Centromeres: getting a grip of chromosomes

On monocentric chromosomes the centromere is thechromosomal site at which the kinetochore complex isassembled. This complex mediates the attachment andmovement of chromosomes along spindle microtubules. Thecentromere is usually the last site to retain cohesion betweensister centromeres. The location of the main sensor fordefective spindle assembly at the kinetochore allows therelease of this cohesion, and thus progression through mitosis,to be held in check until key events have been completed. Theintricate nature of the centromere–kinetochore complexes andthe events they co-ordinate and react to is presently beingdissected by studies in several organisms. In particular, severalnew kinetochore proteins have been identified in manyorganisms over the last year.

AddressesMedical Research Council, Human Genetics Unit, Western GeneralHospital, Crewe Road, Edinburgh EH4 2XU, Scotland, UK*e-mail: [email protected]†e-mail: [email protected]

Current Opinion in Cell Biology 2000, 12:308-319

0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved.

Abbreviationsbp base pairsCENP centromere proteinEM electron microscopykMT kinetochore microtubuleMI meiotic division IMT microtubule

IntroductionThe purpose of mitosis is to ensure that both daughtercells receive a complete set of chromosomes after replica-tion and formation of sister chromatids during S phase.During mitosis, the key chromosomal element responsiblefor directing operations is the centromere and its associat-ed kinetochore complex. In most organisms, thecentromere–kinetochore complex forms at a single pointon the chromosome. The exceptions are holocentric organ-isms, such as the nematode Caenorhabditis elegans, in whichthe kinetochore forms along almost the entire chromosomearm [1–3]. Sister kinetochores must be oriented correctlyto ‘capture’ the microtubules that extend from oppositespindle poles. At the same time, sister-chromatids mustcling to each other and only release this cohesion when allcentromeres have achieved bilateral attachment to themitotic spindle. A sensing mechanism — the spindlecheckpoint — exists at kinetochores, which enforces thisgrip between sister centromeres when a single kinetochoreis unattached or other spindle damage is detected [4,5]. Inthe first, reductional, meiotic division I (MI) sister kineto-chores act as a single unit and the number of chromosomesis reduced to half by ensuring that cohesion between sister

centromeres is maintained and that only paired homolo-gous chromosomes segregate. In the second, equational,meiotic division sister chromatids are released so that fourhaploid gametes are formed.

The contribution of cis-acting DNA to kinetochore assem-bly in different organisms has been discussed in recentreviews [6,7]. Briefly, in Saccharomyces cerevisiae the site atwhich kinetochores are assembled is very precisely fixed;125 base pairs (bp) are sufficient to mediate spindleattachment, sister-chromatid cohesion and monitoring ofthese events by checkpoints. In sharp contrast are themuch larger 40–120 kb, moderately repetitive, structuresdefining Schizosaccharomyces pombe centromeres. It isknown that at least 12 kb is required to provide reasonablesegregation function, although these minimal centromeresare subject to epigenetic regulation. In Drosophila andmammals centromere activity has been mapped to highlyrepetitive regions containing satellite DNA and otherrepetitive elements. The layout of centromeric DNA inthe filamentous fungus Neurospora crassa appears to bevery similar to that of Drosophila, with remnants of trans-posable elements interspersed between different types ofsimple repetitive sequence [8]. The available data suggestthat repetitive sequences, such as alphoid DNA in humancells, are preferred substrates for kinetochore assembly.However, there is clearly an epigenetic component to cen-tromere function, since these repetitive sequences are notalways necessary or sufficient for assembly of an activekinetochore. Kinetochore can be assembled and propogat-ed at novel chromosomal locations, these are known asneocentromeres. The possible factors influencing theestablishment and propagation of sites of centromereactivity have been discussed in several recent reviews[6,7] and will not be dealt with here.

Little is known about the requirements for the assembly ofholocentric kinetochores on mitotic chromosomes innematodes or how they switch to telomere-mediated spin-dle attachment during meiosis [1–3]. However, genomeprojects are stimulating the investigation of proteins innematode and plant kinetochores.

Here, we discuss the substantial progress that has beenmade in identifying new centromere-associated compo-nent and elucidating the kinetochore architecture. We alsoreview the present understanding of kinetochore–micro-tubule (MT) attachment and the release ofsister-chromatid cohesion.

Centromere proteins in budding and fissionyeastSince centromere activity in budding yeast only requires125 bp of cis-acting DNA (the CDE I, II and III elements)

Centromeres: getting a grip of chromosomesAlison L Pidoux* and Robin C Allshire†

they are often considered to be uncomplicated. However,there are now 12 known centromere-associated proteins(Cbf1, Ndc10, Cep3, Ctf13, Skp1, Mif2, Cse4, Ctf19,Mcm21, Okp1, Slk19 and Mtw1) at these supposedly simplecentromeres (Figure 1). The main CDEIII DNA-bindingcomplex is CBF3, which is composed of four of these pro-teins: Ndc10, Cep3, Ctf13 and Skp1. Assembly of theCBF3–CDEIII complex requires association of Skp1 withCtf13, which mediates the phosphorylation-dependent acti-vation of Ctf13. Association of phosphorylated Ctf13–Skp1with dimeric Cep3 protects Ctf13 from ubiquitin-mediateddegradation. This Skp1–Ctf13–Cep3 complex interacts withNdc10 and the resulting complex finally binds to CDEIIIDNA [9••]. The Sgt1 protein also associates with Skp1 and isrequired for regulation of CBF3 activity, presumably by acti-vation of Ctf13 [10•].

Atomic force microscopy suggests that CBF3 binds asym-metrically to the right of CDEIII, which induces somewinding of DNA and a sharp bend in the DNA at the bind-ing site [11]. Ctf19, Mcm21 and Okp1 were recentlyidentified as additional factors that interact with cen-tromeric DNA and other centromeric proteins [12•,13••].These three proteins are thought to form a sub-complex,which might mediate the connection between micro-tubules and the components in close proximity tocentromere DNA [13••].

The Bir1 protein is required for accuracy of chromosometransmission, and it interacts directly or indirectly withthe centromere-associated proteins Cbf1, Ctf19, Ndc10and Skp1 [14]. Mtw1 (which is similar to S. pombeMis12)colocalises with centromeres in vivo, and its association

Figure 1

Esp1

Pds1

APCPds1

Microtubule

Sgt1

Scc1/Cohesin

Centromerespecificnucleosome:Cse4, Histone H4H3, H2A, H2B ?

Slk19

Bir1

Mif2

Cbf1

Skp1

Ctf13

Cep1

Ndc10

Cbf3 complex

S. cerevisiae kinetochore structure

Ub

Ctf19

Okp1

Mcm21

Mtw1

Mad1/2/3Bub1/3Cdc20

Current Opinion in Cell Biology

Scc1

Scc3

Smc1

Smc3

Cohesin complex

Schematic representation of centromere-associated proteins inS. cerevisiae. Ctf13 (dark blue) activation via Skp1-Sgt1 leads to theassembly of the CBF3 complex, which binds CD

EIII [9

•• ,10•]. Aputative Cse4-containing centromere specific nucleosome (black)[16•] provides a protein interface to mediate looping of centromereDNA, which is stabilised by interactions between components of asubcomplex Ctf19, Okp1 and Mcm21 [12• ,13•• ] and components ofCBF3, in addition to Cbf1, Cse4 and Mif2. Bir1 (grey circles) interactswith Ctf19, Cbf1 and CBF3 components [14]. Mtw1 (green oval) is anewly identified centromere-associated protein (G Goshima,M Yanagida, personal communication). Slk19 (grey oval) is probably

associated with centromeres through its interactions with the plusends of microtubules [73]. The spindle checkpoint components Mad1,Mad2, Mad3, Bub1 and Bub3 monitor kinetochore assembly and blockthe metaphase–anaphase transition in response to centromere defectsor spindle damage by inhibition of APC [5,6]. Active centromeresrecruit a high concentration of cohesin [65•–68• ,69•• ]. Activation ofAPC leads to securin/Pds1 degradation through the ubiquitin (ub)pathway and thus liberation of the separin/Esp1-dependent proteaseresulting in double cleavage of the Scc1/Mcd1 cohesin and release ofsister chromatid cohesion concomitant with anaphase [4,5,56•].

with centromeric chromatin is Ndc10 dependent [15•].The histone H3 variant Cse4 is homologous to thehuman centromere protein CENP-A, and Mif2 has simi-larity with mammalian CENP-C [16•,17,18]. These twokinetochore proteins appear to be ubiquitous, with puta-tive counterparts in many organisms.

Current models suggest that at S. cerevisiae kinetochoresa Cse4-containing centromere-specific nucleosome facil-itates the formation of a particular looped DNAconformation, facilitating interactions with factors thatbind specifically to the centromeric CDEIII-bindingcomplex CBF3 (Cep3, Ndc10, Ctf13 and Skp1)[13••,16•]. The composition of this putative centromere-specific nucleosome remains unknown. Genetic analysessuggest that Cse4 interacts with histone H4 but thisremains to be directly demonstrated [19]. Presumably,therefore, at least one H3 subunit of this putative cen-tromeric nucleosome is replaced by Cse4; however, it isnot known whether a complete CSE4–H4–H2A–H2Boctamer is formed. Detailed substitutional analysesreveal that no single residue difference between Cse4and H3 is pivotal in Cse4 function, instead severalamino-acid differences scattered throughout the histone-fold domain confer centromere-specific function on Cse4[20]. Post-translational modifications of histones(e.g. phosphorylation, acetylation, ubiquitination, ribosy-lation) might play an important role in the assembly ofthis type of centromere-specific nucleosome.

Fission yeast have distinct domains within their cen-tromeres, which interact with different proteins [21••]

and have different qualities of transcriptional silencing(Figure 2). The association of Swi6 (the counterpart ofmammalian and fly heterochromatin protein 1 [HP1]) andChp1 chromodomain-containing proteins with the outerrepetitive elements of the centromere DNA is dependenton Rik1 and Clr4 (counterpart of fly and mammalianSuvar39). This association mediates the silencing ofmarker genes placed within these repetitive regions. Incontrast, Mis6 and Mis12 associate only with the centralregion of centromere DNA [22,23•], and Mis6 alleviatessilencing at this region but not at the outer repeats [21••].The role of Clr4 and Swi6 at centromeres may well beconserved in mammals because human Suvar39H1 inter-acts with HP1 and is concentrated around centromeres[24•]. The contribution of HP1 and Suvar39 proteins tomammalian centromere function is not known. However,the fact that human Suvar39H1 is also present at ectopi-cally-activated neocentromeres is suggestive of a role incentromere function [25•]. Recently, 12 additional loci(csp genes) have been described that alleviate silencing inthe outer repeats surrounding S. pombe centromeres [26•].The mutants csp7 to 13 have similar defects in chromo-some segregation as chp1, clr4, rik1 and swi6 mutants butdo not affect the localisation of Swi6 or Chp1. Defectiveproteasome function enhances transcriptional repressionwithin S. pombe centromeres and interferes with chromo-some segregation [27].

Kinetochore structure in multicellular eukaryotesUltrastructureThe familiar electron microscope (EM) image of the ver-tebrate kinetochore is a three-layered disc-shaped

Figure 2

Schematic representation of the centromereand associated proteins in S. pombe. Thelarge (40–120 kb) heterochromaticcentromeres are composed of distinctdomains, which have different qualities oftranscriptional silencing and are associatedwith specific proteins. The central domain has

weak silencing, a unique chromatin structure,and is associated with the Mis6 and Mis12proteins [22,23•]. The outer flanking repeatsexperience strong silencing and areassociated with the chromodomain proteinsSwi6 and Chp1 [21•• ]; Rik1 and thechromo/SET domain protein Clr4 are required

for this association. In support of the notionthat transcriptional silencing reflects theassembly of a functional kinetochore complex,mutations in swi6, chp1, rik1 and clr4 alleviatesilencing in the outer repeats; mutations intwelve csp genes have similar phenotypes andare likely to encode additional centromere-associated factors [26•]. The centromeres infission yeast, like heterochromatic regions invertebrates, are hypoacetylated on histones;transient treatment with the histonedeacetylase inhibitor trichostatin A causes adefective centromere state that is propagatedin an epigenetic fashion [42]. A histone H3variant Cnp1 the likely counterpart of CENP-A, localises to the centromere (B Mellone,RC Allshire, unpublished data; K Takahashi,M Yanagida, personal communication).Likewise, a fission yeast CENP-C-like protein,Cnp3, is found at centromeres (AL Pidoux,RC Allshire, unpublished data). The detailedlocation of these proteins within the fissionyeast centromere is not yet known. The Bub1spindle checkpoint protein is recruited tokinetochores early in mitosis [72•].

Swi6 (HP1)Chp1

Clr4 (Suvar39)Rik1

?

Cnp1 (CENP-A) LocationCnp3 (CENP-C) unknown

Hypoacetylatednucleosomes

Outer repeat Central domain

Mis6Mis12


structure, with the bulk of centromeric chromatin under-neath the inner plate and well separated from the outerplate by an electron translucent zone. Centromere pro-teins, such as CENP-C, are associated with the innerplate, whereas motor proteins such as CENP-E anddynein/dynactin associate with the outer plate/fibrouscorona that contacts the plus ends of microtubules(Figure 3) [17,18]. Alternative fixation protocols haveprovided a modified view of the kinetochore in which a50–75 nm thick, structured fibrous mat (perhaps equiva-lent to the outer and inner plates) lies in close proximityto the underlying chromatin [28•]. Outside this mat is abroad, light-staining zone containing thin fibres, which ispresumably equivalent to the fibrous corona observed byconventional fixation. These preparations also revealthat centromeric chromatin has a distinctive ‘mottled’staining pattern compared with chromosome arms. Thispresumably reflects the unique heterochromatic struc-ture of this region. The distribution of CENP and otherproteins relative to this kinetochore structure willrequire investigation.

HolocentricsCytological and EM studies of conventionally fixedchromosomes have indicated that various insects, plantsand nematodes possess kinetochores that have a diffusedistribution, unlike monocentric chromosomes. Tri-lam-inar kinetochore structures assemble along almost theentire length of sister chromatids, indicating that gross

morphological structure is conserved between monocen-tric and holocentric kinetochores [1–3]. It will beinteresting to determine how such kinetochores arenucleated and assembled and whether particular dis-persed cis-acting elements are involved.

C. elegans provides an excellent system for the study of holo-centric chromosomes. Recently, a homologue of theCENP-A histone H3 variant (HCP-3) and two CENP-Fhomologues (HCP-1 and HCP-2) have been identified inC. elegans [29••,30••]. An interesting question is how kineto-chore proteins are distributed along holocentricchromosomes. Immunolocalisation reveals that at the onsetof mitotic prophase there is a reorganisation of the punctateHCP-3 foci into a single row along each chromosome. Thisrow progresses to form two distinct lines facing oppositepoles on metaphase spindles. At anaphase, the two linesseparate and are oriented parallel to each other as they trav-el towards the spindle poles. HCP-1 shows a similarlocalisation during mitosis, except that immunostaining islost during late anaphase and only reappears duringprophase. Interestingly, both CENP-A and CENP-F homo-logues are only found on the poleward-facing side ofseparated sister chromatids. Thus, holocentric chromosomesmust co-ordinate the attachment of multiple MTs along theentire length of each sister chromatid. This is presumablyfacilitated by orienting all the attachment sites of one chro-matid to face one pole, thus discouraging aberrant bi-polarattachment of kinetochore MTs (kMTs) to one chromatid.

Figure 3

Schematic representation of metazoankinetochore architecture and location ofassociated proteins. The proteins CENP-A,CENP-B, CENP-C, CENP-G and CENP-Hare constitutively present at centromeres[17,18,32,33• ,34• ,35–38]. CENP-C,CENP-G and CENP-A are associated withthe inner plate, whereas CENP-B resides inthe underlying chromatin. Proteins such asCENP-E, dynein/dynactin, ZW10, CLIP-170and Rod [44,45• ,46–48,73] have a dynamicassociation with the kinetochore as it maturesduring the cell cycle, for example, CENP-Eassociates with the outer plate/fibrous coronain late G2/mitosis. The association of somefactors is co-dependent, for example, Rod isrequired for recruitment of ZW10 and dynein[40,41]. Not all of these proteins have beenlocalised at the EM level. Several Mad andBub spindle checkpoint proteins have beenlocalised to the kinetochores byimmunofluorescence. Checkpoint proteins areinvolved in monitoring kinetochore–MTattachment and/or tension. Consistent withsuch roles, BubR1 has been localised to theouter and inner kinetochore plates [51•], andBUB3 has been localised to the kinetochoreand to strands between sister kinetochores[50]. SUV39H1 associates at centromeres in

prometaphase; it interacts with anotherchromodomain protein, HP1, in vitro[24• ,25•]. The kinetochore is depicted as thefamiliar tri-laminar structure; proteins have not

yet been localised with respect to the newview of the vertebrate kinetochore obtained byhigh-pressure freezing [28•].

Heterochromatin:Repetitive DNAHP1/Suvar39?

CENP-BBUB3MCAK

INCENP

Inner plate:CENP-CCENP-GCENP-ABUBR1

Outer Plate:CENP-ECENP-FBUBR1

Fibrous corona:CENP-E

Dynein/dynactin

Microtubules

3F3/2

CLIP-170ZW10Rod

Mad and Bub proteins


In nematodes an amazing reorganisation takes place dur-ing meiosis [1–3]. The classic tri-laminar platekinetochore structure is no longer detected, and chromo-somes make direct contact with spindle MTs viatelomeric chromatin. Thus, during meiosis, chromo-somes align parallel to the spindle. Little is known aboutthe factors required for kinetic activity of telomeres dur-ing meiosis. It will be interesting to determine ifCENP-A associates with telomeric regions during meio-sis. Recently, the coiled-coil PUMA1 protein of thenematode Parascaris univalens was found to associatewith both the continuous holocentric kinetochores dur-ing mitosis and the telomeric spindle attachment sitesduring meiosis [31•].

Centromere proteinsThe inner kinetochore plate protein CENP-C is highlyconserved and represents an essential structural compo-nent of vertebrate kinetochores [17,18]. HumanCENP-C is found only at active centromeres. The exactfunction of CENP-C is unknown, but in vitro experi-ments have indicated that its centromere-targetingdomain overlaps with non sequence-specific DNA-bind-ing regions [32]. The centromere-associated proteinMif2 of S. cerevisiae shares two blocks of similarity withCENP-C. Homologues have been described in maize[33•] and recently a CENP-C-like protein (Cnp3) wassubmitted to the fission yeast database. Maize CENP-Clocalises with the kinetochore throughout the cell cyclebut surprisingly, it does not associate with the neocen-tromeric knob structures activated in particular maizelines. Presumably, these knob structures have an alterna-tive mechanism for interacting with spindlemicrotubules that does not involve CENP-C. The fun-damental kinetochore component CENP-C might havethe ability to nucleate kinetochore assembly. However,when it is overexpressed in chicken cells it does not altereither the association of the kinetochore protein ZW10with centromeres or progression into anaphase, indicat-ing that endogenous kinetochore assembly remainsintact [34•]. CENP-C thus appears necessary, but notsufficient, to induce kinetochore assembly. It was alsofound to be a target for proteasome-mediated degrada-tion induced by infection of cells with herpes simplexvirus 1. Thus, in normal cells CENP-C may be regulatedby ubiquitination or SUMO-1 modification [35].

CENP-G and CENP-H are the most recently identifiedmammalian centromere proteins [36,37]. CENP-G is anuclear matrix associated protein and resides on the innerplate of the kinetochore; the sequence of CENP-G isunknown. CENP-H is a coiled-coil protein and associateswith centromeres throughout all stages of the cell cycle.The function of these proteins is not known.

Centromere identity and chromatin structureEpigenetic processes clearly contribute to determiningthe site at which centromeres are formed in S. pombe,

Drosophila and mammals [6,7]. Like Cse4 in buddingyeast, the histone H3 variant CENP-A must be a keydeterminant for establishing and maintaining a particularlocation as the site of kinetochore assembly on mam-malian chromosomes. CENP-A-like sequences haverecently been submitted to the S. pombe and C. elegansdatabases [29••]. The prevailing hypothesis is thatCENP-A assembles into centromere-specific nucleo-somes that nucleate specialist kinetochore components.How is it regulated so that CENP-A only assembles intothis distinctive chromatin structure? The available datasuggest that the expression of mammalian CENP-A latein S phase might be linked with the late time of kineto-chore DNA replication. This provides a temporalmechanism for assembly of CENP-A at active kinetochores [6,7,38].

Unexpectedly, it has recently been observed that thetranscriptionally silent centromeres of S. pombe appear toreplicate relatively early in S phase (J Hubermann, per-sonal communication). As the S. pombe CENP-A-likeprotein Cnp1 is localised to centromeres (B Mellone,RC Allshire, unpublished data; K Takahashi, M Yanagida,personal communication), a key experiment is the deter-mination of the time of cnp1 gene expression, relative tohistones H3 and H4. Rigorous testing is required todetermine whether incorporation of CENP-A homo-logues into centromeric chromatin is dependent on thetiming of centromeric DNA replication. Histone modifi-cation might also play a role. For example, CENP-A-likeH3 variants might only interact with a particular isoformof H4. The hypo-acetylated state of H3 and H4 in silentcentromeric chromatin has been implicated in the regula-tion of fission yeast centromere function, and defectivehistone deacetylase function has similar consequences forcentromere structure and function ([39–41]; K Ekwall,S Grewal, H Levin, RC Allshire, unpublished data). TheMis6 and Mis12 proteins are essential for centromereactivity in S. pombe and are required for the formation ofthe unusual chromatin that coats the central domain offission yeast centromeres [22,23•]. Mis6 appears to actduring S phase to facilitate the assembly of this distinc-tive centromeric chromatin. It will be of interest todetermine whether fission yeast Cnp1 is a component ofthis chromatin.

Microtubule attachment The kinetochore must capture and attach to MTs.Currently, it is thought that the checkpoint proteins asso-ciated with centromeres assemble a sensory complex thatmonitors defects in kinetochore–MT attachment and/ortension generation across sister kinetochores (resultingfrom bilateral attachment) [4,5,17,18]. Considerableprogress has been made in dissecting the spindle-check-point pathway [4,5,17,18], and components of thespindle-checkpoint pathway have been found to associatewith centromeres in many systems. Many types of dam-age are detected, including aberrant kinetochore

assembly and altered MT stability and dynamics. Suchinsults activate the spindle-checkpoint cascade, whichinhibits the anaphase promoting complex/cyclosome,thereby halting the metaphase to anaphase transition byblocking the degradation of key target proteins, such asthe securins: Pds1 (S. cerevisiae), Cut2 (S. pombe) andPTTG (vertebrates) [5,41,42]. This in turn, at least inS. cerevisiae, blocks the cleavage and release of sister chro-matid cohesion proteins (see below). Thus, thekinetochore-based spindle checkpoint acts as an enforcerof sister-chromatid cohesion in response to defects inspindle assembly.

In mammalian cells, kinetochore attachment to MTs,alignment of chromosomes at metaphase and movementof chromosomes during anaphase is mediated by pro-teins such as the kinesin-related motor CENP-E, the dynein–dynactin complex, and CLIP-170[17,18,43,44•,45]. The recruitment of CENP-E to kine-tochores during mitosis may depend on prior associationof CENP-F with kinetochores [44•,45]. Recruitment ofdynein and dynactin to Drosophila kinetochores requiresZW10, which in turn is dependent on the rough dealprotein Rod [46,47]. CENP-E interacts directly withcheckpoint protein BubR1 and interference withCENP-E function induces a mitotic block that can bebypassed by simultaneously inhibiting BubR1. Thisdirectly links the functions of a kinetochore-associatedmotor, and thus kinetochore–MT interactions, with thespindle checkpoint [44•].

The mammalian checkpoint proteins Bub1, BubR1,Bub3, Mad1 and Mad2 are localised to kinetochores[4,5,17,18]. Detailed analyses of Bub3 localisation indi-cate that it coats the entire surface of the kinetochoreand associates with inter-chromatid bridges on theunderlying centromeric heterochromatin [48]. It is pos-sible that such connections relay signals indicating thatboth sister kinetochores are spindle attached, thusallowing sister-chromatid separation to proceed. BubR1has been localised to the inner and outer kinetochoreplates by immuno-EM [49]. The location of the othercheckpoint proteins has yet to be determined at the EM level.

While the identity of budding yeast kinetochore proteinsthat directly interact with MTs remains elusive, MTattachment has been found to be regulated by phospho-rylation. Defects in either the Ipl1 kinase or the Glc7type 1 phosphatase result in high rates of chromosomeloss and activation of the spindle checkpoint [50•–52•].In vitro studies indicate that Glc7 promotes kineto-chore–MT binding, whereas Ipl1 kinase activity isinhibitory. Dephosphorylation of the kinetochore com-ponent Ndc10 correlates with ability to bind MTs.Ndc10 is phosphorylated by Ipl1 in vitro and is hyper-phosphorylated in glc7 mutant extracts, indicating that itis a downstream target of the phosphatase Glc7p. Thus,

it appears that a balance between kinetochore phospho-rylation and dephosphorylation is important forkinetochore activity in budding yeast in vivo.Homologues of these proteins exist in other species —Ipl1 is similar to aurora kinase [53], and type 1 phos-phatases are known to have roles in chromosomesegregation in many organisms.

Phosphorylation of kinetochore components may alsoplay a role in regulation of MT–kinetochore interactionsin mammals [54,55]. Active MAP kinase (ERK) localisesto mammalian kinetochores during mitosis. Staining isparticularly strong during prophase/prometaphase andweakens as chromosomes congress at the metaphaseplate, and disappears from kinetochores in anaphase.This temporal localisation pattern is similar to that of the3F3/2 phosphoantigen, and experiments on isolatedchromosomes indicate that this 3F3/2 antigen is phos-phorylated upon addition of active MAP kinase.Interestingly, CENP-E can also be phosphorylatedin vitro by MAP kinase at sites that are known to regulateits interaction with MTs. CENP-E and MAP kinase havealso been shown to associate in vivo. The precise mech-anism through which ERK influences MT–kinetochoreinteraction remains to be elucidated.

Centromere and arm cohesionThe sindle checkpoint clearly acts to prevent prematuresister-chromatid separation on aberrant spindles. Thetemporal and reactive regulation of the components ofthe sister-chromatid cohesion complex is important.Most of our knowledge of the role of the conservedcohesin proteins comes from studies in budding yeast.Four proteins, Scc1/Mcd1, Smc1, Smc3 and Scc3, associ-ate to form this cohesin complex [5,38,52•]. Cohesion isestablished during S phase and requires Eco1/Ctf7[56•,57•,58]. A remarkable series of experiments indicat-ed that ubiquitin-mediated degradation of the securinPds1 releases the separin/Esp1-dependent proteaseactivity, which cleaves the cohesin componentScc1/Mcd1, causing dissociation of the cohesin complex([59••]; reviewed in [5,42]). These events occur at theonset of anaphase and sister-chromatid separation. Thus,cleavage of Scc1/Mcd1 appears to be the key event inreleasing the connection between sister-chromatids inS. cerevisiae. In Xenopus, however, the majority of cohesindissociates from chromosomes during prophase, signifi-cantly in advance of the metaphase to anaphasetransition [60•]. Likewise, mouse orthologues of Smc1,Smc3 and Scc1/Mcd1 interact to form a complex, but thisdissociates from chromosomes during prometaphase[61••]. It is therefore important to explore the moleculesand mechanisms responsible for maintaining cohesionuntil anaphase in other systems.

Recently, a vertebrate securin was identified that associ-ates with a separin/Esp1 homologue. Expression of a non-degradable, PTTG-related, securin blocks

sister-chromatid separation in Xenopus extracts [43]. Thissuggests that many regulatory events at the metaphase toanaphase transition are conserved. However, it is notknown whether in other organisms, such as S. pombe,separin (Cut1) and securin (Cut2) act analogously to Esp1and Pds1 to release the Scc1 homologue Rad21. It is pos-sible that the same result is achieved in other systemsthroughout different regulatory events (e.g. phosphoryla-tion). The salient point is that in S. cerevisiae, sisterchromatids separate at metaphase–anaphase, uponrelease of the cohesin complex.

In many metazoans cohesion between sister centromeresis regulated differently from cohesion between chro-matid arms. The most striking indication of this is thefamiliar X configuration taken up by human chromo-somes during metaphase in cells blocked in mitosis byMT-destabilising drugs that activate the spindle check-point (for an excellent discussion see [62••]). Thecentromere is the site where many spindle-checkpointproteins are located, which raises the possibility thatcentromere cohesion is regulated independently ofevents occurring along the arms. In a normal cell cycle,centromeres are separated first, while the arms unfurlfrom each other as the centromeres are pulled towardsthe poles in early anaphase. Whether the arms are actu-ally still physically adhered, or are just lightly associateddue to their close proximity, at the stage when cen-tromeres have separated, is unclear. It is possible that apool of cohesin is retained at the centromere until themoment of anaphase onset. Such a scheme would reca-pitulate the protection afforded to a centromeric pool ofthe meiotic Rec8 cohesin from anaphase I tometaphase II in fission and budding yeasts [63•,64••].

Alternatively, other proteins, such as the condensin sub-units, might substitute for cohesin in holding vertebratesister chromatids together until anaphase. In fission yeast,Mis6 and Mis12 are required to maintain centromerecohesion during mitosis; in their absence sister cen-tromeres separate prematurely but chromosome armcohesion appears to be retained. The Drosophila MEI-S332 protein is loaded onto centromeres duringprometaphase, at approximately the time when cohesin isreleased in Xenopus and mouse. MEI-S332 is required tomaintain sister-chromatid cohesion until anaphase [65•].Cohesin dynamics during Drosophila mitosis has not beenreported, and no homologues of MEI-S332 have beenidentified in vertebrates. However, it is possible that pro-teins such as MEI-S332 protect and retain acentromere-specific pool of cohesin until anaphase.

It is intriguing that only active budding yeast cen-tromeres recruit a high density of cohesin proteins thatassociate with chromatin over several kb on each side ofthe centromere [66•–68•,69••]. The release ofcohesin/Scc1 from centromeres appeared, in initial stud-ies, to correlate with the time of centromere separation.

In addition, small centromeric minichromosomes seemto suffer from premature separation, which can be rescued by insertion of additional cis-acting cohesinrecruitment sites [69••]. However, no significant differ-ence in the timing of centromere and separation arms inS. cerevisiae had been detected. The assay routinely usedto monitor centromere cohesion and separation in bud-ding yeast involves long tandem arrays of Lac or Tetoperators. These are inserted 23 or 35 kb from a cen-tromere and detected in live cells expressing the LacI orTet repressor proteins fused to green fluorescent pro-teins (GFP), which bind and light up their cognatesequences. It was assumed that sequences 23 to 35 kbfrom a centromere would be indicative of centromerebehaviour. Surprisingly, when similar arrays are placed1.8 kb and 3.8 kb from a centromere these truly cen-tromeric markers separate very early, before the arrays 20to 30 kb centromere distal [15•]. Thus, in S. cerevisiae thetiming of the separation of centromeres and arms is alsodistinct. However, unlike in other systems, these cen-tromeres visually separate extremely early relative tochromatid arms, in advance of anaphase onset.Centromeres are frequently separated when cells areblocked early in S phase, close to the time of centromerereplication (G Goshima, M Yanagida, personal communi-cation). This might be explained by the distinctproperties of spindle morphogenesis in budding yeast[70]. Experiments indicate that the release of cen-tromeric cohesin, as monitored by chromatinimmunoprecipitation assays does not correlate with thetime of physical separation of sister centromeres. Howcan these observations be reconciled? Perhaps, althoughvisually separated, the sister centromeres are still physi-cally linked — the high concentration of cohesins mightgive them an elastic property that allows them to stretch1 µm apart. The addition of nocodazole (a microtubuledestabilising drug) may cause sister centromeres tobecome re-opposed because of a loss of tension fromkinetochore MTs. Alternatively, activation of the spindlecheckpoint might trigger the enforcement of tight sistercentromere cohesion. Such possibilities could beaddressed by activating the spindle checkpoint in theabsence of spindle damage and observing whether sistercentromeres are separated. It will be informative to mon-itor centromere dynamics (using the 1.8 and 3.8 kbcentromeric tags) upon nocodazole treatment or spindle-checkpoint activation in pre-anaphase mitotic cells thatcontain a spindle.

Maintaining sister chromatid cohesion duringmeiosis During meiosis, specific proteins associate with cen-tromeres to bring about reductional division. Cohesionbetween sister centromeres must be retained frommetaphase of the first meiotic division until anaphase ofthe second division. Again, cohesion components play animport role in coordinating these events. In particularRec8, a meiosis-specific homologue of the mitotic

cohesin component Scc1sc/Rad21sp has been identifiedin both S. cerevisiae and S. pombe [63•,64••]. Buddingyeast Rec8 is maintained at centromeres from anaphase Ito metaphase II. Similarly, in fission yeast, Rec8 isexpressed prior to pre-meiotic S phase, and bymetaphase I it is restricted to centromeric regions whereit remains until initiation of anaphase II. AlthoughRad21 and Rec8 must act to hold homologues togetheruntil the onset of anaphase I, it appears that Rec8 alonemaintains cohesion between sister chromatids at cen-tromeres, thereby ensuring a reductional division. Thedisappearance of Rec8 at anaphase II is consistent withit maintaining sister-chromatid cohesion up to this point.This is supported by the observation that ectopic expres-sion of Rec8 in mitotic S. pombe cells results inreductional-like divisions and chromosome loss [69••].Mutation of genes encoding an Arabidopsis homologue ofRec8 results in sterility [63•,70] and the human Rec8homologue is specifically expressed in testis, consistentwith a role in meiosis [71].

The spindle-checkpoint proteins also impinge uponmeiotic sister-chromatid separation. Fission yeast pro-tein Bub1 associates with centromeres [72•] and plays adirect role in enforcing cohesion at centromeres duringMI since loss of Bub1, but not Mad2, results in high ratesof equational division. Bub1 is required to maintain Rec8at centromeres at anaphase I, thus underscoring thedirect role played by spindle checkpoint components incentromere functions. During meiosis in fission yeast,Bub1 may play an additional role in promoting the unifi-cation of sister kinetochores, so that they face, and thusattach, to the same spindle pole (P Bernard, J-P Javerzat,personal communication). During Drosophila meiosisMEI-S332 is also required to maintain sister-chromatidcohesion until anaphase II, and it is possible that it alsoacts to protect release of putative centromeric Rec8 [72•].

ConclusionStudies in yeasts, vertebrates and holocentric organismsare opening up new views of centromeres. Although cis-acting DNA elements play a direct role in the assembly ofthe budding yeast kinetochore, the relationship betweenDNA sequence and kinetochore assembly is more com-plex in other eukaryotes, where epigenetic phenomena areclearly important. Histone H3 variants, such as CENP-A,are likely to play fundamental roles in centromere–kineto-chore specification. Investigating the requirements forholocentric activity should shed light on plasticity in kine-tochore form and function. Significant advances have beenmade in understanding the regulation of sister-chromatidcohesion in yeast, although the components and mecha-nisms that maintain sister-chromatid cohesion until theonset of anaphase still need to be dissected in many organ-isms. Chromatid cohesion in meiosis is beginning to beunderstood at a molecular level, and insights have beengained into the regulation of MT–kinetochore association.

UpdateSince this review was written, several interesting papers[74•–76•] have been published.

AcknowledgementsWe thank J Hubermann, G Goshima, K Takahashi, M Yanagida, P Bernardand J-P Javerzat for communicating results prior to publication. ALP issupported by a Caledonian Research Foundation Fellowship. RCA’scentromere research of is made possible by core support from the MedicalResearch Council of Great Britain.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest•• of outstanding interest

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6. Karpen GH, Allshire RC: The case for epigenetic effects oncentromere identity and function. Trends Genet 1997, 13:489-496.

7. Csink AK, Henikoff S: Something from nothing: the evolution andutility of satellite repeats. Trends Genet 1998, 14:200-204.

8. Cambareri EB, Aisner R, Carbon J: Structure of the chromosomeVII centromere region in Neurospora crassa: degeneratetransposons and simple repeats. Mol Cell Biol 1998,18:5465-5477.

9. Russell ID, Grancell AS, Sorger PK: The unstable F-box protein•• p58-Ctf13 forms the structural core of the CBF3 kinetochore

complex. J Cell Biol 1999, 145:933-950.Intricate in vitro analyses provide evidence of the order of events that resultin the assembly of a stable CBF3 complex. The unstable protein Ctf13appears to be pivotal in limiting the amount of CBF3 assembled. Ctf13 acti-vation by Skp1 allows association with Cep3, and subsequent binding toNdc10 protects Ctf13 and results in a stable CBF3 complex. The authorsspeculate that this elaborate assembly pathway might restrict the number ofactive centromeres that can be formed and guard against unwanted, andpotentially lethal, ectopic kinetochore assembly.

10. Kitagawa K, Skowyra D, Elledge SJ, Harper JW, Hieter P: SGT1• encodes an essential component of the yeast kinetochore

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A high dosage of SGT1 rescues alleles of skp1 that are defective in S. cere-visiae kinetochore function. Like SKP1, specific alleles of SGT1 displaydefects in kinetochore function. The Sgt1 protein is conserved and is shownto physically interact with Skp1 and to participate in the activation of Ctf13and formation of the CBF3 complex.

11. Pietrasanta LI, Thrower D, Hsieh W, Rao S, Stemmann O, Lechner J,Carbon J, Hansma H: Probing the Saccharomyces cerevisiaecentromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochorecomplex by using atomic force microscopy. Proc Natl Acad SciUSA 1999, 96:3757-3762.

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Ctf19 is a non-essential protein that specifically associates with S. cere-visiae centromere DNA. Cells lacking Ctf19 have a high rate of chromo-some mis-segregation, and released minichromsomes displayed aweakened ability to interact with MTs in vitro. The localisation of Ctf19 isconsistent with its association with centromeres, however it might alsoassociate with spindle poles.

13. Ortiz J, Stemmann O, Rank S, Lechner J: A putative protein complex•• consisting of Ctf19, Mcm21, and Okp1 represents a missing link

in the budding yeast kinetochore. Genes Dev 1999,13:1140-1155.

A one-hybrid screen with S. cerevisiae CEN3 DNA is used to identifynovel centromere-interacting proteins. Three new putative kinetochoreproteins (Ctf19, Mcm21 and Okp1) were fused to the Gal4-activationdomain and induced strong expression of the HIS3 reporter only whenfunctional centromere DNA was used. Mutations in all three genes result-ed in elevated rates of chromosome loss. Two-hybrid and/or co-immuno-precipitation analyses indicated that Ctf19, Mcm21 and Okp1 proteinsinteract with each other: Ctf19 interacts with Ndc10, Cep3 and Cse4;Mcm21 interacts with Skp1, Mif2, Cbf1 and itself; Okp1 associates withNdc10 and Cep3. Chromatin immunoprecipitation shows that Ctf19,Mcm21 and Okp1 associate with centromere DNA via wild-type CDEIIDNA in a CBF3-dependent manner. It is proposed that CDEII contactsvarious centromere proteins (perhaps Mif2, Cse4/nucleosome) resultingin a DNA–protein complex.

14. Yoon HJ, Carbon J: Participation of Bir1p, a member of theinhibitor of apoptosis family, in yeast chromosome segregationevents. Proc Natl Acad Sci USA 1999, 96:13208-13213.

15. Goshima G, Yanagida M: Establishing biorientation occurs with• precocious separation of the sister kinetochores, but not the

arms, in the early spindle of budding yeast. Cell 2000,100:619-634.

Prior to this paper, budding yeast researchers have assumed that GFP-binding Lac or Tet operator arrays placed 23–35 kb distal from a cen-tromere report the state of cohesion at the centromere. Surprisingly, hereit is demonstrated that similar arrays within 4 kb of a centromere physical-ly separate from each other in the very early stages of spindle assemblyand significantly in advance of the release of arm cohesion sites. Theauthors also demonstrate that Mtw1, the S. cerevisiae homologue ofS. pombe Mis12 protein, is localised at budding yeast centromeres. thenatural precocious separation of S. cerevisiae centromeres raises addi-tional questions about how the spindle checkpoint operates and the highconcentration of cohesion at these centromeres and its temporally regu-lated release during mitosis.

16. Meluh PB, Yang P, Glowczewski L, Koshland D, Smith MM: Cse4p is• a component of the core centromere of Saccharomyces

cerevisiae. Cell 1998, 94:607-613.The first direct demonstration that the S. cerevisiae counterpart of the his-tone H3 variant CENP-A is associated with centromere DNA in vivo. Theauthors present a model to accommodate the folding of centromere DNAaround a centromere-specific nucleosome and its interactions with knowncentromere-associated proteins.

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21 Partridge JF, Borgstrom B, Allshire RC: Distinct protein interaction•• domains and protein spreading in a complex centromere. Genes

Dev 2000, in press.Using chromatin immunoprecipitation, the authors show that Swi6 and Chp1are associated with the outer repeat regions of fission yeast centromere 1,whereas Mis6 is confined to the inner repeats and the central core. Swi6 andChp1 are required to mediate silencing within the outer centromere regions butdefective Mis6 specifically alleviates silencing in the central core. Thus, there areat least two types of silent chromatin at fission yeast centromeres and both arerequired for the assembly of a fully functional kinetochore. In addition, Swi6 andMis6 can spread over and silence genes inserted within cen1, and Swi6 canefficiently coat more than 3 kb of exogenous DNA inserted within this cen-tromere. tRNA genes reside in the region of transition between Swi6 and Mis6associated centromeric chromatin. The ability of these centromere-associatedproteins to spread may have relevance to the processes that lead to neocentromere function.

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mis12+ encodes a protein conserved amongst fungi. Mis12 localises toS. pombe centromeres and is associated with the central regions of centromere1 where it is required for the maintenance of the specialised chromatin.Centromeres appear to separate prematurely and spindle morphogenesis isabnormal in cells defective in either Mis6 or Mis12 function. Spindles are elon-gated at metaphase and may be explained by defective centromere-spindleattachment and associated unbalancing of forces.

24. Aagaard L, Laible G, Selenko P, Schmid M, Dorn R, Schotta G,• Kuhfittig S, Wolf A, Lebersorger A, Singh PB et al.: Functional

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Suvar39 has an amino-terminal chromodomain and carboxy-terminal SETdomain, which is also found in the S. pombe Clr4 protein required for normal cen-tromere structure and function. Mammalian Suvar39H1 localises to centromeresat metaphase and interacts with heterochromatin protein 1 (HP1) in vitro.

25. Aagaard L, Schmid M, Warburton P, Jenuwein T: Mitotic• phosphorylation of SUV39H1, a novel component of active

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The centromeric localisation of SUV39H1 is retained in dicentric humanchromosomes and SUV39H1 is recruited to neocentromeres at novel sites.This suggests that it may have a role in determining centromere activity.SUV39H1 accumulates at centromeres during prometaphase and dissoci-ates at the metaphase to anaphase transition. SUV39H1 displays a mitosis-specific pattern of phosphorylation.

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29. Buchwitz BJ, Ahmad K, Moore LL, Roth MB, Henikoff S: A histone•• H3-like protein in C. elegans. Nature 1999, 401:547-548.The histone H3 variant CENP-A is conserved in C. elegans and localises tothe kinetochore of these holocentric chromosomes. Injection of hcp-3 intoC. elegans (CENP-A) dsRNA resulted in embryonic death, with a high inci-dence of nuclei of unequal size, presumably due to chromosome mis-segre-gation. This confirms that histone H3 variants are fundamental determinants ofkinetochore assembly. How are kinetochores assembled along the length ofsuch chromosome is the next question. These initial observations suggest thatseparate nucleation centres seen in interphase nuclei may be gathered togeth-er into linear structures in mitosis.

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somatic cell mitosis: direct interactions of active ERK withkinetochores and regulation of the mitotic 3F3/2 phosphoantigen.J Cell Biol 1998, 142:1533-1545.

55. Zecevic M, Catling AD, Eblen ST, Renzi L, Hittle JC, Yen TJ,Gorbsky GJ, Weber MJ: Active MAP kinase in mitosis: localizationat kinetochores and association with the motor protein CENP-E.J Cell Biol 1998, 142:1547-1558.

56. Toth A, Ciosk R, Uhlmann F, Galova M, Schleiffer A, Nasmyth K: Yeast• cohesin complex requires a conserved protein, Eco1p(Ctf7), to

establish cohesion between sister chromatids during DNAreplication. Genes Dev 1999, 13:320-333.

The cohesin complex is characterised in detail in this paper. Smc1, Smc3,Scc1/Mcd1 and Scc3 form a complex that binds chromatids during late G1through to anaphase. Scc2 mediates this chromatin binding, and Eco1/Ctf7establishes sister-chromatid cohesion during replication.

57. Skibbens RV, Corson LB, Koshland D, Hieter P: Ctf7p is essential• for sister chromatid cohesion and links mitotic chromosome

structure to the DNA replication machinery. Genes Dev 1999,13:307-319.

The protein Ctf7/Eco1 acts during S phase to establish sister chromatidcohesion in S. cerevisiae. Once cohesion has been established Ctf7/Eco1is no longer required. Genetic interactions between ctf7 and components ofthe replication machinery suggest that it may act at replication forks.

58. Uhlmann F, Nasmyth K: Cohesion between sister chromatids mustbe established during DNA replication. Curr Biol 1998,8:1095-1101.

59. Uhlmann F, Lottspeich F, Nasmyth K: Sister-chromatid separation at•• anaphase onset is promoted by cleavage of the cohesin subunit

Scc1. Nature 1999, 400:37-42.It is demonstrated in vitro that an Esp1-dependent activity is unleashed thatcleaves the S. cerevisiae cohesin Scc1/Mcd1 at two sites, resulting in itsrelease from chromatin. This cleavage by Esp1 is inhibited by Pds1, consis-tent with maximal cleavage and dissociation of cohesin at anaphase onsetwhen Pds1 is degraded. Mutation of the Esp1-dependent cleavage sites inScc1/Mcd1 results in the establishment of cohesion but inhibition of sister-chromatid separation.

60. Losada A, Hirano M, Hirano T: Identification of Xenopus SMC• protein complexes required for sister chromatid cohesion. Genes

Dev 1998, 12:1986-1997.Homologs of Smc1, Smc3 and Scc1/Mcd1 are identified as part of the iso-lated Xenopus cohesin complex. This complex associates with chromatinduring S phase and is required for chromatid cohesion. It dissociates fromchromatin during mitotic prophase.

61. Darwiche N, Freeman LA, Strunnikov A: Characterization of thecomponents of the putative mammalian sister chromatidcohesion complex. Gene 1999, 233:39-47.

62. Rieder CL, Cole R: Chromatid cohesion during mitosis: lessons•• from meiosis. J Cell Sci 1999, 112:2607-2613.The authors present the available data and the arguments for two distinctforms of sister-chromatid cohesion: arm and centromere.

63. Klein F, Mahr P, Galova M, Buonomo SB, Michaelis C, Nairz K,• Nasmyth K: A central role for cohesins in sister chromatid

cohesion, formation of axial elements, and recombination duringyeast meiosis. Cell 1999, 98:91-103.

A comprehensive analysis of the role of cohesin components in sisterchromatid cohesion and recombination during S. cerevisiae meiosis.Smc3 and the meiosis specific Scc1/Mcd1 homologue Rec8 arerequired for sister chromatid cohesion and synatonemal complex forma-tion. Meiotic recombination was reduced in cells lacking Smc3 or Rec8,however, double-strand breaks were formed with normal timing but washyper-resected, consistent with abnormal processing. Rec8 and Smc1coat the entire chromosomal axis during pachytene and subsequentlybecome restricted to a few foci, which coincide with centromeric regionsat anaphase I. By anaphase II, association of Rec8 and Smc3 with chro-mosome spreads is not detected.

64. Watanabe Y, Nurse P: Cohesin Rec8 is required for reductional •• chromosome segregation at meiosis. Nature 1999,

400:461-464.In S. pombe ectopic expression of the meiotic cohesin component Rec8can rescue mitotic defects in cells lacking the mitotic cohesin Rad21, butRad21 cannot substitute for Rec8 during meiosis. In cells lacking Rec8,sister centromere regions undergo equational rather than reductional divi-sion and separate to opposite poles in meiosis I. In meiosis II random,unequal chromosome segregation takes place, resulting in low spore via-bility. During meiotic prophase, Rec8 associates with chromosomes inthe nuclear domain occupied by centromeres but not telomeres. Aftermeiosis I chromatin-associated Rec8 is retained only at centromeres.Rec8 disappears completely at anaphase of meiosis II. Expression ofRec8 in place of Rad21 in mitotic diploid cells induces a reductionalchromosome segregation pattern. The authors argue that Rec8 isrequired to ensure cohesion of chromatids in centromeric regions and toorient sister-kinetochores so that they act as a single functional unit andmove to the same pole during meiosis I.

65. Tang TTL, Bickel SE, Young LM, Orr-Weaver TL: Maintenance of• sister-chromatid cohesion at the centromere by the Drosophila

MEI-S332 protein. Genes Dev 1998, 12:3843-3856.Drosophila Mei-S332 is recruited to centromeres during prometaphase, andthis association is independent of intact microtubules. The carboxyl terminusis essential for chromosomal association. The authors suggest that MEI-S332 is required to maintain sister chromatid cohesion specifically at cen-tromeres, so as to counteract the poleward pulling forces exerted bykinetochores attached to microtubules.

66. Megee PC, Koshland D: A functional assay for centromere• associated sister chromatid cohesion. Science 1999,

285:254-257.An inducible recombination system combined with visual monitoring ofchromatid separation is used to demonstrate that active centromeres aresites of sister chromatid cohesion in S. cerevisiae. The assay is per-formed on cells released into nocodazole, thus maintenance of cen-tromere cohesion and chromatid separation are scored while the spindlecheckpoint is activated.

67. Blat Y, Kleckner N: Cohesins bind to preferential sites along yeast• chromosome III, with differential regulation along arms versus

the centric region. Cell 1999, 98:249-259.The distribution of cohesin was examined over the entire length of S. cere-visiae chromosome III by hybridisation of DNA recovered from chromatinimmunoprecipitations to ordered arrays of chromosome II clones.Scc1/Mcd1 and Smc1 associated preferential with 23 sites but particu-larly strong association with a 50 kb region around the centromere wasfound. The DNA sequences associated with cohesin tended to have a highAT composition.

68. Megee PC, Mistrot C, Guacci V, Koshland D: The centromeric sister• chromatid cohesion site directs Mcd1p binding to adjacent

sequences. Mol Cell 1999, 4:445-450.Chromatin immunoprecipitation is used to demonstrate strong associationof the S. cerevisiae cohesin component Mcd1/Scc1 with at least 2 kb ofcentromere-flanking chromatin. Inducible recombination was used todelete the centromere from a minichromosome in G1 or M phase anddemonstrate that centromeric DNA is required to establish and maintaincohesin association with centromere flanking chromatin. The high AT(60%) content of the sequences adjacent to the centromere promotes effi-cient recruitment of cohesin. A centromere-associated protein may beresponsible for the initial recruitment of cohesin, which might then spreadoutwards along neighbouring nucleosomes. The functional significance ofcentromeric cohesin must be reassessed with the finding that native cen-tromeres separate early during an unadulterated cell cycle (G Goshima,M Yanagida, personal communication).

69. Tanaka T, Cosma MP, Wirth K, Nasmyth K: Identification of cohesin•• association sites at centromeres and along chromosome arms.

Cell 1999, 98:847-858.Extensive analyses using chromatin immunoprecipitation in S. cerevisiaeindicates that Scc1/Mcd1 is associated with specific sites on chromosomearms from S phase until anaphase onset. Short cis-acting sequences wereidentified that confer cohesin association. Scc1/Mcd1 was found to strong-ly associate with CEN3 and CEN6. The entire 6 kb region flanking CEN3appeared to be associated with cohesin. A functional centromere wasshown to be required to efficiently recruit cohesin. Prior inactivation of a cen-tromere was found to be required to abolish recruitment of cohesin in cellswith defective kinetochore components. Minichromosomes with functionalcentromeres apparently separate prematurely in cells arrested at metaphasecompared with chromosomal centromeres. In the light of new data(G Goshima, M Yanagida, personal communication) this may reflect the factthat endogenous centromeres were visualised with Tet operator arrays 35 kbfrom CEN5. Addition of multiple strong-arm cohesion sites delayed separa-tion of minichromosome chromatids. Thus the cohesin-associated siteslocated along chromosome arms confer sister chromatid cohesion. The func-tional significance of centromeric cohesin must be reassessed with the find-ing that native centromeres separate early during a normal cell cycle(G Goshima, M Yanagida, personal communication).

70. Winey M, Mamay CL, O’Toole ET, Mastronarde DN, Giddings TH Jr,McDonald KL, McIntosh JR: Three-dimensional ultrastructuralanalysis of the Saccharomyces cerevisiae mitotic spindle. J CellBiol 1995, 129:1601-1615.

71. Parisi S, McKay MJ, Molnar M, Thompson MA, van der Spek PJ,van Drunen-Schoenmaker E, Kanaar R, Lehmann E, Hoeijmakers JH,Kohli J: Rec8p, a meiotic recombination and sister chromatidcohesion phosphoprotein of the Rad21p family conserved fromfission yeast to humans. Mol Cell Biol 1999, 19:3515-3528.

72. Bernard P, Hardwick K, Javerzat JP: Fission yeast bub1 is a mitotic• centromere protein essential for the spindle checkpoint and the

preservation of correct ploidy through mitosis. J Cell Biol 1998,143:1775-1787.

Mutations in the S. pombe HP1 homologue Swi6 are synthetically lethalwhen the mitotic checkpoint protein Bub1 is deleted. As in multicellulareukaryotes, Bub1 is recruited to kinetochores during the early stages ofmitosis. However, a pool of Bub1 remains centromere associated atmetaphase and even at telophase.

73. Zeng X, Kahana JA, Silver PA, Morphew MK, McIntosh JR, Fitch IT,Carbon J, Saunders WS: Slk19p is a centromere protein thatfunctions to stabilize mitotic spindles. J Cell Biol 1999, 146:415-425.

74. Coweison NP, Partridge JF, Allshire RC, McLaughlin PJ: Dimerisation• of chromo shadow domain and distinction from chromo domain

revealed by structural analysis. Curr Biol 2000, in press.The crystal structure of the chromo shadow domain of fission yeast Swi6(HP1 homologue) is resolved. These analyses reveal a novel dimeric struc-ture formed by two chromo shadow domains. Dimerisation is confirmed byin vitro studies. It is proposed that the deep cleft formed upon the assemblyof this dimeric structure may accomodate an interaction with pentapeptidemotifs found in interacting proteins. The implications of this structure for theformation of silent chromatin are discussed.

75 Saffery R, Irvine DV, Griffiths B, Kalistis P, Wordeman L, Choo KH:• Human centromeres and neocentromeres show identical

distribution patterns of >20 functionally important kinetochore-associated proteins. Hum Mol Genet 200, 9:175-185.

Fluorescence in situ hybridisation using probes that unambiguously deco-rate a neocentromere was combined with immunostaining with reagents thatspecifically recognise the centromere/kinetochore associated proteinsCENP-A, CENP-B, CENP-C, CENP-E, CENP-F, INCENP, CLIP-170,

dynein, dynactin subunits p150 (Glued) and Arp1, MCAK, Tsg24, p55CDC,HZW10,

HBUB1, HBUBR1, BUB3, MAD2, ERK1, 3F3/2, topoisomerase

II and a murine HP1 homologue, M31. All proteins apart from CENP-B werepresent at the neocentromeres. Thus, the components at these extraordinarycentromeres are very similar to normal human centromeres. Thus, the prima-ry DNA sequence at active centromeres seems unimportant once a func-tional kinetochore has been established.

76 Henikoff S, Ahmad K, Platero JS, van Steensel B: Heterochromatic• deposition of centeromeric histone H3-like proteins. Proc Natl

Acad Sci USA 2000, 97:716-721.Cid1, the Drosophila homologue of the histone H3-like protein CENP-A,is shown to localise at fly centromeres. Expression of the human, mouse,and budding yeast centromeric H3-like proteins in flies from the Cid1 pro-moter results in their enrichment in pericentric heterochromatin.Remarkably, the yeast and worm proteins also accumulate at pericentricheterochromatin when expressed in human cells. The authors propose thatcentric heterochromatin assists in the deposition of these centromere spe-cific histone H3-like proteins.

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Centromeres: getting a grip of chromosomes