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

doi:10.1046/j.1432-1033.2002.02886.x

Eur. J. Biochem. 269, 2300–2314 (2002) (cid:1) FEBS 2002

MINIREVIEW

Beyond the ABCs of CKC and SCC Do centromeres orchestrate sister chromatid cohesion or vice versa?

Pamela B. Meluh1 and Alexander V. Strunnikov2 1Memorial Sloan-Kettering Cancer Center, Laboratory of Mechanism and Regulation of Mitosis, New York, USA; 2Unit of Chromosome Structure and Function, NIH, NICHD, Laboratory of Gene Regulation and Development, Bethesda, MD, USA

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.

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

I N T R O D U C T I O N

been proposed [2,3]. We imagine that as for the DNA, replication of chromatin must also be a faithful process.

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 (cid:1)instructions(cid:2) 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

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: p-meluh@ski.mskcc.org 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)

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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

DNA binding activity DNA binding activity

ATPase, coiled-coil domain ATPase, coiled-coil domain HEAT repeats (BIR repeats) HEAT repeats Smc2; Cut14; HCAP-E Smc4; Cut3; HCAP-C Ycs4; Cnd1; HCAP-D2 Ycg1/Ycs5; Cnd3; HCAP-G Brn1; Cnd2; HCAP-H/Barren Barren subunit

Cohesin complex

ATPase, coiled-coil domain ATPase, coiled-coil domain

Smc1, Psm1; hSMC1 Smc3, Psm3; hSMC3 Scc1/Mcd1; Rad21; hRAD21/hSCC1 DNA binding activity DNA binding activity 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

HEAT repeats

Scc2; Mis4; HUMHBC4244 Scc4; ??; ?? Ctf7/Eco1; Eso1; ?? on cohesin; Pds5S.p. interacts with cohesin complex Cohesin complex loading onto chromatin during S phase Cohesin complex loading onto chromatin during S phase Interacts with PCNA; maturation of cohesion; Ctf7/Eco1 C2H2 Zn ﬁnger-like

possesses in vitro lysine acetyltransferase activity toward itself, Scc1/Mcd1, Scc3, and Pds5

domain, Gcn5-related N-acetyl- transferase super family domain; Eso1 also has a polymerase domain (Pol eta) Trf4 & Trf5; (Cid12?); DNA polymerase sigma Cohesion establishment during S phase; Trf4 associates (POLS?)

physically with both Smc1 and Smc2 Cohesion establishment during S phase Accumulates on unattached CKC’s during prophase and Ctf18; (Spbc902.02c?); ?? Mad2; Mad2; MAD2 RFC homology Mitotic checkpoint protein

prometaphase (as do other mitotic checkpoint proteins); Mad2 (or Bub1 and BubR1) can physically associate with APCCdc20 to inhibit its activity CAR (Cohesin Often intergenic A+T-rich region DNA sequence bound by cohesin (in some cases, can promote Associated Region)

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 RING-H2 domain subunit, cullin subunit Promoting Complex’’ or ‘‘Cyclosome’’ Multi-subunit ubiquitin-ligase (E3) for mitotic progression and cyclin B destruction; Cdc20 is the speciﬁcity factor for Pds1; APCCdc20 cyclin B-ubiquitin ligase activity is inhibited by Mad2 Among other things, may phosphorylate Scc1/Mcd1 to enhance Cdc5; Plo1; Polo Polo Kinase (S/T kinase) 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

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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 Essential CKC determinant; may have DNA binding activity; Mif2; Cnp3; CENP-C 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; Nuf2S.c. and Spc25S.c. also interact with Smc1 Non-essential CKC-associated protein; requires separase (Esp1) Slk19; ??; ?? Coiled-coil domain

for cleavage and localization to the spindle midzone in anaphase; null mutants show high frequency of premature sister chromatid separation in meiosis I Non-essential; mutants show equational division (i.e. premature Spo12; (Wis3?); ?? 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

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 (cid:1)back-to-back(cid:2) 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

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

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

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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.

T H E N A T U R E O F C E N T R O M E R I C C H R O M A T I N

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) for the [35–38]. The latter features account (Fig. 1)

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 ((cid:1)Me(cid:2)) histone H3-containing nucleosomes as well as the CENP-A-containing variety ((cid:1)A(cid:2)). The degree of nucleosome interspersion is unresolved and may be species-speciﬁc. Histone H3 methylation by (cid:1)SET(cid:2) domain-containing methyltransferases [e.g. Clr4S.p., Su(var)3–9D.m.; SUV39H1] is critical for recruitment of chromo domain proteins (e.g. HP1 or Swi6S.p..) and cohesin to heterochromatic regions. Recruitment of other centromere proteins (e.g. CENP-C ((cid:1)C(cid:2)); INCENP; and possibly the Ndc80/Hec1 complex) is dependent on CENP-A ((cid:1)A(cid:2)). 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 ((cid:1)P(cid:2)) 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.

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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].

CENP-C, are no longer recruited to the CKC [59–64]. for centromere That said, CENP-A is not sufﬁcient 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].

S I S T E R C H R O M A T I D C O H E S I O N

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 (cid:1)sport(cid:2) a particular histone code.

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]).

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.

Thus, in a few short years, we have gone from regarding SCC as a (cid:1)cytological formalism(cid:2) 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 (cid:1)addresses(cid:2) 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.

P R O T E I N S I N V O L V E D I N E S T A B L I S H M E N T O F S I S T E R C H R O M A T I D C O H E S I O N

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

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

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that

time,

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]).

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 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).

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

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 ((cid:1)Cut(cid:2) phenotype), and/or nondisjunction can result from SCC misregulation. Ac, acetylation; P, phos- phorylation; APC, anaphase promoting complex.

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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- it has not been possible to lishment [94,95]. To date, 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 (cid:1)duplication and assembly(cid:2) 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.

C I S - A C T I N G S I T E S R E Q U I R E D F O R S I S T E R C H R O M A T I D C O H E S I O N

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, and A. 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.

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 (cid:1)spot-welding(cid:2) of homologous sister chromatid domains would be a prerequisite for such models.

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.

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

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

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important to note that functional centromeres, which in yeast are speciﬁed by a (cid:1) 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), it occurs at all, is unknown.

Genetics, University of Colorado Health Sciences Center, DN, USA, personal communication); (c) CARs are (cid:1)port- able(cid:2), 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. (cid:1)breathing(cid:2)) [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 (cid:1)nucleation site(cid:2) 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.

There are several observations that seem at odds with this (cid:1)centromere-directed SCC(cid:2) 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 (cid:1)split apart(cid:2) 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.

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 to studies in S. pombe indicating cohesin is support attracted to heterochromatin and possibly plays an inde- pendent structural role in interphase chromatin [25,39].

R E G U L A T I O N O F S I S T E R C H R O M A T I D C O H E S I O N

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.

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

Another possibility is that we have been misled by the pervasive concept of SCC as (cid:1)molecular glue(cid:2), 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

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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 in mitosis in an APCCdc20-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].

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 (cid:1)breathe(cid:2) ((cid:1) 30 kb from the centromeric DNA) [26,106,115].

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 (cid:1)stronger(cid:2) (i.e. more important in resisting mitotic spindle forces) than centromeric ones.

Work from several labs has revealed that the mechanism whereby such hypothetical (cid:1)strong(cid:2) 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, esp1–1S.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. (cid:1)separase(cid:2)) 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 (cid:1)molecular scissors(cid:2) in the form of Esp1/separase at the onset of anaphase.

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 (cid:1)stronger(cid:2) sites of SCC.

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).

that

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 in all organisms, centromere- proximal cohesion is absolutely critical for establishing stable bipolar attachment, whereas either centromere-

Esp1 exists in a complex with a (cid:1)securin(cid:2) 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-

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[140,141]. Conceivably,

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 thus maintenance of centromere-proximal breakdown, SCC well into mitosis is essential for proper mitotic chromosome segregation.

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) 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, and slk19 mutants [140,145]).

C E N T R O M E R E S R E C R U I T C O H E S I O N P R O T E I N S , B U T D O C O H E S I O N P R O T E I N S R E T U R N T H E F A V O R ?

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 (cid:1)breathe(cid:2) 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).

C E N T R O M E R E S A S S P E C I A L I Z E D S I T E S O F C O H E S I O N

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.

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

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 (cid:1)attracted(cid:2) to (or stabilized at) centromeric heterochromatin via interaction with such chromo domain proteins. In the absence of Swi6, neither Rad21 nor Psc3 (SCC3S.p.) are present at centromeres, and centromeric cohesion, but notably not arm cohesion, is

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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.

3. Csink, A.K. & Henikoﬀ, S. (1998) Something from nothing: the evolution and utility of satellite repeats. Trends Genet. 14, 200–204. 4. Ault, J.G. & Nicklas, R.B.

(1989) Tension, microtubule rearrangements, and the proper distribution of chromosomes in mitosis. Chromosoma 98, 33–39.

5. Skibbens, R.V. & Hieter, P. (1998) Kinetochores and the checkpoint mechanism that monitors for defects in the chromo- some segregation machinery. Annu. Rev. Genet. 32, 307–337. 6. Hirano, T. (2000) Chromosome cohesion, condensation, and separation. Annu. Rev. Biochem. 69, 115–144.

7. Ball, A.R. & Yokomori Jr, K. (2001) The structural mainte- nance of chromosomes (SMC) family of proteins in mammals. Chromosome Res. 9, 85–96.

8. Megee, P.C. & Koshland, D. (1999) A functional assay for centromere-associated sister chromatid cohesion. Science 285, 254–257.

9. Megee, P.C., Mistrot, C., Guacci, V. & Koshland, D. (1999) The centromeric sister chromatid cohesion site directs Mcd1p binding to adjacent sequences. Mol. Cell 4, 445–450.

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].

10. Tanaka, T., Cosma, M.P., Wirth, K. & Nasmyth, K. (1999) Identiﬁcation of cohesin association sites at centromeres and along chromosome arms. Cell 98, 847–858.

11. Sears, D.D., Hegemann, J.H., Shero, J.H. & Hieter, P. (1995) Cis-acting determinants aﬀecting centromere function, sister- chromatid cohesion and reciprocal recombination during meiosis in Saccharomyces cerevisiae. Genetics 139, 1159–1173.

12. Lopez, J.M., Karpen, G.H. & Orr-Weaver, T.L. (2000) Sister- chromatid cohesion via MEI-S332 and kinetochore assembly are separable functions of the Drosophila centromere. Curr. Biol. 10, 997–1000.

13. Losada, A., Hirano, M. & Hirano, T. (1998) Identiﬁcation of Xenopus SMC protein complexes required for sister chromatid cohesion. Genes Dev. 12, 1986–1997.

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.

14. Darwiche, N., Freeman, L.A. & Strunnikov, A. (1999) Char- acterization of the components of the putative mammalian sister chromatid cohesion complex. Gene 233, 39–47.

15. Warren, W.D., Steﬀensen, S., Lin, E., Coelho, P., Loupart, M., Cobbe, N., Lee, J.Y., McKay, M.J., Orr-Weaver, T., Heck, M.M. & Sunkel, C.E. (2000) The Drosophila RAD21 cohesin persists at the centromere region in mitosis. Curr. Biol. 10, 1463– 1466.

16. Waizenegger, I.C., Hauf, S., Meinke, A. & Peters, J.M. (2000) Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in ana- phase. Cell 103, 399–410.

17. Klein, F., Mahr, P., Galova, M., Buonomo, S. B., Michaelis, C., Nairz, K. & Nasmyth, K. (1999) A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell 98, 91–103.

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.

18. Watanabe, Y. & Nurse, P. (1999) Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Nature 400, 461–464.

A C K N O W L E D G E M E N T S

19. Strunnikov, A.V., Larionov, V.L. & Koshland, D. (1993) SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and deﬁnes a new ubiquitous protein family. J. Cell Biol. 123, 1635–1648.

We thank Doug Koshland, Vincent Guacci, Paul Megee, Frank Uhlmann, and Robin Allshire for helpful discussions. 20. Michaelis, C., Ciosk, R. & Nasmyth, K. (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91, 35–45.

R E F E R E N C E S

1. Davey, M.J. & O’Donnell, M. (2000) Mechanisms of DNA 21. Guacci, V., Koshland, D. & Strunnikov, A. (1997) A direct link between sister chromatid cohesion and chromosome condensa- tion revealed through the analysis of MCD1 in S. cerevisiae. Cell 91, 47–57. replication. Curr. Opin. Chem. Biol. 4, 581–586.

22. Hartman, T., Stead, K., Koshland, D. & Guacci, V. (2000) Pds5p is an essential chromosomal protein required for both sister 2. Karpen, G.H. & Allshire, R.C. (1997) The case for epigenetic eﬀects on centromere identity and function. Trends Genet. 13, 489–496.

Centromeric chromatin and sister chromatid cohesion (Eur. J. Biochem. 269) 2311

(cid:1) FEBS 2002

chromatid cohesion and condensation in Saccharomyces cerevi- siae. J. Cell Biol. 151, 613–626.

41. Grewal, S.I., Bonaduce, M.J. & Klar, A.J. (1998) Histone deacetylase homologs regulate epigenetic inheritance of tran- scriptional silencing and chromosome segregation in ﬁssion yeast. Genetics 150, 563–576. 23. Watanabe, Y., Yokobayashi, S., Yamamoto, M. & Nurse, P. (2001) Pre-meiotic S phase is linked to reductional chromosome segregation and recombination. Nature 409, 359–363.

42. Ekwall, K., Cranston, G. & Allshire, R.C. (1999) Fission yeast mutants that alleviate transcriptional silencing in centromeric ﬂanking repeats and disrupt chromosome segregation. Genetics 153, 1153–1169. 24. Biggins, S., Bhalla, N., Chang, A., Smith, D.L. & Murray, A.W. (2001) Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae. Ge- netics 159, 453–470.

43. Hari, K.L., Cook, K.R. & Karpen, G.H. (2001) The Drosophila Su (var) 2–10 locus regulates chromosome structure and function and encodes a member of the PIAS protein family. Genes Dev. 15, 1334–1348. 25. Bernard, P., Maure, J.F., Partridge, J.F., Genier, S., Javerzat, J.P. & Allshire, R.C. (2001) Requirement of heterochromatin for cohesion at centromeres. Science 11, 11.

44. Sullivan, K.F. (2001) A solid foundation: functional special- ization of centromeric chromatin. Curr. Opin. Genet. Dev. 11, 182–188. 26. Tanaka, T., Fuchs, J., Loidl, J. & Nasmyth, K. (2000) Cohesin ensures bipolar attachment of microtubules to sister centro- meres and resists their precocious separation. Nat. Cell Biol. 2, 492–499.

45. Earnshaw, W.C. & Rothﬁeld, N. (1985) Identiﬁcation of a family of human centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma 91, 313–321.

27. Sonoda, E., Matsusaka, T., Morrison, C., Vagnarelli, P., Hoshi, O., Ushiki, T., Nojima, K., Fukagawa, T., Waizenegger, I.C., Peters, J.M., Earnshaw, W.C. & Takeda, S. (2001) Scc1/Rad21/ Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells. Dev. Cell 1, 759–770. 28. Allshire, R.C. (1997) Centromeres, checkpoints and chromatid cohesion. Curr. Opin. Genet. Dev. 7, 264–273. 46. Warburton, P.E., Cooke, C.A., Bourassa, S., Vafa, O., Sullivan, B.A., Stetten, G., Gimelli, G., Warburton, D., Tyler-Smith, C., Sullivan, K.F., Poirier, G.G. & Earnshaw, W.C. (1997) Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres. Curr. Biol. 7, 901–904.

29. Zachariae, W. & Nasmyth, K. (1999) Whose end is destruction: cell division and the anaphase-promoting complex. Genes Dev. 13, 2039–2058.

47. Palmer, D.K., O’Day, K., Wener, M.H., Andrews, B.S. & Margolis, R.L. (1987) A 17-kD centromere protein (CENP-A) copuriﬁes with nucleosome core particles and with histones. J. Cell Biol. 104, 805–815. 30. Nasmyth, K., Peters, J.M. & Uhlmann, F. (2000) Splitting the chromosome: cutting the ties that bind sister chromatids. Science 288, 1379–1385.

31. Copenhaver, G.P., Nickel, K., Kuromori, T., Benito, M.I., Kaul, S., Lin, X., Bevan, M., Murphy, G., Harris, B., Parnell, L.D., McCombie, W.R., Martienssen, R.A., Marra, M. & Preuss, D. (1999) Genetic deﬁnition and sequence analysis of Arabidopsis centromeres. Science 286, 2468–2474.

48. Shelby, R.D., Vafa, O. & Sullivan, K.F. (1997) Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites. J. Cell Biol. 136, 501–513. 49. Yoda, K., Ando, S., Morishita, S., Houmura, K., Hashimoto, K., Takeyasu, K. & Okazaki, T. (2000) Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proc. Natl Acad. Sci. USA 97, 7266–7271. 50. Vafa, O. & Sullivan, K.F. (1997) Chromatin containing CENP-A and alpha-satellite DNA is a major component of the inner kinetochore plate. Curr. Biol. 7, 897–900. 32. Masumoto, H., Sugimoto, K. & Okazaki, T. (1989) Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle. Exp. Cell. Res. 181, 181–196.

51. duSart, D., Cancilla, M.R., Earle, E., Mao, J.I., Saﬀery, R., Tainton, K.M., Kalitsis, P., Martyn, J., Barry, A.E. & Choo, K.H. (1997) A functional neo-centromere formed through acti- vation of a latent human centromere and consisting of non- alpha-satellite DNA. Nat. Genet. 16, 144–153. 33. Sun, X., Wahlstrom, J. & Karpen, G. (1997) Molecular structure of a functional Drosophila centromere. Cell 91, 1007–1019. 34. Tyler-Smith, C. & Floridia, G. (2000) Many paths to the top of the mountain: diverse evolutionary solutions to centromere structure. Cell 102, 5–8.

52. Meeks-Wagner, D. & Hartwell, L.H. (1986) Normal stoichio- metry of histone dimer sets is necessary for high ﬁdelity of mitotic chromosome transmission. Cell 44, 43–52.

53. Saunders, M.J., Yeh, E., Grunstein, M. & Bloom, K. (1990) Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres. Mol. Cell Biol. 10, 5721– 5727. 35. Jeppesen, P., Mitchell, A., Turner, B. & Perry, P. (1992) Anti- bodies to deﬁned histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes. Chromosoma 101, 322–332. 36. Ekwall, K., Olsson, T., Turner, B.M., Cranston, G. & Allshire, R.C. (1997) Transient inhibition of histone deacetylation alters the structural and functional imprint at ﬁssion yeast centromeres. Cell 91, 1021–1032.

37. Van Hooser, A.A., Mancini, M.A., Allis, C.D., Sullivan, K.F. & Brinkley, B.R. (1999) The mammalian centromere: structural domains and the attenuation of chromatin modeling. FASEB J. 13 (Suppl. 2), S216–S220. 54. Smith, M.M. & Stirling, V.B. (1988) Histone H3 and H4 gene deletions in Saccharomyces cerevisiae. J. Cell Biol. 106, 557–566. 55. Halverson, D., Gutkin, G. & Clarke, L. (2000) A novel member of the Swi6p family of ﬁssion yeast chromo domain-containing proteins associates with the centromere in vivo and aﬀects chromosome segregation. Mol. Gen. Genet 264, 492–505.

56. Pinto, I. & Winston, F. (2000) Histone H2A is required for normal centromere function in Saccharomyces cerevisiae. EMBO J. 19, 1598–1612.

38. Nakayama, J., Rice, J.C., Strahl, B.D., Allis, C.D. & Grewal, S.I. (2001) Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110–113. 39. Nonaka, N., Kitajima, T., Yokobayashi, S., Xiao, G., Yama- moto, M., Grewal, S.I. & Watanabe, Y. (2002) Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in ﬁssion yeast. Nat. Cell Biol. 4, 89–93. 57. Smith, M.M., Yang, P., Santisteban, M.S., Boone, P.W., Gold- stein, A.T. & Megee, P.C. (1996) A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmis- sion. Mol. Cell Biol. 16, 1017–1026.

40. Taddei, A., Maison, C., Roche, D. & Almouzni, G. (2001) Reversible disruption of pericentric heterochromatin and cen- tromere function by inhibiting deacetylases. Nat. Cell Biol. 3, 114–120. 58. Glowczewski, L., Yang, P., Kalashnikova, T., Santisteban, M.S. & Smith, M.M. (2000) Histone–histone interactions and centro- mere function. Mol. Cell Biol. 20, 5700–5711.

2312 P. B. Meluh and A. V. Strunnikov (Eur. J. Biochem. 269)

(cid:1) FEBS 2002

78. Kimura, K. & Hirano, T. (2000) Dual roles of the 11S regulatory subcomplex in condensin functions. Proc. Natl Acad. Sci. USA 97, 11972–11977.

59. Stoler, S., Keith, K.C., Curnick, K.E. & Fitzgerald-Hayes, M. (1995) A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev. 9, 573–586. 79. Losada, A. & Hirano, T. (2001) Intermolecular DNA inter- actions stimulated by the cohesin complex in vitro: implications for sister chromatid cohesion. Curr. Biol. 11, 268–272.

60. Meluh, P.B., Yang, P., Glowczewski, L., Koshland, D. & Smith, M.M. (1998) Cse4p is a component of the core centromere of Saccharomyces cerevisiae. Cell 94, 607–613. 80. Hirano, M., Anderson, D.E., Erickson, H.P. & Hirano, T. (2001) Bimodal activation of SMC ATPase by intra- and inter-mole- cular interactions. EMBO J. 20, 3238–3250.

81. deJager, M., vanNoort, J., vanGent, D.C., Dekker, C., Kanaar, R. & Wyman, C. (2001) Human Rad50/Mre11 is a ﬂexible complex that can tether DNA ends. Mol. Cell 8, 1129– 1135. 61. Howman, E.V., Fowler, K.J., Newson, A.J., Redward, S., MacDonald, A.C., Kalitsis, P. & Choo, K.H. (2000) Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice. Proc. Natl Acad. Sci. USA 97, 1148– 1153.

82. Anderson, D.E., Losada, A., Erickson, H.P. & Hirano, T. (2002) Condensin and cohesin display diﬀerent arm conformations with characteristic hinge angles. J. Cell Biol. 156, 419–424. 62. Moore, L.L. & Roth, M.B. (2001) HCP-4, a CENP-C-like pro- tein in Caenorhabditis elegans, is required for resolution of sister centromeres. J. Cell Biol. 153, 1199–1208.

63. Oegema, K., Desai, A., Rybina, S., Kirkham, M. & Hyman, A.A. (2001) Functional analysis of kinetochore assembly in Caenorhabditis elegans. J. Cell Biol. 153, 1209–1226.

64. Blower, M.D. & Karpen, G.H. (2001) The role of Drosophila CID in kinetochore formation, cell-cycle progression and het- erochromatin interactions. Nat. Cell Biol. 3, 730–739. 83. Holt, C.L. & May, G.S. (1996) An extragenic suppressor of the mitosis-defective bimD6 mutation of Aspergillus nidulans codes for a chromosome scaﬀold protein. Genetics 142, 777–787. 84. van Heemst, D., James, F., Poggeler, S., Berteaux-Lecellier, V. & Zickler, D. (1999) Spo76p is a conserved chromosome morpho- genesis protein that links the mitotic and meiotic programs. Cell 98, 261–271.

85. Panizza, S., Tanaka, T., Hochwagen, A., Eisenhaber, F. & Nasmyth, K. (2000) Pds5 cooperates with cohesin in maintaining sister chromatid cohesion. Curr. Biol. 10, 1557–1564. 65. Ortiz, J., Stemmann, O., Rank, S. & Lechner, J. (1999) A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. Genes Dev. 13, 1140–1155.

86. Tanaka, K., Hao, Z., Kai, M. & Okayama, H. (2001) Estab- lishment and maintenance of sister chromatid cohesion in ﬁssion yeast by a unique mechanism. EMBO J. 20, 5779–5790. 66. Yamamoto, A., Guacci, V. & Koshland, D. (1996) Pds1p is required for faithful execution of anaphase in the yeast, Saccharomyces cerevisiae. J. Cell Biol. 133, 85–97.

67. Uhlmann, F., Lottspeich, F. & Nasmyth, K. (1999) Sister-chro- matid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400, 37–42. 87. van Heemst, D., Kafer, E., John, T., Heyting, C., vanAalderen, M. & Zickler, D. (2001) BimD/SPO76 is at the interface of cell cycle progression, chromosome morphogenesis, and recombina- tion. Proc. Natl Acad. Sci. USA 98, 6267–6272.

68. Hauf, S., Waizenegger, I.C. & Peters, J.M. (2001) Cohesin clea- vage by separase required for anaphase and cytokinesis in human cells. Science 293, 1320–1323. 88. Uhlmann, F. & Nasmyth, K. (1998) Cohesion between sister chromatids must be established during DNA replication. Curr. Biol. 8, 1095–1101.

89. Skibbens, R.V., Corson, L.B., Koshland, D. & Hieter, P. (1999) Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery. Genes Dev. 13, 307–319. 69. Rieder, C.L. & Cole, R. (1999) Chromatid cohesion during mitosis: lessons from meiosis. J. Cell Sci. 112, 2607–2613. 70. Holloway, S.L., Glotzer, M., King, R.W. & Murray, A.W. (1993) Anaphase is initiated by proteolysis rather than by the inactiva- tion of maturation-promoting factor. Cell 73, 1393–1402.

71. Guacci, V., Hogan, E. & Koshland, D. (1994) Chromosome condensation and sister chromatid pairing in budding yeast. J. Cell Biol. 125, 517–530.

90. Tanaka, K., Yonekawa, T., Kawasaki, Y., Kai, M., Furuya, K., Iwasaki, M., Murakami, H., Yanagida, M. & Okayama, H. (2000) Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase. Mol. Cell Biol. 20, 3459– 3469.

72. Toth, A., Ciosk, R., Uhlmann, F., Galova, M., Schleiﬀer, A. & Nasmyth, K. (1999) Yeast cohesin complex requires a conserved protein, Eco1p (Ctf7), to establish cohesion between sister chro- matids during DNA replication. Genes Dev 13, 320–333.

91. Ciosk, R., Shirayama, M., Shevchenko, A., Tanaka, T., Toth, A. & Nasmyth, K. (2000) Cohesin’s binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol. Cell 5, 243–254.

73. Tomonaga, T., Nagao, K., Kawasaki, Y., Furuya, K., Mura- kami, A., Morishita, J., Yuasa, T., Sutani, T., Kearsey, S.E., Uhlmann, F., Nasmyth, K. & Yanagida, M. (2000) Character- ization of ﬁssion yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase. Genes Dev. 14, 2757– 2770. 92. Furuya, K., Takahashi, K. & Yanagida, M. (1998) Faithful anaphase is ensured by Mis4, a sister chromatid cohesion mole- cule required in S phase and not destroyed in G1 phase. Genes Dev. 12, 3408–3418.

93. Ivanov, D., Schleiﬀer, A., Eisenhaber, F., Mechtler, K., Haering, C.H. & Nasmyth, K. (2002) Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion. Curr. Biol. 12, 323– 328. 74. Losada, A., Yokochi, T., Kobayashi, R. & Hirano, T. (2000) Identiﬁcation and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes. J. Cell Biol. 150, 405–416.

94. Wang, Z., Castano, I.B., De Las Penas, A., Adams, C. & Christman, M.F. (2000) Pol kappa: a DNA polymerase required for sister chromatid cohesion. Science 289, 774–779. 75. Sumara, I., Vorlaufer, E., Gieﬀers, C., Peters, B.H. & Peters, J.M. (2000) Characterization of vertebrate cohesin complexes and their regulation in prophase. J. Cell Biol. 151, 749–762. 95. Carson, D.R. & Christman, M.F.

(2001) Evidence that replication fork components catalyze establishment of cohesion between sister chromatids. Proc. Natl Acad. Sci. USA 98, 8270– 8275. 76. Prieto, I., Suja, J.A., Pezzi, N., Kremer, L., Martinez, A.C., Rufas, J.S. & Barbero, J.L. (2001) Mammalian STAG3 is a cohesin speciﬁc to sister chromatid arms in meiosis I. Nat Cell Biol. 3, 761–766.

96. Hanna, J.S., Kroll, E.S., Lundblad, V. & Spencer, F.A. (2001) Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion. Mol. Cell Biol. 21, 3144–3158. 77. Revenkova, E., Eijpe, M., Heyting, C., Gross, B. & Jessberger, R. (2001) Novel meiosis-speciﬁc isoform of mammalian SMC1. Mol. Cell Biol. 21, 6984–6998.

Centromeric chromatin and sister chromatid cohesion (Eur. J. Biochem. 269) 2313

(cid:1) FEBS 2002

118. Ciosk, R., Zachariae, W., Michaelis, C., Shevchenko, A., Mann, M. & Nasmyth, K. (1998) An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 93, 1067–1076.

97. Baumgartner, M., Dutrillaux, B., Lemieux, N., Lilienbaum, A., Paulin, D. & Viegas-Pequignot, E. (1991) Genes occupy a ﬁxed and symmetrical position on sister chromatids. Cell 64, 761–766. 98. Stack, S.M. & Anderson, L.K. (2001) A model for chromosome structure during the mitotic and meiotic cell cycles. Chromosome Res. 9, 175–198. 99. Meluh, P.B. & Broach, J.R. (1999) Immunological analysis of 119. Funabiki, H., Kumada, K. & Yanagida, M. (1996) Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large com- plexes. EMBO J. 15, 6617–6628. yeast chromatin. Meth Enzymol 304, 414–430.

120. Uhlmann, F., Wernic, D., Poupart, M.A., Koonin, E.V. & Nasmyth, K. (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103, 375–386. 100. Blat, Y. & Kleckner, N. (1999) Cohesins bind to preferential sites along yeast chromosome III, with diﬀerential regulation along arms versus the centric region. Cell 98, 249–259.

101. Laloraya, S., Guacci, V. & Koshland, D. (2000) Chromosomal addresses of the cohesin component Mcd1p. J. Cell Biol. 151, 1047–1056.

121. Rao, H., Uhlmann, F., Nasmyth, K. & Varshavsky, A. (2001) Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability. Nature 410, 955–959. 122. Baum, P., Yip, C., Goetsch, L. & Byers, B. (1988) A yeast gene essential for regulation of spindle pole duplication. Mol. Cell Biol. 8, 5386–5397. 102. Biggins, S., Severin, F. F., Bhalla, N., Sassoon, I., Hyman, A.A. & Murray, A.W. (1999) The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast. Genes Dev. 13, 532–544.

123. Uzawa, S., Samejima, I., Hirano, T., Tanaka, K. & Yanagida, M. (1990) The ﬁssion yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene. Cell 62, 913–925. 103. Birkenbihl, R.P. & Subramani, S. (1995) The rad21 gene product of Schizosaccharomyces pombe is a nuclear, cell cycle-regulated phosphoprotein. J. Biol. Chem 270, 7703–7711.

124. McGrew, J.T., Goetsch, L., Byers, B. & Baum, P. (1992) Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae. Mol. Biol. Cell 3, 1443–1454.

125. Cohen-Fix, O. & Koshland, D. (1999) Pds1p of budding yeast has dual roles: inhibition of anaphase initiation and regulation of mitotic exit. Genes Dev. 13, 1950–1959. 104. Gregson, H.C., Schmiesing, J.A., Kim, J.S., Kobayashi, T., Zhou, S. & Yokomori, K. (2001) A potential role for human cohesin in mitotic spindle aster assembly. J. Biol. Chem 4, 4. 105. Goshima, G. & Yanagida, M. (2000) Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast. Cell 100, 619–633.

126. Tinker-Kulberg, R.L. & Morgan, D.O. (1999) Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage. Genes Dev. 13, 1936–1949. 106. He, X., Asthana, S. & Sorger, P.K. (2000) Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast. Cell 101, 763–775.

127. Jensen, S., Segal, M., Clarke, D.J. & Reed, S.I. (2001) A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1. J. Cell Biol. 152, 27–40. 107. Pearson, C.G., Maddox, P.S., Salmon, E.D. & Bloom, K. (2001) Budding yeast chromosome structure and dynamics during mitosis. J. Cell Biol. 152, 1255–1266.

128. Zou, H., McGarry, T.J., Bernal, T. & Kirschner, M.W. (1999) Identiﬁcation of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science 285, 418–422.

108. Akhmedov, A.T. Frei, C., Tsai-Pﬂugfelder, M., Kemper, B., Gasser, S.M. & Jessberger, R. (1998) Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure. J. Biol. Chem. 273, 24088–24094. 109. Thrower, D.A. & Bloom, K. (2001) Dicentric chromosome stretching during anaphase reveals roles of sir2/ku in chromatin compaction in budding yeast. Mol Biol. Cell 12, 2800–2812. 110. Volpi, E.V., Sheer, D. & Uhlmann, F. (2001) Cohesion, but not 129. Kumada, K., Nakamura, T., Nagao, K., Funabiki, H., Naka- gawa, T. & Yanagida, M. (1998) Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis. Curr. Biol. 8, 633–641. too close. Curr. Biol. 11, R378.

130. Jallepalli, P.V., Waizenegger, I.C., Bunz, F., Langer, S., Speicher, M.R., Peters, J.M., Kinzler, K.W., Vogelstein, B. & Lengauer, C. (2001) Securin is required for chromosomal stability in human cells. Cell 105, 445–457. 111. Haaf, T., Warburton, P.E. & Willard, H.F. (1992) Integration of human alpha-satellite DNA into simian chromosomes: centro- mere protein binding and disruption of normal chromosome segregation. Cell 70, 681–696.

131. Cohen-Fix, O., Peters, J.M., Kirschner, M.W. & Koshland, D. (1996) Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev. 10, 3081–3093. 112. Warburton, P.E. & Cooke, H.J. (1997) Hamster chromosomes containing ampliﬁed human alpha-satellite DNA show delayed sister chromatid separation in the absence of de novo kinetochore formation. Chromosoma 106, 149–159.

132. Visintin, R., Prinz, S. & Amon, A. (1997) CDC20 and CDH1: a family of substrate-speciﬁc activators of APC-dependent pro- teolysis. Science 278, 460–463. 113. Vagnarelli, P.B. & Earnshaw, W.C. (2001) INCENP loss from an inactive centromere correlates with the loss of sister chromatid cohesion. Chromosoma 110, 393–401.

133. Stemmann, O., Zou, H., Gerber, S.A., Gygi, S.P. & Kirschner, M.W. (2001) Dual inhibition of sister chromatid separation at metaphase. Cell 107, 715–726.

134. Alexandru, G., Uhlmann, F., Mechtler, K., Poupart, M.A. & Nasmyth, K. (2001) Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast. Cell 105, 459–472.

114. Simon, I., Barnett, J., Hannett, N., Harbison, C.T., Rinaldi, N.J., Volkert, T.L., Wyrick, J.J., Zeitlinger, J., Giﬀord, D.K., Jaak- kola, T.S. & Young, R.A. (2001) Serial regulation of transcrip- tional regulators in the yeast cell cycle. Cell 106, 697–708. 115. Goshima, G. & Yanagida, M. (2001) Time course analysis of precocious separation of sister centromeres in budding yeast: continuously separated or frequently reassociated? Genes Cells 6, 765–773. 135. Kingsbury, J. & Koshland, D. (1991) Centromere-dependent binding of yeast minichromosomes to microtubules in vitro. Cell 66, 483–495.

116. Koshland, D.E. & Guacci, V. (2000) Sister chromatid cohesion: the beginning of a long and beautiful relationship. Curr. Opin. Cell Biol. 12, 297–301. 117. Amon, A. (2001) Together until separin do us part. Nat. Cell Biol. 136. Waters, J.C., Skibbens, R.V. & Salmon, E.D. (1996) Oscillating mitotic newt lung cell kinetochores are, on average, under tension and rarely push. J. Cell Sci. 109, 2823–2831. 3, E12–4.

2314 P. B. Meluh and A. V. Strunnikov (Eur. J. Biochem. 269)

(cid:1) FEBS 2002

ponents and has a function in chromosome segregation. J. Cell Biol. 152, 349–360.

149. He, X., Rines, D.R., Espelin, C.W. & Sorger, P.K. (2001) Molecular analysis of kinetochore-microtubule attachment in budding yeast. Cell 106, 195–206. 137. Parisi, S., McKay, M.J., Molnar, M., Thompson, M.A., vanderSpek, P.J., vanDrunen-Schoenmaker, E., Kanaar, R., Lehmann, E., Hoeijmakers, J.H. & Kohli, J. (1999) Recap, a meiotic recombination and sister chromatid cohesion phospho- protein of the Rad21p family conserved from ﬁssion yeast to humans. Mol. Cell Biol. 19, 3515–3528.

138. Buonomo, S.B., Clyne, R.K., Fuchs, J., Loidl, J., Uhlmann, F. & Nasmyth, K. (2000) Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin. Cell 103, 387–398. 150. Peters, A.H., O’Carroll, D., Scherthan, H., Mechtler, K., Sauer, S., Schofer, C., Weipoltshammer, K., Pagani, M., Lachner, M., Kohlmaier, A., Opravil, S., Doyle, M., Sibilia, M. & Jenuwein, T. (2001) Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107, 323–337.

151. Kellum, R. & Alberts, B.M. (1995) Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos. J. Cell Sci. 108, 1419–1431. 139. Krawchuk, M.D., DeVeaux, L.C. & Wahls, W.P. (1999) Meiotic chromosome dynamics dependent upon the rec8(+), rec10(+) and rec11(+) genes of the ﬁssion yeast Schizosaccharomyces pombe. Genetics 153, 57–68.

140. Kamieniecki, R.J., Shanks, R.M. & Dawson, D.S. (2000) Slk19p is necessary to prevent separation of sister chromatids in meiosis I. Curr. Biol. 10, 1182–1190. 152. Ekwall, K., Javerzat, J.P., Lorentz, A., Schmidt, H., Cranston, G. & Allshire, R. (1995) The chromodomain protein Swi6: a key component at ﬁssion yeast centromeres. Science 269, 1429– 1431.

141. Rabitsch, K.P., Toth, A., Galova, M., Schleiﬀer, A., Schaﬀner, G., Aigner, E., Rupp, C., Penkner, A.M., Moreno-Borchart, A.C., Primig, M., Esposito, R.E., Klein, F., Knop, M. & Nas- myth, K. (2001) A screen for genes required for meiosis and spore formation based on whole-genome expression. Curr. Biol. 11, 1001–1009. 153. Ekwall, K., Nimmo, E.R., Javerzat, J.P., Borgstrom, B., Egel, R., Cranston, G. & Allshire, R. (1996) Mutations in the ﬁssion yeast silencing factors clr4+ and rik1+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function. J. Cell Sci. 109, 2637–2648.

142. Bernard, P., Maure, J.F. & Javerzat, J.P. (2001) Fission yeast Bub1 is essential in setting up the meiotic pattern of chromosome segregation. Nat. Cell Biol. 3, 522–526. 154. Aagaard, L., Schmid, M., Warburton, P. & Jenuwein, T. (2000) Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres. J. Cell Sci. 113, 817–829.

143. Moore, D.P., Page, A.W., Tang, T.T., Kerrebrock, A.W. & Orr-Weaver, T.L. (1998) The cohesion protein MEI-S332 loca- lizes to condensed meiotic and mitotic centromeres until sister chromatids separate. J. Cell Biol. 140, 1003–1012.

155. Morishita, J., Matsusaka, T., Goshima, G., Nakamura, T., Tatebe, H. & Yanagida, M. (2001) Bir1/Cut17 moving from chromosome to spindle upon the loss of cohesion is required for condensation, spindle elongation and repair. Genes Cells 6, 743–763. 144. LeBlanc, H.N., Tang, T.T., Wu, J.S. & Orr-Weaver, T.L. (1999) The mitotic centromeric protein MEI-S332 and its role in sister- chromatid cohesion. Chromosoma 108, 401–411.

145. Klapholz, S. & Esposito, R.E. (1980) Recombination and chro- mosome segregation during the single division meiosis in SPO12-1 and SPO13-1 diploids. Genetics 96, 589–611.

156. Yoon, H.J. & Carbon, J. (1999) Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome seg- regation events. Proc. Natl Acad. Sci. USA 96, 13208–13213. 157. Kang, J., Cheeseman, I.M., Kallstrom, G., Velmurugan, S., Barnes, G. & Chan, C.S. (2001) Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)-related protein Sli15 during chromosome segregation. J. Cell Biol. 155, 763–774. 146. Zheng, L., Chen, Y. & Lee, W.H. (1999) Hec1p, an evolutionarily conserved coiled-coil protein, modulates chromosome segrega- tion through interaction with SMC proteins. Mol. Cell Biol. 19, 5417–5428.

147. Van Hooser, A.A., Ouspenski, II, Gregson, H.C., Starr, D.A., Yen, T.J., Goldberg, M.L., Yokomori, K., Earnshaw, W.C., Sullivan, K.F. & Brinkley, B.R. (2001) Speciﬁcation of kineto- chore-forming chromatin by the histone H3 variant CENP-A. J. Cell Sci. 114, 3529–3542. 158. Zeitlin, S.G., Shelby, R.D. & Sullivan, K.F. (2001) CENP-A is phosphorylated by aurora B kinase and plays an unexpected role in completion of cytokinesis. J. Cell Biol. 155, 1147–1157. 159. Zeitlin, S.G., Barber, C.M., Allis, C.D., Sullivan, K.F. & Sulli- van, K. (2001) Diﬀerential regulation of CENP-A and histone H3 phosphorylation in G2/M. J. Cell Sci. 114, 653–661. 148. Wigge, P.A. & Kilmartin, J.V. (2001) The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere com-

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

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Beyond the ABCs of CKC and SCC Do centromeres orchestrate sister chromatid cohesion or vice versa?

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