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ExtratelomericbindingofTRF1andTRF21028npgCellResearch|Vol21No7|July2011ORIGINALARTICLEThehumanTTAGGGrepeatfactors1and2bindtoasubsetofinterstitialtelomericsequencesandsatelliterepeatsThomasSimonet1,Laure-EmmanuelleZaragosi2,ClaudePhilippe3,KevinLebrigand2,ClémentineSchouteden1,AdelineAugereau1,3,SergeBauwens1,JingYe1,3,MarcoSantagostino4,ElenaGiulotto4,FrederiqueMagdinier1,BéatriceHorard1,PascalBarbry2,RainerWaldmann2,EricGilson1,3,51LaboratoiredeBiologieMoléculairedelaCellule-UMR5239CNRS/ENSLyon/UniversitéLyon,EcoleNormaleSupérieuredeLyon,46alléed'Italie,Lyon69364,France;2CNRSandUniversityofNiceSophiaAntipolis,InstitutdePharmacologieMolé-culaireetCellulaire,06560SophiaAntipolis,France;3LaboratoryofBiologyandPathologyofGenomesofUniversityofNiceSophia-Antipolis,CNRSUMR6267/INSERMU998,FacultyofMedicine,Nice,France;4DipartimentodiGeneticaeMicrobiologiaAdrianoBuzzati-Traverso,UniversitàdiPavia,Pavia,Italy;5DepartmentofMedicalGenetics,CHUofNice,Nice,FranceCorrespondence:EricGilsonTel:+33-472728453;Fax:+33-472728080E-mail:Eric.
Gilson@unice.
frReceived3November2010;revised9January2011;accepted11January2011;publishedonline22March2011ThestudyoftheproteinsthatbindtotelomericDNAinmammalshasprovidedadeepunderstandingofthemech-anismsinvolvedinchromosome-endprotection.
However,verylittleisknownonthebindingoftheseproteinstonontelomericDNAsequences.
TheTTAGGGDNArepeatproteins1and2(TRF1andTRF2)bindtomammaliante-lomeresaspartoftheshelterincomplexandareessentialformaintainingchromosomeendstability.
Inthisstudy,wecombinedchromatinimmunoprecipitationwithhigh-throughputsequencingtomapathighsensitivityandresolutionthehumanchromosomalsitestowhichTRF1andTRF2bind.
Whilemostoftheidentifiedsequencescorrespondtotelomericregions,weshowedthatthesetwoproteinsalsobindtoextratelomericsites.
Thevastmajorityoftheseex-tratelomericsitescontainsinterstitialtelomericsequences(orITSs).
However,wealsoidentifiednon-ITSsites,whichcorrespondtocentromericandpericentromericsatelliteDNA.
Interestingly,theTRF-bindingsitesareoftenlocatedintheproximityofgenesorwithinintrons.
WeproposethatTRF1andTRF2couplethefunctionalstateoftelomerestothelong-rangeorganizationofchromosomesandgeneregulationnetworksbybindingtoextratelomericsequences.
Keywords:telomere;TRF1;TRF2;interstitialtelomericsequence;satelliteDNACellResearch(2011)21:1028-1038.
doi:10.
1038/cr.
2011.
40;publishedonline22March2011npgCellResearch(2011)21:1028-1038.
2011IBCB,SIBS,CASAllrightsreserved1001-0602/11$32.
00www.
nature.
com/crIntroductionTheparamountimportanceoftelomerestocellfatelikelystemsfromthegreatdiversityinthefunctionstheyperform[1,2].
Theycontrolthereplicationofchro-mosomalDNAtermini,protectchromosomeendsfromDNArepairandcheckpointactivation,controlthemei-oticspindle,localizethechromosomeendswithinthenuclearspaceandregulatelong-rangechromatinchangesaswellasgeneexpression.
Telomeresconsistofspecificnucleoproteincomplexes[3].
TelomericDNAhassev-eraldistinctivefeatures,includingasequenceformedbyrepetitionsofasmallG-richmotif(TTAGGGinmam-mals)andthepresenceofasingle-strandedtailonthe3′-orientedstrand(Gtail).
TelomericDNAistranscribedintoaUUAGGGrepeat-containingRNAcalledTERRA,whichisbelievedtoplayfundamentalrolesintelomerebiology[4,5].
Akeycomponentofthemammaliantelomereistheshelterincomplex,whichiscomposedofsixpolypep-tides:TRF1,TRF2,Rap1,Tin2,TPP1andPot1[5].
Ofthese,threebindspecificallytoTTAGGGrepeats:TRF1andTRF2,whichrecognizetheduplexDNA,andPot1,whichbindstothesingle-stranded3′overhangs[3,6].
TRF1andTRF2donotexistinbuddingyeast.
Instead,yeastRap1actsasanessentialcappingfactorthatbindstotelomericDNA,whileyeastCdc13bindstothe3′overhangandseemstoperformfunctionsthataresimilartothoseofPot1andTPP1[7].
Telomeresinyeastandmammalscansilenceneigh-www.
cell-research.
com|CellResearchThomasSimonetetal.
1029npgboringgenesbyexertingtelomericpositioneffect(orTPE)[8-10].
TPEisinfluencedbytelomerelengthandstructureaswellasbychromatin-remodelingmachiner-ies[11].
Telomericandsubtelomericchromatindifferfromconstitutiveheterochromatinintermsofstructureanddynamics,specificityofDNAsequences,andbind-ingofspecificfactors[12].
Themechanismsthatinitiatetheformationofheterochromatinattelomeresareun-knownbutlikelyinvolvethebindingofspecificfactorstotelomericDNA.
Forinstance,theN-terminalpartofTRF2mayfacilitateheterochromatinformationbybind-ingtoORC1andTERRA[13].
RepetitionsoftheTTAGGGtelomericunit,calledinterstitialtelomericsequences,orITSs,arealsopresentwithinchromosomes[14].
Inhumans,threeclassesofITSswereidentified[15]:(i)subtelomericITSs,locatedwithinsubtelomericdomainsandcomposedofextendedarrays(usuallyseveralhundredsofbasepairs),includingmanydegenerateunits;theyprobablyarosefromrecom-binationeventsinvolvingchromosometermini[16];(ii)shortinternalITSs,locatedawayfromtelomeresandcomposedofrelativelyfewTTAGGGunits;theseITSsarelikelytohavebeengeneratedduringtherepairofDNAdouble-strandbreaksthatoccurredduringevolu-tion[17];(iii)onefusionITS,locatedin2q14,derivedfromtheendfusionbetweenthetwoancestralchromo-somesthatgaverisetohumanchromosome2[18].
NoclearindicationofanyparticularfunctionofITSshasbeenprovidedsofar.
Emergingevidenceindicatesthattheshelterincom-ponentshavenon-telomericfunctionsinDNArepair[19],Epstein-Barrvirusreplication[20],transcriptionalregulation[21]andNF-κBactivation[22].
Thesenon-telomericfunctionsmightbe,atleastpartially,explainedbytheirbindingtoITSs.
Indeed,thereismountingindi-cationthatshelterincomponentscanbindtointerstitialDNAsequences:(i)TRF1andTRF2bindtotheperi-centromericregionsofhamsterchromosomescontaininglargeblocksofITSs[23,24];(ii)TRF2andTIN2bindtoanITSformedbyararehumanchromosomerear-rangement[25];(iii)TRF2bindstoastretchoftelomericsequencethatisartificiallyinsertedinthemiddleofthelongarmofchromosome4[26];(iv)Rap1bindstoseveralITSsofthemousegenome[27].
However,threenaturallyoccurringITSsofhumanchromosomesdonotappeartobeboundbyTRF2[26].
Therefore,itisstillunclearwhetherTRF1andTRF2reallybindtotheITSsnormallyfoundinhumanchromosomesoreventounre-latedsequences.
Moreover,thereisevidencethatTRF2modulatesgeneexpressionoutsidefromtelomeressinceitinteractswiththerepressorelement1-silencingtran-scriptionfactor(REST),arepressorofgenesdevotedtoneuronalfunctions[21].
Inthisstudy,wemappedthehumanchromosomalsitestowhichTRF1andTRF2bindbycombiningchro-matinimmunoprecipitationwithhigh-throughputDNAsequencing(ChIP-Seq).
ResultsIdentificationofTRFbindingsitesbyChIP-SeqanalysisToestablishglobalbindingprofilesofTRF1andTRF2(collectivelynamedtheTRFproteins),weperformedaChIP-SeqanalysiswithoneantibodyspecificforTRF1andtwoantibodiesspecificforTRF2(onemonoclonalorTRF2m,onepolyclonalorTRF2p).
WeusedtheBJ-HELTRasmctumorcelllinebecauseTRF2isrequiredfortumorigenicitythroughapathwaythatinvolvesuncou-plingoftelomereprotectionandtheDNAdamagere-sponsemechanism,suggestingaroleforextratelomericTRF2bindingsitesinoncogenesis(Biroccioetal,sub-mitted).
Thespecificityoftheanti-TRF1andanti-TRF2antibodieswasconfirmedbyslotblotanalysis(Figure1A).
Wefoundupto50-foldenrichmentoftelomericsequencesintheTRFantibody-immunoprecipitatedsam-pleswhencomparedtoProteinG-Sepharose-precipitatedcontrolsamplesandtotalhistoneH3-immunoprecipi-tation.
ThisresultwasconfirmedbytheanalysisoftheChIP-Seqreads.
InTRFantibody-immunoprecipitatedsampleswedetected90to150timesmoresequencesthatcontainsolelythe(TTAGGG)nmotifthaninthecontrolsamples(Figure1B).
ToidentifyextratelomericbindingsitesfortheTRFproteins,weretainedonlyreadsthatwereuniquelyalignedonthe2006Humangenomeassembly(NCBI36/hg18),andwecheckedthatpure(TTAGGG)nreads,whichlikelyoriginatefromtelomericDNA,hadbeenindeedcompletelydiscarded.
Significantlyread-enrichedpositionsorpeakswereidentifiedusingtheSISSRsoftware[28]withaPvaluethresholdof0.
001usingproteinGimmunoprecipitationasbackground.
Wefur-therremovedtheseeminglyartifactual(non-specific)peaksthroughavisualinspectionofadensityprofileofthematchedreads(seetheexampleforchromosome1,showninSupplementaryinformation,FigureS1).
Fol-lowingthisfiltering,weidentified68peakspresentinallthreeTRFChIP-Seqsamples(TRF1,TRF2mandTRF2p)(Figure2A).
Resultsforchromosome1areshowninFigure2BandthoseforotherchromosomesareshowninSupplementaryinformation,FigureS2.
Notably,18peaksfromtheTRF2mChIP(amongn=90,20%)werenotfoundusingTRF2pantibody,while21peaksidentifiedbytheTRF2pChIP(n=93,22.
5%)werenotpresentinTRF2mChIP.
FormostofExtratelomericbindingofTRF1andTRF21030npgCellResearch|Vol21No7|July2011Figure1(A)SlotblotshowingthetelomericenrichmentofDNAimmunoprecipitatedwithanti-TRF1orTRF2antibodies.
DNAimmunoprecipitatedbyatotalH3antibodyandpulleddownbyproteinGalonewasusedasacontrol.
HalfoftheprecipitatedDNAwasloaded,alongwithaninputscale(2500ngto10ng,correspondingto10%to0.
04%ofthetotalinput),andhybrid-izedsequentiallytoatelomericprobeandagenomicprobe.
Foreachprobe,wequantifiedthefractionoftheimmunoprecipi-tatedDNA.
Theratioofthevalueobtainedforthetelomericprobetothegenomicprobeisthetelomericenrichmentfactor.
(B)Foldenrichmentofthefractionofrawreadscontainingonly(TTAGGG)nsequencesfromTRFChIP-SeqasnormalizedtothereadsobtainedthroughimmunoprecipitationofproteinG.
Figure2(A)TRF1,TRF2m,andTRF2pChIP-Seqpeaks.
Thepeakslargelycoincide,asshownontheVenndiagram.
As-sessmentofoverlapswasperformedbyvisualinspectionintheIntegratedGenomeBrowser.
(B)VisualizationofTRFpeaksandTRFbindingsites.
Regionsofsignificantreadenrichment(PU998)forcriticalreading.
WearealsogratefultoZhouSongyang(BaylorCollegeofMedicine)forsharingunpublishedresults.
ThisworkwassupportedbygrantsfromtheAssociationdelarecherchecontreleCancer(ARC),theInstitutNationalduCancer(programTELOFUN),ANR(programTELOREPandINNATELO)andtheEuropeanCommunity(TELOMARKERHealth-F2-2007-200950).
TSandLEZthanktheFondationdelaRechercheMédicale(FRM)andtheARC,respectively,fortheirfellowships.
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Gilson@unice.
frReceived3November2010;revised9January2011;accepted11January2011;publishedonline22March2011ThestudyoftheproteinsthatbindtotelomericDNAinmammalshasprovidedadeepunderstandingofthemech-anismsinvolvedinchromosome-endprotection.
However,verylittleisknownonthebindingoftheseproteinstonontelomericDNAsequences.
TheTTAGGGDNArepeatproteins1and2(TRF1andTRF2)bindtomammaliante-lomeresaspartoftheshelterincomplexandareessentialformaintainingchromosomeendstability.
Inthisstudy,wecombinedchromatinimmunoprecipitationwithhigh-throughputsequencingtomapathighsensitivityandresolutionthehumanchromosomalsitestowhichTRF1andTRF2bind.
Whilemostoftheidentifiedsequencescorrespondtotelomericregions,weshowedthatthesetwoproteinsalsobindtoextratelomericsites.
Thevastmajorityoftheseex-tratelomericsitescontainsinterstitialtelomericsequences(orITSs).
However,wealsoidentifiednon-ITSsites,whichcorrespondtocentromericandpericentromericsatelliteDNA.
Interestingly,theTRF-bindingsitesareoftenlocatedintheproximityofgenesorwithinintrons.
WeproposethatTRF1andTRF2couplethefunctionalstateoftelomerestothelong-rangeorganizationofchromosomesandgeneregulationnetworksbybindingtoextratelomericsequences.
Keywords:telomere;TRF1;TRF2;interstitialtelomericsequence;satelliteDNACellResearch(2011)21:1028-1038.
doi:10.
1038/cr.
2011.
40;publishedonline22March2011npgCellResearch(2011)21:1028-1038.
2011IBCB,SIBS,CASAllrightsreserved1001-0602/11$32.
00www.
nature.
com/crIntroductionTheparamountimportanceoftelomerestocellfatelikelystemsfromthegreatdiversityinthefunctionstheyperform[1,2].
Theycontrolthereplicationofchro-mosomalDNAtermini,protectchromosomeendsfromDNArepairandcheckpointactivation,controlthemei-oticspindle,localizethechromosomeendswithinthenuclearspaceandregulatelong-rangechromatinchangesaswellasgeneexpression.
Telomeresconsistofspecificnucleoproteincomplexes[3].
TelomericDNAhassev-eraldistinctivefeatures,includingasequenceformedbyrepetitionsofasmallG-richmotif(TTAGGGinmam-mals)andthepresenceofasingle-strandedtailonthe3′-orientedstrand(Gtail).
TelomericDNAistranscribedintoaUUAGGGrepeat-containingRNAcalledTERRA,whichisbelievedtoplayfundamentalrolesintelomerebiology[4,5].
Akeycomponentofthemammaliantelomereistheshelterincomplex,whichiscomposedofsixpolypep-tides:TRF1,TRF2,Rap1,Tin2,TPP1andPot1[5].
Ofthese,threebindspecificallytoTTAGGGrepeats:TRF1andTRF2,whichrecognizetheduplexDNA,andPot1,whichbindstothesingle-stranded3′overhangs[3,6].
TRF1andTRF2donotexistinbuddingyeast.
Instead,yeastRap1actsasanessentialcappingfactorthatbindstotelomericDNA,whileyeastCdc13bindstothe3′overhangandseemstoperformfunctionsthataresimilartothoseofPot1andTPP1[7].
Telomeresinyeastandmammalscansilenceneigh-www.
cell-research.
com|CellResearchThomasSimonetetal.
1029npgboringgenesbyexertingtelomericpositioneffect(orTPE)[8-10].
TPEisinfluencedbytelomerelengthandstructureaswellasbychromatin-remodelingmachiner-ies[11].
Telomericandsubtelomericchromatindifferfromconstitutiveheterochromatinintermsofstructureanddynamics,specificityofDNAsequences,andbind-ingofspecificfactors[12].
Themechanismsthatinitiatetheformationofheterochromatinattelomeresareun-knownbutlikelyinvolvethebindingofspecificfactorstotelomericDNA.
Forinstance,theN-terminalpartofTRF2mayfacilitateheterochromatinformationbybind-ingtoORC1andTERRA[13].
RepetitionsoftheTTAGGGtelomericunit,calledinterstitialtelomericsequences,orITSs,arealsopresentwithinchromosomes[14].
Inhumans,threeclassesofITSswereidentified[15]:(i)subtelomericITSs,locatedwithinsubtelomericdomainsandcomposedofextendedarrays(usuallyseveralhundredsofbasepairs),includingmanydegenerateunits;theyprobablyarosefromrecom-binationeventsinvolvingchromosometermini[16];(ii)shortinternalITSs,locatedawayfromtelomeresandcomposedofrelativelyfewTTAGGGunits;theseITSsarelikelytohavebeengeneratedduringtherepairofDNAdouble-strandbreaksthatoccurredduringevolu-tion[17];(iii)onefusionITS,locatedin2q14,derivedfromtheendfusionbetweenthetwoancestralchromo-somesthatgaverisetohumanchromosome2[18].
NoclearindicationofanyparticularfunctionofITSshasbeenprovidedsofar.
Emergingevidenceindicatesthattheshelterincom-ponentshavenon-telomericfunctionsinDNArepair[19],Epstein-Barrvirusreplication[20],transcriptionalregulation[21]andNF-κBactivation[22].
Thesenon-telomericfunctionsmightbe,atleastpartially,explainedbytheirbindingtoITSs.
Indeed,thereismountingindi-cationthatshelterincomponentscanbindtointerstitialDNAsequences:(i)TRF1andTRF2bindtotheperi-centromericregionsofhamsterchromosomescontaininglargeblocksofITSs[23,24];(ii)TRF2andTIN2bindtoanITSformedbyararehumanchromosomerear-rangement[25];(iii)TRF2bindstoastretchoftelomericsequencethatisartificiallyinsertedinthemiddleofthelongarmofchromosome4[26];(iv)Rap1bindstoseveralITSsofthemousegenome[27].
However,threenaturallyoccurringITSsofhumanchromosomesdonotappeartobeboundbyTRF2[26].
Therefore,itisstillunclearwhetherTRF1andTRF2reallybindtotheITSsnormallyfoundinhumanchromosomesoreventounre-latedsequences.
Moreover,thereisevidencethatTRF2modulatesgeneexpressionoutsidefromtelomeressinceitinteractswiththerepressorelement1-silencingtran-scriptionfactor(REST),arepressorofgenesdevotedtoneuronalfunctions[21].
Inthisstudy,wemappedthehumanchromosomalsitestowhichTRF1andTRF2bindbycombiningchro-matinimmunoprecipitationwithhigh-throughputDNAsequencing(ChIP-Seq).
ResultsIdentificationofTRFbindingsitesbyChIP-SeqanalysisToestablishglobalbindingprofilesofTRF1andTRF2(collectivelynamedtheTRFproteins),weperformedaChIP-SeqanalysiswithoneantibodyspecificforTRF1andtwoantibodiesspecificforTRF2(onemonoclonalorTRF2m,onepolyclonalorTRF2p).
WeusedtheBJ-HELTRasmctumorcelllinebecauseTRF2isrequiredfortumorigenicitythroughapathwaythatinvolvesuncou-plingoftelomereprotectionandtheDNAdamagere-sponsemechanism,suggestingaroleforextratelomericTRF2bindingsitesinoncogenesis(Biroccioetal,sub-mitted).
Thespecificityoftheanti-TRF1andanti-TRF2antibodieswasconfirmedbyslotblotanalysis(Figure1A).
Wefoundupto50-foldenrichmentoftelomericsequencesintheTRFantibody-immunoprecipitatedsam-pleswhencomparedtoProteinG-Sepharose-precipitatedcontrolsamplesandtotalhistoneH3-immunoprecipi-tation.
ThisresultwasconfirmedbytheanalysisoftheChIP-Seqreads.
InTRFantibody-immunoprecipitatedsampleswedetected90to150timesmoresequencesthatcontainsolelythe(TTAGGG)nmotifthaninthecontrolsamples(Figure1B).
ToidentifyextratelomericbindingsitesfortheTRFproteins,weretainedonlyreadsthatwereuniquelyalignedonthe2006Humangenomeassembly(NCBI36/hg18),andwecheckedthatpure(TTAGGG)nreads,whichlikelyoriginatefromtelomericDNA,hadbeenindeedcompletelydiscarded.
Significantlyread-enrichedpositionsorpeakswereidentifiedusingtheSISSRsoftware[28]withaPvaluethresholdof0.
001usingproteinGimmunoprecipitationasbackground.
Wefur-therremovedtheseeminglyartifactual(non-specific)peaksthroughavisualinspectionofadensityprofileofthematchedreads(seetheexampleforchromosome1,showninSupplementaryinformation,FigureS1).
Fol-lowingthisfiltering,weidentified68peakspresentinallthreeTRFChIP-Seqsamples(TRF1,TRF2mandTRF2p)(Figure2A).
Resultsforchromosome1areshowninFigure2BandthoseforotherchromosomesareshowninSupplementaryinformation,FigureS2.
Notably,18peaksfromtheTRF2mChIP(amongn=90,20%)werenotfoundusingTRF2pantibody,while21peaksidentifiedbytheTRF2pChIP(n=93,22.
5%)werenotpresentinTRF2mChIP.
FormostofExtratelomericbindingofTRF1andTRF21030npgCellResearch|Vol21No7|July2011Figure1(A)SlotblotshowingthetelomericenrichmentofDNAimmunoprecipitatedwithanti-TRF1orTRF2antibodies.
DNAimmunoprecipitatedbyatotalH3antibodyandpulleddownbyproteinGalonewasusedasacontrol.
HalfoftheprecipitatedDNAwasloaded,alongwithaninputscale(2500ngto10ng,correspondingto10%to0.
04%ofthetotalinput),andhybrid-izedsequentiallytoatelomericprobeandagenomicprobe.
Foreachprobe,wequantifiedthefractionoftheimmunoprecipi-tatedDNA.
Theratioofthevalueobtainedforthetelomericprobetothegenomicprobeisthetelomericenrichmentfactor.
(B)Foldenrichmentofthefractionofrawreadscontainingonly(TTAGGG)nsequencesfromTRFChIP-SeqasnormalizedtothereadsobtainedthroughimmunoprecipitationofproteinG.
Figure2(A)TRF1,TRF2m,andTRF2pChIP-Seqpeaks.
Thepeakslargelycoincide,asshownontheVenndiagram.
As-sessmentofoverlapswasperformedbyvisualinspectionintheIntegratedGenomeBrowser.
(B)VisualizationofTRFpeaksandTRFbindingsites.
Regionsofsignificantreadenrichment(PU998)forcriticalreading.
WearealsogratefultoZhouSongyang(BaylorCollegeofMedicine)forsharingunpublishedresults.
ThisworkwassupportedbygrantsfromtheAssociationdelarecherchecontreleCancer(ARC),theInstitutNationalduCancer(programTELOFUN),ANR(programTELOREPandINNATELO)andtheEuropeanCommunity(TELOMARKERHealth-F2-2007-200950).
TSandLEZthanktheFondationdelaRechercheMédicale(FRM)andtheARC,respectively,fortheirfellowships.
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