÕ (i.e. more important in resisting
mitotic spindle forces) than centromeric ones.

As mentioned previously, the apparent dichotomy of
cohesin sites at arms and centromeres found in yeast is
paralleled in Drosophila and mammalian cells. In these
metazoans, the bulk of cohesin dissociates from chromo-
some arms during the late stages of mitotic chromosome
condensation, hinting that only a minor, virtually invisible,
fraction of cohesin remains on chromosomes to maintain
SCC through metaphase [14,15,75]. Experiments both in vivo
and in vitro showed that such dissociation of cohesin in
prometaphase is not accompanied by proteolysis of hRad21
[16,74]. The residual pool of chromatin-associated cohesin,
revealed by overexpressing a tagged form of hRad21,
localizes to centromere-proximal regions [16]. As in yeast,
this fraction of hRad21 is apparently removed from
chromatin in an Esp1-dependent fashion, because cleavage
of tagged hRad21 was observed [16]. Indeed, in subsequent
experiments, overexpression of a noncleavable mutant of
hRad21 was shown to block anaphase and cytokinesis [68].
Thus, during mitosis in higher eukaryotes, cohesin is
removed from sister chromatids by at least two distinct
mechanisms such that centromeres, which selectively retain
cohesin, appear to be the ÔstrongerÕ sites of SCC.
While yeast and metazoans both regulate centromeric
vs. arm SCC differentially, they seem to do so in opposite
ways (Fig. 2). One wonders if this is really the case, given
that cohesion proteins and cell cycle machinery are widely
conserved. The apparent differences might simply reflect
the timing with which different organisms assemble their
mitotic spindles and establish bipolar chromosome attach-
ment. We suggest that in all organisms, centromere-

proximal cohesion is absolutely critical for establishing
stable bipolar attachment, whereas either centromere-
2308 P. B. Meluh and A. V. Strunnikov (Eur. J. Biochem. 269) Ó FEBS 2002
proximal SCC or arm SCC can resist outward pulling
spindle forces once bipolar attachment is achieved. In
yeast, the spindle forms in S phase and centromeres (which
have measurable MT binding activity at this point [135])
can, in principle, establish stable bipolar attachment soon
after their replication. In contrast, plant and animal
spindles form in M-phase, following nuclear envelope
breakdown, thus maintenance of centromere-proximal
SCC well into mitosis is essential for proper mitotic
chromosome segregation.
Due to SCC, stable bipolar spindle attachment places
centromeric chromatin under tension. Conceivably this
tension is transduced (mechanically or chemically) to
centromeric cohesin, such that interchromatid cohesion is
weakened without dissociation or cleavage of cohesin. This
would explain why in budding yeast, sister centromeres can
ÔbreatheÕ soon after replication, whereas a similar change in
centromeric cohesion would not occur in higher eukaryotes
until bipolar spindle attachment is established during
prometaphase. Notably, intersister kinetochore distance
does in fact increase in a MT-dependent fashion at this stage
in vertebrate cells [136]. The notion that centromeric
cohesion is by design tension-sensitive seems inconsistent
with observations that cohesin association is favored and
perhaps enhanced at or near centromeres. Indeed, as
discussed above, mitotic centromeres in yeast and metazo-
ans are preferential sites of cohesin binding. However, in

budding yeast, cohesin binding at centromeres has been
most often examined in nocodazole-arrested cells, but to our
knowledge, such results have never been directly compared
to cells synchronously traversing an unperturbed cell cycle.
Thus it is possible that cohesin binding at centromeres is
enhanced as a consequence of mitotic checkpoint activation.
Alternatively, the persistence of cohesin at centromeres
despite loss of cohesion might reflect a second noncohesion-
related role for cohesin at centromeres (see below).
CENTROMERES AS SPECIALIZED
SITES OF COHESION
As highlighted in the preceding sections, SCC at or near
centromeres somehow differs from that on sister chromatid
arms. One simple explanation for this is that all cohesin
complexes are not created equally. Perhaps distinct modes
of regulation reflect differences in the subunit composition
and/or post-translation modification of cohesin complexes.
Indeed, genome sequencing projects have revealed potential
variant cohesin subunits within a single organism, and
distinct cohesin complexes have been identified biochemi-
cally [74,75].
Meiosis is one natural situation where differences in
centromere-proximal vs. centromere-distal SCC are both
functionally significant and might reflect cohesin complexes
of distinct composition. To ensure reductional division in
meiosis I, it is imperative that sister chromatids remain
paired at their centromeres. Meiotic cells express a variant
of the essential cohesin subunit Scc1/Mcd1, called Rec8,
that is thought to replace Scc1/Mcd1 in a meiosis-specific
cohesin complex [17,18,137–139]. Despite significant func-

tional redundancy between these two complexes, they are
likely to play distinct roles in meiosis. The most fascinating
property of Rec8 is its persistence at sister centromeres until
anaphase of meiosis II, whereas Scc1 and the bulk of Rec8
are removed prior to the onset of anaphase I to facilitate
dissolution of chiasmata. This pericentromeric fraction
of Rec8 is critical for kinetochore attachment and proper
chromosome alignment on both the meiosis I and
meiosis II spindles. Presumably, pericentromeric Rec8 is
spared from cleavage and degradation at anaphase of
meiosis I via interaction with an unknown protective
factor(s) [140,141]. Conceivably, this protective factor
simultaneously stabilizes centromeric Rec8 and suppresses
or alters centromere segregation functions. Candidates for
such factors might be defective in mutants that exhibit
precocious sister chromatid separation in meiosis I (e.g.
yeast bub1 mutants [142] or Drosophila mei-S332 mutants
[143,144]) or that seem to bypass meiosis I altogether (e.g.
spo12, spo13,andslk19 mutants [140,145]).
CENTROMERES RECRUIT COHESION
PROTEINS, BUT DO COHESION
PROTEINS RETURN THE FAVOR?
As discussed above, functional centromeres in budding
yeast are necessary and sufficient to promote cohesin
binding over an extended chromosomal domain and in
metazoans, cohesin is preferentially retained at centromeres
until anaphase. It follows that centromeric proteins would
be implicated in cohesin recruitment [10,25,39,146,147].
However, it is too soon to know which, if any, centromere
protein directly mediates cohesin targeting because the

dependency relationships for centromere assembly have not
been fully elucidated. Nonetheless, examples of potential
cross-talk between cohesin and centromeres have been
described (summarized in Fig. 1). When experimentally
mistargeted to noncentromeric chromatin in HeLa cells,
CENP-A, but not CENP-C, causes corecruitment (or
stabilization) hSMC1 [147]. This suggests that CENP-A
might interact with cohesin. The conserved and essential
Ndc80/Hec1 complex (comprised of Ndc80/Hec1, Nuf2,
Spc24, and Spc25 [148,149]); might also serve to recruit
cohesion proteins to centromeric regions. Components of
the Ndc80/Hec1 complex colocalize with centromeres and
impact chromosome segregation in budding and fission
yeast, as well as in human cells [146,148]. Intriguingly,
Ndc80/Hec1 interacts both physically and genetically with
the cohesin subunit Smc1. It remains to be tested whether
cohesin deposition (or selective retention) at centromeres
requires Ndc80/Hec1, or vice versa.
It is possible that cohesin assembly at centromeres is not
directed by a specific centromere protein, but rather is an
indirect consequence of the overall state of histone modifi-
cation. Two properties of heterochromatin, namely, histone
hypoacetylation [35,36,40] and histone H3 lysine 9 methyla-
tion [38,150], are required for faithful chromosome segrega-
tion. As noted earlier, centromeres in many organisms are
heterochromatic. Moreover, several heterochromatin pro-
teins that localize to centromeres via their chromo domains
(which bind lysine-9-methylated histone H3) have also been
implicated in centromere function (i.e. SUV39H1, HP-1,
Clr4, Swi6 [40,55,151–154]). Recent studies in fission yeast

indicate that cohesin may be ÔattractedÕ to (or stabilized at)
centromeric heterochromatin via interaction with such
chromo domain proteins. In the absence of Swi6, neither
Rad21 nor Psc3 (SCC3
S.p.
) are present at centromeres, and
centromeric cohesion, but notably not arm cohesion, is
Ó FEBS 2002 Centromeric chromatin and sister chromatid cohesion (Eur. J. Biochem. 269) 2309
compromised [25,39]. An important and unanswered ques-
tion is whether the centromere directs the specific pattern of
histone modification, formation of pericentric heterochro-
matin, and subsequent cohesin binding or vice versa.
In this regard, several yeast cohesion mutants exhibit a
mitotic checkpoint-dependent delay in the onset of ana-
phase [89,96]. While this could reflect the lack of tension
between the two sister chromatids, it could also indicate that
cohesion contributes to the proper architecture and function
of the kinetochore. Indeed, recent findings suggest that
cohesin components might be required for recruitment and/
or anchoring of some kinetochore proteins. Kinetochore
assembly in the absence of Scc1 in chicken cells appears
to be normal with respect to CENP-C, CENP-H, and
Mad2 targeting. However, without Scc1, the chromosomal
passenger protein INCENP fails to localize properly
to centromeric regions [27]. Similarly, Rad21-depleted
S. pombe cells also show defective targeting of chromoso-
mal passengers (i.e. aurora B kinase and Bir1/Cut17) to
mitotic chromosomes [155]. Although cohesin-dependent
localization of the corresponding proteins in budding yeast
(i.e. Sli15, Ipl1, and Bir1) has not been demonstrated, these

proteins are implicated in centromere function and/or
localize to the centromere [102,156,157].
Interestingly, aurora B, which phosphorylates histone H3
in mitosis, was recently shown to phosphorylate the histone
H3-related centromere protein CENP-A [158]. CENP-A
phosphorylation begins early in prophase, peaks in pro-
metaphase and is lost during anaphase, a pattern reminis-
cent of that of centromeric cohesin [159]. A domin- ant
negative phosphorylation site mutant of CENP-A disrupts
the dynamic localization of INCENP and aurora B. Thus,
centromeric localization of cohesin is dependent (directly or
indirectly) on CENP-A [10,147], and localization of aurora
kinase and its cohort is dependent on cohesin. Once
recruited, aurora kinase may act on CENP-A in a way that
affects both its own localization and that of cohesin. It is
currently unknown if CENP-A phosphorylation by aurora
in vivo requires cohesin or otherwise affects cohesin.
Taken together, these observations hint at a complicated
interplay between centromere and cohesion proteins, both
for their localization and activity. Until recently, the focus
has been on how centromeres might direct local cohesion,
but it seems probable that centromere-proximal cohesion
also coordinates aspects of centromere function. By a design
for which we have only mastered the ABC and perhaps a
first level primer, these interdependencies serve to coordi-
nate the irreversible separation of sister chromatids at the
onset of anaphase with directional chromosome movement
on the spindle and timely cytokinesis.
ACKNOWLEDGEMENTS
We thank Doug Koshland, Vincent Guacci, Paul Megee, Frank

Uhlmann, and Robin Allshire for helpful discussions.
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2314 P. B. Meluh and A. V. Strunnikov (Eur. J. Biochem. 269) Ó FEBS 2002