染色质研究技术与原理

染色质研究原理与技术翁杰敏教授目前一页\总数一百零五页\编于十九点一、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、体外染色质重组目前二页\总数一百零五页\编于十九点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等发现核小体目前三页\总数一百零五页\编于十九点目前四页\总数一百零五页\编于十九点A.InterphasechromatinB.amitoticchromosome,whichisduplicatedalreadyCompactionofchromatiniscell-stagedependent目前五页\总数一百零五页\编于十九点A)30nmfibersB)beadsonastring-nucleosomeFrominterphasenucleusNucleosomeistherepeatingunitofchromatin目前六页\总数一百零五页\编于十九点ChromatindigestionwithMNase目前七页\总数一百零五页\编于十九点Nucleosomecoreparticle2.8AcrystalstructureoftheMono-nucleosome目前八页\总数一百零五页\编于十九点Nucleosome=anucleosomecoreparticle+linker（核小体）DNA+alinkerhistoneDNAlength:180-200bpNucleosomecoreparticle=histoneoctamer+146bpDNANomenclature目前九页\总数一百零五页\编于十九点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

目前十页\总数一百零五页\编于十九点Histonefold-3alphahelicesand2folds目前十一页\总数一百零五页\编于十九点目前十二页\总数一百零五页\编于十九点Histoneself-assembly目前十三页\总数一百零五页\编于十九点Nucleosome目前十四页\总数一百零五页\编于十九点Nucleosome目前十五页\总数一百零五页\编于十九点TheNucleosomeH3H3ThecorehistonesarepredominantlyglobularexceptfortheN-terminal“tails”,whichareunstructured.目前十六页\总数一百零五页\编于十九点Differentstatesofchromatin目前十七页\总数一百零五页\编于十九点FunctionStorageofgeneticinformation（储存）PrecisesegregationofreplicatedDNAintotwodaughtercells（精确分离到不同）Platformfortranscription,replication,recombinationandDNArepair（平台）目前十八页\总数一百零五页\编于十九点二、DNA甲基化修饰和组蛋白修饰三、研究染色质修饰的技术目前十九页\总数一百零五页\编于十九点如何进行调控？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目前二十页\总数一百零五页\编于十九点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目前二十一页\总数一百零五页\编于十九点DNAmethylationhistonevariantsATP-drivenchromatinremodelingcomplexesHistonemodificationsNon-codingRNAsMechanismsforepigeneticregulation目前二十二页\总数一百零五页\编于十九点目前二十三页\总数一百零五页\编于十九点目前二十四页\总数一百零五页\编于十九点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.目前二十五页\总数一百零五页\编于十九点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目前二十六页\总数一百零五页\编于十九点GeneralfunctionofDNAmethylationSuppressinggeneexpressionInactivatingtheX-chromosomeinfemaleRegulatingtheimprintedgeneexpressionSilencingtherepetitivesequencesandtransposonelementsRegulatingtissue-specificgeneexpression

AberrantDNAmethylationisassociatedwithmanyhumandiseasessuchascancers目前二十七页\总数一百零五页\编于十九点3humanDNAmethyltransferases

DNMT1

DNMT3A

DNMT3Bdenovomethyltransferases–highlyexpressedatembryoimplantationwhenwavesofdenovomethylationareoccurringinthegenomemaintenancemethyltransferases目前二十八页\总数一百零五页\编于十九点MammalianDNMTs(DNA甲基化酶）Dnmt1thoughttobeprimarilyamaintenancemethylasewitha10-40foldpreferenceforhemimethylatedDNA.Localizestoreplicationfoci.Dnmt3Aand3Barethoughttobedenovomethylases.DNMT3familycontainsimilarcatalyticdomain,butdifferentregulatorydomains.ContainsaPHD(planthomeodomain)foundinmanychromatinassociatedproteins.目前二十九页\总数一百零五页\编于十九点JonesandLiang,NatureReview2009MammalianDNAdonovoandmaintenanceenzymesDNMT3A,DNMT3BandDNMT1DNA甲基化酶体系：起始与维持性甲基化酶目前三十页\总数一百零五页\编于十九点DNAMethylationandCancerDNA甲基化异常与肿瘤的关系RecentwholegenomebisulfitesequencinganalysesofvariouscancersampleshaveconfirmedprevalentglobalhypomethylationincancersbuthaveonlyidentifiedinfrequentCpGislandhypermethylation.目前三十一页\总数一百零五页\编于十九点IsDNAhypomethylationacausalfactorfortumorigenesis?DNA低甲基化是否可以导致肿瘤发生？目前三十二页\总数一百零五页\编于十九点NatureGenetics2011

MutationsinDNMT3Awereidentifiedin23of112(20.5%)cases.Atotalof62outof281patients(22.1%)hasDNMT3AmutationsPNAS2011DeletionandmutationofDNMT3Ahavebeenlinkedtotumorigenesis目前三十三页\总数一百零五页\编于十九点DiseasesassociatedwithabnormalmethylationDiseases

GenesinvolvedICF DNMT3BRett MeCP2FragileXSyndrome FMRα-Thalassaemia ATRXCancer Tumorsuppressorgenes目前三十四页\总数一百零五页\编于十九点ＤＮＡ甲基化酶敲除小鼠表型TheKnock-outofanyofthemethyltransferasegenesislethalduringdevelopment(Dnmt1or3B)orshortlythereafter(3A;about4weeksofage).MutationsinDnmt3BcauseICFsyndrome(Imunodeficiency,centromericinstability,facialanomalies).ThefunctionofDNMT2andDNMT3Larelessunderstood.DNMT3Litselfdoesnothaveactivity,butformscomplexwithDNMT3aorDNMT3bandpotentiatetheiractivity.目前三十五页\总数一百零五页\编于十九点

CpGmethylation–howdoesitaffecttranscription?CpGmethylationpreventsbindingofsometranscriptionfactors(SP1) Allowbindingofmethylated-DNAbindingproteins(MBPsuchasMeCP2)MeCP2recruitsacomplexofhistonedeacetylasesandSIN3Ainducesaclosedchromatinstructure→genesilencinglongtermrepressiongene目前三十六页\总数一百零五页\编于十九点Methylcytosine-bindingproteins(DNAmethylationreaders),includingMeCP2,recognize5mCandrecruitcorepressorcomplexes(e.g.,Sin3a-HDAC1/2),resultinginchromatincompactionandgenerepressionMethylcytosine-bindingproteins目前三十七页\总数一百零五页\编于十九点检测ＤＮＡ甲基化的方法

１.

Detectionofgenomicmethylationbymethylation-sensitiverestrictionenzymes

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

HpaIIXBosticketal.,2007Science目前三十八页\总数一百零五页\编于十九点２.DetectionofMethylcytosines:BisulfiteSequencing（亚硫酸氢盐测序法）目前三十九页\总数一百零五页\编于十九点PrincipleofBisulfiteModificationGgg gcg gac cgcGgg gug gau uguGgg gcmg gacm cmgcmGgg gcmg gacm cmgcmBisulfitemodificationBisulfitemodificationUnmethylatedDNAMethylatedDNA目前四十页\总数一百零五页\编于十九点(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。目前四十一页\总数一百零五页\编于十九点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。目前四十二页\总数一百零五页\编于十九点目前四十三页\总数一百零五页\编于十九点DNADemethylationActivedemethylation(byenzymes)

AlkBlikeproteins?RepairdamagedDNAbuthasnoconvincingevidencethatthisfamilyproteinsworkon5-methylcytosine.

InArabidopsisasubfamilyofDNAglycosylasesfunctiontopromoteDNAdemethylationthroughabaseexcision-repairpathway.RecentlyactiveDNAdemethylationinmammaliancellshasbeenreportedbyabaseexcisionrepairpathwaywheretheAIDfamilyofdeaminasesconvert5-methylcytosinetothyminefollowedbymismatchrepairbyDNAglycosylaseMBD4orTDG.

DNMT3A/DNMT3BConversionof5-methylcytosine(5mC)to5-hydroxymethylcytosine(5hmC)byTetfamilyproteins.2.Passivedemethylation(DNAreplicatesbutwithoutmethylation)目前四十四页\总数一百零五页\编于十九点DNAMethylationReprogrammingDuringMammalianDevelopment目前四十五页\总数一百零五页\编于十九点DNAdemethylationofearlyembryos3h6hPMPMPMPM8hAphidicolinFirstmet.22h2cells45h4cells目前四十六页\总数一百零五页\编于十九点TheyidentifiedthemammalianTETproteinsashomologsofthetrypanosomeproteinsJBP1andJBP2,whichhavebeenproposedtooxidizethe5-methylgroupofthymine.目前四十七页\总数一百零五页\编于十九点ExpressionofTET1inHEK293Tcellsresultsindecreased5mCstaining目前四十八页\总数一百零五页\编于十九点TETfamilyproteinsoxidize5mCsequentiallytogenerate5hmC,5fCand5caC目前四十九页\总数一百零五页\编于十九点Active(主动)DNADemethylationBER:BaseExcisionRepair(DNA损伤修复的一种方式）目前五十页\总数一百零五页\编于十九点TET1andTranscriptionalRegulation目前五十一页\总数一百零五页\编于十九点5hmCDetection1.5hmCspecificantibody2.5hmCchemicalproperty3.Massanalysis目前五十二页\总数一百零五页\编于十九点DNA甲基化概述目前五十三页\总数一百零五页\编于十九点HistoneModificationsTypeofmodificationsUniquepropertyEnzymesFunctionMechanism目前五十四页\总数一百零五页\编于十九点Thereareatleast10differenttypesofmodificationsandover60residuesaremodifiedbyoneormoretypesofmodifications.TypesofCovalentModificationsonHistones+LysinePropionylationandButyrylation目前五十五页\总数一百零五页\编于十九点目前五十六页\总数一百零五页\编于十九点MethodsforIdentificationofHistoneModificationsChemicallabelingModification-and/orsite-specificantibodies

chromatinimmunoprecipitationassay(ChIP)

3.Massspectrometry目前五十七页\总数一百零五页\编于十九点目前五十八页\总数一百零五页\编于十九点众多的组蛋白化学修饰（种类和位点）目前五十九页\总数一百零五页\编于十九点HowHistoneModificationswork?AffectthenucleosomestructureAffecthigherorderchromatinstructureAffectotherhistonemodificationsHistonecode---alloworpreventbindingofspecifichistonebindingoreffectorproteins目前六十页\总数一百零五页\编于十九点HistoneAcetylationFirstreportedbyAllfreyat1964(PNAS).Correlateswithtranscriptionalactivationandisdynamic.AcetylationneutralizespositivechargeonLys.BreakthroughTheidentificationoffirstnuclearhistoneacetyltransferase(HAT)byDavidAllis’group

Browelletal.,Cell1996

TetrahymenahistoneacetyltransferaseA:ahomologtotheyeastGcn5plinkinghistoneacetylationtogeneactivationTheidentificationoffirsthistonedeacetylase(HDAC)byShreiber’sgroup

TauntonetalScience1996

AmammalianhistonedeacetylaserelatedtoayeasttranscriptionalregulatorRpd3p目前六十一页\总数一百零五页\编于十九点

HighlyacetylatedhistonesareassociatedwithactivelytranscribedchromatinIncreasinghistoneacetylationcanturnonsomegenes.ImmunoprecipitationofDNAcross-linkedtochromatinwithantibodiesagainstAc-histonesenrichesforactivelytranscribedgenes.Theacetylatedchromatinismore“open”DNasesensitiveaccessibletotranscriptionfactorsandpolymerases目前六十二页\总数一百零五页\编于十九点HistoneAcetylationbyAcetyltransferases(HATs)TypeAnuclearHATs:acetylatehistonesinchromatin.TypeBcytoplasmicHATs:acetylatefreehistones

priortotheirassemblyintochromatin.AcetylateK5andK12inhistoneH4Useacetyl-coAasdonorcoA目前六十三页\总数一百零五页\编于十九点SomecommoncoactivatorsarenuclearHATsGcn5pisatranscriptionalactivatorofmanygenesinyeast.ItisalsoaHAT.PCAF(P300/CBPassociatedfactor)isaHATandishomologoustoyeastGcn5p.P300andCBParesimilarproteinsthatinteractwithmanytranscriptionfactors(e.g.CREB,AP1andMyoD).P300/CBPareneededforactivationbymanytranscriptionfactors,andthusareconsideredasgeneralcoactivators.P300/CBPhasintrinsicHATactivityaswellasbindingtotheHATPCAF.目前六十四页\总数一百零五页\编于十九点目前六十五页\总数一百零五页\编于十九点RolesofhistoneacetylationIncreaseaccessoftranscriptionfactorstoDNAinnucleosomes.Decondense30nmchromatinfibersServeasmarkersforbindingofnon-histoneproteins(e.g.bromodomainproteins).AcetylationneutralizespositivechargeonLysandweakenshistone-DNAinteractionandthereforeenhanceschromatindynamics.Acetylationalsoreduceshistone-histoneinteraction.目前六十六页\总数一百零五页\编于十九点Histonedeacetylationiscatalyzedbyhistonedeacetylases(HDACs)Threeclasses,about20identifiedmembers.canberecruitedbytranscriptionalrepressorstospecifictargetgenesand/ordeacetylatehistonesinchromatininanon-targeting,globalfashion.AcetylationanddeacetylationareverydynamiceventsAberranthistonedeacetylationhasbeenlinkedtocancer目前六十七页\总数一百零五页\编于十九点目前六十八页\总数一百零五页\编于十九点目前六十九页\总数一百零五页\编于十九点EffectofHistoneAcetylationon

ChromatinStructureandTranscriptionActivationRepression目前七十页\总数一百零五页\编于十九点HDACinhibitorscaninducecelldifferentiationpossiblythroughinductionofp21andcyclinD1andhasbeentestedascancertreatmentdrugsinclinicaltrials目前七十一页\总数一百零五页\编于十九点HistoneMethylationTwotypes:arginine-

andlysine-specificHMTs.Argcanbemono-anddi-(asymmetricorsymmetric)methylated,whereLyscanbemono-,di-ortri-methylated.Histonemethylationisbelievedtobestableuntilrecently.Turnoverrateofhistonemethylationissimilartothatofhistoneturnover.MethylationdoesnotneutralizepositivechargeonLysandArg.Methylgroupisthoughttobetoosmalltohaveadirecteffectonchromatinstructure.Thefunctionofhistonemethylationisbelievetobemediatedthroughspecificmethylatedhistonebindingproteins(histonecodehypothesis)目前七十二页\总数一百零五页\编于十九点HistoneLysineMethylationTheretypes:Mono-,diandtri-methylationFoundinallfourcorehistonesandlinkerhistones.Multiplesitesoneachhistones.Enzymesappeartobesite-specificInvolvedinallDNAtemplatedprocesses.目前七十三页\总数一百零五页\编于十九点Lysinemethylation目前七十四页\总数一百零五页\编于十九点HistoneLysineMethyltransferases(KMTs)目前七十五页\总数一百零五页\编于十九点HistoneLysineMethylationandTranscriptionH3K4,H3K36andH3K79ActivationRepressionH3K9,H3K27andH4K20目前七十六页\总数一百零五页\编于十九点Howdoeshistonemethylationaffectchromatinfunction?H3-K9methylationcreatesabindingsiteforHP1andHP1isknowntoassociatewithHDACsandinvolvedinheterochromatinformation(whyitisinvolvedinrepression)H3-K4methylationpreventsbindingofcorepressorcomplexNuRDH3-K27methylationrecruitsrepressivepolycomecomplexPRC1.Theunderliningmechanismformanyothermodificationsisnotclear目前七十七页\总数一百零五页\编于十九点ReversibilityofLysineMethylationTheLSD1family(ShiYangGroup)TheJmjcdomainfamily(ZhangYiGroup)Passivedemethylation

1.Histonetailclipping.2.Histonereplacementbyunmodifiedhistonesorhistonevariants.Activedemethylation

目前七十八页\总数一百零五页\编于十九点LysdemethylationbyLSD1KubicekSandJenuweinT.Cell,Vol119,903-906,Dec.29,2004Minireview:ACrackinHistoneLysineMethylationCofactor:FAD(flavin-adeninedinucleotide)Shietal.,Cell2004目前七十九页\总数一百零五页\编于十九点LysdemethylationbyJHDM1Cofactors:Fe(II)anda-ketoglutarateTsukadaetal.,Nature2006目前八十页\总数一百零五页\编于十九点InterplayBetweenDifferentHistoneModificationsAtthelevelofmodificationAttheleveloffunctionP-S10inhibitsbindingofHP1toH3K9(Me)2/3.Fischleetal.,Nature.2005Dec22;438(7071):1116-22目前八十一页\总数一百零五页\编于十九点

Somethingiswritingthiscode….

andSomethingisreadingthiscode…HistoneCodeHypothesisMultiplehistonemodifications,actingincombinatorialorsequentialfashionononeormultiplehistonetails,specifyuniquedownstreamfunctions.目前八十二页\总数一百零五页\编于十九点HistoneCodeHypothesisWritersErasersReaders/Effectors目前八十三页\总数一百零五页\编于十九点Formore,read:目前八十四页\总数一百零五页\编于十九点WhatistheconnectionbetweenDNAmethylationandhistonemodification?YesorNo?Ifyes,how?目前八十五页\总数一百零五页\编于十九点ApproachesforidentificationandcharacterizationofHMTsEducatedguess+experimentalverification

SUV39H1byReaetal.,Nature2000(RegulationofchromatinstructurebysitespecifichistoneH3methyltransferases)2.BiochemicalpurificationofHMTactivitiesusinginvitroHMTassay.

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

SETdomains(e.g.,G9a)目前八十六页\总数一百零五页\编于十九点1.通过同源顺序分析方法寻找组蛋白修饰酶目前八十七页\总数一百零五页\编于十九点顺序分析比较目前八十八页\总数一百零五页\编于十九点2.利用体外反应检测组蛋白修饰酶活性目前八十九页\总数一百零五页\编于十九点目前九十页\总数一百零五页\编于十九点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目前九十一页\总数一百零五页\编于十九点进一步的酶活性鉴定（体外与体内实验）目前九十二页\总数一百零五页\编于十九点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目前九十三页\总数一百零五页\编于十九点5.体外利用WesternBlot和特异组蛋白修饰抗体检测组蛋白修饰目前九十四页\总数一百零五页\编于十九点6.利用质谱方法精确分析组蛋白修饰目前九十五页\总数一百零五页\编于十九点目前九十六页\总数一百零五页\编于十九点Genome-widestructuralorganizationofPICsChIP-exoapproachtoexaminethepreciselocationof6045PICsinyeastgenome.PICswerepositionedwithinpromotersandexcludedfromcodingregions.Rhee&PughNature2013目前九十七页\总数一百零五页\编于十九点四、体外染色质重组

目前九十八页\总数一百零五页\编于十九点ThemajorityofchromatinassemblyiscoupledtoDNAreplicationinthecellIncorporationoftheH3-H4tetramerbyhistonechaperonesAdditionoftwoH2A-H2Bdimerstoformaco