染色质研究技术与原理演示文稿

染色质研究技术与原理演示文稿当前1页，总共104页。染色质研究技术与原理当前2页，总共104页。一、染色质和核小体二、DNA甲基化修饰和组蛋白修饰三、研究染色质修饰的技术四、体外染色质重组当前3页，总共104页。染色质研究的历史性成果年W.Flemming提出“Chromatin”这个名称1884年A.Kosse发现Histone组蛋白1944年Avery等证明DNA是遗传物质1953年Watson&Crick提出DNAdoublehelix1964年组蛋白修饰（乙酰化和甲基化acetylationandmethylation)1973年Olins等电镜观察到染色质的念珠重复；1974年Kornberg等发现核小体当前4页，总共104页。当前5页，总共104页。A.InterphasechromatinB.amitoticchromosome,whichisduplicatedalreadyCompactionofchromatiniscell-stagedependent当前6页，总共104页。A)30nmfibersB)beadsonastring-nucleosomeFrominterphasenucleusNucleosomeistherepeatingunitofchromatin当前7页，总共104页。ChromatindigestionwithMNase当前8页，总共104页。Nucleosomecoreparticle2.8AcrystalstructureoftheMono-nucleosome当前9页，总共104页。Nucleosome=anucleosomecoreparticle+linker（核小体）DNA+alinkerhistoneDNAlength:180-200bpNucleosomecoreparticle=histoneoctamer+146bpDNANomenclature当前10页，总共104页。Histones-highlybasic(+)proteinsProtein

Molecular

weight

Major

Aminoacid

H1

21

Lys++

H2a

13.8

Lys

H2b

13.8

Lys

H3

15.4

Arg/LysH4

11.4

Arg/Lys

当前11页，总共104页。Histonefold-3alphahelicesand2folds当前12页，总共104页。当前13页，总共104页。Histoneself-assembly当前14页，总共104页。Nucleosome当前15页，总共104页。Nucleosome当前16页，总共104页。TheNucleosomeH3H3ThecorehistonesarepredominantlyglobularexceptfortheN-terminal“tails”,whichareunstructured.当前17页，总共104页。Differentstatesofchromatin当前18页，总共104页。FunctionStorageofgeneticinformation（储存）PrecisesegregationofreplicatedDNAintotwodaughtercells（精确分离到不同）Platformfortranscription,replication,recombinationandDNArepair（平台）当前19页，总共104页。二、DNA甲基化修饰和组蛋白修饰三、研究染色质修饰的技术当前20页，总共104页。如何进行调控？染色质结构=DNA+组蛋白八聚体DNA是遗传信息携带者，染色质是遗传信息存在形式当前21页，总共104页。1.不能降解DNA或者组蛋白2.

通过化学修饰调控

DNA甲基化，组蛋白修饰3.通过改变结构调控

更换不同类型组蛋白

通过ATP依赖性重塑酶改变结构答案当前22页，总共104页。DNAmethylationhistonevariantsATP-drivenchromatinremodelingcomplexesHistonemodificationsNon-codingRNAsMechanismsforepigeneticregulation当前23页，总共104页。当前24页，总共104页。当前25页，总共104页。DNAMethylationDNAmethylationoccursatC5positionofCwithinCpGdinucleotides.5mCconstitutes<1%ofnucleotides.TheDNAofmostvertebratesisdepletedinCpGdinucleotides.TheremainingCpGstendtoclusterinregionsreferredtoasCpGislands(CGI).AformaldefinitionofaCpGislandwasprovidedby

Gardiner-GardenandFrommer(1987).ACpGislandisdefinedasaregionofatleast200bp,withtheproportionofGsorCs,referredtoas“GCcontent,”greaterthan50%,andobservedtoexpectedCpGratio(O/E)greaterthan0.6..Thereareestimatedtobeabout26,000-45,000CpGislands,manyofwhichtendtobelocatedinthepromoterregionsofhousekeepinggenesandsometissuespecificgenes.当前26页，总共104页。Fig.2.Agenomicregionof40000basesfromchromosome1isshown.Theticksonthe

x-axisrepresentCpGlocations.ThepointsrepresentCpGratesinsegmentsoflength256basesThecurveistheresultofakernelsmootherofthepoints.Approximately20%ofthegenomeareCsand20%areGs.Thus,weexpectabout4%ofdinucleotidestobeCpG.However,mostpointsarewellbelowrates4%with2clusterswellabove4%.

ThelatterareCGI.Wuetal.,Biostatistics.2010Jul;11(3):499–514.CGICGICGshore当前27页，总共104页。GeneralfunctionofDNAmethylationSuppressinggeneexpressionInactivatingtheX-chromosomeinfemaleRegulatingtheimprintedgeneexpressionSilencingtherepetitivesequencesandtransposonelementsRegulatingtissue-specificgeneexpression

AberrantDNAmethylationisassociatedwithmanyhumandiseasessuchascancers当前28页，总共104页。3humanDNAmethyltransferases

DNMT1

DNMT3A

DNMT3Bdenovomethyltransferases–highlyexpressedatembryoimplantationwhenwavesofdenovomethylationareoccurringinthegenomemaintenancemethyltransferases当前29页，总共104页。MammalianDNMTs(DNA甲基化酶）Dnmt1thoughttobeprimarilyamaintenancemethylasewitha10-40foldpreferenceforhemimethylatedDNA.Localizestoreplicationfoci.Dnmt3Aand3Barethoughttobedenovomethylases.DNMT3familycontainsimilarcatalyticdomain,butdifferentregulatorydomains.ContainsaPHD(planthomeodomain)foundinmanychromatinassociatedproteins.当前30页，总共104页。JonesandLiang,NatureReview2009MammalianDNAdonovoandmaintenanceenzymesDNMT3A,DNMT3BandDNMT1DNA甲基化酶体系：起始与维持性甲基化酶当前31页，总共104页。DNAMethylationandCancerDNA甲基化异常与肿瘤的关系RecentwholegenomebisulfitesequencinganalysesofvariouscancersampleshaveconfirmedprevalentglobalhypomethylationincancersbuthaveonlyidentifiedinfrequentCpGislandhypermethylation.当前32页，总共104页。IsDNAhypomethylationacausalfactorfortumorigenesis?DNA低甲基化是否可以导致肿瘤发生？当前33页，总共104页。NatureGenetics2011

MutationsinDNMT3Awereidentifiedin23of112(20.5%)cases.Atotalof62outof281patients(22.1%)hasDNMT3AmutationsPNAS2011DeletionandmutationofDNMT3Ahavebeenlinkedtotumorigenesis当前34页，总共104页。DiseasesassociatedwithabnormalmethylationDiseases

GenesinvolvedICF DNMT3BRett MeCP2FragileXSyndrome FMRα-Thalassaemia ATRXCancer Tumorsuppressorgenes当前35页，总共104页。ＤＮＡ甲基化酶敲除小鼠表型TheKnock-outofanyofthemethyltransferasegenesislethalduringdevelopment(Dnmt1or3B)orshortlythereafter(3A;about4weeksofage).MutationsinDnmt3BcauseICFsyndrome(Imunodeficiency,centromericinstability,facialanomalies).ThefunctionofDNMT2andDNMT3Larelessunderstood.DNMT3Litselfdoesnothaveactivity,butformscomplexwithDNMT3aorDNMT3bandpotentiatetheiractivity.当前36页，总共104页。

CpGmethylation–howdoesitaffecttranscription?CpGmethylationpreventsbindingofsometranscriptionfactors(SP1) Allowbindingofmethylated-DNAbindingproteins(MBPsuchasMeCP2)MeCP2recruitsacomplexofhistonedeacetylasesandSIN3Ainducesaclosedchromatinstructure→genesilencinglongtermrepressiongene当前37页，总共104页。Methylcytosine-bindingproteins(DNAmethylationreaders),includingMeCP2,recognize5mCandrecruitcorepressorcomplexes(e.g.,Sin3a-HDAC1/2),resultinginchromatincompactionandgenerepressionMethylcytosine-bindingproteins当前38页，总共104页。检测ＤＮＡ甲基化的方法

１.

Detectionofgenomicmethylationbymethylation-sensitiverestrictionenzymes

-----CCGG----------GGCC----------CCGG----------GGCC-----CH3CH3HpaII,MspIMspI√

HpaIIXBosticketal.,2007Science当前39页，总共104页。２.DetectionofMethylcytosines:BisulfiteSequencing（亚硫酸氢盐测序法）当前40页，总共104页。PrincipleofBisulfiteModificationGgg gcg gac cgcGgg gug gau uguGgg gcmg gacm cmgcmGgg gcmg gacm cmgcmBisulfitemodificationBisulfitemodificationUnmethylatedDNAMethylatedDNA当前41页，总共104页。(3)联合亚硫酸氢钠限制性内切酶分析法(combinedbisulfiterestrictionanalysis,COBRA)该法的优点是：方法相对简单，不需预先知道CpG位点及样本序列；可进行甲基化水平的定量研究；需要样本量少，可用于石蜡包埋样本的分析。缺点是：只能获得特殊酶切位点甲基化情况，因此检测阴性不能排除样品DNA中甲基化存在的可能；由于酶和PCR的使用，序列分析受到限制。当前42页，总共104页。４.高效液相层析(highperformanceliquidchromatography,HPLC)高效液相层析能够定量测定基因组整体甲基化水平，其过程是：先将DNA样品经盐酸或氢氟酸水解成碱基，水解产物通过色谱柱，将结果与标准品比较，紫外光测定吸收峰值，计算5mC/(5mC+5C)的积分面积得出基因组整体的甲基化水平。Fraga等运用高效毛细管电泳法(highperformancecapillaryelectrophoresis,HPCE)处理DNA水解产物确定5mC的水平，相比HPLC，HPCE更简便、快速、经济。HPLC及HPCE测定基因组整体DNA甲基化水平的敏感性均较高。当前43页，总共104页。当前44页，总共104页。DNADemethylationActivedemethylation(byenzymes)

AlkBlikeproteins?RepairdamagedDNAbuthasnoconvincingevidencethatthisfamilyproteinsworkon5-methylcytosine.

InArabidopsisasubfamilyofDNAglycosylasesfunctiontopromoteDNAdemethylationthroughabaseexcision-repairpathway.RecentlyactiveDNAdemethylationinmammaliancellshasbeenreportedbyabaseexcisionrepairpathwaywheretheAIDfamilyofdeaminasesconvert5-methylcytosinetothyminefollowedbymismatchrepairbyDNAglycosylaseMBD4orTDG.

DNMT3A/DNMT3BConversionof5-methylcytosine(5mC)to5-hydroxymethylcytosine(5hmC)byTetfamilyproteins.2.Passivedemethylation(DNAreplicatesbutwithoutmethylation)当前45页，总共104页。DNAMethylationReprogrammingDuringMammalianDevelopment当前46页，总共104页。DNAdemethylationofearlyembryos3h6hPMPMPMPM8hAphidicolinFirstmet.22h2cells45h4cells当前47页，总共104页。TheyidentifiedthemammalianTETproteinsashomologsofthetrypanosomeproteinsJBP1andJBP2,whichhavebeenproposedtooxidizethe5-methylgroupofthymine.当前48页，总共104页。ExpressionofTET1inHEK293Tcellsresultsindecreased5mCstaining当前49页，总共104页。TETfamilyproteinsoxidize5mCsequentiallytogenerate5hmC,5fCand5caC当前50页，总共104页。Active(主动)DNADemethylationBER:BaseExcisionRepair(DNA损伤修复的一种方式）当前51页，总共104页。TET1andTranscriptionalRegulation当前52页，总共104页。5hmCDetection1.5hmCspecificantibody2.5hmCchemicalproperty3.Massanalysis当前53页，总共104页。DNA甲基化概述当前54页，总共104页。HistoneModificationsTypeofmodificationsUniquepropertyEnzymesFunctionMechanism当前55页，总共104页。Thereareatleast10differenttypesofmodificationsandover60residuesaremodifiedbyoneormoretypesofmodifications.TypesofCovalentModificationsonHistones+LysinePropionylationandButyrylation当前56页，总共104页。当前57页，总共104页。MethodsforIdentificationofHistoneModificationsChemicallabelingModification-and/orsite-specificantibodies

chromatinimmunoprecipitationassay(ChIP)

3.Massspectrometry当前58页，总共104页。当前59页，总共104页。众多的组蛋白化学修饰（种类和位点）当前60页，总共104页。HowHistoneModificationswork?AffectthenucleosomestructureAffecthigherorderchromatinstructureAffectotherhistonemodificationsHistonecode---alloworpreventbindingofspecifichistonebindingoreffectorproteins当前61页，总共104页。HistoneAcetylationFirstreportedbyAllfreyat1964(PNAS).Correlateswithtranscriptionalactivationandisdynamic.AcetylationneutralizespositivechargeonLys.BreakthroughTheidentificationoffirstnuclearhistoneacetyltransferase(HAT)byDavidAllis’group

Browelletal.,Cell1996

TetrahymenahistoneacetyltransferaseA:ahomologtotheyeastGcn5plinkinghistoneacetylationtogeneactivationTheidentificationoffirsthistonedeacetylase(HDAC)byShreiber’sgroup

TauntonetalScience1996

AmammalianhistonedeacetylaserelatedtoayeasttranscriptionalregulatorRpd3p当前62页，总共104页。

HighlyacetylatedhistonesareassociatedwithactivelytranscribedchromatinIncreasinghistoneacetylationcanturnonsomegenes.ImmunoprecipitationofDNAcross-linkedtochromatinwithantibodiesagainstAc-histonesenrichesforactivelytranscribedgenes.Theacetylatedchromatinismore“open”DNasesensitiveaccessibletotranscriptionfactorsandpolymerases当前63页，总共104页。HistoneAcetylationbyAcetyltransferases(HATs)TypeAnuclearHATs:acetylatehistonesinchromatin.TypeBcytoplasmicHATs:acetylatefreehistones

priortotheirassemblyintochromatin.AcetylateK5andK12inhistoneH4Useacetyl-coAasdonorcoA当前64页，总共104页。SomecommoncoactivatorsarenuclearHATsGcn5pisatranscriptionalactivatorofmanygenesinyeast.ItisalsoaHAT.PCAF(P300/CBPassociatedfactor)isaHATandishomologoustoyeastGcn5p.P300andCBParesimilarproteinsthatinteractwithmanytranscriptionfactors(e.g.CREB,AP1andMyoD).P300/CBPareneededforactivationbymanytranscriptionfactors,andthusareconsideredasgeneralcoactivators.P300/CBPhasintrinsicHATactivityaswellasbindingtotheHATPCAF.当前65页，总共104页。当前66页，总共104页。RolesofhistoneacetylationIncreaseaccessoftranscriptionfactorstoDNAinnucleosomes.Decondense30nmchromatinfibersServeasmarkersforbindingofnon-histoneproteins(e.g.bromodomainproteins).AcetylationneutralizespositivechargeonLysandweakenshistone-DNAinteractionandthereforeenhanceschromatindynamics.Acetylationalsoreduceshistone-histoneinteraction.当前67页，总共104页。Histonedeacetylationiscatalyzedbyhistonedeacetylases(HDACs)Threeclasses,about20identifiedmembers.canberecruitedbytranscriptionalrepressorstospecifictargetgenesand/ordeacetylatehistonesinchromatininanon-targeting,globalfashion.AcetylationanddeacetylationareverydynamiceventsAberranthistonedeacetylationhasbeenlinkedtocancer当前68页，总共104页。当前69页，总共104页。当前70页，总共104页。EffectofHistoneAcetylationon

ChromatinStructureandTranscriptionActivationRepression当前71页，总共104页。HDACinhibitorscaninducecelldifferentiationpossiblythroughinductionofp21andcyclinD1andhasbeentestedascancertreatmentdrugsinclinicaltrials当前72页，总共104页。HistoneMethylationTwotypes:arginine-

andlysine-specificHMTs.Argcanbemono-anddi-(asymmetricorsymmetric)methylated,whereLyscanbemono-,di-ortri-methylated.Histonemethylationisbelievedtobestableuntilrecently.Turnoverrateofhistonemethylationissimilartothatofhistoneturnover.MethylationdoesnotneutralizepositivechargeonLysandArg.Methylgroupisthoughttobetoosmalltohaveadirecteffectonchromatinstructure.Thefunctionofhistonemethylationisbelievetobemediatedthroughspecificmethylatedhistonebindingproteins(histonecodehypothesis)当前73页，总共104页。HistoneLysineMethylationTheretypes:Mono-,diandtri-methylationFoundinallfourcorehistonesandlinkerhistones.Multiplesitesoneachhistones.Enzymesappeartobesite-specificInvolvedinallDNAtemplatedprocesses.当前74页，总共104页。Lysinemethylation当前75页，总共104页。HistoneLysineMethyltransferases(KMTs)当前76页，总共104页。HistoneLysineMethylationandTranscriptionH3K4,H3K36andH3K79ActivationRepressionH3K9,H3K27andH4K20当前77页，总共104页。Howdoeshistonemethylationaffectchromatinfunction?H3-K9methylationcreatesabindingsiteforHP1andHP1isknowntoassociatewithHDACsandinvolvedinheterochromatinformation(whyitisinvolvedinrepression)H3-K4methylationpreventsbindingofcorepressorcomplexNuRDH3-K27methylationrecruitsrepressivepolycomecomplexPRC1.Theunderliningmechanismformanyothermodificationsisnotclear当前78页，总共104页。ReversibilityofLysineMethylationTheLSD1family(ShiYangGroup)TheJmjcdomainfamily(ZhangYiGroup)Passivedemethylation

1.Histonetailclipping.2.Histonereplacementbyunmodifiedhistonesorhistonevariants.Activedemethylation

当前79页，总共104页。LysdemethylationbyLSD1KubicekSandJenuweinT.Cell,Vol119,903-906,Dec.29,2004Minireview:ACrackinHistoneLysineMethylationCofactor:FAD(flavin-adeninedinucleotide)Shietal.,Cell2004当前80页，总共104页。LysdemethylationbyJHDM1Cofactors:Fe(II)anda-ketoglutarateTsukadaetal.,Nature2006当前81页，总共104页。InterplayBetweenDifferentHistoneModificationsAtthelevelofmodificationAttheleveloffunctionP-S10inhibitsbindingofHP1toH3K9(Me)2/3.Fischleetal.,Nature.2005Dec22;438(7071):1116-22当前82页，总共104页。

Somethingiswritingthiscode….

andSomethingisreadingthiscode…HistoneCodeHypothesisMultiplehistonemodifications,actingincombinatorialorsequentialfashionononeormultiplehistonetails,specifyuniquedownstreamfunctions.当前83页，总共104页。HistoneCodeHypothesisWritersErasersReaders/Effectors当前84页，总共104页。Formore,read:当前85页，总共104页。WhatistheconnectionbetweenDNAmethylationandhistonemodification?YesorNo?Ifyes,how?当前86页，总共104页。ApproachesforidentificationandcharacterizationofHMTsEducatedguess+experimentalverification

SUV39H1byReaetal.,Nature2000(RegulationofchromatinstructurebysitespecifichistoneH3methyltransferases)2.BiochemicalpurificationofHMTactivitiesusinginvitroHMTassay.

SET7byWangetal,MolCell2001(PurificationandfunctionalcharacterizationofahistoneH3-lysine4-specificmethyltransferase)Sequencesimilarity:testingtheproteinscontainingaSETdomain.

SETdomains(e.g.,G9a)当前87页，总共104页。1.通过同源顺序分析方法寻找组蛋白修饰酶当前88页，总共104页。顺序分析比较当前89页，总共104页。2.利用体外反应检测组蛋白修饰酶活性当前90页，总共104页。当前91页，总共104页。3.通过体外酶活检测结合蛋白质分离纯化方法寻找组蛋白修饰酶Wangetal.,2001Science293:853-857当前92页，总共104页。进一步的酶活性鉴定（体外与体内实验）当前93页，总共104页。4.利用特异组蛋白修饰抗体免疫染色研究细胞内组蛋白修饰当前94页，总共104页。5.体外利用WesternBlot和特异组蛋白修饰抗体检测组蛋白修饰当前95页，总共104页。6.利用质谱方法精确分析组蛋白修饰当前96页，总共104页。当前97页，总共104页。Genome-widestructuralorganizationofPICsChIP-exoapproachtoexaminethepreciselocationof6045PICsinyeastgenome.PICswerepositionedwithinpromotersandexcludedfromcodingregions.Rhee&PughNature2013当前98页，总共104页。四、体外染色质重组

当前99页，总共104页。Themajorityofchromatinassemblyisc