10.1101/gad.226118.113
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Accessthemostrecentversionatdoi:201327:2489-2499GenesDev.
XavierTadeo,JiyongWang,ScottP.
Kallgren,etal.
heterochromatinassemblyEliminationofshelterincomponentsbypassesRNAiforpericentricMaterialSupplementalhttp://genesdev.
cshlp.
org/content/suppl/2013/11/14/27.
22.
2489.
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cshlp.
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full.
html#ref-list-1Thisarticlecites62articles,27ofwhichcanbeaccessedfreeat:LicenseCommonsCreative.
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xhtmlsixmonthsafterthefull-issuepublicationdate(seeThisarticleisdistributedexclusivelybyColdSpringHarborLaboratoryPressforthefirstServiceEmailAlertingclickhere.
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;PublishedbyColdSpringHarborLaboratoryPressColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
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cshlp.
orgDownloadedfromEliminationofshelterincomponentsbypassesRNAiforpericentricheterochromatinassemblyXavierTadeo,1JiyongWang,1ScottP.
Kallgren,1JinqiangLiu,2BharatD.
Reddy,1FengQiao,2andSongtaoJia1,31DepartmentofBiologicalSciences,ColumbiaUniversity,NewYork,NewYork10027,USA;2DepartmentofBiologicalChemistry,UniversityofCaliforniaatIrvine,Irvine,California92697,USATheRNAipathwayisrequiredforheterochromatinassemblyatrepetitiveDNAelementsindiverseorganisms.
Infissionyeast,lossofRNAicausespericentricheterochromatindefects,compromisinggenesilencingandchromosomesegregation.
HereweshowthatdeletionoftelomereshelterincomponentsrestorespericentricheterochromatinanditsfunctionsinRNAimutants.
Wefurtherisolatedaseparation-of-functionmutantofPoz1andrevealedthatdefectivetelomeresilencing,butnottelomerelengthcontrol,iscriticalforbypassingRNAi.
Furtheranalysesdemonstratedthatcompromisingshelterin-mediatedheterochromatinassemblyinRNAimutantsreleasesheterochromatinproteinSwi6,whichisredistributedtopericentricregionsthroughRNAi-independentheterochromatinassemblypathways.
GiventhehighmobilityofSwi6proteinandthatincreasedlevelsofSwi6facilitatesheterochromatinspreadingaswellasectopicheterochromatinassembly,ourresultssuggestthatconstitutiveheterochromatindomainsusemultiplepathwaystoformhigh-affinityplatformstorestrainSwi6,thuslimitingitsavailabilityandavoidingpromiscuousheterochromatinformation.
[Keywords:RNAi;heterochromatinassembly;telomere;shelterin;centromere]Supplementalmaterialisavailableforthisarticle.
ReceivedJuly5,2013;revisedversionacceptedOctober9,2013.
EukaryoticgenomespossesslargestretchesofrepetitiveDNAelementsthatformcompactchromatinstructurestermedheterochromatin.
Theseregionsarerelativelygene-poorbutoccupycrucialchromosomeregions,suchascentromeresandtelomeres,whichplaycriticalrolesinchromosomesegregationandthree-dimensionalgenomeorganization.
Heterochromatinalsoinhibitstranscrip-tionandrecombinationoftheunderlyingDNArepeats,renderingitindispensableformaintaininggenomein-tegrity(GrewalandJia2007;AlmouzniandProbst2011).
GenomicDNAisfoldedwithhistonestoformchroma-tin.
Eachchromosomalregionisassociatedwithdistinctprofilesofhistonepost-translationalmodifications,whichplayessentialrolesinregulatingchromatinstructureandfunction.
Histonesatheterochromaticregionsaregener-allydevoidofacetylationandaremethylatedatH3Lys9(H3K9me).
H3K9meallowsforthebindingofheterochro-matinprotein1(HP1),whichcompactschromatinthroughitsself-associationandtherecruitmentofotherchroma-tin-modifyingactivities(GrewalandJia2007).
Althoughheterochromatinhastraditionallybeenconsideredsilent,stable,andstatic,recentbiochemicalandimagingstudiesshowthatthebindingofHP1proteinstoheterochromatinregionsissurprisinglydynamic(Cheutinetal.
2003,2004;Festensteinetal.
2003;Sadaieetal.
2008).
Moreover,althoughheterochromatinnormallyexcludesthetran-scriptionalmachinery,theDNArepeatswithinhetero-chromatinaretranscribed,andthetranscriptsarepro-cessedbytheRNAipathwayintosiRNAs,whichtargethistone-modifyingcomplexestorepetitiveDNAele-ments(Moazed,2009;LejeuneandAllshire2011;CastelandMartienssen2013).
RNAi-dependentheterochromatinassemblyisbeststudiedinthefissionyeastSchizosaccharomycespombe(forreview,seeMoazed2009;LejeuneandAllshire,2011;GotoandNakayama2012;CastelandMartienssen2013).
Inthisorganism,constitutiveheterochromatinislocalizedatthecentromeres,telomeres,andthesilentmatingtyperegion,allofwhichsharesimilarrepetitiveDNAele-mentscomposedofdganddhrepeats.
TheDNArepeats2013Tadeoetal.
ThisarticleisdistributedexclusivelybyColdSpringHarborLaboratoryPressforthefirstsixmonthsafterthefull-issuepublicationdate(seehttp://genesdev.
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xhtml).
Aftersixmonths,itisavailableunderaCreativeCommonsLicense(Attribution-NonCommercial3.
0Unported),asdescribedathttp://creativecommons.
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3CorrespondingauthorE-mailjia@biology.
columbia.
eduArticleisonlineathttp://www.
genesdev.
org/cgi/doi/10.
1101/gad.
226118.
113.
GENES&DEVELOPMENT27:2489–2499PublishedbyColdSpringHarborLaboratoryPress;ISSN0890-9369/13;www.
genesdev.
org2489ColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfromaretranscribedbyRNApolymeraseII(PolII)duringtheSphaseofthecellcycle,producingdsRNAswiththehelpofaRNA-dependentRNApolymerasecomplex(RDRC).
TheribonucleaseDicer(Dcr1)processesthesedsRNAsintosiRNAs,whichareloadedontotheArgonautesiRNAchaperonecomplex(ARC)andthentransferredtoRNAi-inducedtranscriptionalsilencingcomplex(RITS).
TheArgonauteprotein(Ago1)withinRITSbindssiRNAsandtargetsRITStonascentRNAtranscriptsfromrepeatregions.
RITSthenrecruitstheCLRCcomplex,whichcontainstheH3K9methyltransferaseClr4.
H3K9mere-cruitschromodomainproteinsSwi6andChp2togetherwiththeirassociatedhistonedeacetylasesandchromatinremodelingactivitiestoachievetranscriptionalsilencing.
H3K9mealsostabilizesthebindingofRITStochromatinthroughthechromodomainproteinChp1,whichinturnrecruitsRDRCtogeneratemoredsRNAsandsiRNAs,thusformingaself-reinforcingloopbetweensiRNApro-ductionandheterochromatinassembly.
InadditiontoRNAi-basedmechanisms,DNA-basedmechanismsalsofunctionatrepeatregionstorecruitCLRC.
Forexample,ATF/CREBfamilyproteinsAtf1andPcr1cooperatewithRNAitoestablishheterochromatinatthesilentmatingtyperegion(Jiaetal.
2004;Kimetal.
2004).
Similarly,telomereshelterincomponentTaz1andthetelomere-associatedsequence(TAS)functiontogetherwithRNAitoestablishheterochromatinattelomeres(Kanohetal.
2005).
Asaresult,lossofRNAiseverelyaffectspericentricheterochromatinfunctionsbuthaslittleeffectonheterochromatinstructuresatthesilentmatingtyperegionortelomeres.
However,evenatpericentricregions,H3K9meandSwi6arestillpresentinRNAimutants,albeitatlowerlevels,suggestingtheexistenceofRNAi-independentmechanismstoestablishhetero-chromatin(Sadaieetal.
2004).
ThesepathwaysinvolvetheClr3andSir2histonedeacetylasessinceH3K9melevelsarefurtherreducedfrompericentricregionsinclr3Ddcr1Dandsir2Ddcr1Dcells,althoughSir2seemstofunctioninaseparatepathwayfromClr3(Yamadaetal.
2005;Alperetal.
2013;Buscainoetal.
2013;Marinaetal.
2013).
Interestingly,eliminationoftheMst2histoneH3K14acetyltransferasecomplexactivity,theJmjCdomainpro-teinEpe1,orRNAqualitycontrolfactorMlo3bypassestherequirementofRNAiforpericentricheterochromatinassembly(Trewicketal.
2007;Reddyetal.
2011;Reyes-Turcuetal.
2011).
AlthoughthemechanismsbywhichthesemutantsregulateheterochromatinassemblyintheabsenceofRNAidiffer,theseresultsnonethelessindicatethatRNAiisnotobligatoryforpericentricheterochro-matinformationundercertainconditions.
Tofurtherunderstandheterochromatinassemblypathwaysatperi-centricregions,weperformedahigh-throughputscreenofthefissionyeastdeletionlibraryformutantsthatsupportpericentricheterochromatinassemblyintheab-senceofRNAi.
Wediscoveredthatlossofthetelomereshelterincomponentsinvolvedinpropertelomeremain-tenancealsobypassedRNAiforpericentricheterochro-matinformation.
WefurtherisolatedamutantofshelterincomponentPoz1thatspecificallyaffectedtelomerichet-erochromatinassemblywithoutaffectingtelomerelengthandshowedthatlossofshelterininanRNAimutantbackgroundresultedintheredistributionofheterochro-matinproteinSwi6fromtelomericheterochromatintopericentricregionsthroughRNAi-independenthetero-chromatinassemblypathwaysinvolvingClr3andSir2.
Furthermore,weshowedthatincreasingoverallSwi6levelswassufficienttobypassRNAiforpericentricheterochromatinassembly.
Ourresultsdemonstrateady-namicequilibriumofdifferentheterochromatindomainsthroughthehighmobilityofSwi6andsuggestthatconstitutiveheterochromatinregions,suchascentro-meresandtelomeres,playimportantrolesinbalancingtheavailabilityofsilencingfactorstomaintaintheoverallepigeneticlandscapeofthegenome.
ResultsAhigh-throughputscreenformutantsthatbypasstherequirementoftheRNAimachineryforpericentricheterochromatinassemblyInordertounderstandthemechanismofRNAiandheterochromatinassembly,weperformedascreenofthefissionyeastdeletionlibraryformutantsthatallowrestorationofpericentricheterochromatinintheabsenceofRNAifunction.
Weusedaquerystraincontainingaura4+reportergeneinsertedattheouterrepeatofcentro-mereI(otrTura4+)(SupplementalFig.
S1A).
Inwild-typecells,thisreportergeneissilencedduetotheformationofheterochromatin.
Asaresult,thereislittlegrowthonmediumlackinguracilbutrobustgrowthoncounter-selectivemediumcontaining5-fluorooroticacid(FOA),whichistoxictocellsexpressingura4+(Fig.
1A).
Muta-tionsthataffectheterochromatinstructureatpericentricregions,suchasdcr1D,resultedinstrongcellgrowthonmediumlackinguracilbutlittlegrowthonmediumcontainingFOA(Fig.
1A).
Throughtwosuccessivecrosses,weintroducedtheotrTura4+reporteranddcr1Dintothedeletionlibraryandobtainedhaploidcells,eachcontain-ingotrTura4+,dcr1D,andasinglegenedeletion(Supple-mentalFig.
S1B).
TheresultingcellsweregrownonmediumcontainingFOAtomeasurecellgrowth(Supple-mentalFig.
S1C;SupplementalTableS1).
OurscreenidentifiedmutantsknowntobypassRNAiforpericentricheterochromatinassembly,suchasMst2complexcompo-nentsmst2D,nto1D,andptf2D(Reddyetal.
2011;Wangetal.
2012),validatingtheeffectivenessofourscreen.
Ourscreenalsoidentifiedpoz1D,atelomereshelterincompo-nentinvolvedintelomerelengthcontrol(Miyoshietal.
2008),asshowingthestrongestrescueofheterochromaticsilencing(SupplementalFig.
S1C;SupplementalTableS1).
WealsoperformedsimilarscreenswithanotrTade6+reporter(SupplementalFig.
S2A).
Thisreportergeneissilencedinwild-typecells,resultingintheformationofredcoloniesonlow-adeninemediumduetotheaccumu-lationofmetabolicintermediates.
InRNAimutants,suchasdcr1D,lossofpericentricheterochromatinresultedintheexpressionofAde6,givingrisetowhitecolonies(SupplementalFig.
S2A).
Wealsoidentifiedpoz1DasoneTadeoetal.
2490GENES&DEVELOPMENTColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfromofthestrongestsuppressorsofdcr1Dandago1DwiththeotrTade6+reporter(SupplementalFig.
S2B,C;Supplemen-talTableS1).
Altogether,weidentifiedatotalof33genedeletionsthatbypassedRNAiinbothotrTura4+andotrTade6+reporterscreens.
EliminationofPoz1bypassesRNAiforpericentricheterochromatinfunctionsToconfirmthatlossofPoz1canindeedrestorepericentricheterochromatininRNAimutants,weconstructedapoz1Ddcr1DdoublemutantstraincontainingotrTura4+.
Serialdilutionanalysesconfirmedthatpoz1Ddcr1Dgrewweaklyonmediumwithouturacilbutrobustlyonme-diumcontainingFOA(Fig.
1A).
Chromatinimmunopre-cipitation(ChIP)analysesshowedthatinpoz1Ddcr1Dcells,heterochromatinhallmarksatpericentricdhrepeats,suchasH3K9me2andSwi6,wererestoredtonearwild-typelevels(Fig.
1B,C).
Moreover,ChIPanalysesshowedthatPolIIwasstronglyexcludedfrompericentricrepeats(Fig.
1D),suggestingthatheterochromatinformedinpoz1Ddcr1Dcellsiscapableoftranscriptionalgenesilencing.
Pericentricheterochromatinisrequiredfortheaccu-mulationofhighlevelsofcohesins,whicharecriticalforchromosomesegregationduringmitosis(Bernardetal.
2001;Nonakaetal.
2002;Yamagishietal.
2008).
Whenheterochromatiniscompromised,suchasindcr1D,lossofcohesinresultsindefectiveattachmentofcentromeresbymicrotubulesforbiorientation.
Asaresult,cellsareverysensitivetothemicrotubulepoisonthiabendazole(TBZ)(Halletal.
2003;Volpeetal.
2003).
Inpoz1Ddcr1Dcells,bothcohesinlocalization,asmeasuredbyChIPanalysesofcohesinsubunitRad21-Flag,andTBZsensi-tivityweresignificantlyrescued(Fig.
1A,E).
Takento-gether,theseresultsindicatethatpericentricheterochro-matinformedinpoz1Ddcr1Dcellsisfunctionalinregulatingchromosomesegregation.
BypassingRNAirequiresRNAi-independentheterochromatinassemblypathwaysatpericentricregionsInfissionyeast,theRNAipathwayinvolvesseveraldiffer-entcomplexesinadditiontoDicer(GotoandNakayama2012).
Wealsofoundthatpoz1DrescuedsilencingdefectsatotrTura4+andTBZsensitivityofallRNAimutantstested,suchasthoseinRITS(ago1Dandchp1D)andRDRC(rdp1D)(Fig.
2A).
However,poz1Dcouldnotrescuesilenc-ingdefectsandTBZsensitivityofheterochromatinmutantsinvolvedinhistonemodificationsortheirrecog-nition,suchasclr4D,swi6D,orclr3D(Fig.
2B),suggestingthathistonemodificationsarestillessentialforpericentricheterochromatinassemblyinpoz1Ddcr1Dcells.
RNAiisrequiredforboththeestablishmentandmain-tenanceofheterochromatinatpericentricregions(Sadaieetal.
2004).
Todistinguishwhetherpoz1Ddcr1Drescuesheterochromatinmaintenanceorestablishment,wein-troducedotrTura4+ineitherasilencedstate(fromwild-typecells)oradesilencedstate(fromclr4Dcells)intoFigure1.
EliminationofPoz1bypassestherequirementofDicerforpericentricheterochromatinassemblyandfunction.
(A)TenfoldserialdilutionanalysesoftheindicatedyeaststrainsgrownontheindicatedmediumtomeasuretheexpressionofotrTura4+andsensitivitytoTBZ.
(B–E)ChIPanalysisofH3K9me2,Swi6,PolII(Rpb1),andRad21-Flaglevelsatpericen-tromericdhrepeats,normalizedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
Figure2.
EliminationofPoz1bypassestheentireRNAipathway,butnothistonemodifications,forpericentrichetero-chromatinassemblyandfunction.
(A,B)TenfoldserialdilutionanalysesoftheindicatedyeaststrainsgrownontheindicatedmediumtomeasuretheexpressionofotrTura4+andsensitivitytoTBZ.
ShelterinregulatespericentricheterochromatinGENES&DEVELOPMENT2491ColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfrompoz1Ddcr1Dcellsbygeneticcrosses(Fig.
3A).
Interest-ingly,poz1Ddcr1DcellsefficientlymaintainedsilencingofotrTura4+whenitwasinheritedfromawild-typecellbutfailedtoestablishsilencingwhenitwasinheritedfromaclr4Dcell(Fig.
3B).
ChIPanalysesofH3K9me2alsoconfirmedthisconclusion(Fig.
3B).
InRNAimutants,althoughsilencingofreportergenesiscompletelylost,significantamountsofH3K9mearestillretainedatpericentricrepeats(Fig.
3C;Sadaieetal.
2004).
ThehistonedeacetylasesClr3andSir2functioncooperativelywithRNAitoestablishheterochromatinatpericentricrepeats,andH3K9me2iscompletelyabol-ishedinclr3Ddcr1Dandsir2Ddcr1Dcells(Yamadaetal.
2005;Alperetal.
2013;Buscainoetal.
2013;Marinaetal.
2013).
Theresultsthatpoz1Ddcr1Dcellsmaintainedpre-existingheterochromatinbutfailedtoestablishhetero-chromatindenovosuggestthattheresidualH3K9meindcr1Dcellsisnecessaryforpoz1Ddcr1Dcellstomaintainpericentricheterochromatin.
Indeed,neitherpoz1Ddcr1Dclr3Dnorpoz1Ddcr1Dsir2DcellscanmaintainsilencingofotrTura4+orH3K9melevelsatpericentricrepeats(Fig.
3C),suggestingthattheRNAi-independentpathwayiscriticalfortheabilityofpoz1Ddcr1Dcellstomaintainpericentricheterochromatin.
EliminationofothershelterincomponentsalsobypassesRNAiforpericentricheterochromatinassemblyPoz1isamemberofthetelomere-associatedshelterincomplex(Miyoshietal.
2008)thatprotectschromosomeendsfrombeingrecognizedasDNAdamagesitesandpreventsDNAlossduringreplication(PalmanddeLange2008;JainandCooper2010).
TelomericDNAconsistsofshorttandemDNArepeatsfollowedbyasingle-strandedoverhang(Fig.
4A).
Infissionyeast,Taz1(homologofmammalianTRF1/2)bindstothedsDNArepeats(Cooperetal.
1997),andthePot1–Tpz1complex(homologoustomammalianPOT1–TPP1)bindstothessDNAoverhang(BaumannandCech2001;Miyoshietal.
2008).
Rap1andPoz1formabridgethatconnectsTaz1andPot1–Tpz1complexes(Miyoshietal.
2008).
Pot1–Tpz1associateswithCcq1torecruittelomeraseTrt1,whichusesassoci-atedRNAasatemplatetoelongatethetelomereover-hang(Nakamuraetal.
1997;Miyoshietal.
2008;TomitaandCooper2008;Moseretal.
2011;Yamazakietal.
2012).
ByChIPanalyses,wefoundthatPoz1wasenrichedattelomeres,asexpected,butnotdetectedatpericentricrepeats,evenindcr1Dcells(Fig.
4B).
Furthermore,North-ernblotanalysesshowedthatnosiRNAsderivedfrompericentricrepeatsweredetectedinpoz1Ddcr1Dcells(Fig.
4C),suggestingthatPoz1regulatespericentricheterochro-matinassemblyindirectlythroughRNAi-independentmechanisms.
SinceourscreensofmutantsthatbypassRNAialsoconsistentlyidentifiedrap1D(SupplementalTableS1),wetestedwhetherothershelterinmutantsbehavedsimi-larly.
Wefoundthatrap1D,taz1D,andccq1DalsorescuedRNAimutants'defectsinotrTura4+silencingandTBZsensitivity(Fig.
4D;SupplementalFig.
S3).
Similarly,rap1Ddcr1Dclr3Dandtaz1Ddcr1Dclr3Dfailedtomaintainpericentricheterochromatinsilencing(SupplementalFig.
S4),indicatingtherequirementofRNAi-independentpericentricheterochromatinpathways.
Incontrast,trt1Dandaccq1-T93Amutant,whichattenuatedrecruitmentofTrt1totelomeres(Moseretal.
2011;Yamazakietal.
2012),hadnoeffectonthisprocess(Fig.
4D).
Wereasonedthatthedifferentbehaviorofccq1Dandccq1-T93AisbecauseCcq1isalsopartoftheSHRECcomplexinvolvedintelomeresilencing(Sugiyamaetal.
2007;MotamediFigure3.
EliminationofPoz1bypassesRNAiforpericentricheterochromatinmaintenancebutnotforitsestablishment.
(A)Schematicdiagramoftheexperimentaldesigntoexamineheterochro-matinmaintenanceandestablishment.
(B,C,left)TenfoldserialdilutionanalysesoftheindicatedyeaststrainstomeasuretheexpressionofotrTura4+.
(Right)ChIPanalysisofH3K9me2levelsatura4+orpericentricdhrepeats,normalizedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
Tadeoetal.
2492GENES&DEVELOPMENTColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfrometal.
2008),whichisnotaffectedbytheccq1-T93Amutation(Moseretal.
2011;Yamazakietal.
2012).
Con-sistentwiththisidea,wealsofoundthatsde2D,whichresultedinselectivelossoftelomeresilencing(Sugioka-SugiyamaandSugiyama2011),wasabletobypassRNAiforpericentricheterochromatinformation(SupplementalTableS1;Fig.
4D).
Pot1andTpz1arerequiredfortelomereendprotection.
Asaresult,pot1Dortpz1Dcellslosetelomeresquicklyandcanonlysurvivebycircularizingallthreechromosomes(BaumannandCech2001;Miyoshietal.
2008).
Nonetheless,pericentricheterochromatinwasefficientlymaintainedinpot1Ddcr1Dandtpz1Ddcr1Dcells,asindicatedbyotrTura4+silencingandTBZsensi-tivity(Fig.
4D).
TheabilityofshelterintoregulatetelomericheterochromatiniskeytobypassingRNAiOneofthecommonphenotypesofthetelomere-relatedmutantsthatbypassRNAiisthattheyallaffecttelomeresilencing(Cooperetal.
1997;Nimmoetal.
1998;KanohandIshikawa2001;Sugiyamaetal.
2007;Moseretal.
2009).
However,themechanismbywhichshelterinnu-cleatesheterochromatinassemblyisunknown.
BecauseTaz1andPot1contributeindependentlytoshelterinre-cruitment,itisdifficulttoexaminethesequencebywhichthesefactorsarerecruitedatnativetelomeres.
Wecon-structedRap1fusedwithaGal4DNA-bindingdomain(Rap1-GBD)atitsendogenouschromosomelocation.
Rap1-GBDissufficienttoinduceectopicheterochromatinassemblyatareportergenewithadjacentGal4-bindingsites(3xgbs-ade6+)undertheconditionofmildSwi6overexpression(SupplementalFig.
S5).
SilencingdependsonPoz1butnotTaz1(Fig.
5A),suggestingthatPoz1functionsdownstreamfromTaz1andRap1intelomericheterochromatinassembly.
TodistinguishthefunctionoftelomerelengthcontrolandtelomericsilencinginbypassingRNAi,wemutatedsevenconservedresiduesofPoz1individuallyattheendogenouspoz1locusandscreenedformutationsthatspecificallyaffectedtelomericsilencingwithoutaffectingtelomerelengthhomeostasis(SupplementalFig.
S6).
Intheend,weisolatedaW209AmutationthathadnoeffectonPoz1proteinlevels,asindicatedbyWesternblotanalysis(Fig.
5B),andhadlittleeffectontelomerelengthhomeostasis,asindicatedbySouthernblotanalyses(Fig.
5C).
However,itcauseddefectsinRap1-GBD-mediatedheterochromatinassembly,likeinpoz1Dcells(Fig.
5A).
Wealsotestedtheeffectofpoz1-W209Aonsilencingneartelomeres.
Sincemultiplepathwayscooperativelyregu-latetelomericheterochromatinassembly,includingshel-terin,RNAi(througharepeatsequencewithinthetlh1+gene),andanunknownmechanismatTAS(Kanohetal.
2005),weusedaura4+reporterinsertedneartelomericrepeatsonaminichromosome(TELTura4+)(Nimmoetal.
1994)thatlacksthetlh1+geneandTAS,makingtheroleofshelterinonsilencingmoreapparent(SupplementalFig.
S7).
Indeed,poz1-W209AresultedinastronglossofFigure4.
EliminationofothershelterincomponentsalsobypassesRNAiforperi-centricheterochromatinassembly.
(A)Sche-maticdiagramofthetelomerecomplex.
(B)ChIPanalysisofPoz1-Flaglevelsatsubtelo-mericandpericentricregions.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
(C)North-ernblotanalysesofsiRNAsderivedfrompericentricdhrepeats.
(D)Tenfoldserialdi-lutionsoftheindicatedyeaststrainsweregrownontheindicatedmediumtomeasuretheexpressionofotrTura4+andsensitivitytoTBZ.
Thetrt1Ddcr1Dandccq1-T93Adcr1Dcellswerefreshlygerminatedtoavoidsenes-cence-associatedtelomererearrangement.
ShelterinregulatespericentricheterochromatinGENES&DEVELOPMENT2493ColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfromsilencingatthisreporter,concomitantwiththelossofH3K9meatthislocation,toadegreesimilartopoz1Dandclr4Dcells(Fig.
5D).
Remarkably,poz1-W209Aalsores-cuedpericentricheterochromatinassemblyindcr1Dcells(Fig.
5E),asindicatedbytherestorationofsilencingattheotrTura4+reporterandH3K9meatpericentricdhrepeats.
Thus,compromisedtelomericsilencingisthereasonwhylossofshelterinrescuespericentricheterochromatinas-semblydefectsassociatedwithRNAimutants.
ShelterinandRNAimodulatethedistributionofSwi6betweenpericentricregionsandtelomeresWethenperformedChIP–chipanalysisofSwi6withanAgilentmicroarray.
WedesignedadditionalprobesthatprovidehighcoverageofcentromereI,whichwasorigi-nallyabsentinthearrayduetoitsrepetitivenature.
Indcr1Dcells,Swi6levelswerelower,butnotabolished,attheentirepericentricregion,asexpected(Fig.
6A).
Un-expectedly,Swi6levelsincreasedsignificantlyacrossthesubtelomericheterochromatindomain,withsignificantspreadingintoeuchromaticregions(Fig.
6A).
Inpoz1-W209Adcr1Dcells,Swi6isnotonlyrestoredtowild-typelevelsatpericentricregionsbutalsoreducedatthesub-telomereregionscomparedwithdcr1D.
ThetotallevelsofSwi6remainconstantinthesecells(Fig.
6B),suggestingthatthedifferencesinSwi6localizationaretheresultofSwi6redistributionattheseregions.
ToexaminewhetherthisredistributionofSwi6hasanyphysiologicalconsequences,wemeasuredRNAtranscriptlevelsderivedfromthepericentricdhrepeatandsub-telomerictlh1.
Indcr1Dcells,highlevelsofdhrepeattranscriptsweredetected,andinpoz1-W209Adcr1Dcells,dhtranscriptlevelsdecreasedsignificantly,consistentwithourresultsthatthesecellsformnormalpericentricheterochromatin.
Incontrast,tlh1transcriptslevelswerehighinpoz1-W209Acellsbutsignificantlyreducedinpoz1-W209Adcr1Dcells(Fig.
6C).
Thus,poz1-W209Aanddcr1Dmutuallysuppressedtheother'sphenotypes.
IfmobilizationofsilencingproteinsfromtelomeresisthereasonthatshelterinmutantsbypassRNAiforperi-centricheterochromatinassembly,weexpectthatsimplyincreasingtheconcentrationofsilencingproteinsissufficienttobypassRNAi.
Asexpected,introducingtheOE-swi6+transgene,whichresultedinaboutfourfoldexcessSwi6proteininthecells(SupplementalFig.
S5),wassufficienttorescuesilencingdefectsandTBZsensi-tivityofallRNAimutantstested,suchasdcr1D,ago1D,andrdp1D(Fig.
6D;SupplementalFig.
S8A).
TheOE-swi6+transgenealsocomplementedswi6DbutcouldnotrescuethesilencingphenotypeandTBZsensitivityofclr4D(SupplementalFig.
S8A).
Moreover,OE-swi6+dcr1Dclr3DandOE-swi6+dcr1Dsir2Dfailedtomaintainpericentricsilencing(SupplementalFig.
S4),suggestingthattheabilityofSwi6overexpressiontorescueRNAidefectsalsode-pendsonRNAi-independentheterochromatinassemblyFigure5.
Lossofshelterin-mediatedsilencingattelo-meresisrequiredforbypassingRNAiforpericentricheterochromatinassembly.
(A)Tenfoldserialdilutionsoftheindicatedyeaststrainsweregrownonalow-adeninemediumtomeasuretheexpressionof3xgbs-ade6+.
(B)WesternblotanalyseswereperformedtomeasurePoz1-mycproteinlevels.
(C)Southernblotanalyseswereperformedtomeasuretelomerelength.
ThegenomicDNAwasdigestedwithEcoRI,andthetelomericrepeatsequencewasusedasaprobe.
(*)Controlpol1fragment.
(D,E)Tenfoldserialdilutionsoftheindicatedyeaststrainsweregrownonthein-dicatedmediumtomeasuretheexpressionofTELTura4+andChIPanalysisofH3K9me2levelsatura4+orpericentricdhrepeat,normalizedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
Tadeoetal.
2494GENES&DEVELOPMENTColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
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Weobtainedsimilarre-sultswithcellscontainingoneadditionalcopyofSwi6insertedatthears1locusdrivenundertheendogenousswi6promoter,whichisexpectedtoincreaseSwi6levelsbytwofold(SupplementalFig.
S8B;Sadaieetal.
2008).
Wealsotestedwhetheroverexpressionofotherhetero-chromatinfactorscouldalsobypassRNAi.
Chp2isanotherHP1homologandisenrichedatallheterochromatindomains(ThonandVerhein-Hansen2000;Sadaieetal.
2004;Motamedietal.
2008;Fischeretal.
2009).
However,incorporatinganadditionalcopyofchp2+underthecontrolofitsendogenouspromoterintothears1locus(Sadaieetal.
2008)couldnotrescuedcr1D(SupplementalFig.
S8B).
Moreover,introducingChp2onamulticopyplasmiddrivenunderaninduciblenmt1promotercouldnotrescuedcr1Deither(SupplementalFig.
S8C).
SimilarresultswereobtainedwithoverexpressionofClr4orClr3(Supplemen-talFig.
S8C).
However,wecautionthatbothClr4andClr3arepresentinmultisubunitcomplexes,sotheirover-expressionmightnotresultinsignificantup-regulationoftheirenzymaticactivitiesinvivo.
Nonetheless,thedifferentresultsobtainedwithHP1homologssuggestthatSwi6,butnotChp2,playsamajorroleinbalancingthestrengthofdifferentheterochromatindomains.
DiscussionRNAiplaysessentialrolesinheterochromatinassemblyindiverseorganisms.
Infissionyeast,lossofRNAiselec-tivelyimpairsheterochromatinassemblyatpericentricregions.
WefoundthateliminationoftelomereshelterincomponentsbypassedtherequirementofRNAiforperi-centricheterochromatinassembly.
Theseparation-of-functionmutantofPoz1allowedustodemonstratethatlossofshelterinandRNAiresultsinthereleasefromtelomeresofsilencingfactorssuchasSwi6,whicharethencompetitivelyrecruitedtopericentricregionsthroughRNAi-independentheterochromatinassemblypathways(Fig.
7).
Inwild-typecells,thehistonedeacetylaseClr3/Sir2functionscooperativelywithRNAitoregulatepericentricheterochromatinassembly,whereasshelterinandTASFigure6.
ShelterinandRNAiaffectthedistributionofSwi6betweenpericentricandtelomericregions.
(A)ChIP–chipanaly-sesofSwi6levelsatcentromereI(leftpanel)andtheleftsideoftelomereI(rightpanel).
(B)WesternblotanalyseswereperformedtomeasureSwi6proteinlevels.
(C)RT–PCRanalyseswereperformedtomeasureperi-centricdh(left)ortelomerictlh1(right)transcriptlevels,normalizedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
(D)TenfoldserialdilutionsoftheindicatedyeaststrainsweregrownontheindicatedmediumtomeasuretheexpressionofotrTura4+andChIPanalysisofH3K9me2levelsatura4+orpericentricdhrepeat,normal-izedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
TheOE-swi6+strainsalsocontainawild-typecopyofswi6+.
Figure7.
AmodelforshelterinandRNAiinregulatinghetero-chromatinassembly.
ShelterinregulatespericentricheterochromatinGENES&DEVELOPMENT2495ColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
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Theseparallelpathwaysformhigh-affinityplatformsfortherecruitmentofheterochro-matinproteinSwi6.
Indcr1Dcells,pericentricheterochro-matinisseverelycompromised,andtheClr3/Sir2pathwayrecruitsSwi6atalowerefficiency.
Therelativelyhigheraffinityoftelomericheterochromatin,whichisduetothepresenceofshelterinandTAS-mediatedheterochromatinassemblypathways,absorbstheextraSwi6,resultinginhigherSwi6levelsattelomeresandthespreadingofSwi6intoeuchromatinatsubtelomericregions.
Inpoz1Ddcr1Dcells,telomericheterochromatinassemblypathwaysarefurthercompromised,andtheClr3/Sir2pathwayatperi-centricregionsisabletocompetewithTASsequencesforSwi6.
IncreasedSwi6mightfacilitatetherecruitmentofCLRCandtherestorationofH3K9meatpericentricheterochromatin.
Thesubtelomereregion'sloweraffin-ityforSwi6inpoz1Ddcr1DcellsresultsinareductionofSwi6levelsandheterochromatinspreadingcomparedwithdcr1Dcells.
Wesuspectthattheabilityofpot1Dandtpz1Dtobypassdcr1Disduetothelossoftelomereandsubtelomeresequences,whichconsequentlyaffectssilencingattelomeres.
SuchamodelisconsistentwithrecentimagingandbiochemicalstudiesshowingthatHP1/Swi6proteinsarehighlymobileonchromatin(Cheutinetal.
2003,2004;Festensteinetal.
2003;Sadaieetal.
2008).
OurresultsthatincreasingSwi6levelsbypassesRNAiforpericentricheterochromatinassemblysuggestthattheavailabilityofheterochromatinproteinsinthenucleus,suchasSwi6,islimiting.
SustaininglowSwi6availabilityisessentialformaintainingtheepigeneticlandscapeofthegenome,ashigherlevelsofSwi6havebeenassociatedwithincreasedefficiencyofheterochromatinassemblyatendogenousandectopicsites.
Forexample,artificialtargetingofClr4toDNAfailstoinduceheterochroma-tinassemblyunlessSwi6isoverexpressed(Supplemen-talFig.
S5B)orendogenousheterochromatinstructuresarecompromisedtoreleasesilencingproteins(Kaganskyetal.
2009).
Inaddition,exogenouslyintroducedsiRNAsfailtoestablishheterochromatinunlessSwi6isoverex-pressed(Iidaetal.
2008).
Moreover,overexpressionofSwi6increasestheconversionrateofalessstableheterochro-matindomainatthematingtyperegion(Nakayamaetal.
2000)andenhancesthespreadingofheterochromatinwhenchromatinboundariesarecompromised(Nomaetal.
2001;Wangetal.
2013).
Thus,themultiplepathwaysthatregulateheterochromatinassemblyatconstitutiveheterochromatinregions,suchastelomeric,pericentric,andthesilentmatingtyperegions,mightbekeytotheformationofhigher-affinityplatformsforheterochromatinproteins,thusbalancingtheneedforproperheterochro-matinassemblyattheseregionsandpreventinghetero-chromatinassemblyatcrypticsites.
Whenanyofthesemechanismsarecompromised,Swi6redistributesaccord-ingtothestrengthofheterochromatinassemblypathwaysatdifferentregionsandismorelikelytoberecruitedtositesthatotherwisecannotformheterochromatin.
Heterochromatinismainlypresentatpericentricandsubtelomericregionsindiverseorganisms.
Althoughtheroleofpericentricheterochromatininregulatingchro-mosomesegregationhasbeenwellstudied,thefunctionsoftelomericheterochromatinarelessclearlydefined.
IncertainorganismssuchasDrosophila,telomeresarecomposedofretrotransposonsinsteadoftelomericre-peats,whicharepackagedintoheterochromatintorecruitendprotectionfactors(Masonetal.
2008).
Infissionyeast,amplificationofsubtelomereheterochromatinisalsoassociatedwithcellsurvivalintheabsenceoftelomerase(Jainetal.
2010).
Certainheterochromatinfactors,suchasClr4andRik1,alsoregulatetelomereclusteringduringmeiosis(Tuzonetal.
2004).
However,heterochromatinfactorshavenoeffectontelomerelengthcontrolanddonotprotectagainsttelomereendfusions(Tuzonetal.
2004).
Comparedwithotherheterochromatinregions,thetelomereandsubtelomereregionsareuniqueinbufferingthechangesinheterochromatinlevelsbecausetherearenodefinedheterochromatinboundaries,andalargetran-sitionzoneisformed,withSwi6levelsgraduallydeclining(Fig.
6A).
Incontrast,atcentromeresandthesilentmatingtyperegion,silencingfactorconcentrationsarehigh,andwell-definedheterochromatinboundariespreventspread-ing(Cametal.
2005).
Thus,theseregionsmightalreadybesaturatedbysilencingfactors,andthebufferingcapacityislimited.
Thisisexemplifiedindcr1D,inwhichSwi6proteinlevelsweresignificantlyincreasedacrosstheentiresubtelomericregion,withsignificantamountsofspreadingintoeuchromaticregions,whereasinpoz1Dcells,therewerelittlechangesofSwi6levelsatpericentricregionsbutenhancedheterochromatinassemblybyGBD-Clr4(Fig.
6A;SupplementalS5B).
Conversely,cellscon-tainingtheminichromosomeCh10(Niwaetal.
1989),whichismainlycomposedofpericentricrepeatsandcentromereandtelomeresequences,showreducedlevelsofSwi6selectivelyatsubtelomericregions(Chikashigeetal.
2007).
Itisworthnotingthatthebuddingyeast,whichusesSir2/3/4proteinstoassembleheterochromatininsteadoftheH3K9me-HP1system,alsousestelomerestolimitsilencingfactors,andreleasingthesefactorsfromtelomeressimilarlyincreasessilencingatinternalsites(Mailletetal.
1996;Marcandetal.
1996;Taddeietal.
2009).
Thus,thesequestrationofsilencingfactorsthroughconstitutiveheterochromatindomainsmightbeespeciallyimportantforgenomesthatarehighlyactivetopreventectopicheterochromatinassemblyinordertomaintaintheepigeneticlandscape.
MaterialsandmethodsFissionyeaststrainsandgeneticanalysesYeaststrainscontainingpoz1D,rap1D,andtrt1DwerederivedfromtheBioneerfissionyeastdeletionlibrary,verifiedviaPCR,andbackcrossed.
ThestraincontainingOE-swi6+wasconstructedbyintegratingaPCRfragmentcontainingtheade6promoter,swi6codingregion,and39untranslatedregionandahphMX6cassetteintotheendogenousade6+locus.
Poz1mutantswereconstructedbyintegratingaPCRfragmentcontainingthemutations,amyctag,andaKanMX6cassetteintotheendog-enouspoz1+locus.
Rap1-Flagandtaz1DwereconstructedbyaPCR-basedmodulemethod.
Geneticcrosseswereusedtocon-structallotherstrains.
Strainscontainingtpz1Dorpot1DwereTadeoetal.
2496GENES&DEVELOPMENTColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
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orgDownloadedfromfirstgeneratedindiploidcells,followedbytetraddissectiontoobtaindesiredgenotypes.
Forserialdilutionplatingassays,10-folddilutionsofalog-phaseculturewereplatedontheindicatedmediumandgrownfor3dat30°C.
ScreensformutationsthatsuppressthesilencingdefectsofRNAimutantsQuerystrainswerematedwiththefissionyeastdeletionlibrarywiththeaidoftheSingerRoToRHDApinningrobotaspre-viouslydescribed(Roguevetal.
2007).
ThefissionyeastdeletionlibrarywasconstructedwithaKanMX4cassettethatconfersresistancetogeneticin(Bioneer).
Tworeporterstrains(otrTura4+orotrTade6+)wereconstructedbyinsertingaNatMX6cassette,whichconfersresistancetonourseothricin,;400basepairs(bp)totherightsideboundaryofcentromereIheterochromatin.
Thedcr1Dandago1DquerystrainswereconstructedwithanhphMX6cassette,conferringresistancetohygromycin.
TheotrTura4+orotrTade6+reporteranddcr1Dweresequentiallyintroducedintothedeletionlibrary,andtheresultinghaploidcellswereselectedandpinnedtoselectivemediumtomeasurecellgrowthorcolonycolor.
ChIPanalysisChIPanalyseswereperformedasdescribedpreviously(Houetal.
2010).
AntibodiesusedwereH3K9me2(Abcam),Swi6(Reddyetal.
2011),PolII(Covance),myc(Covance),andFlag(Sigma).
Quantitativereal-timePCR(qPCR)wasperformedwithMaximaSYBRGreenqPCRmastermix(Fermentas)inanABI7300Real-TimePCRSystem.
DNAserialdilutionswereusedastemplatestogenerateastandardcurveofamplificationforeachpairofprimers,andtherelativeconcentrationoftargetsequencewascalculatedaccordingly.
Anact1fragmentwasusedasareferencetocalculatetheenrichmentofChIPoverwhole-cellextractforeachtargetsequence.
ChIP–chipanalysiswasperformedaccord-ingtotheAgilentYeastChIP-on-chipanalysisprotocol.
ThemicroarrayusedwasanAgilentS.
pombeWhole-GenomeChIP-on-chipmicroarray(G4810A)withadditionalprobesthaten-compasscentromeres,whichwereoriginallyabsentfromthearrayduetotherepetitivenatureoftheseDNAsequences.
RNAanalysesTotalcellularRNAwasisolatedfromlog-phasecellsusingaMasterPureYeastRNApurificationkit(Epicentre)accordingtothemanufacturer'sprotocol.
Quantificationwithreal-timeRT–PCRwasperformedwithPowerSYBRGreenRNA-to-CTOne-Stepkit(AppliedBiosystems).
RNAserialdilutionswereusedastemplatetogeneratethestandardcurveofamplificationforeachpairofprimers,andtherelativeconcentrationoftargetsequencewascalculatedaccordingly.
Anact1fragmentservedasareferencetonormalizetheconcentrationofsamples.
Theconcentrationofeachtargetgeneinwildtypewasarbitrarilysetto1andservedasreferenceforothersamples.
NorthernblotofsiRNAswasperformedasdescribedpreviously(Reddyetal.
2011).
TelomerelengthanalysisForpoz1mutantandpoz1Dstrains,asinglecolonyfromthefirstrestreakafterthemutationordeletionwasgrownovernight.
GenomicDNAwasisolated,digestedwithEcoRI,andseparatedona1%agarosegel.
HybridizationwithradiolabeledtelomericDNAandpol1(control)probeswasperformedaspreviouslydescribed(Junetal.
2013).
AcknowledgmentsWethankJunichiNakayama,RobinAllshire,MichaelKeogh,andToruNakamuraforyeaststrains,plasmids,andantibodies,andAllisonCohenfortechnicalassistance.
ThisworkwassupportedbyNationalInstitutesofHealth(NIH)grantsR01-GM085145(toS.
J.
)andR01-GM098943to(F.
Q.
).
X.
T.
wassup-portedbytheFulbrightScholarProgram.
B.
RandS.
P.
K.
weresupportedbyNIHtraininggrantT32-GM008798.
F.
Q.
wassup-portedbyaBasilO'ConnorStarterScholarResearchAwardfromMarchofDimesandaBeginningGrant-in-aidfromtheAmericanHeartAssociation.
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TheJmjCdomainproteinEpe1preventsunregulatedassem-blyanddisassemblyofheterochromatin.
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ShelterinregulatespericentricheterochromatinGENES&DEVELOPMENT2499ColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloaded
XavierTadeo,JiyongWang,ScottP.
Kallgren,etal.
heterochromatinassemblyEliminationofshelterincomponentsbypassesRNAiforpericentricMaterialSupplementalhttp://genesdev.
cshlp.
org/content/suppl/2013/11/14/27.
22.
2489.
DC1.
htmlReferenceshttp://genesdev.
cshlp.
org/content/27/22/2489.
full.
html#ref-list-1Thisarticlecites62articles,27ofwhichcanbeaccessedfreeat:LicenseCommonsCreative.
http://creativecommons.
org/licenses/by-nc/3.
0/CreativeCommonsLicense(Attribution-NonCommercial3.
0Unported),asdescribedat).
Aftersixmonths,itisavailableunderahttp://genesdev.
cshlp.
org/site/misc/terms.
xhtmlsixmonthsafterthefull-issuepublicationdate(seeThisarticleisdistributedexclusivelybyColdSpringHarborLaboratoryPressforthefirstServiceEmailAlertingclickhere.
rightcornerofthearticleorReceivefreeemailalertswhennewarticlescitethisarticle-signupintheboxatthetophttp://genesdev.
cshlp.
org/subscriptionsgoto:Genes&DevelopmentTosubscribeto2013Tadeoetal.
;PublishedbyColdSpringHarborLaboratoryPressColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfromColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfromEliminationofshelterincomponentsbypassesRNAiforpericentricheterochromatinassemblyXavierTadeo,1JiyongWang,1ScottP.
Kallgren,1JinqiangLiu,2BharatD.
Reddy,1FengQiao,2andSongtaoJia1,31DepartmentofBiologicalSciences,ColumbiaUniversity,NewYork,NewYork10027,USA;2DepartmentofBiologicalChemistry,UniversityofCaliforniaatIrvine,Irvine,California92697,USATheRNAipathwayisrequiredforheterochromatinassemblyatrepetitiveDNAelementsindiverseorganisms.
Infissionyeast,lossofRNAicausespericentricheterochromatindefects,compromisinggenesilencingandchromosomesegregation.
HereweshowthatdeletionoftelomereshelterincomponentsrestorespericentricheterochromatinanditsfunctionsinRNAimutants.
Wefurtherisolatedaseparation-of-functionmutantofPoz1andrevealedthatdefectivetelomeresilencing,butnottelomerelengthcontrol,iscriticalforbypassingRNAi.
Furtheranalysesdemonstratedthatcompromisingshelterin-mediatedheterochromatinassemblyinRNAimutantsreleasesheterochromatinproteinSwi6,whichisredistributedtopericentricregionsthroughRNAi-independentheterochromatinassemblypathways.
GiventhehighmobilityofSwi6proteinandthatincreasedlevelsofSwi6facilitatesheterochromatinspreadingaswellasectopicheterochromatinassembly,ourresultssuggestthatconstitutiveheterochromatindomainsusemultiplepathwaystoformhigh-affinityplatformstorestrainSwi6,thuslimitingitsavailabilityandavoidingpromiscuousheterochromatinformation.
[Keywords:RNAi;heterochromatinassembly;telomere;shelterin;centromere]Supplementalmaterialisavailableforthisarticle.
ReceivedJuly5,2013;revisedversionacceptedOctober9,2013.
EukaryoticgenomespossesslargestretchesofrepetitiveDNAelementsthatformcompactchromatinstructurestermedheterochromatin.
Theseregionsarerelativelygene-poorbutoccupycrucialchromosomeregions,suchascentromeresandtelomeres,whichplaycriticalrolesinchromosomesegregationandthree-dimensionalgenomeorganization.
Heterochromatinalsoinhibitstranscrip-tionandrecombinationoftheunderlyingDNArepeats,renderingitindispensableformaintaininggenomein-tegrity(GrewalandJia2007;AlmouzniandProbst2011).
GenomicDNAisfoldedwithhistonestoformchroma-tin.
Eachchromosomalregionisassociatedwithdistinctprofilesofhistonepost-translationalmodifications,whichplayessentialrolesinregulatingchromatinstructureandfunction.
Histonesatheterochromaticregionsaregener-allydevoidofacetylationandaremethylatedatH3Lys9(H3K9me).
H3K9meallowsforthebindingofheterochro-matinprotein1(HP1),whichcompactschromatinthroughitsself-associationandtherecruitmentofotherchroma-tin-modifyingactivities(GrewalandJia2007).
Althoughheterochromatinhastraditionallybeenconsideredsilent,stable,andstatic,recentbiochemicalandimagingstudiesshowthatthebindingofHP1proteinstoheterochromatinregionsissurprisinglydynamic(Cheutinetal.
2003,2004;Festensteinetal.
2003;Sadaieetal.
2008).
Moreover,althoughheterochromatinnormallyexcludesthetran-scriptionalmachinery,theDNArepeatswithinhetero-chromatinaretranscribed,andthetranscriptsarepro-cessedbytheRNAipathwayintosiRNAs,whichtargethistone-modifyingcomplexestorepetitiveDNAele-ments(Moazed,2009;LejeuneandAllshire2011;CastelandMartienssen2013).
RNAi-dependentheterochromatinassemblyisbeststudiedinthefissionyeastSchizosaccharomycespombe(forreview,seeMoazed2009;LejeuneandAllshire,2011;GotoandNakayama2012;CastelandMartienssen2013).
Inthisorganism,constitutiveheterochromatinislocalizedatthecentromeres,telomeres,andthesilentmatingtyperegion,allofwhichsharesimilarrepetitiveDNAele-mentscomposedofdganddhrepeats.
TheDNArepeats2013Tadeoetal.
ThisarticleisdistributedexclusivelybyColdSpringHarborLaboratoryPressforthefirstsixmonthsafterthefull-issuepublicationdate(seehttp://genesdev.
cshlp.
org/site/misc/terms.
xhtml).
Aftersixmonths,itisavailableunderaCreativeCommonsLicense(Attribution-NonCommercial3.
0Unported),asdescribedathttp://creativecommons.
org/licenses/by-nc/3.
0/.
3CorrespondingauthorE-mailjia@biology.
columbia.
eduArticleisonlineathttp://www.
genesdev.
org/cgi/doi/10.
1101/gad.
226118.
113.
GENES&DEVELOPMENT27:2489–2499PublishedbyColdSpringHarborLaboratoryPress;ISSN0890-9369/13;www.
genesdev.
org2489ColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfromaretranscribedbyRNApolymeraseII(PolII)duringtheSphaseofthecellcycle,producingdsRNAswiththehelpofaRNA-dependentRNApolymerasecomplex(RDRC).
TheribonucleaseDicer(Dcr1)processesthesedsRNAsintosiRNAs,whichareloadedontotheArgonautesiRNAchaperonecomplex(ARC)andthentransferredtoRNAi-inducedtranscriptionalsilencingcomplex(RITS).
TheArgonauteprotein(Ago1)withinRITSbindssiRNAsandtargetsRITStonascentRNAtranscriptsfromrepeatregions.
RITSthenrecruitstheCLRCcomplex,whichcontainstheH3K9methyltransferaseClr4.
H3K9mere-cruitschromodomainproteinsSwi6andChp2togetherwiththeirassociatedhistonedeacetylasesandchromatinremodelingactivitiestoachievetranscriptionalsilencing.
H3K9mealsostabilizesthebindingofRITStochromatinthroughthechromodomainproteinChp1,whichinturnrecruitsRDRCtogeneratemoredsRNAsandsiRNAs,thusformingaself-reinforcingloopbetweensiRNApro-ductionandheterochromatinassembly.
InadditiontoRNAi-basedmechanisms,DNA-basedmechanismsalsofunctionatrepeatregionstorecruitCLRC.
Forexample,ATF/CREBfamilyproteinsAtf1andPcr1cooperatewithRNAitoestablishheterochromatinatthesilentmatingtyperegion(Jiaetal.
2004;Kimetal.
2004).
Similarly,telomereshelterincomponentTaz1andthetelomere-associatedsequence(TAS)functiontogetherwithRNAitoestablishheterochromatinattelomeres(Kanohetal.
2005).
Asaresult,lossofRNAiseverelyaffectspericentricheterochromatinfunctionsbuthaslittleeffectonheterochromatinstructuresatthesilentmatingtyperegionortelomeres.
However,evenatpericentricregions,H3K9meandSwi6arestillpresentinRNAimutants,albeitatlowerlevels,suggestingtheexistenceofRNAi-independentmechanismstoestablishhetero-chromatin(Sadaieetal.
2004).
ThesepathwaysinvolvetheClr3andSir2histonedeacetylasessinceH3K9melevelsarefurtherreducedfrompericentricregionsinclr3Ddcr1Dandsir2Ddcr1Dcells,althoughSir2seemstofunctioninaseparatepathwayfromClr3(Yamadaetal.
2005;Alperetal.
2013;Buscainoetal.
2013;Marinaetal.
2013).
Interestingly,eliminationoftheMst2histoneH3K14acetyltransferasecomplexactivity,theJmjCdomainpro-teinEpe1,orRNAqualitycontrolfactorMlo3bypassestherequirementofRNAiforpericentricheterochromatinassembly(Trewicketal.
2007;Reddyetal.
2011;Reyes-Turcuetal.
2011).
AlthoughthemechanismsbywhichthesemutantsregulateheterochromatinassemblyintheabsenceofRNAidiffer,theseresultsnonethelessindicatethatRNAiisnotobligatoryforpericentricheterochro-matinformationundercertainconditions.
Tofurtherunderstandheterochromatinassemblypathwaysatperi-centricregions,weperformedahigh-throughputscreenofthefissionyeastdeletionlibraryformutantsthatsupportpericentricheterochromatinassemblyintheab-senceofRNAi.
Wediscoveredthatlossofthetelomereshelterincomponentsinvolvedinpropertelomeremain-tenancealsobypassedRNAiforpericentricheterochro-matinformation.
WefurtherisolatedamutantofshelterincomponentPoz1thatspecificallyaffectedtelomerichet-erochromatinassemblywithoutaffectingtelomerelengthandshowedthatlossofshelterininanRNAimutantbackgroundresultedintheredistributionofheterochro-matinproteinSwi6fromtelomericheterochromatintopericentricregionsthroughRNAi-independenthetero-chromatinassemblypathwaysinvolvingClr3andSir2.
Furthermore,weshowedthatincreasingoverallSwi6levelswassufficienttobypassRNAiforpericentricheterochromatinassembly.
Ourresultsdemonstrateady-namicequilibriumofdifferentheterochromatindomainsthroughthehighmobilityofSwi6andsuggestthatconstitutiveheterochromatinregions,suchascentro-meresandtelomeres,playimportantrolesinbalancingtheavailabilityofsilencingfactorstomaintaintheoverallepigeneticlandscapeofthegenome.
ResultsAhigh-throughputscreenformutantsthatbypasstherequirementoftheRNAimachineryforpericentricheterochromatinassemblyInordertounderstandthemechanismofRNAiandheterochromatinassembly,weperformedascreenofthefissionyeastdeletionlibraryformutantsthatallowrestorationofpericentricheterochromatinintheabsenceofRNAifunction.
Weusedaquerystraincontainingaura4+reportergeneinsertedattheouterrepeatofcentro-mereI(otrTura4+)(SupplementalFig.
S1A).
Inwild-typecells,thisreportergeneissilencedduetotheformationofheterochromatin.
Asaresult,thereislittlegrowthonmediumlackinguracilbutrobustgrowthoncounter-selectivemediumcontaining5-fluorooroticacid(FOA),whichistoxictocellsexpressingura4+(Fig.
1A).
Muta-tionsthataffectheterochromatinstructureatpericentricregions,suchasdcr1D,resultedinstrongcellgrowthonmediumlackinguracilbutlittlegrowthonmediumcontainingFOA(Fig.
1A).
Throughtwosuccessivecrosses,weintroducedtheotrTura4+reporteranddcr1Dintothedeletionlibraryandobtainedhaploidcells,eachcontain-ingotrTura4+,dcr1D,andasinglegenedeletion(Supple-mentalFig.
S1B).
TheresultingcellsweregrownonmediumcontainingFOAtomeasurecellgrowth(Supple-mentalFig.
S1C;SupplementalTableS1).
OurscreenidentifiedmutantsknowntobypassRNAiforpericentricheterochromatinassembly,suchasMst2complexcompo-nentsmst2D,nto1D,andptf2D(Reddyetal.
2011;Wangetal.
2012),validatingtheeffectivenessofourscreen.
Ourscreenalsoidentifiedpoz1D,atelomereshelterincompo-nentinvolvedintelomerelengthcontrol(Miyoshietal.
2008),asshowingthestrongestrescueofheterochromaticsilencing(SupplementalFig.
S1C;SupplementalTableS1).
WealsoperformedsimilarscreenswithanotrTade6+reporter(SupplementalFig.
S2A).
Thisreportergeneissilencedinwild-typecells,resultingintheformationofredcoloniesonlow-adeninemediumduetotheaccumu-lationofmetabolicintermediates.
InRNAimutants,suchasdcr1D,lossofpericentricheterochromatinresultedintheexpressionofAde6,givingrisetowhitecolonies(SupplementalFig.
S2A).
Wealsoidentifiedpoz1DasoneTadeoetal.
2490GENES&DEVELOPMENTColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfromofthestrongestsuppressorsofdcr1Dandago1DwiththeotrTade6+reporter(SupplementalFig.
S2B,C;Supplemen-talTableS1).
Altogether,weidentifiedatotalof33genedeletionsthatbypassedRNAiinbothotrTura4+andotrTade6+reporterscreens.
EliminationofPoz1bypassesRNAiforpericentricheterochromatinfunctionsToconfirmthatlossofPoz1canindeedrestorepericentricheterochromatininRNAimutants,weconstructedapoz1Ddcr1DdoublemutantstraincontainingotrTura4+.
Serialdilutionanalysesconfirmedthatpoz1Ddcr1Dgrewweaklyonmediumwithouturacilbutrobustlyonme-diumcontainingFOA(Fig.
1A).
Chromatinimmunopre-cipitation(ChIP)analysesshowedthatinpoz1Ddcr1Dcells,heterochromatinhallmarksatpericentricdhrepeats,suchasH3K9me2andSwi6,wererestoredtonearwild-typelevels(Fig.
1B,C).
Moreover,ChIPanalysesshowedthatPolIIwasstronglyexcludedfrompericentricrepeats(Fig.
1D),suggestingthatheterochromatinformedinpoz1Ddcr1Dcellsiscapableoftranscriptionalgenesilencing.
Pericentricheterochromatinisrequiredfortheaccu-mulationofhighlevelsofcohesins,whicharecriticalforchromosomesegregationduringmitosis(Bernardetal.
2001;Nonakaetal.
2002;Yamagishietal.
2008).
Whenheterochromatiniscompromised,suchasindcr1D,lossofcohesinresultsindefectiveattachmentofcentromeresbymicrotubulesforbiorientation.
Asaresult,cellsareverysensitivetothemicrotubulepoisonthiabendazole(TBZ)(Halletal.
2003;Volpeetal.
2003).
Inpoz1Ddcr1Dcells,bothcohesinlocalization,asmeasuredbyChIPanalysesofcohesinsubunitRad21-Flag,andTBZsensi-tivityweresignificantlyrescued(Fig.
1A,E).
Takento-gether,theseresultsindicatethatpericentricheterochro-matinformedinpoz1Ddcr1Dcellsisfunctionalinregulatingchromosomesegregation.
BypassingRNAirequiresRNAi-independentheterochromatinassemblypathwaysatpericentricregionsInfissionyeast,theRNAipathwayinvolvesseveraldiffer-entcomplexesinadditiontoDicer(GotoandNakayama2012).
Wealsofoundthatpoz1DrescuedsilencingdefectsatotrTura4+andTBZsensitivityofallRNAimutantstested,suchasthoseinRITS(ago1Dandchp1D)andRDRC(rdp1D)(Fig.
2A).
However,poz1Dcouldnotrescuesilenc-ingdefectsandTBZsensitivityofheterochromatinmutantsinvolvedinhistonemodificationsortheirrecog-nition,suchasclr4D,swi6D,orclr3D(Fig.
2B),suggestingthathistonemodificationsarestillessentialforpericentricheterochromatinassemblyinpoz1Ddcr1Dcells.
RNAiisrequiredforboththeestablishmentandmain-tenanceofheterochromatinatpericentricregions(Sadaieetal.
2004).
Todistinguishwhetherpoz1Ddcr1Drescuesheterochromatinmaintenanceorestablishment,wein-troducedotrTura4+ineitherasilencedstate(fromwild-typecells)oradesilencedstate(fromclr4Dcells)intoFigure1.
EliminationofPoz1bypassestherequirementofDicerforpericentricheterochromatinassemblyandfunction.
(A)TenfoldserialdilutionanalysesoftheindicatedyeaststrainsgrownontheindicatedmediumtomeasuretheexpressionofotrTura4+andsensitivitytoTBZ.
(B–E)ChIPanalysisofH3K9me2,Swi6,PolII(Rpb1),andRad21-Flaglevelsatpericen-tromericdhrepeats,normalizedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
Figure2.
EliminationofPoz1bypassestheentireRNAipathway,butnothistonemodifications,forpericentrichetero-chromatinassemblyandfunction.
(A,B)TenfoldserialdilutionanalysesoftheindicatedyeaststrainsgrownontheindicatedmediumtomeasuretheexpressionofotrTura4+andsensitivitytoTBZ.
ShelterinregulatespericentricheterochromatinGENES&DEVELOPMENT2491ColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfrompoz1Ddcr1Dcellsbygeneticcrosses(Fig.
3A).
Interest-ingly,poz1Ddcr1DcellsefficientlymaintainedsilencingofotrTura4+whenitwasinheritedfromawild-typecellbutfailedtoestablishsilencingwhenitwasinheritedfromaclr4Dcell(Fig.
3B).
ChIPanalysesofH3K9me2alsoconfirmedthisconclusion(Fig.
3B).
InRNAimutants,althoughsilencingofreportergenesiscompletelylost,significantamountsofH3K9mearestillretainedatpericentricrepeats(Fig.
3C;Sadaieetal.
2004).
ThehistonedeacetylasesClr3andSir2functioncooperativelywithRNAitoestablishheterochromatinatpericentricrepeats,andH3K9me2iscompletelyabol-ishedinclr3Ddcr1Dandsir2Ddcr1Dcells(Yamadaetal.
2005;Alperetal.
2013;Buscainoetal.
2013;Marinaetal.
2013).
Theresultsthatpoz1Ddcr1Dcellsmaintainedpre-existingheterochromatinbutfailedtoestablishhetero-chromatindenovosuggestthattheresidualH3K9meindcr1Dcellsisnecessaryforpoz1Ddcr1Dcellstomaintainpericentricheterochromatin.
Indeed,neitherpoz1Ddcr1Dclr3Dnorpoz1Ddcr1Dsir2DcellscanmaintainsilencingofotrTura4+orH3K9melevelsatpericentricrepeats(Fig.
3C),suggestingthattheRNAi-independentpathwayiscriticalfortheabilityofpoz1Ddcr1Dcellstomaintainpericentricheterochromatin.
EliminationofothershelterincomponentsalsobypassesRNAiforpericentricheterochromatinassemblyPoz1isamemberofthetelomere-associatedshelterincomplex(Miyoshietal.
2008)thatprotectschromosomeendsfrombeingrecognizedasDNAdamagesitesandpreventsDNAlossduringreplication(PalmanddeLange2008;JainandCooper2010).
TelomericDNAconsistsofshorttandemDNArepeatsfollowedbyasingle-strandedoverhang(Fig.
4A).
Infissionyeast,Taz1(homologofmammalianTRF1/2)bindstothedsDNArepeats(Cooperetal.
1997),andthePot1–Tpz1complex(homologoustomammalianPOT1–TPP1)bindstothessDNAoverhang(BaumannandCech2001;Miyoshietal.
2008).
Rap1andPoz1formabridgethatconnectsTaz1andPot1–Tpz1complexes(Miyoshietal.
2008).
Pot1–Tpz1associateswithCcq1torecruittelomeraseTrt1,whichusesassoci-atedRNAasatemplatetoelongatethetelomereover-hang(Nakamuraetal.
1997;Miyoshietal.
2008;TomitaandCooper2008;Moseretal.
2011;Yamazakietal.
2012).
ByChIPanalyses,wefoundthatPoz1wasenrichedattelomeres,asexpected,butnotdetectedatpericentricrepeats,evenindcr1Dcells(Fig.
4B).
Furthermore,North-ernblotanalysesshowedthatnosiRNAsderivedfrompericentricrepeatsweredetectedinpoz1Ddcr1Dcells(Fig.
4C),suggestingthatPoz1regulatespericentricheterochro-matinassemblyindirectlythroughRNAi-independentmechanisms.
SinceourscreensofmutantsthatbypassRNAialsoconsistentlyidentifiedrap1D(SupplementalTableS1),wetestedwhetherothershelterinmutantsbehavedsimi-larly.
Wefoundthatrap1D,taz1D,andccq1DalsorescuedRNAimutants'defectsinotrTura4+silencingandTBZsensitivity(Fig.
4D;SupplementalFig.
S3).
Similarly,rap1Ddcr1Dclr3Dandtaz1Ddcr1Dclr3Dfailedtomaintainpericentricheterochromatinsilencing(SupplementalFig.
S4),indicatingtherequirementofRNAi-independentpericentricheterochromatinpathways.
Incontrast,trt1Dandaccq1-T93Amutant,whichattenuatedrecruitmentofTrt1totelomeres(Moseretal.
2011;Yamazakietal.
2012),hadnoeffectonthisprocess(Fig.
4D).
Wereasonedthatthedifferentbehaviorofccq1Dandccq1-T93AisbecauseCcq1isalsopartoftheSHRECcomplexinvolvedintelomeresilencing(Sugiyamaetal.
2007;MotamediFigure3.
EliminationofPoz1bypassesRNAiforpericentricheterochromatinmaintenancebutnotforitsestablishment.
(A)Schematicdiagramoftheexperimentaldesigntoexamineheterochro-matinmaintenanceandestablishment.
(B,C,left)TenfoldserialdilutionanalysesoftheindicatedyeaststrainstomeasuretheexpressionofotrTura4+.
(Right)ChIPanalysisofH3K9me2levelsatura4+orpericentricdhrepeats,normalizedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
Tadeoetal.
2492GENES&DEVELOPMENTColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfrometal.
2008),whichisnotaffectedbytheccq1-T93Amutation(Moseretal.
2011;Yamazakietal.
2012).
Con-sistentwiththisidea,wealsofoundthatsde2D,whichresultedinselectivelossoftelomeresilencing(Sugioka-SugiyamaandSugiyama2011),wasabletobypassRNAiforpericentricheterochromatinformation(SupplementalTableS1;Fig.
4D).
Pot1andTpz1arerequiredfortelomereendprotection.
Asaresult,pot1Dortpz1Dcellslosetelomeresquicklyandcanonlysurvivebycircularizingallthreechromosomes(BaumannandCech2001;Miyoshietal.
2008).
Nonetheless,pericentricheterochromatinwasefficientlymaintainedinpot1Ddcr1Dandtpz1Ddcr1Dcells,asindicatedbyotrTura4+silencingandTBZsensi-tivity(Fig.
4D).
TheabilityofshelterintoregulatetelomericheterochromatiniskeytobypassingRNAiOneofthecommonphenotypesofthetelomere-relatedmutantsthatbypassRNAiisthattheyallaffecttelomeresilencing(Cooperetal.
1997;Nimmoetal.
1998;KanohandIshikawa2001;Sugiyamaetal.
2007;Moseretal.
2009).
However,themechanismbywhichshelterinnu-cleatesheterochromatinassemblyisunknown.
BecauseTaz1andPot1contributeindependentlytoshelterinre-cruitment,itisdifficulttoexaminethesequencebywhichthesefactorsarerecruitedatnativetelomeres.
Wecon-structedRap1fusedwithaGal4DNA-bindingdomain(Rap1-GBD)atitsendogenouschromosomelocation.
Rap1-GBDissufficienttoinduceectopicheterochromatinassemblyatareportergenewithadjacentGal4-bindingsites(3xgbs-ade6+)undertheconditionofmildSwi6overexpression(SupplementalFig.
S5).
SilencingdependsonPoz1butnotTaz1(Fig.
5A),suggestingthatPoz1functionsdownstreamfromTaz1andRap1intelomericheterochromatinassembly.
TodistinguishthefunctionoftelomerelengthcontrolandtelomericsilencinginbypassingRNAi,wemutatedsevenconservedresiduesofPoz1individuallyattheendogenouspoz1locusandscreenedformutationsthatspecificallyaffectedtelomericsilencingwithoutaffectingtelomerelengthhomeostasis(SupplementalFig.
S6).
Intheend,weisolatedaW209AmutationthathadnoeffectonPoz1proteinlevels,asindicatedbyWesternblotanalysis(Fig.
5B),andhadlittleeffectontelomerelengthhomeostasis,asindicatedbySouthernblotanalyses(Fig.
5C).
However,itcauseddefectsinRap1-GBD-mediatedheterochromatinassembly,likeinpoz1Dcells(Fig.
5A).
Wealsotestedtheeffectofpoz1-W209Aonsilencingneartelomeres.
Sincemultiplepathwayscooperativelyregu-latetelomericheterochromatinassembly,includingshel-terin,RNAi(througharepeatsequencewithinthetlh1+gene),andanunknownmechanismatTAS(Kanohetal.
2005),weusedaura4+reporterinsertedneartelomericrepeatsonaminichromosome(TELTura4+)(Nimmoetal.
1994)thatlacksthetlh1+geneandTAS,makingtheroleofshelterinonsilencingmoreapparent(SupplementalFig.
S7).
Indeed,poz1-W209AresultedinastronglossofFigure4.
EliminationofothershelterincomponentsalsobypassesRNAiforperi-centricheterochromatinassembly.
(A)Sche-maticdiagramofthetelomerecomplex.
(B)ChIPanalysisofPoz1-Flaglevelsatsubtelo-mericandpericentricregions.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
(C)North-ernblotanalysesofsiRNAsderivedfrompericentricdhrepeats.
(D)Tenfoldserialdi-lutionsoftheindicatedyeaststrainsweregrownontheindicatedmediumtomeasuretheexpressionofotrTura4+andsensitivitytoTBZ.
Thetrt1Ddcr1Dandccq1-T93Adcr1Dcellswerefreshlygerminatedtoavoidsenes-cence-associatedtelomererearrangement.
ShelterinregulatespericentricheterochromatinGENES&DEVELOPMENT2493ColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
cshlp.
orgDownloadedfromsilencingatthisreporter,concomitantwiththelossofH3K9meatthislocation,toadegreesimilartopoz1Dandclr4Dcells(Fig.
5D).
Remarkably,poz1-W209Aalsores-cuedpericentricheterochromatinassemblyindcr1Dcells(Fig.
5E),asindicatedbytherestorationofsilencingattheotrTura4+reporterandH3K9meatpericentricdhrepeats.
Thus,compromisedtelomericsilencingisthereasonwhylossofshelterinrescuespericentricheterochromatinas-semblydefectsassociatedwithRNAimutants.
ShelterinandRNAimodulatethedistributionofSwi6betweenpericentricregionsandtelomeresWethenperformedChIP–chipanalysisofSwi6withanAgilentmicroarray.
WedesignedadditionalprobesthatprovidehighcoverageofcentromereI,whichwasorigi-nallyabsentinthearrayduetoitsrepetitivenature.
Indcr1Dcells,Swi6levelswerelower,butnotabolished,attheentirepericentricregion,asexpected(Fig.
6A).
Un-expectedly,Swi6levelsincreasedsignificantlyacrossthesubtelomericheterochromatindomain,withsignificantspreadingintoeuchromaticregions(Fig.
6A).
Inpoz1-W209Adcr1Dcells,Swi6isnotonlyrestoredtowild-typelevelsatpericentricregionsbutalsoreducedatthesub-telomereregionscomparedwithdcr1D.
ThetotallevelsofSwi6remainconstantinthesecells(Fig.
6B),suggestingthatthedifferencesinSwi6localizationaretheresultofSwi6redistributionattheseregions.
ToexaminewhetherthisredistributionofSwi6hasanyphysiologicalconsequences,wemeasuredRNAtranscriptlevelsderivedfromthepericentricdhrepeatandsub-telomerictlh1.
Indcr1Dcells,highlevelsofdhrepeattranscriptsweredetected,andinpoz1-W209Adcr1Dcells,dhtranscriptlevelsdecreasedsignificantly,consistentwithourresultsthatthesecellsformnormalpericentricheterochromatin.
Incontrast,tlh1transcriptslevelswerehighinpoz1-W209Acellsbutsignificantlyreducedinpoz1-W209Adcr1Dcells(Fig.
6C).
Thus,poz1-W209Aanddcr1Dmutuallysuppressedtheother'sphenotypes.
IfmobilizationofsilencingproteinsfromtelomeresisthereasonthatshelterinmutantsbypassRNAiforperi-centricheterochromatinassembly,weexpectthatsimplyincreasingtheconcentrationofsilencingproteinsissufficienttobypassRNAi.
Asexpected,introducingtheOE-swi6+transgene,whichresultedinaboutfourfoldexcessSwi6proteininthecells(SupplementalFig.
S5),wassufficienttorescuesilencingdefectsandTBZsensi-tivityofallRNAimutantstested,suchasdcr1D,ago1D,andrdp1D(Fig.
6D;SupplementalFig.
S8A).
TheOE-swi6+transgenealsocomplementedswi6DbutcouldnotrescuethesilencingphenotypeandTBZsensitivityofclr4D(SupplementalFig.
S8A).
Moreover,OE-swi6+dcr1Dclr3DandOE-swi6+dcr1Dsir2Dfailedtomaintainpericentricsilencing(SupplementalFig.
S4),suggestingthattheabilityofSwi6overexpressiontorescueRNAidefectsalsode-pendsonRNAi-independentheterochromatinassemblyFigure5.
Lossofshelterin-mediatedsilencingattelo-meresisrequiredforbypassingRNAiforpericentricheterochromatinassembly.
(A)Tenfoldserialdilutionsoftheindicatedyeaststrainsweregrownonalow-adeninemediumtomeasuretheexpressionof3xgbs-ade6+.
(B)WesternblotanalyseswereperformedtomeasurePoz1-mycproteinlevels.
(C)Southernblotanalyseswereperformedtomeasuretelomerelength.
ThegenomicDNAwasdigestedwithEcoRI,andthetelomericrepeatsequencewasusedasaprobe.
(*)Controlpol1fragment.
(D,E)Tenfoldserialdilutionsoftheindicatedyeaststrainsweregrownonthein-dicatedmediumtomeasuretheexpressionofTELTura4+andChIPanalysisofH3K9me2levelsatura4+orpericentricdhrepeat,normalizedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
Tadeoetal.
2494GENES&DEVELOPMENTColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
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Weobtainedsimilarre-sultswithcellscontainingoneadditionalcopyofSwi6insertedatthears1locusdrivenundertheendogenousswi6promoter,whichisexpectedtoincreaseSwi6levelsbytwofold(SupplementalFig.
S8B;Sadaieetal.
2008).
Wealsotestedwhetheroverexpressionofotherhetero-chromatinfactorscouldalsobypassRNAi.
Chp2isanotherHP1homologandisenrichedatallheterochromatindomains(ThonandVerhein-Hansen2000;Sadaieetal.
2004;Motamedietal.
2008;Fischeretal.
2009).
However,incorporatinganadditionalcopyofchp2+underthecontrolofitsendogenouspromoterintothears1locus(Sadaieetal.
2008)couldnotrescuedcr1D(SupplementalFig.
S8B).
Moreover,introducingChp2onamulticopyplasmiddrivenunderaninduciblenmt1promotercouldnotrescuedcr1Deither(SupplementalFig.
S8C).
SimilarresultswereobtainedwithoverexpressionofClr4orClr3(Supplemen-talFig.
S8C).
However,wecautionthatbothClr4andClr3arepresentinmultisubunitcomplexes,sotheirover-expressionmightnotresultinsignificantup-regulationoftheirenzymaticactivitiesinvivo.
Nonetheless,thedifferentresultsobtainedwithHP1homologssuggestthatSwi6,butnotChp2,playsamajorroleinbalancingthestrengthofdifferentheterochromatindomains.
DiscussionRNAiplaysessentialrolesinheterochromatinassemblyindiverseorganisms.
Infissionyeast,lossofRNAiselec-tivelyimpairsheterochromatinassemblyatpericentricregions.
WefoundthateliminationoftelomereshelterincomponentsbypassedtherequirementofRNAiforperi-centricheterochromatinassembly.
Theseparation-of-functionmutantofPoz1allowedustodemonstratethatlossofshelterinandRNAiresultsinthereleasefromtelomeresofsilencingfactorssuchasSwi6,whicharethencompetitivelyrecruitedtopericentricregionsthroughRNAi-independentheterochromatinassemblypathways(Fig.
7).
Inwild-typecells,thehistonedeacetylaseClr3/Sir2functionscooperativelywithRNAitoregulatepericentricheterochromatinassembly,whereasshelterinandTASFigure6.
ShelterinandRNAiaffectthedistributionofSwi6betweenpericentricandtelomericregions.
(A)ChIP–chipanaly-sesofSwi6levelsatcentromereI(leftpanel)andtheleftsideoftelomereI(rightpanel).
(B)WesternblotanalyseswereperformedtomeasureSwi6proteinlevels.
(C)RT–PCRanalyseswereperformedtomeasureperi-centricdh(left)ortelomerictlh1(right)transcriptlevels,normalizedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
(D)TenfoldserialdilutionsoftheindicatedyeaststrainsweregrownontheindicatedmediumtomeasuretheexpressionofotrTura4+andChIPanalysisofH3K9me2levelsatura4+orpericentricdhrepeat,normal-izedtoact1.
Thenumbersareaveragesofthreeexperiments,anderrorbarsrepresentstandarddeviation.
TheOE-swi6+strainsalsocontainawild-typecopyofswi6+.
Figure7.
AmodelforshelterinandRNAiinregulatinghetero-chromatinassembly.
ShelterinregulatespericentricheterochromatinGENES&DEVELOPMENT2495ColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
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Theseparallelpathwaysformhigh-affinityplatformsfortherecruitmentofheterochro-matinproteinSwi6.
Indcr1Dcells,pericentricheterochro-matinisseverelycompromised,andtheClr3/Sir2pathwayrecruitsSwi6atalowerefficiency.
Therelativelyhigheraffinityoftelomericheterochromatin,whichisduetothepresenceofshelterinandTAS-mediatedheterochromatinassemblypathways,absorbstheextraSwi6,resultinginhigherSwi6levelsattelomeresandthespreadingofSwi6intoeuchromatinatsubtelomericregions.
Inpoz1Ddcr1Dcells,telomericheterochromatinassemblypathwaysarefurthercompromised,andtheClr3/Sir2pathwayatperi-centricregionsisabletocompetewithTASsequencesforSwi6.
IncreasedSwi6mightfacilitatetherecruitmentofCLRCandtherestorationofH3K9meatpericentricheterochromatin.
Thesubtelomereregion'sloweraffin-ityforSwi6inpoz1Ddcr1DcellsresultsinareductionofSwi6levelsandheterochromatinspreadingcomparedwithdcr1Dcells.
Wesuspectthattheabilityofpot1Dandtpz1Dtobypassdcr1Disduetothelossoftelomereandsubtelomeresequences,whichconsequentlyaffectssilencingattelomeres.
SuchamodelisconsistentwithrecentimagingandbiochemicalstudiesshowingthatHP1/Swi6proteinsarehighlymobileonchromatin(Cheutinetal.
2003,2004;Festensteinetal.
2003;Sadaieetal.
2008).
OurresultsthatincreasingSwi6levelsbypassesRNAiforpericentricheterochromatinassemblysuggestthattheavailabilityofheterochromatinproteinsinthenucleus,suchasSwi6,islimiting.
SustaininglowSwi6availabilityisessentialformaintainingtheepigeneticlandscapeofthegenome,ashigherlevelsofSwi6havebeenassociatedwithincreasedefficiencyofheterochromatinassemblyatendogenousandectopicsites.
Forexample,artificialtargetingofClr4toDNAfailstoinduceheterochroma-tinassemblyunlessSwi6isoverexpressed(Supplemen-talFig.
S5B)orendogenousheterochromatinstructuresarecompromisedtoreleasesilencingproteins(Kaganskyetal.
2009).
Inaddition,exogenouslyintroducedsiRNAsfailtoestablishheterochromatinunlessSwi6isoverex-pressed(Iidaetal.
2008).
Moreover,overexpressionofSwi6increasestheconversionrateofalessstableheterochro-matindomainatthematingtyperegion(Nakayamaetal.
2000)andenhancesthespreadingofheterochromatinwhenchromatinboundariesarecompromised(Nomaetal.
2001;Wangetal.
2013).
Thus,themultiplepathwaysthatregulateheterochromatinassemblyatconstitutiveheterochromatinregions,suchastelomeric,pericentric,andthesilentmatingtyperegions,mightbekeytotheformationofhigher-affinityplatformsforheterochromatinproteins,thusbalancingtheneedforproperheterochro-matinassemblyattheseregionsandpreventinghetero-chromatinassemblyatcrypticsites.
Whenanyofthesemechanismsarecompromised,Swi6redistributesaccord-ingtothestrengthofheterochromatinassemblypathwaysatdifferentregionsandismorelikelytoberecruitedtositesthatotherwisecannotformheterochromatin.
Heterochromatinismainlypresentatpericentricandsubtelomericregionsindiverseorganisms.
Althoughtheroleofpericentricheterochromatininregulatingchro-mosomesegregationhasbeenwellstudied,thefunctionsoftelomericheterochromatinarelessclearlydefined.
IncertainorganismssuchasDrosophila,telomeresarecomposedofretrotransposonsinsteadoftelomericre-peats,whicharepackagedintoheterochromatintorecruitendprotectionfactors(Masonetal.
2008).
Infissionyeast,amplificationofsubtelomereheterochromatinisalsoassociatedwithcellsurvivalintheabsenceoftelomerase(Jainetal.
2010).
Certainheterochromatinfactors,suchasClr4andRik1,alsoregulatetelomereclusteringduringmeiosis(Tuzonetal.
2004).
However,heterochromatinfactorshavenoeffectontelomerelengthcontrolanddonotprotectagainsttelomereendfusions(Tuzonetal.
2004).
Comparedwithotherheterochromatinregions,thetelomereandsubtelomereregionsareuniqueinbufferingthechangesinheterochromatinlevelsbecausetherearenodefinedheterochromatinboundaries,andalargetran-sitionzoneisformed,withSwi6levelsgraduallydeclining(Fig.
6A).
Incontrast,atcentromeresandthesilentmatingtyperegion,silencingfactorconcentrationsarehigh,andwell-definedheterochromatinboundariespreventspread-ing(Cametal.
2005).
Thus,theseregionsmightalreadybesaturatedbysilencingfactors,andthebufferingcapacityislimited.
Thisisexemplifiedindcr1D,inwhichSwi6proteinlevelsweresignificantlyincreasedacrosstheentiresubtelomericregion,withsignificantamountsofspreadingintoeuchromaticregions,whereasinpoz1Dcells,therewerelittlechangesofSwi6levelsatpericentricregionsbutenhancedheterochromatinassemblybyGBD-Clr4(Fig.
6A;SupplementalS5B).
Conversely,cellscon-tainingtheminichromosomeCh10(Niwaetal.
1989),whichismainlycomposedofpericentricrepeatsandcentromereandtelomeresequences,showreducedlevelsofSwi6selectivelyatsubtelomericregions(Chikashigeetal.
2007).
Itisworthnotingthatthebuddingyeast,whichusesSir2/3/4proteinstoassembleheterochromatininsteadoftheH3K9me-HP1system,alsousestelomerestolimitsilencingfactors,andreleasingthesefactorsfromtelomeressimilarlyincreasessilencingatinternalsites(Mailletetal.
1996;Marcandetal.
1996;Taddeietal.
2009).
Thus,thesequestrationofsilencingfactorsthroughconstitutiveheterochromatindomainsmightbeespeciallyimportantforgenomesthatarehighlyactivetopreventectopicheterochromatinassemblyinordertomaintaintheepigeneticlandscape.
MaterialsandmethodsFissionyeaststrainsandgeneticanalysesYeaststrainscontainingpoz1D,rap1D,andtrt1DwerederivedfromtheBioneerfissionyeastdeletionlibrary,verifiedviaPCR,andbackcrossed.
ThestraincontainingOE-swi6+wasconstructedbyintegratingaPCRfragmentcontainingtheade6promoter,swi6codingregion,and39untranslatedregionandahphMX6cassetteintotheendogenousade6+locus.
Poz1mutantswereconstructedbyintegratingaPCRfragmentcontainingthemutations,amyctag,andaKanMX6cassetteintotheendog-enouspoz1+locus.
Rap1-Flagandtaz1DwereconstructedbyaPCR-basedmodulemethod.
Geneticcrosseswereusedtocon-structallotherstrains.
Strainscontainingtpz1Dorpot1DwereTadeoetal.
2496GENES&DEVELOPMENTColdSpringHarborLaboratoryPressonNovember16,2013-Publishedbygenesdev.
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orgDownloadedfromfirstgeneratedindiploidcells,followedbytetraddissectiontoobtaindesiredgenotypes.
Forserialdilutionplatingassays,10-folddilutionsofalog-phaseculturewereplatedontheindicatedmediumandgrownfor3dat30°C.
ScreensformutationsthatsuppressthesilencingdefectsofRNAimutantsQuerystrainswerematedwiththefissionyeastdeletionlibrarywiththeaidoftheSingerRoToRHDApinningrobotaspre-viouslydescribed(Roguevetal.
2007).
ThefissionyeastdeletionlibrarywasconstructedwithaKanMX4cassettethatconfersresistancetogeneticin(Bioneer).
Tworeporterstrains(otrTura4+orotrTade6+)wereconstructedbyinsertingaNatMX6cassette,whichconfersresistancetonourseothricin,;400basepairs(bp)totherightsideboundaryofcentromereIheterochromatin.
Thedcr1Dandago1DquerystrainswereconstructedwithanhphMX6cassette,conferringresistancetohygromycin.
TheotrTura4+orotrTade6+reporteranddcr1Dweresequentiallyintroducedintothedeletionlibrary,andtheresultinghaploidcellswereselectedandpinnedtoselectivemediumtomeasurecellgrowthorcolonycolor.
ChIPanalysisChIPanalyseswereperformedasdescribedpreviously(Houetal.
2010).
AntibodiesusedwereH3K9me2(Abcam),Swi6(Reddyetal.
2011),PolII(Covance),myc(Covance),andFlag(Sigma).
Quantitativereal-timePCR(qPCR)wasperformedwithMaximaSYBRGreenqPCRmastermix(Fermentas)inanABI7300Real-TimePCRSystem.
DNAserialdilutionswereusedastemplatestogenerateastandardcurveofamplificationforeachpairofprimers,andtherelativeconcentrationoftargetsequencewascalculatedaccordingly.
Anact1fragmentwasusedasareferencetocalculatetheenrichmentofChIPoverwhole-cellextractforeachtargetsequence.
ChIP–chipanalysiswasperformedaccord-ingtotheAgilentYeastChIP-on-chipanalysisprotocol.
ThemicroarrayusedwasanAgilentS.
pombeWhole-GenomeChIP-on-chipmicroarray(G4810A)withadditionalprobesthaten-compasscentromeres,whichwereoriginallyabsentfromthearrayduetotherepetitivenatureoftheseDNAsequences.
RNAanalysesTotalcellularRNAwasisolatedfromlog-phasecellsusingaMasterPureYeastRNApurificationkit(Epicentre)accordingtothemanufacturer'sprotocol.
Quantificationwithreal-timeRT–PCRwasperformedwithPowerSYBRGreenRNA-to-CTOne-Stepkit(AppliedBiosystems).
RNAserialdilutionswereusedastemplatetogeneratethestandardcurveofamplificationforeachpairofprimers,andtherelativeconcentrationoftargetsequencewascalculatedaccordingly.
Anact1fragmentservedasareferencetonormalizetheconcentrationofsamples.
Theconcentrationofeachtargetgeneinwildtypewasarbitrarilysetto1andservedasreferenceforothersamples.
NorthernblotofsiRNAswasperformedasdescribedpreviously(Reddyetal.
2011).
TelomerelengthanalysisForpoz1mutantandpoz1Dstrains,asinglecolonyfromthefirstrestreakafterthemutationordeletionwasgrownovernight.
GenomicDNAwasisolated,digestedwithEcoRI,andseparatedona1%agarosegel.
HybridizationwithradiolabeledtelomericDNAandpol1(control)probeswasperformedaspreviouslydescribed(Junetal.
2013).
AcknowledgmentsWethankJunichiNakayama,RobinAllshire,MichaelKeogh,andToruNakamuraforyeaststrains,plasmids,andantibodies,andAllisonCohenfortechnicalassistance.
ThisworkwassupportedbyNationalInstitutesofHealth(NIH)grantsR01-GM085145(toS.
J.
)andR01-GM098943to(F.
Q.
).
X.
T.
wassup-portedbytheFulbrightScholarProgram.
B.
RandS.
P.
K.
weresupportedbyNIHtraininggrantT32-GM008798.
F.
Q.
wassup-portedbyaBasilO'ConnorStarterScholarResearchAwardfromMarchofDimesandaBeginningGrant-in-aidfromtheAmericanHeartAssociation.
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