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: 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) 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 [...]... 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... reminiscent 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... 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 coordinate 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,... (2001) Serial regulation of transcriptional 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, 765–773 116 Koshland, D.E & Guacci, V (2000) Sister chromatid cohesion: the beginning of a long and beautiful relationship... precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast Cell 100, 619–633 106 He, X., Asthana, S & Sorger, P.K (2000) Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast Cell 101, 763–775 107 Pearson, C.G., Maddox, P.S., Salmon, E.D & Bloom, K (2001) Budding yeast chromosome structure and dynamics during... essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery Genes Dev 13, 307–319 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 91 Ciosk, R., Shirayama, M.,... 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, 459–472 135 Kingsbury, J & Koshland, D (1991) Centromere-dependent binding of yeast minichromosomes to microtubules...2310 P B Meluh and A V Strunnikov (Eur J Biochem 269) compromised [25,39] An important and unanswered question is whether the centromere directs the specific pattern of histone modification, formation of pericentric heterochromatin, and subsequent cohesin binding or vice versa In this regard, several yeast cohesion mutants exhibit a mitotic checkpoint-dependent delay in the onset of anaphase [89,96]... T.L (2000) Sisterchromatid 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 14 Darwiche, N., Freeman, L.A & Strunnikov, A (1999) Characterization of the components of the putative . 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. 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. 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;