Implication of DNA Demethylation and Bivalent Hist.docx
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ImplicationofDNADemethylationandBivalentHist
ImplicationofDNADemethylationandBivalentHistoneModificationforSelectiveGeneRegulationinMousePrimordialGermCells
KentaroMochizuki,1MakotoTachibana,2MitinoriSaitou,3,4YukoTokitake,1andYasuhisaMatsui1,*
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Abstract
Primordialgermcells(PGCs)sequentiallyinducespecificgenesrequiredfortheirdevelopment.WefocusedonepigeneticchangesthatregulatePGC-specificgeneexpression.mil-1,Blimp1,andStellaarepreferentiallyexpressedinPGCs,andtheirexpressionisupregulatedduringPGCdifferentiation.Here,wefirstdeterminedDNAmethylationstatusofmil-1,Blimp1,andStellaregulatoryregionsinepiblastandinPGCs,andfoundthattheywerehypomethylatedindifferentiatingPGCsafterE9.0,inwhichthosegeneswerehighlyexpressed.WeusedsiRNAtoinhibitamaintenanceDNAmethyltransferase,Dnmt1,inembryonicstem(ES)cellsandfoundthattheflankingregionsofallthreegenesbecamehypomethylatedandthatexpressionofeachgeneincreased1.5-to3-fold.Inaddition,wealsofound1.5-to5-foldincreaseofthePGCgenesinthePGCLCs(PGC-likecells)inducedformEScellsbyknockdownofDnmt1.Wealsoobtainedevidenceshowingthatmethylationoftheregulatoryregionofmil-1resultedin2.5-folddecreaseinexpressioninareporterassay.Together,theseresultssuggestedthatDNAdemethylationdoesnotplayamajorroleoninitialactivationofthePGCgenesinthenascentPGCsbutcontributedtoenhancementoftheirexpressioninPGCsafterE9.0.However,wealsofoundthatrepressionofrepresentativesomaticgenes,Hoxa1andHoxb1,andatissue-specificgene,Gfap,inPGCswasnotdependentonDNAmethylation;theirflankingregionswerehypomethylated,buttheirexpressionwasnotobservedinPGCsatE13.5.TheirpromoterregionsshowedthebivalenthistonemodificationinPGCs,thatmaybeinvolvedinrepressionoftheirexpression.OurresultsindicatedthatepigeneticstatusofPGCgenesandofsomaticgenesinPGCsweredistinct,andsuggestedcontributionofepigeneticmechanismsinregulationoftheexpressionofaspecificgenesetinPGCs.
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Introduction
Germcellsaretheonlycellscapableofgivingrisetotrulytotipotentcells,viadifferentiationtosperms/eggsandsubsequentfertilization.Inmouseembryosataroundembryonicday(E)7.25,asmallpopulationofprimordialgermcells(PGCs)intheextraembryonicmesodermandderivedfromtheepiblastsisfirst“fate-determined”.ShortlybeforePGCfatedetermination,celltype-specificexpressionofBlimp1/Prdm1andPrdm14initiatesinPGCprecursors;theseproteinsarekeytranscriptionalregulatorsofPGCdevelopment.Blimp1/Prdm1andPrdm14repressthesomaticmesodermalprogram[1]–[3].Furthermore,manypluripotency-relatedgenes,includingOct4,Nanog,andSox2,areexpressedspecificallyinPGCs[2],[4].Oct4playsessentialrolesinPGCsfatedetermination[5].Oncefate-determined,PGCsstarttomigratetogenitalridges,thefuturegonads;there,theyrapidlyproliferateandincreaseinnumber.AportionofPGCsiseliminatedbyapoptosisduringthisperiod[6].Nanos3[7],[8]isinitiallyexpressedspecificallyinPGCsataroundPGCfatedetermination,anditsupportssurvivalofmigratingPGCsalongwithOct4[9]andNanog[10],[11].
Genome-wideepigeneticchangesalsooccurinmigratingPGCs[summarizedin12],andPrdm14isinvolvedinthisprocess[3].Afterarrivalatgenitalridges,PGCsundergofurtherepigeneticchangessuchasDNAdemethylationofimprintedgenesandtherepetitivesequences[13].GermcellsthereafterstopproliferationatE14.5,andmalegermcellsarearrestedinG1phaseofcell-cycle,resumeproliferationatthetimeofbirthasspermatogonialstemcellsandpartofthemstarttodifferentiatetowardsspermatozoa.AtE14.5,femalegermcellsimmediatelyentermeiosis,aresoonarrestedatmeioticprophaseI.Apartofoocytesthenresumesmeiosisaccordingtoestruscycleinadultovaryandundergofurthermaturation.PGC-specificexpressionofthemouseVasahomolog(Mvh/Ddx4)andDazl(Daz-like)isinitiatedindifferentiatingPGCsatE11.5[14],andthesegenesarerequiredforprogressionthroughmeioticprophaseIinmalegermcells[15]andforsex-specificdifferentiationoffetalgermcells[16],[17],respectively.Inaddition,Nanos2isspecificallyexpressedinmalePGCsafterPGCcolonizationofthegenitalridges,anditsuppressesmeiosisandpromotesmalegermcelldifferentiation[7],[18].Furthermore,ourpreviousinvestigationrevealedthatthemeiosis-specifichistonemethyltransferaseMeisetz/Prdm9isspecificallyexpressedinearlymeioticgermcellsbothintestesandovariesandplaysanessentialroleinproperprogressionthroughearlymeioticprophase[19].Overthecourseofseveraldevelopmentalstages,PGCssequentiallyinducemanyspecificgenesthatarerequiredfortheproperprogressionofmultipleuniquedevelopmentalevents[summarizedin20];therefore,itisimportanttoelucidatethemechanismthatcontrolPGC-specificgeneexpressiontounderstandregulationofPGCdevelopment.
Severalexperimentshavebeenperformedtoidentifycis-regulatoryelementswithintheflankingregionsofPGCgenesusingtransgenicmicecarryingreportergenes,suchaslacZandgreenfluorescentprotein(GFP),fusedtotheseflankingregions.Forexample,18.0kbpoftheflankingsequencesofOct4geneissufficientforreproducingtheendogenousOct4expressionpattern,i.e.specificexpressioninblastomereandinnercellmassinpre-implantationembryos,andinepiblastandPGCafterimplantationintransgenicmice[21],[22].Withinthis18.0kbpregion,theproximalenhancer(PE),whichislocated1.4kbpto0.3kbpupstreamfromatranscriptionstartsite(TSS),directsepiblast-specificexpression,whereasthedistalenhancer(DE),located4.6kbpto2.0kbpupstreamfromaTSS,isnecessaryforexpressioninPGCs[21],[22].Inaddition,mil-1(fragilis/Ifitm3),isarepresentativePGCgenethatisfirstexpressedinPGCprecursors[23],[24].A3.0kbpsequenceinthe5′-flankingregionofmil-1isnecessaryforPGC-specificexpressionatthetimeoftheirspecificationonward,andthecis-regulatoryelement,Ifitmgenesconsensuselement(ICE)wasparticularlyimportantforitsPGC-specificexpression.ICEisapproximately190bpinlengthandcontainsa90bpshortinterspersedtransposableelement(SINE)-likesequencethatislocatedat2kbpupstreamfromaTSS.ICEconsensussequenceswerealsofoundwithinregionsflankingotherPGCgenes[25].Similarly,reporterconstructionsofotherPGCgenes(e.g.Blimp1,Prdm14,Stella/Dppa3,Mvh,andDazl),mimickedtheendogenousexpressionpatternsintransgenicmice[1],[3],[26]–[28],butthecriticalcis-regulatorysequencesintheseconstructshavenotbeenidentifiedyet.ThemolecularmechanismscontrollingPGC-specificgeneexpressionhavebeenrarelystudied;nevertheless,itisclearthatsomePGC-specificgenes(e.g.MvhandDazl)areinitiallyexpressedafterPGCcolonizethegenitalridgesandthattheregionsflankingthesegenesareconcomitantlydemethylatedinPGCs[14].
EpigeneticmechanismsarealsoinvolvedinrepressingexpressionofPGCgenesinsomaticcells.TherepressivetranscriptionfactorE2F6maybenecessarytosilenceseveralPGCgenesinsomaticcellsviaDNAhypermethylationthatlocksthetargetpromotersintranscriptionallyinactivestates[29]–[31].Inaddition,suppressionofOct4expressioninsomaticcellsbyanorphannuclearreceptor,germcellnuclearfactor(GCNF),dependsonDNAhypermethylationoftheOct4flankingregion[32],[33].Interestingly,invarioustypesofhumantumors,manytestis-specificgenesandPGC-specificgenesareectopicallyexpressed,andCpGintheflankingregionsareCpG-hypomethylated[34],[35].Reportedly,theflankingregionsofPGC-specificgenes(e.g.VASAandSCP1/SYCP1)aregenerallyCpG-hypermethylatedinnormalsomatictissues;thesefindingsindicatethatDNAdemethylationactivatesectopicexpressionintumors[36]–[39].Takentogether,thesefindingsindicatethatDNAmethylationpreventsectopicexpressionofPGC-specificand/orpluripotent-relatedgenesinnormalsomaticcells.Inaddition,genome-wideDNAmethylationanalysisrevealedthatDNAmethylationtargetedtorepressthegermcellrelatedgenesinpre-andpost-implantationepiblast[40];therefore,itislikelythatthereareepigeneticactivatingmechanismsthatinducenormalexpressionofspecificgenesinPGCs.
Here,wefocusedondetailedepigeneticchangesofrepresentativegenespreferentiallyexpressedinPGCsandsomaticgenes,andapossibleroleofDNAdemethylationintheexpressionofPGCgenesthatareinitiallyexpressedaroundthetimeofPGCfatedeterminationwasalsoinvestigated.OurfindingsindicatedthattheregionsflankingPGCgenesthatcontaintheconsensuselement,ICE,commonlyunderwentDNAdemethylatedindifferentiatingPGCsafterE9.0inwhichtheexpressionofthosegeneswasupregulated.WealsoshowedthatrepressionoftheHoxgenes,representativesomaticgenes,aswellasaneuralcell-specificGfapgeneinPGCswasnotdependentonDNAmethylation,butmayberegulatedbythebivalenthistonemodification.
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Results
mil-1RegulatoryRegionswereHypomethylatedinDifferentiatingPGCs
Wepreviouslyreportedthat3.0kbpofthe5′-flankingregionofmil-1genewasnecessaryforPGC-specificexpression[25],butthemechanismsthatconferPGC-specificexpressionarenotfullycharacterized.DNAmethylationisoneofthemostwell-knownepigeneticmechanismsregulatinggeneexpression,andmethylationofCpGsitesoftenrepressesgeneexpressi