染色質(zhì)研究技術(shù)與原理

染色質(zhì)研究原理與技術(shù)翁杰敏教授當(dāng)前第1頁\共有105頁\編于星期四\18點(diǎn)一、染色質(zhì)和核小體二、DNA甲基化修飾和組蛋白修飾三、研究染色質(zhì)修飾的技術(shù)四、體外染色質(zhì)重組當(dāng)前第2頁\共有105頁\編于星期四\18點(diǎn)染色質(zhì)研究的歷史性成果年W.Flemming提出“Chromatin”這個(gè)名稱1884年A.Kosse發(fā)現(xiàn)Histone組蛋白1944年Avery等證明DNA是遺傳物質(zhì)1953年Watson&Crick提出DNAdoublehelix1964年組蛋白修飾（乙酰化和甲基化acetylationandmethylation)1973年Olins等電鏡觀察到染色質(zhì)的念珠重復(fù)；1974年Kornberg等發(fā)現(xiàn)核小體當(dāng)前第3頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第4頁\共有105頁\編于星期四\18點(diǎn)A.InterphasechromatinB.amitoticchromosome,whichisduplicatedalreadyCompactionofchromatiniscell-stagedependent當(dāng)前第5頁\共有105頁\編于星期四\18點(diǎn)A)30nmfibersB)beadsonastring-nucleosomeFrominterphasenucleusNucleosomeistherepeatingunitofchromatin當(dāng)前第6頁\共有105頁\編于星期四\18點(diǎn)ChromatindigestionwithMNase當(dāng)前第7頁\共有105頁\編于星期四\18點(diǎn)Nucleosomecoreparticle2.8AcrystalstructureoftheMono-nucleosome當(dāng)前第8頁\共有105頁\編于星期四\18點(diǎn)Nucleosome=anucleosomecoreparticle+linker（核小體）DNA+alinkerhistoneDNAlength:180-200bpNucleosomecoreparticle=histoneoctamer+146bpDNANomenclature當(dāng)前第9頁\共有105頁\編于星期四\18點(diǎn)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

當(dāng)前第10頁\共有105頁\編于星期四\18點(diǎn)Histonefold-3alphahelicesand2folds當(dāng)前第11頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第12頁\共有105頁\編于星期四\18點(diǎn)Histoneself-assembly當(dāng)前第13頁\共有105頁\編于星期四\18點(diǎn)Nucleosome當(dāng)前第14頁\共有105頁\編于星期四\18點(diǎn)Nucleosome當(dāng)前第15頁\共有105頁\編于星期四\18點(diǎn)TheNucleosomeH3H3ThecorehistonesarepredominantlyglobularexceptfortheN-terminal“tails”,whichareunstructured.當(dāng)前第16頁\共有105頁\編于星期四\18點(diǎn)Differentstatesofchromatin當(dāng)前第17頁\共有105頁\編于星期四\18點(diǎn)FunctionStorageofgeneticinformation（儲(chǔ)存）PrecisesegregationofreplicatedDNAintotwodaughtercells（精確分離到不同）Platformfortranscription,replication,recombinationandDNArepair（平臺(tái)）當(dāng)前第18頁\共有105頁\編于星期四\18點(diǎn)二、DNA甲基化修飾和組蛋白修飾三、研究染色質(zhì)修飾的技術(shù)當(dāng)前第19頁\共有105頁\編于星期四\18點(diǎn)如何進(jìn)行調(diào)控？染色質(zhì)結(jié)構(gòu)=DNA+組蛋白八聚體DNA是遺傳信息攜帶者，染色質(zhì)是遺傳信息存在形式當(dāng)前第20頁\共有105頁\編于星期四\18點(diǎn)1.不能降解DNA或者組蛋白2.

通過化學(xué)修飾調(diào)控

DNA甲基化，組蛋白修飾3.通過改變結(jié)構(gòu)調(diào)控

更換不同類型組蛋白

通過ATP依賴性重塑酶改變結(jié)構(gòu)答案當(dāng)前第21頁\共有105頁\編于星期四\18點(diǎn)DNAmethylationhistonevariantsATP-drivenchromatinremodelingcomplexesHistonemodificationsNon-codingRNAsMechanismsforepigeneticregulation當(dāng)前第22頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第23頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第24頁\共有105頁\編于星期四\18點(diǎn)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.當(dāng)前第25頁\共有105頁\編于星期四\18點(diǎn)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當(dāng)前第26頁\共有105頁\編于星期四\18點(diǎn)GeneralfunctionofDNAmethylationSuppressinggeneexpressionInactivatingtheX-chromosomeinfemaleRegulatingtheimprintedgeneexpressionSilencingtherepetitivesequencesandtransposonelementsRegulatingtissue-specificgeneexpression

AberrantDNAmethylationisassociatedwithmanyhumandiseasessuchascancers當(dāng)前第27頁\共有105頁\編于星期四\18點(diǎn)3humanDNAmethyltransferases

DNMT1

DNMT3A

DNMT3Bdenovomethyltransferases–highlyexpressedatembryoimplantationwhenwavesofdenovomethylationareoccurringinthegenomemaintenancemethyltransferases當(dāng)前第28頁\共有105頁\編于星期四\18點(diǎn)MammalianDNMTs(DNA甲基化酶）Dnmt1thoughttobeprimarilyamaintenancemethylasewitha10-40foldpreferenceforhemimethylatedDNA.Localizestoreplicationfoci.Dnmt3Aand3Barethoughttobedenovomethylases.DNMT3familycontainsimilarcatalyticdomain,butdifferentregulatorydomains.ContainsaPHD(planthomeodomain)foundinmanychromatinassociatedproteins.當(dāng)前第29頁\共有105頁\編于星期四\18點(diǎn)JonesandLiang,NatureReview2009MammalianDNAdonovoandmaintenanceenzymesDNMT3A,DNMT3BandDNMT1DNA甲基化酶體系：起始與維持性甲基化酶當(dāng)前第30頁\共有105頁\編于星期四\18點(diǎn)DNAMethylationandCancerDNA甲基化異常與腫瘤的關(guān)系RecentwholegenomebisulfitesequencinganalysesofvariouscancersampleshaveconfirmedprevalentglobalhypomethylationincancersbuthaveonlyidentifiedinfrequentCpGislandhypermethylation.當(dāng)前第31頁\共有105頁\編于星期四\18點(diǎn)IsDNAhypomethylationacausalfactorfortumorigenesis?DNA低甲基化是否可以導(dǎo)致腫瘤發(fā)生？當(dāng)前第32頁\共有105頁\編于星期四\18點(diǎn)NatureGenetics2011

MutationsinDNMT3Awereidentifiedin23of112(20.5%)cases.Atotalof62outof281patients(22.1%)hasDNMT3AmutationsPNAS2011DeletionandmutationofDNMT3Ahavebeenlinkedtotumorigenesis當(dāng)前第33頁\共有105頁\編于星期四\18點(diǎn)DiseasesassociatedwithabnormalmethylationDiseases

GenesinvolvedICF DNMT3BRett MeCP2FragileXSyndrome FMRα-Thalassaemia ATRXCancer Tumorsuppressorgenes當(dāng)前第34頁\共有105頁\編于星期四\18點(diǎn)ＤＮＡ甲基化酶敲除小鼠表型TheKnock-outofanyofthemethyltransferasegenesislethalduringdevelopment(Dnmt1or3B)orshortlythereafter(3A;about4weeksofage).MutationsinDnmt3BcauseICFsyndrome(Imunodeficiency,centromericinstability,facialanomalies).ThefunctionofDNMT2andDNMT3Larelessunderstood.DNMT3Litselfdoesnothaveactivity,butformscomplexwithDNMT3aorDNMT3bandpotentiatetheiractivity.當(dāng)前第35頁\共有105頁\編于星期四\18點(diǎn)

CpGmethylation–howdoesitaffecttranscription?CpGmethylationpreventsbindingofsometranscriptionfactors(SP1) Allowbindingofmethylated-DNAbindingproteins(MBPsuchasMeCP2)MeCP2recruitsacomplexofhistonedeacetylasesandSIN3Ainducesaclosedchromatinstructure→genesilencinglongtermrepressiongene當(dāng)前第36頁\共有105頁\編于星期四\18點(diǎn)Methylcytosine-bindingproteins(DNAmethylationreaders),includingMeCP2,recognize5mCandrecruitcorepressorcomplexes(e.g.,Sin3a-HDAC1/2),resultinginchromatincompactionandgenerepressionMethylcytosine-bindingproteins當(dāng)前第37頁\共有105頁\編于星期四\18點(diǎn)檢測ＤＮＡ甲基化的方法

１.

Detectionofgenomicmethylationbymethylation-sensitiverestrictionenzymes

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

HpaIIXBosticketal.,2007Science當(dāng)前第38頁\共有105頁\編于星期四\18點(diǎn)２.DetectionofMethylcytosines:BisulfiteSequencing（亞硫酸氫鹽測序法）當(dāng)前第39頁\共有105頁\編于星期四\18點(diǎn)PrincipleofBisulfiteModificationGgg gcg gac cgcGgg gug gau uguGgg gcmg gacm cmgcmGgg gcmg gacm cmgcmBisulfitemodificationBisulfitemodificationUnmethylatedDNAMethylatedDNA當(dāng)前第40頁\共有105頁\編于星期四\18點(diǎn)(3)聯(lián)合亞硫酸氫鈉限制性內(nèi)切酶分析法(combinedbisulfiterestrictionanalysis,COBRA)該法的優(yōu)點(diǎn)是：方法相對簡單，不需預(yù)先知道CpG位點(diǎn)及樣本序列；可進(jìn)行甲基化水平的定量研究；需要樣本量少，可用于石蠟包埋樣本的分析。缺點(diǎn)是：只能獲得特殊酶切位點(diǎn)甲基化情況，因此檢測陰性不能排除樣品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。當(dāng)前第41頁\共有105頁\編于星期四\18點(diǎn)４.高效液相層析(highperformanceliquidchromatography,HPLC)高效液相層析能夠定量測定基因組整體甲基化水平，其過程是：先將DNA樣品經(jīng)鹽酸或氫氟酸水解成堿基，水解產(chǎn)物通過色譜柱，將結(jié)果與標(biāo)準(zhǔn)品比較，紫外光測定吸收峰值，計(jì)算5mC/(5mC+5C)的積分面積得出基因組整體的甲基化水平。Fraga等運(yùn)用高效毛細(xì)管電泳法(highperformancecapillaryelectrophoresis,HPCE)處理DNA水解產(chǎn)物確定5mC的水平，相比HPLC，HPCE更簡便、快速、經(jīng)濟(jì)。HPLC及HPCE測定基因組整體DNA甲基化水平的敏感性均較高。當(dāng)前第42頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第43頁\共有105頁\編于星期四\18點(diǎn)DNADemethylationActivedemethylation(byenzymes)

AlkBlikeproteins?RepairdamagedDNAbuthasnoconvincingevidencethatthisfamilyproteinsworkon5-methylcytosine.

InArabidopsisasubfamilyofDNAglycosylasesfunctiontopromoteDNAdemethylationthroughabaseexcision-repairpathway.RecentlyactiveDNAdemethylationinmammaliancellshasbeenreportedbyabaseexcisionrepairpathwaywheretheAIDfamilyofdeaminasesconvert5-methylcytosinetothyminefollowedbymismatchrepairbyDNAglycosylaseMBD4orTDG.

DNMT3A/DNMT3BConversionof5-methylcytosine(5mC)to5-hydroxymethylcytosine(5hmC)byTetfamilyproteins.2.Passivedemethylation(DNAreplicatesbutwithoutmethylation)當(dāng)前第44頁\共有105頁\編于星期四\18點(diǎn)DNAMethylationReprogrammingDuringMammalianDevelopment當(dāng)前第45頁\共有105頁\編于星期四\18點(diǎn)DNAdemethylationofearlyembryos3h6hPMPMPMPM8hAphidicolinFirstmet.22h2cells45h4cells當(dāng)前第46頁\共有105頁\編于星期四\18點(diǎn)TheyidentifiedthemammalianTETproteinsashomologsofthetrypanosomeproteinsJBP1andJBP2,whichhavebeenproposedtooxidizethe5-methylgroupofthymine.當(dāng)前第47頁\共有105頁\編于星期四\18點(diǎn)ExpressionofTET1inHEK293Tcellsresultsindecreased5mCstaining當(dāng)前第48頁\共有105頁\編于星期四\18點(diǎn)TETfamilyproteinsoxidize5mCsequentiallytogenerate5hmC,5fCand5caC當(dāng)前第49頁\共有105頁\編于星期四\18點(diǎn)Active(主動(dòng))DNADemethylationBER:BaseExcisionRepair(DNA損傷修復(fù)的一種方式）當(dāng)前第50頁\共有105頁\編于星期四\18點(diǎn)TET1andTranscriptionalRegulation當(dāng)前第51頁\共有105頁\編于星期四\18點(diǎn)5hmCDetection1.5hmCspecificantibody2.5hmCchemicalproperty3.Massanalysis當(dāng)前第52頁\共有105頁\編于星期四\18點(diǎn)DNA甲基化概述當(dāng)前第53頁\共有105頁\編于星期四\18點(diǎn)HistoneModificationsTypeofmodificationsUniquepropertyEnzymesFunctionMechanism當(dāng)前第54頁\共有105頁\編于星期四\18點(diǎn)Thereareatleast10differenttypesofmodificationsandover60residuesaremodifiedbyoneormoretypesofmodifications.TypesofCovalentModificationsonHistones+LysinePropionylationandButyrylation當(dāng)前第55頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第56頁\共有105頁\編于星期四\18點(diǎn)MethodsforIdentificationofHistoneModificationsChemicallabelingModification-and/orsite-specificantibodies

chromatinimmunoprecipitationassay(ChIP)

3.Massspectrometry當(dāng)前第57頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第58頁\共有105頁\編于星期四\18點(diǎn)眾多的組蛋白化學(xué)修飾（種類和位點(diǎn)）當(dāng)前第59頁\共有105頁\編于星期四\18點(diǎn)HowHistoneModificationswork?AffectthenucleosomestructureAffecthigherorderchromatinstructureAffectotherhistonemodificationsHistonecode---alloworpreventbindingofspecifichistonebindingoreffectorproteins當(dāng)前第60頁\共有105頁\編于星期四\18點(diǎn)HistoneAcetylationFirstreportedbyAllfreyat1964(PNAS).Correlateswithtranscriptionalactivationandisdynamic.AcetylationneutralizespositivechargeonLys.BreakthroughTheidentificationoffirstnuclearhistoneacetyltransferase(HAT)byDavidAllis’group

Browelletal.,Cell1996

TetrahymenahistoneacetyltransferaseA:ahomologtotheyeastGcn5plinkinghistoneacetylationtogeneactivationTheidentificationoffirsthistonedeacetylase(HDAC)byShreiber’sgroup

TauntonetalScience1996

AmammalianhistonedeacetylaserelatedtoayeasttranscriptionalregulatorRpd3p當(dāng)前第61頁\共有105頁\編于星期四\18點(diǎn)

HighlyacetylatedhistonesareassociatedwithactivelytranscribedchromatinIncreasinghistoneacetylationcanturnonsomegenes.ImmunoprecipitationofDNAcross-linkedtochromatinwithantibodiesagainstAc-histonesenrichesforactivelytranscribedgenes.Theacetylatedchromatinismore“open”DNasesensitiveaccessibletotranscriptionfactorsandpolymerases當(dāng)前第62頁\共有105頁\編于星期四\18點(diǎn)HistoneAcetylationbyAcetyltransferases(HATs)TypeAnuclearHATs:acetylatehistonesinchromatin.TypeBcytoplasmicHATs:acetylatefreehistones

priortotheirassemblyintochromatin.AcetylateK5andK12inhistoneH4Useacetyl-coAasdonorcoA當(dāng)前第63頁\共有105頁\編于星期四\18點(diǎn)SomecommoncoactivatorsarenuclearHATsGcn5pisatranscriptionalactivatorofmanygenesinyeast.ItisalsoaHAT.PCAF(P300/CBPassociatedfactor)isaHATandishomologoustoyeastGcn5p.P300andCBParesimilarproteinsthatinteractwithmanytranscriptionfactors(e.g.CREB,AP1andMyoD).P300/CBPareneededforactivationbymanytranscriptionfactors,andthusareconsideredasgeneralcoactivators.P300/CBPhasintrinsicHATactivityaswellasbindingtotheHATPCAF.當(dāng)前第64頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第65頁\共有105頁\編于星期四\18點(diǎn)RolesofhistoneacetylationIncreaseaccessoftranscriptionfactorstoDNAinnucleosomes.Decondense30nmchromatinfibersServeasmarkersforbindingofnon-histoneproteins(e.g.bromodomainproteins).AcetylationneutralizespositivechargeonLysandweakenshistone-DNAinteractionandthereforeenhanceschromatindynamics.Acetylationalsoreduceshistone-histoneinteraction.當(dāng)前第66頁\共有105頁\編于星期四\18點(diǎn)Histonedeacetylationiscatalyzedbyhistonedeacetylases(HDACs)Threeclasses,about20identifiedmembers.canberecruitedbytranscriptionalrepressorstospecifictargetgenesand/ordeacetylatehistonesinchromatininanon-targeting,globalfashion.AcetylationanddeacetylationareverydynamiceventsAberranthistonedeacetylationhasbeenlinkedtocancer當(dāng)前第67頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第68頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第69頁\共有105頁\編于星期四\18點(diǎn)EffectofHistoneAcetylationon

ChromatinStructureandTranscriptionActivationRepression當(dāng)前第70頁\共有105頁\編于星期四\18點(diǎn)HDACinhibitorscaninducecelldifferentiationpossiblythroughinductionofp21andcyclinD1andhasbeentestedascancertreatmentdrugsinclinicaltrials當(dāng)前第71頁\共有105頁\編于星期四\18點(diǎn)HistoneMethylationTwotypes:arginine-

andlysine-specificHMTs.Argcanbemono-anddi-(asymmetricorsymmetric)methylated,whereLyscanbemono-,di-ortri-methylated.Histonemethylationisbelievedtobestableuntilrecently.Turnoverrateofhistonemethylationissimilartothatofhistoneturnover.MethylationdoesnotneutralizepositivechargeonLysandArg.Methylgroupisthoughttobetoosmalltohaveadirecteffectonchromatinstructure.Thefunctionofhistonemethylationisbelievetobemediatedthroughspecificmethylatedhistonebindingproteins(histonecodehypothesis)當(dāng)前第72頁\共有105頁\編于星期四\18點(diǎn)HistoneLysineMethylationTheretypes:Mono-,diandtri-methylationFoundinallfourcorehistonesandlinkerhistones.Multiplesitesoneachhistones.Enzymesappeartobesite-specificInvolvedinallDNAtemplatedprocesses.當(dāng)前第73頁\共有105頁\編于星期四\18點(diǎn)Lysinemethylation當(dāng)前第74頁\共有105頁\編于星期四\18點(diǎn)HistoneLysineMethyltransferases(KMTs)當(dāng)前第75頁\共有105頁\編于星期四\18點(diǎn)HistoneLysineMethylationandTranscriptionH3K4,H3K36andH3K79ActivationRepressionH3K9,H3K27andH4K20當(dāng)前第76頁\共有105頁\編于星期四\18點(diǎn)Howdoeshistonemethylationaffectchromatinfunction?H3-K9methylationcreatesabindingsiteforHP1andHP1isknowntoassociatewithHDACsandinvolvedinheterochromatinformation(whyitisinvolvedinrepression)H3-K4methylationpreventsbindingofcorepressorcomplexNuRDH3-K27methylationrecruitsrepressivepolycomecomplexPRC1.Theunderliningmechanismformanyothermodificationsisnotclear當(dāng)前第77頁\共有105頁\編于星期四\18點(diǎn)ReversibilityofLysineMethylationTheLSD1family(ShiYangGroup)TheJmjcdomainfamily(ZhangYiGroup)Passivedemethylation

1.Histonetailclipping.2.Histonereplacementbyunmodifiedhistonesorhistonevariants.Activedemethylation

當(dāng)前第78頁\共有105頁\編于星期四\18點(diǎn)LysdemethylationbyLSD1KubicekSandJenuweinT.Cell,Vol119,903-906,Dec.29,2004Minireview:ACrackinHistoneLysineMethylationCofactor:FAD(flavin-adeninedinucleotide)Shietal.,Cell2004當(dāng)前第79頁\共有105頁\編于星期四\18點(diǎn)LysdemethylationbyJHDM1Cofactors:Fe(II)anda-ketoglutarateTsukadaetal.,Nature2006當(dāng)前第80頁\共有105頁\編于星期四\18點(diǎn)InterplayBetweenDifferentHistoneModificationsAtthelevelofmodificationAttheleveloffunctionP-S10inhibitsbindingofHP1toH3K9(Me)2/3.Fischleetal.,Nature.2005Dec22;438(7071):1116-22當(dāng)前第81頁\共有105頁\編于星期四\18點(diǎn)

Somethingiswritingthiscode….

andSomethingisreadingthiscode…HistoneCodeHypothesisMultiplehistonemodifications,actingincombinatorialorsequentialfashionononeormultiplehistonetails,specifyuniquedownstreamfunctions.當(dāng)前第82頁\共有105頁\編于星期四\18點(diǎn)HistoneCodeHypothesisWritersErasersReaders/Effectors當(dāng)前第83頁\共有105頁\編于星期四\18點(diǎn)Formore,read:當(dāng)前第84頁\共有105頁\編于星期四\18點(diǎn)WhatistheconnectionbetweenDNAmethylationandhistonemodification?YesorNo?Ifyes,how?當(dāng)前第85頁\共有105頁\編于星期四\18點(diǎn)ApproachesforidentificationandcharacterizationofHMTsEducatedguess+experimentalverification

SUV39H1byReaetal.,Nature2000(RegulationofchromatinstructurebysitespecifichistoneH3methyltransferases)2.BiochemicalpurificationofHMTactivitiesusinginvitroHMTassay.

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

SETdomains(e.g.,G9a)當(dāng)前第86頁\共有105頁\編于星期四\18點(diǎn)1.通過同源順序分析方法尋找組蛋白修飾酶當(dāng)前第87頁\共有105頁\編于星期四\18點(diǎn)順序分析比較當(dāng)前第88頁\共有105頁\編于星期四\18點(diǎn)2.利用體外反應(yīng)檢測組蛋白修飾酶活性當(dāng)前第89頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第90頁\共有105頁\編于星期四\18點(diǎn)3.通過體外酶活檢測結(jié)合蛋白質(zhì)分離純化方法尋找組蛋白修飾酶Wangetal.,2001Science293:853-857當(dāng)前第91頁\共有105頁\編于星期四\18點(diǎn)進(jìn)一步的酶活性鑒定（體外與體內(nèi)實(shí)驗(yàn)）當(dāng)前第92頁\共有105頁\編于星期四\18點(diǎn)4.利用特異組蛋白修飾抗體免疫染色研究細(xì)胞內(nèi)組蛋白修飾當(dāng)前第93頁\共有105頁\編于星期四\18點(diǎn)5.體外利用WesternBlot和特異組蛋白修飾抗體檢測組蛋白修飾當(dāng)前第94頁\共有105頁\編于星期四\18點(diǎn)6.利用質(zhì)譜方法精確分析組蛋白修飾當(dāng)前第95頁\共有105頁\編于星期四\18點(diǎn)當(dāng)前第96頁\共有105頁\編于星期四\18點(diǎn)Genome-widestructuralorganizationofPICsChIP-exoapproachtoexaminethepreciselocationof6045PICsinyeastgenome.PICswerepositionedwithinpromotersandexcludedfromcodingregions.Rhee&PughNature2013當(dāng)前第97頁\共有105頁\編于星期四\18點(diǎn)四、體外染色質(zhì)重組

當(dāng)前第98頁\共有105頁\編于星期四\18點(diǎn)ThemajorityofchromatinassemblyiscoupledtoDNAreplicationinthecellIncorporationoftheH3-H4tetramerbyhistonechaperonesAdditionoftwoH2A-H2Bdimerst