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The Journal of Cell Biology

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Alison L.Pidoux, and segregation required forcentromeric silencing Sim4: anovel fissionyeast protein plate andtheirmovementtooppositepolesrequireMT actions. Congressionofchromosomestothemetaphase same directiontopromoteproperbilateralspindleinter- with allMT-bindingsitesofeachkinetochorefacingthe anaphase. Theresultingsistercentromeresmustbebioriented 2002). Centromeresmustreplicatebutremainlinkeduntil and Allshire,2000a;McIntoshetal.,2002;Nasmyth, equal segregationofchromosomesatcelldivision(Pidoux (MTs)* ofthemitoticandmeioticspindlesensure chore assembles.Kinetochoresinteractwithmicrotubules The centromereisthechromosomalsiteatwhichkineto- display severe chromosome segregationdefects. sim 2 1 Introduction silencing andkinetochore assemblyorstructure. The resulting exploited toidentifycomponentsinvolved incentral core meric . Here,transcriptional silencingwas of afunctionalkinetochore complexandflanking centro- region aretranscriptionally silenced,reflecting the formation -specific proteins.Genesplacedwithineither to underliethekinetochore itself,asitisassociatedwith central corehasadistinctchromatin structureandislikely  segregation Key words: kinetochore; ;centromere; silencing; chromosome TBZ, thiabendazole. IP, immunoprecipitate;MNase,micrococcal nuclease;MT,microtubule; *Abbreviations usedinthispaper:ChIP, chromatinimmunoprecipitation; [email protected] UK. Tel.:44-131-650-7117.Fax:44-131-650-7778.E-mail: 3JR, Building, UniversityofEdinburgh,MayfieldRoad,EdinburghEH9 for CellBiology,InstituteofandMolecular6.34Swann Address correspondencetoRobinC.Allshire,WellcomeTrustCentre The onlineversionofthisarticleincludessupplementalmaterial. http://www.jcb.org/cgi/doi/10.1083/jcb.200212110 The Journal ofCellBiology

Wellcome Trust Centre forCellBiology, Institute ofCellandMolecular Biology, University ofEdinburgh, Edinburgh EH93JR,UK Medical Research CouncilHuman Unit, Western General Hospital, Edinburgh EH42XU,UK

TheRockefeller University Press, 0021-9525

Article

(silencinginthemiddleofcentromere)mutants regions arerequiredforfullcentromerefunction,the the central coreandtheouterrepeats. Although both ission yeast centromeresarecomposedoftwo domains:

1,2 , Volume 161, Number 2,April 28,2003295–307 William Richardson, William /2003/04/295/13 $8.00 2 andRobin C.Allshire sim2 the spindlecheckpoint(MusacchioandHardwick,2002). chores. Thekinetochoreisamonitoringandeffectorsitefor dynamics andMTmotorproteinsactingatthekineto- inverted repeatelements(innermost repeats; Mis6 and,likeMis6,affectsCnp1 coimmunoprecipitates withthecentral corecomponent the unusualchromatin structureofthisregion.Sim4 associated withthecentral coreregionandisrequired for novel essentialcoiled-coilprotein,which isspecifically build afunctionalkinetochore. as apowerful approach foridentifyingproteinsrequiredto and kinetochore function,andestablishdefective silencing fundamental linkbetweensilencing,chromatin structure, calization atthekinetochore. Ouranalysesillustrate the central domain.FunctionalMis6isrequiredforSim4 lo- specific histoneH3variant Cnp1 encodes aknown kinetochore protein,the centromere- centromere, acentralcoreregion ( Takahashi etal.,1992;Pidoux andAllshire,2000b).Ateach structural organization(Fig.1 A;Hahnenbergeretal.,1991; 2000b). Thethreefissionyeastcentromeressharethesame structurally complexcentromeres(PidouxandAllshire, function becauseitcombinesgenetictractabilitywith excellent systemfortheinvestigationofcentromere–kinetochore The fissionyeast, simple organisms(Heetal.,2001;Cheeseman2002). consists ofaverylargecomplexmanyproteinsevenin identification ofitsproteincomponents.Thekinetochore Sullivan etal.,2001). the siteofcentromereactivity(KarpenandAllshire,1997; protein modificationscontributetothemaintenanceof epigenetic mechanismsbasedonproteinheritabilityand An activecentromereisalsoresponsibleforitsownpropagation; specific toeachcentromere.These areflankedbytheouter Understanding thesekinetochorepropertiesrequiresthe 1,2 Schizosaccharomyces pombe CENP-A CENP-A cnt associationwiththe ) issurroundedby . sim4 imr ,an provides ), whichare encodesa brought toyouby provided byPubMedCentral 295 CORE The Journal of Cell Biology 296 viate centralcoresilencingare sociates with kaseko etal.,2001).However, anotherMAP,Alp14/Mtc1,as- with thecentralcoreregionin amitosis-specificmanner(Na- of suchamodel,theMT-associated proteinDis1isassociated conformation inadditiontocentromeric cohesion.Insupport important butauxiliaryrole, possiblyaffectingkinetochore assembles atthecentralcore,and theouterrepeatsprovidean etal.,2000).Itislikelythatthe“kinetochoreproper” (Takahashi corporation ofnewlysynthesizedCnp1–GFPatcentromeres CENP-A; Takahashietal.,2000).Mis6isrequiredforthein- the of centralcorechromatinisalsodistinct,asevidencedby Takahashi etal.,2000;Jin2002).Thecomposition chromatin structure(Saitohetal.,1997;Goshima1999; core–associated proteinsdisruptthiscentralcore–specific Clarke, 1991;Takahashietal.,1992).Mutantsincentral nucleosomal laddertypicalofmostchromatin(Polizziand micrococcal nuclease(MNase)generatesasmearratherthan a tromere (Bernardetal.,2001;Nonaka2002). recruitment ofahighdensityRad21-cohesintothecen- al., 2002;Volpeet2002,2003),whichisrequiredforthe (Bannister etal.,2001;NakayamaPartridge and theassemblyoftranscriptionallysilentheterochromatin activity, methylateshistoneH3,promotingSwi6binding observations suggestamodelinwhichClr4,guidedbyRNAi wall etal.,1997)andmethylatedonlysine9ofH3.Recent matin, areunderacetylatedonhistoneH3andH4tails(Ek- (Kniola etal.,2001). tural studiesalsorevealtwophysicallydistinctdomains al., 1997;Partridgeet2000;Jin2002).Ultrastruc- and Mal2associatewiththecentralcoreregion(Saitohet al., 2001;Nakaseko etal.,2001).Eachfission yeastkineto- not alleviate and includemutantsin tween thethreecentromeres.Thecentralcoresof Most oftheseaffectonlytheheterochromatic al., 1995;Ekwallet1995,1997;Partridge2000). and concomitantlyaffectcentromerefunction(Allshireet have beenidentifiedthatalleviatetranscriptionalsilencing the assemblyofkinetochorecomplex.Severalmutants this islikelytoreflectpackagingintoheterochromatinand transcriptionally silenced(Allshireetal.,1994,1995),and metazoa. Genesplacedwithinfissionyeastcentromeresare matin, whichresemblescentromericheterochromatinin mains (Fig.1A).Theouterrepeatsareassembledinchro- quences) andtheouterrepeatregionsformtwodistinctdo- domain protein(HP1homologue;Ekwalletal.,1995), clr4 cen3 repeat elements( are specificallyassociatedwiththe proteins associate(Partridgeetal.,2000).Swi6andChp1 the regionsofcentromericDNAwithwhichencoded 2002). Theexistenceoftwoclassesmutantsisreflectedin 2000), and served in Central corechromatinisunusual;limiteddigestionwith Nucleosomes oftheouterrepeats,likeotherheterochro- The centralregion(includingtheinnerpartof The JournalofCellBiology specific associationoftheH3variantCnp1(fissionyeast sharea4-kbelement, , whichencodesahistonemethyltransferase(Reaetal., cen2 rik1 otr otr (Woodetal.,2002).

and

silencing(Partridgeetal.,2000;Jin

. Theonlymutantsshowntostronglyalle- otr

imr ), theorganizationofwhichdiffersbe-

inmitosis,but not with

swi6

|

cnt1

Volume 161,Number2,2003

mis6 / , whichencodesachromo-

cnt3

otr

and

, whichispartiallycon-

region,whereasMis6

mal2

, andtheydo

cnt

otr

(Garciaet

cen1 domain

imr

and

se-

lencing andallowedfastgrowthon et al.,1997;Partridge2000)alleviatedcentralcoresi- respectively (Fig.1C);the (Fig. 1C).Thusthe silencing(Allshireetal.,1995),hadlittleeffect telomeric nine ( referred toaswildtype. were designatedFY3027and FY3033 andwillbehenceforth be usefulforscreeningpurposes.Strainswiththisgenotype crippled background forscreening.Toovercomethis,apromoter- growth onmediumlackinguracil,givinganunacceptable containing segregation. Theidentificationof tromere function,chromatinstructure,andchromosome fission yeastcentromerestoisolatemutantsthataffectcen- components. Wehaveexploitedtranscriptionalsilencingat effective toolforthedirectidentificationofkinetochore sets themapartfromthoseofbuddingyeastandprovidesan tion (forreviewseePidouxandAllshire,2000b). affecting cohesion,replication,aswellkinetochorefunc- formed inbuddingandfissionyeastshavegarneredmutants 2002). Screensbasedonminichromosomestabilityper- ganisms (WiggeandKilmartin,2001;Nishihashietal., homology toproteinsdiscoveredingeneticallytractableor- the identificationofmammaliankinetochoreproteinsisby autoimmune sera(Plutaetal.,1995).Analternativerouteto screens, andmuchknowledgehasstemmedfromtheuseof in aconcertedfashiontowardthesamepole. plex kinetochoressothatallMTattachmentsitesareoriented additional leveloforganizationmustoperateatthemorecom- associatewithoneMT(Wineyetal.,1995).An binding sites(Dingetal.,1993).Incontrast,buddingyeast chore, likethoseofmanymetazoa,containsmultipleMT- growing arg Wild-type strains weremutagenized(Fig. 1 B)andfast- core silencing Isolation ofmutantsthatalleviate central and ura4 ( Transcription ismoreweaklyrepressedinthecentralcore A sensitiveassaytomonitorcentralcoresilencing Results the genotype monitor silencingatothersites,strainswereconstructedwith sensitivity andspecificitytobeusableinageneticscreen.To yeast centromeres(Allshireetal.,1994);insertionofthe central corecomponentMis6. tromere centralcoreregion,andtoformacomplexwiththe also beenidentifiedandshowntobeassociatedwiththecen- dates thisapproach.Anovelkinetochoreprotein,Sim4,has mutations inthe B; Allshireetal.,1994,1995;Nimmo1998).The tion at25

cnt

The phenomenonofsilencingatfissionyeastcentromeres Metazoan kinetochoresarenoteasilydissectedbygenetic

) comparedwiththeouterrepeatregions(

his3

geneinthecentralcoreof

arg), formingtinycoloniesafterseveraldaysincuba-

arg3

genesarestronglysilencedat

C (Fig.1C).Aspredicted,the

cnt1:arg3

cnt1:arg3

coloniespicked (Fig.1C).Becausegenesen- genewasinsertedinto

rik1 grewveryslowlyonmedialackingargi- cnt1:arg3

otr2:ura4 gene,requiredforouterrepeatand cnt3:ade6 insertionprovidedsufficient

cen1 cnt3:ade6 sim2 insertionprovednotto ( cnt1 cnt1:ura4 otr2 arg medium,whereas mis6 asCnp1 (Fig.1A).Strains

tel1L:his3 andthetelomere, mutant(Saitoh otr ) allowsgood CENP-A ) offission (Fig.1 ura4 vali- The Journal of Cell Biology ura4 Mellone, B.G.,personalcommunication). E). Littleornoalleviationof silencing wasobservedat type atpermissiveandrestrictive temperatures(Fig.1,Dand strains withthe from the RT-PCR. Therewasasignificant increasein (Allshire etal.,1994;Partridge etal.,2000)wereanalyzedby 2002). Goshima etal.,1999;Partridge2000;Jin dicted, toalleviatecentralcoresilencing(Saitohetal.,1997; sim minigene control( and thetelomere(Fig.1C).Noneof sim1 mutants wereplacedintofourcomplementationgroups, ing drugthiabendazole(TBZ).17oftheconditionallethal thermo- orcryosensitiveand/orsensitivetotheMT-disrupt- screened forconditionallethality:55of180mutantswere coding centralcore–associatedproteinsareessential,we ated silencingat content/full/jcb.200212110/DC1). Thesemutantsallevi- Fig. S1,availableathttp://www.jcb.org/cgi/ tromere (see lelic to random integranteuchromaticcontrollocus( To quantifydefectivecentromeresilencing, mutantswasspecific tothecentralcoreregion. . Thesedataconfirm thatthealleviationof silencing in , 2 sim2 , mis6 3 cnt1:ura4 , and isallelicto , mis12 ura4 4 cnt1:arg3 , forsilencinginthemiddleofcen- ura4-DSE sitein geneinsertedateither , or cnp1 mal2 sim butmaintainedsilencingat ) attheendogenous CENP-A mutantscomparedwithwild , whichareknown,orpre- (Takahashietal.,2000; sim Rint mutantsareal- cnt1 ura4 ) anda sim ura4 , otr1 message mutant locus , ora ura4 otr1: otr nofluorescence with 1996). Neither Star orV-shapedspindleswereseenfrequentlyin spindles withhypercondensedchromatinwerecommon. 2, B–D)andunevensegregationofchromosomes.Short played laggingchromosomesonlateanaphasespindles(Fig. polar spindle. tants, whichmayindicatedefectsintheorganizationofabi- tivity toTBZthanwildtype,butlowersensitivity some (Fig.S1). sim mutants suchas sim in chromosomesegregation sim temperature (36 TBZ. Toinvestigatethechromosomesegregationdefects, even segregation andlaggingchromosomes areseeninthe even mutations ofchromosome IIIsegregation (Fig. 2F).Un- totheribosomalDNArepeatsrevealed allper- hybridizes ging .FISHperformed withaprobethat gation, 24%hadunevensegregation, and40%showedlag- 36% oflateanaphasecellsshowed apparentlynormalsegre- hypercondensed chromosomes, rarelyseeninwild-typecells. There wasahighproportionof cellswithshortspindles The mutantsweregrownatpermissive(25 mutantsshowedenhancedlossratesofaminichromo- mutantsdisplayseveredefects sim4 mutantwasanalyzedingreaterdetail(Fig.2E). Fission yeastkinetochoreprotein,Sim4| sim1 rik1 sim2 C for6h),fixed,andprocessedimmu-

, ,

nor

clr4 -tubulin antibody.

sim3

and innerpartof with a cnt1 scripts fromrandomintegrant( TBZ at25 and YEScontaining0,10,or15mg/ml Assessment ofgrowthonYESat36 (Bottom) Structureofthe the twodomains(Partridgeetal.,2000). tRNA genesatthetransitionpointbetween Vertical linesindicatethepositionof surrounded byouterrepeatregions( centromere 1.(Top)Centralcore( centromere centralcore. mutants thatalleviatesilencinginthe Figure 1. 25 expressed relativetothewildtypeat scripts normalizedto RT-PCR showninD.Thelevelsoftran- 5720, and5688.(E)Quantificationof 5717, 5674,5695,5719,5683,5714, lyzed wereFY4835,4837,4841,5711, at theendogenouslocus.Strainsana- Plates wereincubatedat25 cnt1 10 various loci.Thefirstspotcontains5 of used toassaysilencing.(C)Serialdilutions showing insertionsitesofmarkergenes defective incentralcoresilencing, strain FY3027usedtoisolatemutants NcoI; C,ClaI;E,EcoRI.(B)Diagramof at thecentralcore.Restrictionsites:N,

, and S. pombe 3

C foreach cellsfollowedbyfivefolddilutions.

, and mis6

, and , and

ura4

swi6

showedsupersensitivityto

otr1 otr1

C. (D)RT-PCRof sim4

A geneticscreentoidentify minigenecontrol(

strainstoassaysilencingat

(Fig.1C;Ekwalletal., insertionsites,compared .

ura4

showedgreatersensi-

imr1L

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and

Pidouxetal.

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3

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The Journal of Cell Biology 298 data). Preliminary observationssuggestthat both areprimary defectsin first mitosisafterreleasefrom aG1block,indicatingthat Figure 2. body (green)andDAPIstainingofDNA(red).Bar,5 (H) cellsincubatedat36 DAPI [green]).(GandH)Spindle lengthin ribosomal DNAclustersontheends ofchromosome3(rDNA[red], makes tothetotalofmitoticcells. (F)FISHwithaprobetothe diagrammed. Thebottomfiguresindicatethepercentageeachcategory early anaphaseB,lateB)thatdisplayedthephenotype category (earlymitosis[prometaphase,metaphase,anaphaseA], temperature. Thetopsetofnumbersisthepercentagecellsinthat segregation phenotypesinwildtypeandthe segregation andlaggingchromosomes.(E)Tableofchromosome types andstar-shapedspindles(firsttwocells).(D) segregation andlaggingchromosomes.(C) chromosome segregationinwild-typecells.(B) before fixationandimmunofluorescencewithTAT1 Strains (FY3027,4484,4502,and4536)wereshiftedto36 The JournalofCellBiology Chromosome segregationdefectsin C for4h(

|

Volume 161,Number2,2003 sim4 -tubulin [green],DAPI[red]). mutants(unpublished sim3 cut9 sim4 sim1 (G)and - mutantatrestrictive sim 143 sim4-193 sim4 mutants. - , similarpheno- 106 -tubulin anti- m. (A)Equal isnotde- cut9sim4 , uneven C for6h , uneven (A–D)

and wasreplacedbyan sity whenSim4wasexpressedfromamulticopyplasmid, vealed abandof rified. Westernanalysisoftotal only fourtosixtimes. (2.68 12% increaseinspindlelengthcomparedwithwildtype in metaphase(Heetal.,1997). ponent Mad2wasusedasanalternativemethodofblocking 0.54 sim4 data). S2;unpublished due todecreasedproteinstability (Fig. Sim4 presentina analysis showedthattherewas nochangeintheamountof the polyclonalantibodiesarespecificforSim4.Western 2000; Jinetal.,2002). (Saitoh etal.,1997;Goshima1999;Takahashi in otherstrainsthathavedefectscentralcoreproteins strictive temperatures.Similarobservationshavebeenmade coiled coil(Fig.S2).The to encodeanovel31.7-kDproteinwithlargeregionsof placed byaladderinthe and Clarke,1991;Takahashietal.,1992)waslostre- unique centralcoresmearpatternseeninwildtype(Polizzi hybridization witha Chromatin wasanalyzedbylimitedMNasedigestionand in the Central corechromatinstructureisdisrupted ORF hasthereforebeendesignated www.jcb.org/cgi/content/full/jcb.200212110/DC1). This Fig. S2,availableathttp:// O94494 (SPBC18E5.03c; ORF thionine tolysinechangeataminoacid230in sim4-193 menting genomicfragmentis35kbfrom closely linkedtothematingtypelocus,andcomple- sim4 A genomicplasmidwasisolatedbycomplementationofthe to centromeres sim4 reminiscent of clusteredcentromeres(Fig. 4A).Several spot wasseen in thenucleusoflivingcells ininterphase, nomic locuswithGFP(Bahler etal.,1998).AsingleGFP sim4 Spindle lengthwasmeasuredinmetaphase-arrested netochore-to-pole tensionortoadefectinMTfunction. 1999; Jinetal.,2002);thisispresumablyduetoreducedki- longer metaphasespindlesthannormal(Goshimaetal., 1997). Mutantsdefectiveinkinetochorefunctionhave clustering ofcentromeresseenin fective incentromericcohesion,nordoesitdisplaythede- netochore–spindle interactions. observations suggestthatthe sim4cut9 G andH).Therewasa13%increaseinspindlelength cut9sim4 Antibodies wereraisedagainstGST–Sim4andaffinitypu- The temperature-sensitivephenotype.The –GFP fusiongeneatthe sporesfromtetraddissectionsgerminatedbutdivided encodesanovelproteinthatlocalizes sim4 sim4 m). Overexpressionofthespindlecheckpointcom- 0.37 (2.63 cellsthathadbeenshiftedto36 allelewasfoundtohaveamutationcausingme- mutant ORFwasCOOH-terminally taggedatitsge- m and2.40 sim4 0.94 30 kD(Fig.S2)thatincreasedininten- cnt1 strain,indicatingthatitsdefect isnot sim4 probe.AsshowninFig.3,the m) comparedwith sim4 60-kD bandinastrainwith sim4 sim4 0.31 mutantatpermissiveandre- S. pombe gene is essential (Fig. S2); geneisessential(Fig. mis6 mutanthasdefectsinki- sim4-193 locus(seebelow).Thus, sim4 m, respectively).These mutants(Saitohetal., proteinextractsre- C for4h(Fig.2, andispredicted sim4 mat2 cellsshoweda cut9 mutationis / mat3 (2.32 cut9 . The and The Journal of Cell Biology fbp1 quences wereenriched 6.5-and6.8-fold,respectively, rela- A, centralcore ( repeats of gions withinthecentralcore of multiplex PCR.Theseprimer pairsspecificallyamplifyre- iments wereperformed.DNA presentincrudeextractsand is associated,chromatinimmunoprecipitation(ChIP)exper- To determinewithwhichregion(s)ofthecentromereSim4 central coreregion Sim4 isassociatedwiththecentromere Mis6–HA (Saitohetal.,1997)werefixedandstainedwith ter nuclei(Fig.4A).CellsexpressingbothSim4–GFPand cells, spotswereobservedattheleadingedgesofdaugh- (or replicated)centromeres(Fig.4,AandB);inanaphaseB spots wereseeninearlymitoticcells,likelytobeindividual Bottom, Southernblothybridizedwith stained gelwithladderindicativeofpartialMNasedigestion. grown at25 mutant. Figure 3. the the smearpatterninwildtypeisreplacedbyladder-like tromere-associated protein. netochore proteinMis6indicatethatSim4isanovelcen- localization patternandcolocalizationwiththebonafideki- and Mis6–HAwasalsoobserved(Fig.4C).Thus,theSim4 two epitopes(Fig.4B).ColocalizationofendogenousSim4 -Sim4 ChIPswasanalyzedusing fourprimerpairsina -GFP and sim4 , acontroleuchromaticgene ( MNasedigestionofchromatinfromcells(FY3027and4536) mutant. Central corechromatinstructureisdisruptedina cen1 C orshiftedto36 -HA antibodies,revealingcolocalizationofthe ( cnt imr ) sequencesand innerrepeat( ), theouterrepeatof C for6h.Top,ethidiumbromide– cen1 cnt1 fbp and ). AsshowninFig.5 probe,showingthat 3 ( cen1 cnt ), theinner ( otr imr ), and sim4 ) se-

FY2929. Bar,5 with Mis6–HA(red),themergedimageisshownonright.Strain strain containing inserted atthecentralcore,Sim4ChIPwasperformedon a termine whetherSim4iscapableofcoatingexogenousDNA the fissionyeastcentromere(Partridgeetal.,2000).Tode- coating, orspreadingover,noncentromericDNAinsertedin and Swi6,butnotChp1,havebeenshowntobecapableof outer repeatregions. the centraldomainofcentromerebutisabsentfrom periments indicatethatSim4isspecificallyassociatedwith Outer repeatsequences( tive to control ( matic locus.Bothstrainsalso containedthe in Sim4ChIPs,but row). image ( ization ofSim4–GFP(green)withMis6–HA(red)andmerged late anaphaseB(centromeresareatthespindlepoles).(B) or anaphaseA),fourcentromericspotsarevisible;early,mid,and centromeres areclustered);earlymitosis(prometaphaseormetaphase Sim4–GFP (FY5077),showingfromlefttoright:interphase(thethree Figure 4. that onsurrounding Sim4 assembledover to anaverageof500–1,000bp, thisenrichmentwasdueto pared with with Sim4 isassociated withthecentraldomainand alsoindicates Several centromere-associatedproteins,includingMis6 Strain FY5237.(C)ColocalizationofSim4( ura4 right) ininterphase(toprow)andearlymitosis(bottom fbp ura4-DSE Sim4 colocalizeswithcentromeres. inSim4ChIPscomparedwiththeinputcontrol. Fission yeastkinetochoreprotein,Sim4| atacontrolrandomintegrant( ura4-DSE m. ura4 ). Therewasnoenrichmentof cnt1 ura4 (Fig.5B).Aschromatinwas sheared cnt1 insertedat : ura4 otr sequences.This confirmsthat sequencesandnotsimplydue to ) werenotenriched.Theseex- wasenriched11.9-foldcom- cnt1 , andacontrolstrain (A)Cellsexpressing -Sim4, green) ura4 Pidouxetal. Rint minigene ) euchro- Rint:ura4 Colocal- 299 The Journal of Cell Biology 300 enrichment of a controleuchromaticlocus.Enrichmentof Sim4 wasgreatly reducedatthepermissive temperature calization atthe centromere.Centromere localizationof we investigatedwhetherthey are requiredforeachother’slo- Because Mis6andSim4exist inakinetochoresubcomplex, Mis6 isrequiredforkinetochore localizationofSim4 other kinetochorecomponents.Immunoprecipitationwith chore, weaskedwhetherSim4coimmunoprecipitateswith tigate protein–proteininteractionsatthefissionyeastkineto- protein complexmadeupofsmallersubcomplexes.Toinves- The fissionyeastkinetochoreislikelytobeamassivemulti- kinetochore component,Mis6 Sim4 coimmunoprecipitateswiththecentralcore be incorporatedintostructurescloselyassociatedwithSim4. that noncentromericDNAinsertedintothecentralcorecan fbp in ChIPsiscomparedwiththeinputPCRandexpressedrelativeto (A) Figure 5. this site.Enrichmentof indicating thatSim4cancoatnoncentromericDNAinsertedat the endogenouslocus.Only PCR analysis.Thepositionsofprimersin with input isindicated. teins, suchasMis12(unpublished data;Goshimaetal.,1999). does notcoimmunoprecipitatewithotherkinetochorepro- disrupt it(unpublisheddata).Thiscomplexisspecific,asSim4 concentrations ofsalt,urea,ornonionicdetergentsfailedto peared tobeverytightlyassociated,aswashingIPswithhigh clearly coimmunoprecipitated(Fig.6A).Thiscomplexap- with complementaryantibodies.Mis6–HAandSim4–GFP munoprecipitates (IPs)wereanalyzedbyWesternblotting tracts fromastraincontainingMis6–HAandSim4–GFP.Im- -HA, -HA, (right).(B) cnt The JournalofCellBiology ura4 and -GFP, or insertedat Sim4 isassociatedwiththecentromerecentralcore. imr ura4 sequencesareenrichedin -Sim4 ChIPperformedonstrains(FY4835and4837) sequencesrelativetothe Rint -Sim4 antibodieswasperformedonex- ura4 or ura4

inthe | cnt1

Volume 161,Number2,2003 at . PCRwith cnt1 -Sim4 ChIPsrelativetothe isenrichedinSim4ChIPs, cen1 -Sim4 ChIP.Multiplex cnt ura4-DSE ura4 areindicated; and primersassays imr minigeneat sequences fbp is and wild typeand localization inwildtypeand indicated atright,andDAPIstaining (red).Bar,5 and processingforimmunolocalization withtheantibodies(green) 5900) weregrownat25 standards areshown.(B–E)Strains (FY3027,5691,4536,5903,and The positionsofMis6–HA(M),Sim4–GFP (S),IgG(asterisk),and multiplex PCRquantificationmethod).Westernblotting multiplex proximately fivefoldreduction,withinthelimitsof quences inthe large reductioninSim4associatedwith experiments wereperformed(Fig.7A).At36 in a (25 analyzed onWesternblotswitheither indicated antibodies,orbeadsonlyasanegativecontrol.IPswere Mis6–HA andSim4–GFP(FY5237).IPswereperformedwiththe relationships. Figure 6. sim4 C) andabolishedattherestrictivetemperature(36 mis6 mutant.(E)Sim4 localizationinwildtypeand mutant(Fig.6B).Toquantifythiseffect,ChIP Sim4–Mis6 complexandkinetochoredependency (A)Extractswerepreparedfromcellsexpressing sim4 mis6 mutant.(D)Mis6–HA localizationinwildtype mutantcomparedwithwildtype(ap- C orshiftedto36 mis6 mutant.(C)Cnp1localizationin -HA or C for6hbeforefixation -Sim4 antibodies. cnt m. (B)Sim4 C, therewasa and sim4 imr mutant. se- C) The Journal of Cell Biology rum raisedtoaCnp1NH fission yeast,wealsoperformedCnp1ChIP,usingantise- been proposedtofunctionasaloadingfactorforCnp1in in wild-typeand confirmed thatthereweresimilaramountsofSim4protein ciated with 1–19). AdecreaseintheamountofendogenousCnp1asso- with HA-taggedCnp1(Takahashi etal.,2000). cubated at36 compared with wildtype.Associationof Mis6–HA with domain sequences wasreducedat36 C) andbyChIP(Fig.7B). Cnp1 associationwithcentral reduction inlocalizationwas apparentcytologically(Fig.6 Cnp1 localizationwasinvestigated ina cnt C (Fig.7A),consistentwith results obtained and mis6 imr cells(unpublisheddata).AsMis6has wasobservedinthe 2 -terminal peptide(aminoacids C inthe mis6 sim4 sim4 mutantin- mutant;a mutant pression ofSim2/Cnp1 and mutantsinotherknown kinetochoreproteins.Overex- Genetic interactionswereinvestigated between Genetic interactionsbetween I). Overexpression ofSim4suppressed sensitivity ofthe other Cnp1 andMis6. that Sim4isrequiredforlocalizationofwild-typelevels localization ofSim4tothecentromerecentraldomain,and observations indicatethatfunctionalMis6isrequiredfor duced centromereassociation(Fig.6E;Fig.7C).These D; Fig.7B).ThemutantSim4protein,itself,showedre- the centraldomainwasreducedin Fission yeastkinetochoreprotein,Sim4| sim measured relativetothe PCR data. of ChIPs.Right,quantificationChIP antibodies. Left,multiplexPCRanalysis 36 5900) weregrownat25 Strains (FY3027,5691,4536,5903,and relationships forkinetochorelocalization. Figure 7. sim4 36 in wildtypeand separate ChIPs.(A)Sim4andCnp1ChIP ments, anddatainCarefromthree from fourtosixseparateChIPmeasure- the wild-typevalue.DatainAandBare mutant valuehasbeennormalizedto PCR. Foreachsite/temperature,the control andnormalizedtotheinput enrichment of input, andtherefore,thereisarelative The ChIPPCRiscomparedwiththis chromatin (little extent, at25 of wild-typeinputat36 wild typeand and 36 CENP-A mutantstovarying extents(Table C for6hChIPwiththeindicated C. (C)Sim4ChIPinwildtypeand mutantat25 suppressedthetemperature C. (B)Mis6–HAandCnp1in sim cnt ChIP analysisofdependency C showsbiasofextracted and sim4 cnt mutants cnt mis6 and sim4 imr C and36 mis6 mutantat25 and mutantat25 enrichmentis imr mutant(Fig.6 C and,toalesser and fbp Pidouxetal. imr C orshiftedto sequences. euchromatic sim chromatin). C. ThePCR sim3 mutants C and , but C 301

The Journal of Cell Biology 302 mis6-302 sim4-193 sim3-143 sim2-76 sim1-106 sim3 teraction with single mutants. mutants wereseverelygrowthimpairedcomparedwiththe published data). temperature butdidnotreimposecentralcoresilencing(un- pressors allowedonlyimprovedgrowthattherestrictive lencing at complemented thetemperaturesensitivityandreimposedsi- that for theidentificationofkinetochore components.Thefact of silencingscreenshavenot beenusedinotherorganisms such asDNAreplicationand spindlefunction.Alleviation variety offactorsimportantforchromosomemaintenance, of mutantsthataffectkinetochorefunction,ratherthan a transmission isthatitleadsimmediatelytotheidentification vantage ofthisscreenoverthosebasedonminichromosome tants thataffectcentromere–kinetochorefunction.Thead- yeast centromeretofacilitatethedirectidentificationofmu- We haveexploitedtranscriptionalsilencingatthefission Discussion sim1 not “ 36 gene wereassayedbyserialdilutionon Mutants containingmulticopygenomicplasmidsbearingtheindicated Table I. chore. suchasSim3,arelikelytofunctionatthekineto- identified, ponents. TheyalsosuggestthatSimproteinsyettobe teractions betweenSimproteinsandotherkinetochorecom- paired. sim3bub1 mad3 and the promote thedeposition ofCnp1 lead totheidentification ofchromatinassembly factorsthat central coreregionvalidatesthis approach.Thisscreencould chore proteinspecificallyassociated withthecentromere sim3 Overexpression ofMis6partiallysuppressed mutant withwhich tants weresyntheticallylethal(TableII). and thespindlecheckpointmutants synthetic lethalityobservedbetween particularly for notype (TableII;unpublisheddata).All All doublemutantcombinationsbetweentwo These geneticinteractionsareindicativeoffunctionalin- ”) andtogrowthwithbonafideORFforeachmutant( C. Growthwascomparedtomutantwithemptyplasmids(equivalent The JournalofCellBiology and or sim1 sim2 . Exceptionstothispatternwerethe ORF(unpublisheddata)alsopartiallysuppressed High-copy suppressionof sim4 or doublemutants,whichwerehighlygrowthim- -tubulin mutant sim4 encodesCnp1 cnt1 . Inallcasestested,thebonafide sim2 rik1 andslightlysuppressed +++(+) +++ + + +++ + ++ ++ ++++ sim1 i1Cp i4Mis6 Sim4 Cnp1 Sim1 : sim1 arg3 . Agenomicclonebearingtheputative mis6 . Doublemutantsbetween and and , whereasmulticopyextragenicsup- or

CENP-A sim4 | nda3 sim4

mis12 Volume 161,Number2,2003 ++++ +++ showedaslightsyntheticin- sim . Surprisingly,therewasno showedgrowthimpairment, andSim4isanovelkineto- CENP-A showedanysyntheticphe- mutants leu platesattemperatures25– bub1 sim . Factorsthatregulate mis6 mutants,or ++++ , sim1 mal2sim bub3 , butnot sim1bub1 sim1 ++++ wastheonly sim , sim mad2 , butnot , bold). mutants sim double gene mu- sim2 mis6 , and ++++ ND +(+) and . , whereas 11allelesof data). Itisalsoclearthatthe least threemore J., personalcommunication;unpublisheddata).Thereareat addition, theSim1proteiniscentromereassociated(Abbott, as ithasalreadyidentifiedtwokinetochorecomponents.In a components beforecentromereassociation,mightalsohave would transientlyassociatewiththecentromereormodify modifying enzymessuchasdeacetylasesandmethylasesthat centromere–kinetochore function,forinstance,chromatin- paired comparedwithsinglemutants. spores aftertetraddissection,butsubsequentgrowthwasonlyslightlyim- mad3 mad2 bub3 bub1 compared withsinglemutants. mutants; S-,S--,S---,syntheticinteraction,degreeofgrowthimpairment SL, syntheticallylethal; rik1 nda3 mal2 mis12 Table II. al., 2002).Although nodefiniteSim4homologues canbe in kinetochore complexesinbuddingyeast (Cheesemanet Sim4 isasmallcoiled-coilprotein; manysuchproteinsexist dent onMis6,andtheconverse istruetoalesserdegree. Association ofSim4withthe centromereisstronglydepen- plex, andoverexpressionofSim4 stronglysuppresses ochore proteinMis6;Sim4and Mis6areinthesamecom- gene insertedinthecentralcore. mis6 sim4 sim3 sim2 sim1 sim4 when defective.However,the components canbeinordertoalleviatecentromeresilencing known whatdistancefromcentromericDNAkinetochore viate centralcoresilencing,werenotrecovered.Itisyet DNA, itisofinterestthatSim4capablecoatinga sociation ofkinetochorecomponentswithnoncentromeric neocentromere formationinotherorganismsrequirestheas- that Sim4isrequiredforkinetochorefunction.Given junction ofsisterchromatids.Thesephenotypesindicate quently aberrant,withlaggingchromosomesandnondis- and increasedsensitivitytoMTpoisons.Mitosisisfre- core chromatinstructure,elevatedratesofchromosomeloss, yeast centromeres.The with Mis6andisrestrictedtothecentraldomainoffission with kinetochoresduringmitosis(Nakasekoetal.,2001). tify components,suchasDis1,thatonlytransientlyassociate a sim1mis6 sim Sim4 displaysmanyfunctional interactionswiththekinet- Our characterizationofSim4showsthatitcolocalizes wasrecovered. phenotype.The Synthetic interactionsof doublemutantsinitiallygrewverypoorlyaftergerminationof S--- --S -S- ND ND S--- ND S-- S--- S- SL/S--- S--- S- S--- S-- S- S--- S--- S-- LS SL SL SL S-- SL SL SL i1sm i3sm mis6 sim4 sim3 sim2 sim1 a sim complementationgroups(unpublished mis6 , nogrowthimpairmentcomparedwithsingle sim1 sim sim4 and screenislikelytobeveryfruitful, wereisolated,onlyonealleleof mutantexhibitsalteredcentral mal2 sim sim sim S--- DS-- ND S-- mutants screenisunlikelytoiden- screenisnotsaturated; mutants,knowntoalle- /S- ura4 mis6 ND ND /S- . The Journal of Cell Biology ular nucleosomal spacingorloadingofhistone H3.What- transcription factors, butalsofromactivities thatinducereg- chore protectsthecentralcore chromatin,notonlyfrom nonregularly spaced.Perhaps thefullyassembledkineto- gion maynothavearegularspacing of ing nucleosomes.Alternatively, thenucleosomesinthisre- DNA maybewrappedmore loosely aroundCnp1-contain- nized insuchawaythataccessibility toMNaseisaltered; smear patternsuggeststhatcentralcorechromatinisorga- there isareducedlevelofCnp1 heterologous promoter)attherestrictivetemperature,and of newlysynthesizedGFP-taggedCnp1(expressedfroma ogy betweenthetwo. Sim4 andchickenCENP-Haddweighttotheweakhomol- plex. Theobservedsimilaritiesinbehaviorbetween hashi etal.,2002),suggestingthattheyarepartofacom- tween CENP-HandCENP-Ihasalsobeenreported(Nishi- required forSim4localization.Atwo-hybridinteractionbe- localization ofCENP-H(Nishihashietal.,2002),asMis6is the Mis6homologue,CENP-I,isrequiredforkinetochore 1999, 2000;Fukagawaetal.,2001).InchickenDT40cells, Fig. S2;Sugataetal., vertebrate CENP-H(25%similarity; weak similarityinstructureandsequencebetweenSim4 identified inmetazoaorotherorganisms,wehavenoticeda Cnp1 the proposalthatMis6actsasaloadingfactorfor to its endogenouspromoter).Theseobservationshaveled that both sim3 tion, tromere siteandspecification. it couldplayapartintheepigeneticinheritanceofcen- (Mellone andAllshire,2003).Suchamodelisattractive,as tachments producetensionatfunctionalkinetochores incorporated atmitosiswhenproperkinetochore–MTat- cleosomes ofthisregion. central coreandmaytaketheplaceofhistoneH3innu- 2000; Jinetal.,2002).Cnp1ispresentathighlevelsinthe (Saitoh etal.,1997;Goshima1999;Takahashi cycle. OnepossibilityisthatCnp1 fect Cnp1 ferent organisms.However,severalcentralcoremutantsaf- reflect differencesinorganizationanddetailsbetweendif- 2001; Nishihashietal.,2002).Theseinconsistenciesmay it isrequiredforCENP-Clocalization(Fukagawaetal., quired forthelocalizationofCENP-A,but,likeCENP-H, counterpart. Inaddition,chickenCENP-I/Mis6isnotre- for loadingofCse4p(Measdayetal.,2002),theCENP-A the buddingyeastMis6homologue,Ctf3p,isnotrequired that actsasaspecificloadingfactorforCnp1.However, preted asevidenceforaMis6/Sim4-containingcomplex tion ofCnp1withthecentromere,andthiscouldbeinter- sized Cnp1 netochore thatdirectstheincorporationofnewlysynthe- probably exist,itisthepresenceofafullyfunctionalki- that althoughspecificloadingorassemblyfactorsforCnp1 published data)aswell others, havereducedCnp1 A Similar toothermutantsthataffectcentraldomainfunc- , and mis6 sim4 CENP-A mutantisdefectiveincentromereincorporation mutantsdisruptitsuniquechromatinstructure mis6 CENP-A sim7 CENP-A (Takahashietal.,2000).Wehaveobserved and (Abbott,J.,personalcommunication;un- centromereassociation,including intothecentromere,cellcycleafter sim4 mutantsdisplayreducedassocia- mis6 sim4 CENP-A and and CENP-A inthecentraldomain.The mis6 sim4 CENP-A –HA (expressedfrom 150 bp,butmaybe . Wefavortheidea mutants,amongst isonlycorrectly S. pombe sim1 , transcriptional silencing,normallevelsofCnp1 ever thecauseofunusualchromatin,itcorrelateswith mutants istypifiedby type sensitivitytoMTdrugs.Asecondclassofcentralcore action, perse,whichisconsistentwiththeirobservedwild- mutants wouldnothaveadefectinkinetochore–MTinter- chores faceinoppositedirections(Saitohetal.,1997); tion ofthekinetochore,ensuringthatsisterkineto- proposed thatMis6isrequiredforthebiorientationfunc- segregation, withfewlaggingchromosomes.Ithasbeen class, e.g., the centralcorefallintotwoclasses.Mutantsoffirst alleviate centralcoresilencingorproteinsthatarelocatedat sue culturecells(Ciminietal.,2001,2002).Mutantsthat mechanism contributingtoaneuploidyinmammaliantis- 2002). Merotelicattachmenthasbeenshowntobeamajor (Pidoux etal.,2000;Bernard2001;Nonaka may alsohavearoleinpreventingmerotelicattachment the outerrepeatsthatisrequiredforcentromericcohesion and Roth,2002).TheSwi6-containingheterochromatinof tain, 1986;Pidouxetal.,2000;YuandDawe,Stear emanating frombothspindlepoles(LadrachandLaFoun- chores, inwhichasinglekinetochoreinteractswithMTs chromosomes areduetomerotelicattachmentofkineto- binding sitesonsinglekinetochores.Itislikelythatlagging of defectsinMTinteractionorthecoordinationMT- MT-disrupting drugs(Ekwalletal.,1996,1999),indicative levels oflaggingchromosomesandaresupersensitiveto kinetochore integrity. tion ofthiscomplex kinetochore. to buildupadetailed pictureofthearchitecture andfunc- tions anddependencyrelationships forlocalization,weaim interactions inconjunction with protein–proteininterac- additional kinetochorecomponents. Byinvestigatinggenetic is clearlyaveryeffectiveapproach fortheidentificationof sensitive toMTdrugs. both unevensegregationandlaggingchromosomesare that theirfunctionsoverlapbutarenotidentical; and maycooperatefunctionally,geneticevidencesuggests spindle. AlthoughMis6andSim4areinthesamecomplex, together sothattheyinteractcorrectlywiththemitotic chore inwhichthemultipleMT-bindingsitesarelocked andforassemblingafullyfunctionalkineto- of thisclass,isrequiredbothforthebiorientationsister does thetic interactionswiththe in nents suggeststhatthespindlecheckpointmaybeimpaired of Mis6.Thelackinteractionwithcheckpointcompo- mis6 mis6 synthetically lethalwith Cnp1 assay ofkinetochoreassembly andsuccessfullyidentified (Warren etal.,2002). kinetochores independentofitsroleincheckpointfunction of Bub1havesuggestedthatitmayplayadditionalroles at strong arrestatmetaphaseisconsistentwiththis.Analyses Mutants thatalleviateouterrepeatsilencinghavehigh We haveusedcentromericsilencinginfissionyeastasan sim , butnotviceversa,suggeststhatSim4actsupstream isnot.ThefactthatoverexpressionofSim4suppresses cnp1 CENP-A mutants;thefactthatthesemutantsdonotexhibita / mis6 sim2 andanovelkinetochorecomponent, Sim4.This Fission yeastkinetochoreprotein,Sim4| and . WeproposethatSim4,andothermembers cnp1 sim1 sim1 / sim2 sim1 , and sim3 , displayunevenchromosome , -tubulin mutant 4 sim2 , and alsodisplaystrongersyn- / cnp1 sim4 , and . Thesedisplay Pidouxetal. CENP-A nda3 sim3 sim4 than , but , and mis6 303 is The Journal of Cell Biology 304 5958 3990 5711 1895 4636 5688 5720 5714 5683 5719 5695 5674 5717 4005 4835 4575 5903 5900 Genotypes ofstrainsused.For markedwithanasterisk(*),onlytherelevantgenotypeislisted. 2919 1645 972 Strain Table III. 845 5959 5895 2930 5077 1317 4025 4015 2929 3272 5251 4022 4841 3606 5691 4540 4536 4502 4461 4485 1869 944 2221 1646 3828 3119 5237 5254 4837 4504 4462 3027 382 1891 1648 1647 4484 3033 The JournalofCellBiology List ofstrainsusedinthisstudy

| h/h h h sim1-106Rint:ura4ura4-DSE h h h sim4-193otr1R h sim4-193cnt1 h sim4-193Rint:ura4ura4-DSE h sim3-143otr1R h sim3-143cnt1 h sim3-143Rint:ura4ura4-DSE h sim1-106otr1R h sim1-106cnt1 h sim1-106cnt1:arg3otr2:ura4tel1:his3[Ch16ade6-216LEU2 h h mal2-1cnt1:arg3cnt3:ade6otr2:ura4tel1 h h h h h h h/h h h h h h sim4-193cnt1:arg3otr2:ura4tel1:his3[Ch16ade6-216LEU2 h sim2-76cnt1:arg3otr2:ura4tel1:his3[Ch16ade6-216LEU2 h h mis6-302cnt1:ura4ura4-DSEleu1-32 h h sim3-143cnt1:arg3otr2:ura4tel1:his3[Ch16ade6-216LEU2 h h h mis6-302cnt1:arg3cnt3:ade6otr2:ura4tel1 h h h h h h h h h h h h h h h h h h h h h h h

A 90 Volume 161,Number2,2003 rik1::LEU2+cnt1:arg3cnt3:ade6otr2:ura4tel1 mis12-537leu1-32 nda3-km311leu1-32 mis6::mis6-3HA-LEU2 sim4-193mis6::mis6-3HA-LEU2 sim4-193mis6::mis6-3HA-LEU2 mis6::mis6-3HA-LEU2 mis6::mis6-3HA-LEU2 mis6-302cnt1:ura4ura4-DSEleu1-32 mis6-302leu1-32 sim4::sim4-GFP-kanMX6mis6::mis6-3HA-LEU2 sim4::sim4GFP-kanMX6ade6-210leu1-32ura4-D18arg3-D4his3-D1 sim4::sim4GFP-kanMX6cnt1:arg3cnt3:ade6otr2:ura4tel1 swi6::his1 cnt1:arg3otr2:ura4tel1:his3[Ch16ade6-216LEU2 sim4-193cut9-665 cut9-665leu1-32ura4-DSEade6-210his1-102 otr1R cnt1 Rint:ura4ura4-DSEleu1-32his3-D1arg3-D4 sim4-193cnt1:arg3cnt3:ade6otr2:ura4tel1 sim4-193cnt1:arg3cnt3:ade6otr2:ura4tel1 sim3-143cnt1:arg3cnt3:ade6otr2:ura4tel1 sim3-143cnt1:arg3cnt3:ade6otr2:ura4tel1 sim2-76cnt1:arg3cnt3:ade6otr2:ura4tel1 sim2-76cnt1:arg3cnt3:ade6otr2:ura4tel1 sim1-106cnt1:arg3cnt3:ade6otr2:ura4tel1 sim1-106cnt1:arg3cnt3:ade6otr2:ura4tel1 cnt1 cnt1 cnt3 rik1::LEU2cnt3:ura4ade6-210arg3-D4his3-D1leu1-32ura4-D18/-DSE otr2 cnt1 cnt1 ade6-216arg3-D4his3-D1leu1-32ura4-D18 ade6-216arg3-D4his3-D1leu1-32ura4-D18 ade6-210arg3-D4his3-D1leu1-32ura4-D18 otr1R ade6-210 arg3-D4his3-D1leu1-32ura4-D18 sim4 sim4 (HindIII): (NcoI): (NcoI): (NcoI): (NcoI): (NcoI): (NcoI): (SphI): (SphI): /sim4::ura4 ade6-210/ade6-216leu1-32/leu1-32ura4-D18/ura4-D18arg3-D4/arg3-D4 his3-D1/his3-D1 /sim4::sim4GFP-kanMX6 ade6-210/ade6-216leu1-32/leu1-32ura4-D18/ura4-D18 arg3-D4/arg3-D4his3-D1/his3-D1 ura4 ura4-DSEleu1-32his3-D1arg3-D4 arg3 cnt3 arg3 cnt3 ade6 ade6-DNNleu1-32ura4-D18 arg3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 ura4 ade6-210arg3-D4his3-D1leu1-32ura4-D18 ade6-210his1-102leu1-32ura4-DSE[Ch16ade6-216LEU2 ura4 ura4-DSEleu1-32his3-D1arg3-D4 ade6 tel1 ura4 ade6-210leu1-32ura4-DSE (NcoI): (NcoI): (NcoI): (SphI): (SphI): (SphI): * ura4 ura4-DSE ura4 ura4-DSE ura4 ura4-DSE ura4 ura4-DSE ura4 ura4-DSE ura4 ura4-DSE (NcoI): (NcoI): L: his3 ade6-210his3-D1leu1-32ura4-DSE cnt1:ura4ura4-DSEleu1-32 cnt1:ura4ura4-DSEade6-210leu1-32 RInt:ura4ura4-DSEade6-210leu1-32 ade6 otr2 ade6 otr2 * * * cnt1:ura4ura4-DSEleu1-32 RInt:ura4ura4-DSEleu1-32 * * * * * * * (HindIII): (HindIII): * L: his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 L: L: L: L: L: L: L: L: L: his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 ura4 tel1 ura4 tel1 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 L: his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 ade6-210leu1-32ura4-D18his3-D1cnt1:arg3orarg3 * ] ade6-210ura4-D18leu1-32his3-D1arg3-D4 L: L: his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 Genotype L: ] ade6-210ura4-D18leu1-32his3-D1arg3-D4 his3 ade6-210leu1-32ura4-D18arg3-D4his3-D1 ] ade6-210ura4-D18leu1-32his3-D1arg3-D4 ] ade6-210ura4-D18leu1-32his3-D1arg3-D4 ] ade6-210ura4-D18leu1-32his3-D1arg3-D4 * * ] The Journal of Cell Biology and 36 pressed relativetowildtypeat25 mutants wasassessedonYESphloxineBat25 fixed for30minat 18 min inanice-water bathand22minat18 presented (Fig.1E).The times ateachtemperature,andaveragevaluesfromtheseexperimentsare tem and1DImageAnalysissoftware.wasperformedtwotofour tion ofbandswasperformedusingtheEastmanKodakCo.EDAS290sys- (Molecular Probes)accordingtothemanufacturer’sinstructions.Quantita- 1.5% agarosegelsandpoststainedwithethidiumbromideorSYBRgreen et al.,1997).The 36 which containsa5.2-kbEcoRI ements, wasPCRamplifiedandclonedintoNcoI-digestedpKS-cnt1, stream oftheATG,includingTATAboxbutlackingotherpromoterel- KS. GrowthofserialdilutionswasassessedonPMGphloxineBand bated at25 fragment bearingthe type withagenomiclibrary(pDB). were identifiedbycomplementationofthetemperature-sensitivepheno- 3033. with strainsFY944,1895,382,and1869createdFY3027 An FOA-resistant,arg 1:300; bodies forWesternblottingweredilutedinPBS-Tweenasfollows: a sheep. Biosciences). GST–Sim4fusionproteinwaspurifiedandusedtoimmunize plasmids (pDB)bearing formaldehyde, incubationwascontinued at36 strictive temperature,cellswereshifted to36 Jin etal.,2002),exceptforthefollowing modifications.Forgrowthatre- ChIP wasperformedaspreviouslydescribed (EkwallandPartridge,1999; ChIP sim4 and immunoprecipitations Production ofantibodies,Westernblotting, Total RNAwaspreparedfromstrainsgrowninYESat25 RT-PCR YES andspheroplastedwithzymolyase-100T(ICNBiomedicals). previously described(Allshireetal.,1994),exceptthatcellsweregrownin MNase digestionofchromatininpermeabilizedcellswasperformedas Micrococcal nucleasedigestionofchromatin Synthetic lethality. Analysis ofgeneticinteractions ing at forsupersensitivitytoTBZ,andmaintenanceofsilenc- cryosensitivity, onies. Reproduciblyfastgrowerson growing colonieswerepickedfromthebackgroundofslower-growingcol- 3–5 mJUV(50–80%killing),andincubatedat25 FY3027 or3033cellswerespreadon Screen formutantsthatalleviatecentralcoresilencing moter-crippled S. pombe Yeast strains netics, andmanipulations(Morenoetal.,1991). Standard procedureswereusedforbacterialandfissionyeastgrowth,ge- Chemicals wereobtainedfromSigma-Aldrichunlessstatedotherwise. Standard techniques Materials andmethods arg3 wick, 2002). cipitations wereperformedaspreviouslydescribed(MillbandandHard- were developedusingECLreagents(AmershamBiosciences).Immunopre- times andplacedincomplementationgroups. arg andcomparedwiththerelevantemptyplasmid. Multicopy suppression. C for6h,andRT-PCRwasperformedaspreviouslydescribed(Ekwall fragmentwastransformedintostrainFY1891(Allshireetal.,1994). cDNAwasPCRamplifiedandclonedintopGEX-4T1(Amersham otr2:ura4 C. -HA, 1:300; strainsusedinthisstudyareshownTableIII.Toinsertthepro- -Sim4 antibodieswereaffinitypurifiedonnitrocellulose.Anti- C. Thegrowthofviabledoublemutantscomparedwithsingle arg3 and At least30asciweredissectedonYESplatesandincu- ura4 mis6 geneat tel1L:his3 C). Cellswerespheroplasted at10 -GFP, 1:2,000(in1%milk;MolecularProbes).Blots colonywasanalyzedbySouthernblotting.Crosses and sim4 sim ura4 ORFwasPCRamplifiedandclonedintopAL- mutantsweretransformedwithgenomic ura4-DSE , to cnt1 . Mutantswerebackcrossedatleastthree cnp1 cnt1 ura4-DSE C foreachsite, , an fragment(Fig.1A).The / sim2 PCRproductswereseparatedon arg3 arg weretestedforthermo-and arg PMGplates,irradiatedwith ratiowasdeterminedandex- , and C (cellsgrownat 25 fragmentwith181bpup- C for6h.Afteradditionof C, 28 C for5min,followedby3 sim2 sim1 Rint C for5–20d,andfast- C, 30 , cnt1 and ORFs.Agenomic 8 cells/mlinPEMS C orshiftedto C, 32 , or sim4 cnt1 otr1 C, 34 C were (Nc genes . -Sim4, o C, I) : (1:15), andaffinity-purifiedsheep 2000) orusingthefollowingsetup:100 1996). Microscopywasperformedaspreviouslydescribed(Pidouxetal., or 1:1,000.FISHwasperformedaspreviouslydescribed(Ekwalletal., (Molecular Probes)-conjugatedsecondaryantibodieswereusedat1:100 cells foreachstrain. IPLab orMetamorphsoftware.Spindlelengthwasmeasuredin100–300 arated chromosomes,wherebothspindlepoleswereinfocus,usingeither 7 hinthesamemedium.Spindlelengthwasmeasuredcellswithunsep- pared formaldehydeforstainingwith al., 2000),exceptthatcellswerefixedfor5–10minin3.7%freshlypre- Immunofluorescence wasperformedaspreviouslydescribed(Pidouxet Cytology Allshire, R.C.,J.-P. Javerzat, N.J.Redhead,andG.Cranston. 1994.Positioneffect References Accepted: 7March2003 Revised: 5March2003 Submitted: 18December2002 low ofTheWellcomeTrust. Britain, andTheWellcomeTrust.R.C.AllshireisaPrincipalResearchFel- for InternationalCancerResearch,theMedicalResearchCouncilofGreat lowship (RoyalSocietyofEdinburgh)toA.L.PidouxandbytheAssociation ing discussions. manuscript, andpastpresentmembersoftheAllshirelabforstimulat- bott, K.Hardwick,B.Earnshaw,andMelloneforcriticalreadingofthe bodies, andreagents.WethankPaulPerryformicroscopysupport,J.Ab- Gull, B.Mellone,T.Nakamura,S.Sazer,andM.Yanagidaforstrains,anti- We areverygratefultoW.Earnshaw,U.Fleig,I.Hagan,K.Hardwick, K. minichromosome lossdata, //www.jcb.org/cgi/content/full/jcb.200212110/DC1. Thesefiguresinclude The supplementalfigures(Figs. S1andS2)forthisarticleareavailableathttp: Online supplementalmaterial Texasred(JacksonImmunoResearchLaboratories),orAlexa Aldrich), antiserum (1:300),mouse12CA5 0.05% glutaraldehyde.Thefollowingantibodieswereused:sheep beling ofMTs,cellswerefixedfor10–15minin3.7%formaldehyde, described (Ekwalletal.,1997). (100 mMPipes,pH7,1EDTA,MgCl as wellWesternblottingdataanddescriptionofthe and cellpelletswerefrozenat ml zymolyase-100Tfor25minat36 in theabsenceofthiaminefor16h,andthencellswereshiftedto36 sim4 ware (UniversalImagingCorp.). (Roper Scientific).ImageacquisitionwascontrolledusingMetamorphsoft- wheel (PriorScientific),andPhotometricsCoolSnapHQCCDcamera equipped withChroma83000and86000filtersets,PriorProScanfilter- on aCarlZeissMicroImaging,Inc.Axioplan2IEfluorescencemicroscope (Kniola etal.,2001),30 dure wasfollowed(EkwallandPartridge,1999).10 grown at25 described forRT-PCR.FortheinputPCR, as previouslydescribed(Jinetal.,2002).PCRproductswerequantified normalized tothe with mutants wereconsistentbetweenexperiments.ChIPperformedonstrains periments (producingtheerrorbarsinFig.7),foldreductionsseen was variationintheactualvaluesobtainedbetweenindividualChIPex- tions ofinputandChIP’dsamplesgaveverysimilarresults.Althoughthere justed toensurethatthiswasthecase.PCRperformedondifferentdilu- present andifnecessaryexposuretimes,oramountoftemplate,weread- ChIPs. Thequantificationdependsonaquantifiable check thatthemultiplexPCRwasagoodmethodforquantificationof corrected fortheratioobtainedininputPCR.Stepsweretakento and -HA antibody wasusedinChIPs.MultiplexPCRanalysisperformed -HA This researchwassupportedbyaCaledonianResearchFoundationFel- For measurementofspindlelengths, otr cellscontainingpREP3X- ura4 variegation atfission yeastcentromeres. bandsintheChIPswascalculatedrelativeto insertionsat C andshiftedto36 Fission yeastkinetochoreprotein,Sim4| fbp value,givingtheinputratio.Enrichmentof Rint sim4 l affinity-purified or mad2 20 cnt1 sequenceandalignmentwithHsCENP-H, C for4h.Alternatively,wild-typeand C. ThereafterthestandardChIPproce- (Heetal.,1997)weregrownat25 -Sim4 antibody(1:30).FITC(Sigma- wasanalyzedbyPCRaspreviously C. 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