Multivariate statistical analysis of epigenetic regulation with application to the analysis of human embryonic stem cells [Elektronische Ressource] / Lukas W. Chavez Wurm

Multivariate statistical analysis of epigenetic regulation with application to the analysis of human embryonic stem cells [Elektronische Ressource] / Lukas W. Chavez Wurm

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GMultivaereicriate(Dr.statisticalUnivanalysisderofFepigeneticInformatikregulationErlangungwitheinesapplicationhaftetonat)thehanalysisMathematikofFhumanBerlinembdesryradesonicDoktorsstemNaturwissensccellsnLukrer.asamW.acChabvhezundWderurmreien2010ersit?tDissertationzurMolecularSupdisputation:ervisor:alterProf.VingronDr.Prof.MartinSaarlandes)VingronDr.(Max-Planck-Institutek-InstitutenforJ?rnMolecularersit?tGeofnProf.etics)MartinA(Max-Plancdvisor:forDr.GeRalfetics)HerwigDr.(Max-PlancWk-Institute(UnivfordesMolecularDateGenetics)theReview15.12.2010er:43Contents.1.Introvdu.c.t.iCpGo.n.53.3.51.162Humanorkem.bry.onic.stem.cells.(hES.Cs)......of...man.and.................er..........5eth1.2.T.ranscri56ptional.regulation.ofagepluri.pdepotency4.1.68...con...deriv..........................6441.3.DNA.meth.ylation3.3.2.......3.3.3.......3.3.4.......score.....of...57.ylated...w.......4.dermal.stem.core.trolling.tal.....4.39.1.4.In.troMeDIP-Seqductionprolesto.metho3.2.2dsanalysisand.limitations.for.d.etec.ti.ng40DNAhmenmeth.ylation...10.1.5.Aims.of.theNormalizationthesis................Reads.(rpm).............factors...............curv...............e12score2.Exp.erimental.techniques.16.2.1methDNA.arra.ys......Ev.data.......Iden.tially.(DMRs).....MEDIPS.pac...............and.during.of.ry.ells.tifying.net.OCT4.otency.Exp.ced.metho..16.2.2.Statistical.testingqualitometricsf.dieren.tial.expression....4.4.W.meth...77...33.Co.erage..............19.2.3.Second.generation.sequen.c.i.ng3.2.

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GMultivaereicriate(Dr.statisticalUnivanalysisderofFepigeneticInformatikregulationErlangungwitheinesapplicationhaftetonat)thehanalysisMathematikofFhumanBerlinembdesryradesonicDoktorsstemNaturwissensccellsnLukrer.asamW.acChabvhezundWderurmreien2010ersit?tDissertationzurMolecularSupdisputation:ervisor:alterProf.VingronDr.Prof.MartinSaarlandes)VingronDr.(Max-Planck-Institutek-InstitutenforJ?rnMolecularersit?tGeofnProf.etics)MartinA(Max-Plancdvisor:forDr.GeRalfetics)HerwigDr.(Max-PlancWk-Institute(UnivfordesMolecularDateGenetics)theReview15.12.2010er:43Contents.1.Introvdu.c.t.iCpGo.n.53.3.51.162Humanorkem.bry.onic.stem.cells.(hES.Cs)......of...man.and.................er..........5eth1.2.T.ranscri56ptional.regulation.ofagepluri.pdepotency4.1.68...con...deriv..........................6441.3.DNA.meth.ylation3.3.2.......3.3.3.......3.3.4.......score.....of...57.ylated...w.......4.dermal.stem.core.trolling.tal.....4.39.1.4.In.troMeDIP-Seqductionprolesto.metho3.2.2dsanalysisand.limitations.for.d.etec.ti.ng40DNAhmenmeth.ylation...10.1.5.Aims.of.theNormalizationthesis................Reads.(rpm).............factors...............curv...............e12score2.Exp.erimental.techniques.16.2.1methDNA.arra.ys......Ev.data.......Iden.tially.(DMRs).....MEDIPS.pac...............and.during.of.ry.ells.tifying.net.OCT4.otency.Exp.ced.metho..16.2.2.Statistical.testingqualitometricsf.dieren.tial.expression....4.4.W.meth...77...33.Co.erage..............19.2.3.Second.generation.sequen.c.i.ng3.2.3.enric.t.............................3.3................24.2.4.Chromatin.imm.unoprecipitation.(ChIP)........3.3.1.p.million.........................4425Coupling2.5.Meth.ylated.DNA.imm.unoprecipitation.(MeDIP)................45.Calibration.e........27.2.6.Alternativ.e.metho.ds.for.detecting.DNA.meth48ylationRelativ.m.ylation.(rms)..................28523AbsoluteMoylationdelling(ams)of.MeDIP-Seq.data.30.3.1.Genome.v.ector....3.3.6.aluation.MeDIP-Seq.normalization.............3.4.tication.dieren.meth.regions...........3.5.soft.are.k................30.3.2.Qualit.y.con.trol65metricsGenetic.epigenetic.endencies.endo.dierentiation.hu-.emb.onic.c.68.Iden.a.regulatory.w.of.con.plurip...4.2.erimen.pro.ures.analysis.ds..........3.3.3.2.170SaturationMeDIP-Seqanalysisy.trol.......................74.Comparing.and.GSBS.ed.ylation.......3.endixContiten.ts.4.5ciatesPromoter.meth.ylationBibliography...histone.....alterations.on...110...hmen.en...........ylation...8.net...91.Abb.111.endix...4.10.analysis.ylation79to4.6dicationsMeth.ylation.patterns.of.transcrip.tion.factor.binding.si4.11telsgene.......Epigenetic.OCT4.ork......79Conclusion4.7NotationIden109ticationcknoof112dieren114tially116meth.ylated.regions85(DMRs)Enric.t.asso.de-meth.ev.ts.functional.mo....79.4.8.Genome.wide.distri.bution.of.DMRs................85.Dieren.a.meth.and.expression...........8.4.12.eects83the4.9regulatoryDierenwtial.meth.ylation.at.TFBSs......5.94.98.and.reviations.Publications.A.wledgments.Zusammenfassung.App.1.App.2.4.stem1patienIntrohromatinductionthe1.1fromHumanedembtheryeonicadultstemwcellscell-replacemen(hESCs)donor-hostInhES1989,aThstudoformson[96]andindepcolleaguestheirpresenyteddiforfuturetheyrstcausedtimealternativthebderivcolleaguesationedofotenhESCspluripfromtran-themolecularinnerakcelltimemassSubsequenofpresenhBothumanharacteristicblativainstotcystsfor[98].First,Fareorcthis,incleaikvgenomicageline.stageforhcellsumandevem-abryeos,canprowithoutduceddevbotherysomaticincellsvitrofos)fertilizationcellsforeclinical1.1).purpsoses,demonstratedwberebcultured2007,tooratoriesblastoiPSCscysttiatedstageeandthetheorinnertiationcellthreemassesiPSCswgeneeremethisolatedo(seecuFiguree1.1).therapiThomsonputativandapplications,colleaguesallodenedbthreetaminatedessentstialculture.careharacteristicstoofyprimatekgroundsemandbryortunatelyonicapproacstemationcellshES-lik[98].vTheserecenareed.(i)hand,derivaahatiwnohnefromsingletheterferingpreimplbryatalnOntationsevoronptoeriimplanttationhaemthatbryao,factors(ii)sucienprolongeddierenun-todierensimilartibrya(seeted2006,proliferationh(andstemnessv),theandiPSCs(iii)generatedstablebdevinelopmen,talwptotenvtialhtoerivformideriv[95,ativhaeswnofcellsallotencythreehESCs.emtheybrySCsonictogermoflagermyFersbleevmorphologyen,afterandprolon5gwedmainculture(ltiespluripputativotencyfuture).tHumanes.ESCsforoereinsighclinicaltshESCsinnottowdevtoelopmenetalonevbenreagentsusedwhiccellhSecond,cannotrejectionsbleelystudiedarisedibredieringctlbacyofintthecellinFtact,heumanhesemderivbryofoandbutethathahaeveenetlyimpelopoOnrtanonetKilmanskconsequencesyiandn[50]clinicalvareas.shoFthortexample,ESCsunbderstandingderivthefrommecblastomershanismsinthatwithconemtrolonicdierenelopmentiationpenablestial.directedthedif-hand,fereneraltiiesareprogrammingticellsoinducednotenofstemhES(iPSCs)cellsvtodemonstratedsptransductioneciconlycellfewtscriptionyp(TFesis[98].tHoresettingwtiatedeviner,aderivstateationtoofmESCsonicfromcellshFigureumanInblastoTcystsaforaclinicalhiapplicationscolleag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InnettroofductionecicFiguredirect1.2:erimenOCT4dierencoregenesregulatory2,netincorpwSOolines)rk.additiCore34].OCT4andtranscriptionalxesreguleingatorhESCsy5].net-orkwinformationorkgenesiden(redtiedANOGbhipyandanlevinregulationtegrativwithetiation,analysrediosindicateofbChspIforP-on-Chip,[6,RNAi75,knoThecwkdoalsownoratesandonsetargetqfromuencX2e-basedlines)motifNmapping(blue[17].ChIP-on-cGreenexpbtsoanxesonalrepreseneltgenegenes[68,asso8ciateddescrib1toInyltransferasetrohomologductionylationFigurey1.3:elopmenCytosinestimmeth)ylation.anDNAtheirmethTheyltransferasesnovomediateforthetherevtranscriptionalersibletattactheh-DNAmenDNAtseofhand,aylationmethonsibleylisgroupylation(CHand3er,)directattheirtmethhedieren5'revpylosition5'ofatecytosines.cTisakTheenmediatedtogether,1.3).pluripommonlyotencyOismegopreservvcellularernedtenancebDNAystrandsththeesetsTFemOCT4thoughandmethcriticalmethregulatoryproteinact-bing.proteinyltransferases-DNAbindinactivitteractionsvhaemvmethesbattaceenain(CHvtestigated.ositionBesideyrimidinetheseDNAtranscriptionesfthaitself,ctorconsideredmediatedmoregulations,cessitylationisysu(seeppyltransosedarethataccordingthereac-existtheepitenancegylationennecessaryeDNAtievcreplicationmecosedhanisyltransferasemcopsylationindaughvDNAolv[43].edhand,inyla-regulationDNAofinstemoniccellItdierenthattiationareandrespdevdeelopmenMoreot,isincludinghhistoneothermohasdiactivitDNMT3LcationsnovoandyDNAymethDNAylation.thHo[43].wiseveder,durisuconih9epigenylationticemecthehanismsersibleofhmenhESCofdierenmethtiationgroupha3vaethenotpyofetpbderiveencytosine.inmethvdoestigatednotonhangeaefullsequencegenomeandlevthereforeel.asThisepigeneticthesisdication.revproealsofformeththeisrstbtimemethglobalenzymesepigenFigureticMethmoferadicationsenzymesemergingcupclassiedontoinducedenzymaticltivities.onssoneofmainpluripofotencythaactivitnisdtoduringedierenmethtiafteraerytiDNAocycle.npropofmainhESCsmethalongresptheforendoyingdermalmethlineage.patternsMoreothevterer,duringimpreplicationortanDNMT1tOninotherteractionsdebmethettionwupeenmethDNApatternsmethearlyylation,brytranscriptiondevfactort.binding,isandtgeneDNMT3aexpressionDNMT3balterationstheareyltransferasesrevonsibleealed.thisFinallynovo,ylation.epigeneticvdepDNMT3LendenciesaduringwhicendoisdermaltodierenDNMT3stiationutarenopre-catalyticsenytedInstead,inassistsdetaildeformeththebcoreincreasingOCT4abilitntoettowandorkulatingthateirconytrolsDNApluriylationpinotencyolv.in1.3regulationDNAngmethylationbryIncmammals,ti-DNAgenome1DNAInandtroreceivduction1.2ationan[72]appliedandthisreprogrammingfofofsomaticecellssinDNAtotheinduceddetailedplurip(WotenqtSCsstemissuecellsb[26,medium-,16promoters.]dy.toAboerranbisulptylationmethethylationlocanthatbsequencingeaassoeenciatedcost-eectivwithrstsevrepererepreseneects,DNAfylationoDNArhigh-,examtoploleothertheedinductionpof[85].cancerlimitations[47,there41].methoFOnurthermore,dsdistinctprogenomeatwideunoprecipita-methmoreylationlevpatternsFdistinguishindividdif-mferenhaptertshotguncell-tmeyplesha[30,suc85].yInscreenmamAsmals,y-DNAwmethcostylation[64primarily(RRBS)otheccurssequencedatofCpGcorre-siteswbutthaexpressionrecenalsotconstudynotions,hasenshodensitwnththatRaknon-Cpal.Ggene-bmeththylationelyaccoungenetsIntrofordsapprorximatelyIn25%tofjorallformethyla-ylateonedbasedcytosinesbinorderhDNAumanformemresolution.bryhand,onicbasedstemdscellse[64].methItareisersuprpofosedtecthatbmethtionedylationseeaec.tswholegenephiteexpressiondetectbylationyleveitherAlthoughinresolutionterferingewith[63,bindingtecofestrabnappliedscriptionsetsfactorsororexample,mogenomedifyingofcbasehromatinrecenstructureattoabasequencerepressiv.ebisulphitestateaddress[43].yMeissnertetbal.the[72]reducedhawidev10elationshoetwneenthmeaylationtgenemethatylatedbutCpGsatareanddynamictraryepigeneticpreviousmarkatsvthatsomeuw-CpGndergoyextensivOneechand,hangesyduringetcellularobservdierenthattiation,oparticularlymeinylationregulatoryositivregionscorrelateoutsidewithofexpressioncore1.4promoters.ductionTheirmethodataandsuppfoortdetectingthemethylationnotionprinciple,thatarebwothmaCpG-richigh-thoughputhdsanddetecting-pmethotion.ortheregulatoryhand,elemenhitetsmethoundergocandistinctemoindestoofduceepigeneticmethregulation.inRegulatoryationelemenbasetsOninotherCpG-pimmotionormsequenceoconaretextscost-eectivseembuttoedundergoylationextensivelseofandwdynamicresolution.methoya-descriptionlationtheandualde-methhniquesylation.willHenecene,intheirsection,studycconrms2theBisulphiteconceptorofgenomedistinctbisulregulatorysequencingmecGSBS)hanismscytosineofthDNAonmethbase-pairylation,ewhere.methwholeylationsingle-baseofmapsisolatedvCpGsbmageneratedy64]conhtributehnitouccannothromatinetcondensationeoredirectlytoinlargeterfereowithsequencestranscriptionsamples.factoranbinding.theMore-fullomethvlomeer,hEhonypresolutionermethereylationtlyofortedhigh-CpG-densitayofpromotersout(HCPs)billionleadsreadsto]irrevReducedersibletationgenesequencingsilencing[72][72].thisAbccordingreducingtoamountheseofndings,toRakyeanonetcostal.a[85]viewobservgenomeedmethaproles.negative