分子生物学(双语)10-Chromatin课件

Chapter10ChromatinIndividualnucleosomesarereleasedbydigestionofchromatinwithmicrococcalnuclease.Thebaris100nmReprintedfromCell,vol.4,P.Oudet,M.Gross-Bellard,andP.Chambon,Electronmicroscopicandbiochemicalevidence...,pp.281-300.Copyright1975,withpermissionfromElsevier[/science/journal/00928674].PhotocourtesyofPierreChambon,CollegeofFrance.BasicStructuresnucleosome–Thebasicstructuralsubunitofchromatin,consistingof~200bpofDNAandanoctamerofhistoneproteins.histonetails–Flexibleamino-orcarboxy-terminalregionsofthecorehistonesthatextendbeyondthesurfaceofthenucleosome.Histonetailsaresitesofextensiveposttranslationalmodification.10nmfiber–Alineararrayofnucleosomesgeneratedbyunfoldingfromthenaturalconditionofchromatin.linkerhistones–Afamilyofhistones(suchashistoneH1)thatarenotcomponentsofthenucleosomecore.Linkerhistonesbindnucleosomesand/orlinkerDNAandpromote30nmfiberformation.

BasicStructuresThe10nmfiberinpartiallyunwoundstatecanbeseentoconsistofastringofnucleosomesPhotocourtesyofBarbaraHamkalo,UniversityofCalifornia,Irvine.The10nmfiberisacontinuousstringofnucleosomes30nmfiber–Acoilofnucleosomes.Itisthebasicleveloforganizationofnucleosomesinchromatin.nonhistone–Anystructuralproteinfoundinachromosomeexceptoneofthehistones.BasicStructuresThe30nmfiberhasacoiledstructurePhotocourtesyofBarbaraHamkalo,UniversityofCalifornia,Irvine.The30nmfiberisatwostarthelixconsistingoftworowsofnucleosomescoiledintoasolenoidReprintedfromCell,vol.128,D.J.Tremethick,Higher-orderstructureofchromatin...,pp.651-654.Copyright2007,withpermissionfromElsevier[/science/journal/00928674].DNAIsOrganizedinArraysofNucleosomesMNase(micrococcalnuclease)cleaveslinkerDNAandreleasesindividualnucleosomesfromchromatin.>95%oftheDNAisrecoveredinnucleosomesormultimerswhenMNasecleavesDNAinchromatin.ThelengthofDNApernucleosomevariesforindividualtissuesorspeciesinarangefrom154to260bp.NucleosomalDNAisdividedintothecoreDNAandlinkerDNAdependingonitssusceptibilitytoMNase.MicrococcalnucleasedigestschromatininnucleiintoamultimericseriesofDNAbandsthatcanbeseparatedbygelelectrophoresis.EachmultimerofnucleosomescontainstheappropriatenumberofunitlengthsofDNAPhotocourtesyofMarkusNoll,UniversitätZürich.Footprinting:CharacterizingRNAPolymerase–PromoterandDNA–ProteinInteractionsfootprinting–AtechniqueforidentifyingthesiteonDNAboundbysomeproteinbyvirtueoftheprotectionofbondsinthisregionagainstattackbynucleases.AproteinprotectsaseriesofbondsagainstnucleaseattackFIGURE05:MicrococcalnucleaseinitiallycleavesbetweennucleosomesThenucleosomeisacylinderwithDNAorganizedinto~12/3turnsaroundthesurfaceDNAoccupiesmostoftheoutersurfaceofthenucleosomeSequencesontheDNAthatlieondifferentturnsaroundthenucleosomemaybeclosetogetherThenucleosomeconsistsofapproximatelyequalmassesofDNAandhistones(includingH1)PossiblemodelfortheinteractionofhistoneH1withthenucleosome202122TheNucleosomeIstheSubunitofAllChromatinEachhistoneisextensivelyinterdigitatedwithitspartner.Allcorehistoneshavethestructuralmotifofthehistonefold.N-andC-terminalhistonetailsextendoutofthenucleosome.H1isassociatedwithlinkerDNAandmaylieatthepointwhereDNAentersorexitsthenucleosome.PhotoscourtesyofE.N.Moudrianakis,JohnsHopkinsUniversity.FIGURE10ab:Thecrystalstructureofthehistonecoreoctamerisrepresentedinaspace-fillingmodelThecrystalstructureofthehistonecoreoctamerisrepresentedinaspace-fillingmodelPhotoscourtesyofE.N.Moudrianakis,JohnsHopkinsUniversity.LinkerDNAistheregionof8to114bp,andthisistheregionthatissusceptibletoearlycleavagebytheenzyme.ChangesinthelengthoflinkerDNAaccountforthevariationintotallengthofnucleosomalDNA.H1isassociatedwithlinkerDNAandmaylieatthepointwhereDNAentersandleavesthenucleosome.25Thehistonefoldconsistsoftwoshorta-helicesflankingalongera-helixStructuresfromProteinDataBank1HIO.G.Arents,etal.,Proc.Natl.Acad.Sci.USA88(1991):10145-10152.Histonepairs(H3+H4andH2A+H2B)interacttoformhistonedimersStructuresfromProteinDataBank1HIO.G.Arents,etal.,Proc.Natl.Acad.Sci.USA88(1991):10145-10152.ThecrystalstructureofthehistonecoreoctamerisrepresentedinaribbonmodelStructuresfromProteinDataBank1AOI.K.Luger,etal.,Nature389(1997):251-260.ThecrystalstructureofthehistonecoreoctamerisrepresentedinaribbonmodelStructuresfromProteinDataBank1AOI.Luger,K.,etal.,Nature389(1997):251-260.FIGURECO:ChromatinStructurefromProteinDataBase1ZBB.T.Schalch,etal.,Nature436(2005):138-141.PhotocourtesyofChrisNelson,UniversityofVictoria.The30nmfiberisatwostarthelixconsistingoftworowsofnucleosomescoiledintoasolenoidReprintedfromCell,vol.128,D.J.Tremethick,Higher-orderstructureofchromatin...,pp.651-654.Copyright2007,withpermissionfromElsevier[/science/journal/00928674].ThehistonetailsaredisorderedandexitfrombothfacesofthenucleosomeandbetweenturnsoftheDNAStructurefromProteinDataBank1AOI.K.Luger,etal.,Nature389(1997):251-260.FIGURE13:ThehistonefolddomainsofthehistonesarelocatedinthecoreofthenucleosomeFigure29.20:Thestructuresofhistonetailsarenotdefined.Figure29.21:HistonetailsemergebetweenDNAturns.3738HistonetailshavemanysitesofmodificationAdaptedfromTheScientist17(2003):p.27.Thepositivechargeonlysineisneutralizeduponacetylation,whilemethylatedlysineandarginineretaintheirpositivecharges41FIGURE20:Mostmodifiedsitesinhistoneshaveasingle,specifictypeofmodification,butsomesitescanhavemorethanonetypeofmodification43FIGURE18:AcetylationduringreplicationoccursonspecificsitesonhistonesbeforetheyareincorporatedintonucleosomesFIGURE19:AcetylationassociatedwithgeneactivationoccursbydirectlymodifyingspecificsitesonhistonesthatarealreadyincorporatedintonucleosomesFigure2.ProposedmechanismbywhichDNAmethylationleadstotranscriptionalrepression.(a)Transcriptionallyactivechromatinispredominantlyunmethylatedandhashighlevelsofacetylatedhistonetails(shortblacksquiggles).(b)MethylationatCpGdinucleotidescanbecarriedoutbyoneofthethreeknownhumanDNAmethyltransferases(DNMT1,3aand3b),resultinginDNAwithhighlevelsofCpGmethylation(purplecircles),butstillcontainingpredominantlyacetylatedhistonetails.DNAinthisformwouldstillbeexpectedtobetranscriptionallycompetent.(c)MethylatedDNAistargetedbymethyl-bindingdomain(MBD)proteinssuchasMBD2andMeCP2,whicharefoundassociatedwithlargeproteincomplexessuchastheNuRDcomplex(MBD2)andtheSin3acomplex(MeCP2).Histonedeacetylase(HDAC1and2)andchromatin-remodellingactivities(Mi-2andSin3a)withinthesecomplexesresultinalterationsinchromatinstructure,producingchromatinthatisrefractorytotranscriptionalactivation(pinkstreaksrepresentdeacetylatedhistonetails).Thefunctionalrolesofothercomponentsinthesecomplexesarenotyetknown.Abbreviations:MTA2,metastasis-associatedprotein2;RbAp46/48,retinoblastoma-associatedprotein46/48;RNApolII,RNApolymeraseII;SAP18/30,Sin3-associatedpolypeptides18/30(fig002gsb).ProposedmechanismbywhichDNAmethylationleadstotranscriptionalrepressionG.StrathdeeandR.Brown4647CovalentModificationofHistonesPhotocourtesyofSeanD.Taverna,JohnsHopkinsUniversitySchoolofMedicine,andHaitaoLi,MemorialSloan-KetteringCancerCenter.AdditionalinformationatS.D.Taverna,etal.,Nat.Struct.Mol.Biol.14(2007):1025-1040.NumerousproteinmotifsrecognizemethylatedlysinesBromodomainsareproteinmotifsthatbindacetyllysinesStructurefromProteinDataBank1E6I.D.J.Owen,etal.,EMBOJ.19(2000):6141-6149.NumerousproteinmotifsrecognizemethylatedlysinesStructurefromProteinDataBank1KNE.S.A.JacobsandS.Khorasanizadeh,Science295(2002):2080-2083.NumerousproteinmotifsrecognizemethylatedlysinesStructurefromProteinDataBank2GFA.Y.Huang,etal.,Science12(2006):748-751.HistoneVariantsProduceAlternativeNucleosomes,andpotentiallyAlternativeNucleosomalOrganizationAllcorehistonesexceptH4aremembersoffamiliesofrelatedvariants.Histonevariantscanbecloselyrelatedorhighlydivergentfromcanonicalhistones.Differentvariantsservedifferentfunctionsinthecell.Themajorcorehistonescontainaconservedhistone-folddomainAdaptedfromK.SarmaandD.Reinberg,Nat.Rev.Mol.CellBiol.6(2005):139-149.5455γ-H2AXisdetectedbyanantibody(yellow)andappearsalongthepathtracedbyalaserthatproducesdouble-strandbreaks.H2AXhasSEQL/YinC-termwhichcanbephosphorylated.©Rogakouetal.,1999.OriginallypublishedinTheJournalofCellBiology,146:905-915.PhotocourtesyofWilliamM.Bonner,NationalCancerInstitute,NIH.ThenucleosomeisacylinderwithDNAorganizedinto~12/3turnsaroundthesurface

Organizedbendingofchainsaroundasphericalorcircularobject.....nucleosomesandbicyclechains.58Nicksindouble-strandedDNAarerevealedbyfragmentswhentheDNAisdenaturedtogivesinglestrandsSitesfornickinglieatregularintervalsalongcoreDNA,asseeninaDNaseIdigestofnucleiThemostexposedpositionsonDNArecurwithaperiodicitythatreflectsthestructureofthedoublehelixRotationalpositioningdescribestheexposureofDNAonthesurfaceofthenucleosomeDNAStructureVariesontheNucleosomalSurfaceDNAiswrapped1.65timesaroundthehistoneoctamer.ThestructureoftheDNAisalteredsothatithas:anincreasednumberofbasepairs/turninthemiddleadecreasednumberattheends62ThePeriodicityofDNAChangesontheNucleosome∼0.6negativeturnsofDNAareabsorbedbythechangeinbp/turn:from10.5insolutiontoanaverageof10.2onthenucleosomalsurfaceThisexplainsthelinking-numberparadox.63DNAiswrapped1.67timesaroundthehistoneoctamer.DNAonthenucleosomeshowsregionsofsmoothcurvatureandregionsofabruptkinks.ThestructureoftheDNAisalteredsothatithasanincreasednumberofbasepairs/turninthemiddle,butadecreasednumberattheends.DNAstructureinnucleosomalDNAStructuresfromProteinDataBank:1P34.U.M.Muthurajan,etal.,EMBOJ.23(2004):260-271.~0.6negativeturnsofDNAareabsorbedbythechangeinbp/turnfrom10.5insolutiontoanaverageof10.2onthenucleosomalsurface,whichexplainsthelinking-numberparadox.DNAstructureinnucleosomalDNAAdaptedfromT.J.RichmondandC.A.Davey,Nature423(2003):145-150.ThesupercoilsoftheSV40minichromosomecanberelaxedtogenerateacircularstructure,whoselossofhistonesthengeneratessupercoilsinthefreeDNAChromatinIsFundamentallyDividedintoEuchromatinandHeterochromatinIndividualchromosomescanbeseenonlyduringmitosis.Duringinterphase,thegeneralmassofchromatinisintheformofeuchromatin,whichisslightlylesstightlypackedthanmitoticchromosomes.RegionsofcompactheterochromatinareclusterednearthenucleolusandnuclearmembranePhotocourtesyofEdmundPuvion,CentreNationaldelaRechercheScientifiqueNucleosomepositioningplacesrestrictionsitesatuniquepositionsrelativetothelinkersitescleavedbymicrococcalnucleaseNucleosomesmayformatspecificpositionsastheresultofeither:thelocalstructureofDNAproteinsthatinteractwithspecificsequencesThemostcommoncauseofnucleosomepositioningiswhenproteinsbindingtoDNAestablishaboundary.Intheabsenceofnucleosomepositioning,arestrictionsitecanliesatanypossiblelocationindifferentcopiesofthegenomeTranslationalpositioningdescribesthelinearpositionofDNArelativetothehistoneoctamerFigure29.33:PhasingcontrolsexposureoflinkerDNA.71IfnucleosomesarenotrandomlydepositedontheDNA(a)AretheyarelocatedbysitespecificinteractionbetweenspecificDNAsequencesandnucleosomeproteins(INTRINSIC).(b)Isthelocationofcertainhistonespreferentiallyassembledgivingrisetoclearboundarypatterns(e.g.atapromoter)atthe5'endofthegeneandsubsequentnucleosomesarespacedoutevenlyfromthatpoint

(EXTRINSIC).7273AreTranscribedGenesOrganizedinNucleosomes?Nucleosomesarefoundatthesamefrequencywhenonedigestswithmicrococcalnuclease:transcribedgenesnontranscribedgenesSomeheavilytranscribedgenesappeartobeexceptionalcasesthataredevoidofnucleosomes.Figure29.3674IndividualrDNAtranscriptionunitsalternatewithnontranscribedDNAsegmentsReproducedfromO.L.MillerandB.R.Beatty,Science164(1969):955-957.PhotocourtesyofOscarMiller.RNApolymeraseiscomparableinsizetothenucleosomeandmightencounterdifficultiesinfollowingtheDNAaroundthehistoneoctamerTopphotocourtesyofE.N.Moudrianakis,JohnsHopkinsUniversity.BottomphotocourtesyofRogerKornberg,StanfordUniversitySchoolofMedicine.77AnexperimenttotesttheeffectoftranscriptiononnucleosomesshowsthatthehistoneoctamerisdisplacedfromDNAandrebindsatanewpositionHistoneoctamersaredisassembledaheadoftranscriptiontoremovenucleosomesAncillaryfactorsarerequiredboth:forRNApolymerasetodisplaceoctamersduringtranscriptionforthehistonestoreassembleintonucleosomesaftertranscriptionIndirectend-labelingidentifiesthedistanceofaDNasehypersensitivesitefromarestrictioncleavagesiteBasal/unactivatedHIS3chromatinisstaticandshowsasingledominantnucleosomalarraywithlittleremodelingactivity(toppanel)Basal/unactivatedHIS3chromatinisstaticandshowsasingledominantnucleosomalarraywithlittleremodelingactivity(toppanel)ReproducedfromMol.CellBiol.,2006,vol.26,pp.8252-8266,DOIandreproducedwithpermissionfromtheAmericanSocietyofMicrobiology.PhotocourtesyofDavidJ.Clark,NationalInstitutesofHealth.Heat-shock-inducedpuffingatmajorheatshockloci87AandCPhotocourtesyofVictorG.Corces,EmoryUniversity.The87Aand87Cloci,containingheatshockgenes,expanduponheatshockinDrosophilapolytenechromosomesDNAaseHypersensitiveSitesReflectChangesinChromatinStructureHypersensitivesitesarefoundatthepromotersofexpressedgenes.Theyaregeneratedbythebindingoftranscriptionfactorsthatdisplacehistoneoctamers.84SensitivitytoDNaseIcanbemeasuredbydeterminingtherateofdisappearanceofthematerialhybridizingwithaparticularprobeInadulterythroidcells,theadultb-globingeneishighlysensitivetoDNaseIdigestionPhotocourtesyofHaroldWeintraub,FredHutchinsonCancerResearchCenter.UsedwithpermissionofMarkGroudineNucleosomeassemblyinvivoAnenhanceractivatesapromoterinitsvicinity,butcanbeblockedfromdoingsobyaninsulatorlocatedbetweenthemAninsulatormayblockheterochromatinAproteinthatbindstotheinsulatorscs'islocalizedatinterbandsinDrosophilapolytenechromosomesReprintedfromCell,vol.81,K.Zhao,C.M.Hart,andU.K.Laemmli,Visualizationofchromosomaldomains...,pp.879-889.Copyright1995,withpermissionfromElsevier[/science/journal/00928674].PhotocourtesyofUlrichK.TheinsulatorofthegypsytransposonblockstheactionofanenhancerwhenitisplacedbetweentheenhancerandthepromoterSu(Hw)/mod(mdg4)complexesarefoundinclustersatthenuclearperipheryFab-7isaboundaryelementthatisnecessaryfortheindependenceofregulatoryelementsiab-6andiab-7AnLCRMayControlaDomainLocuscontrolregions(LCRs)arelocatedatthe5′endofachromosomaldomainandtypicallyconsistofmultipleDNAsehypersensitivesites.LCRsregulategeneclusters.LCRsusuallyregulatelocithatshowcomplexdevelopmentalorcell-typespecificpatternsofgeneexpression.FIGURE59:Thebeta-globinlocusismarkedbyhypersensitivesitesateitherendChromosomeconformationcapture(3C)isonemethodtodetectphysicalinteractionsbetweenregionsofchromatininvivoAdaptedfromA.MieleandJ.Dekker,Mol.Biosyst.4(2008):1046-1057Chromosomeconformationcapture(3C)isonemethodtodetectphysicalinteractionsbetweenregionsofchromatininvivoAdaptedfromA.MieleandJ.Dekker,Mol.Biosyst.4(2008):1046-1058Chromosomeconformationcapture(3C)isonemethodtodetectphysicalinteractionsbetweenregionsofchromatininvivoAdaptedfromA.MieleandJ.Dekker,Mol.Biosyst.4(2008):1046-1059ChromatinIsFundamentallyDividedintoEuchromatinandHeterochromatinIndividualchromosomescanbeseenonlyduringmitosis.Duringinterphase,thegeneralmassofch