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The Journal of Cell Biology
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Alison L.Pidoux, and chromosome segregation required forcentromeric silencing Sim4: anovel fissionyeast kinetochore 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 heterochromatin. Here,transcriptional silencingwas of afunctionalkinetochore complexandflanking centro- region aretranscriptionally silenced,reflecting the formation centromere-specific proteins.Genesplacedwithineither to underliethekinetochore itself,asitisassociatedwith central corehasadistinctchromatin structureandislikely segregation Key words: kinetochore; chromatin;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 Genetics Unit, Western General Hospital, Edinburgh EH42XU,UK
TheRockefeller University Press, 0021-9525
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(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).
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