植物表观遗传学课件

植物表观遗传学巩志忠中国农业大学生物学院Tel:6273-3733Email:gongzz@

植物表观遗传学巩志忠1Epigenetics（表观遗传学）:是指以不涉及到DNA序列的改变、但可以通过有丝分裂和减数分裂进行遗传的生物现象。Epigenetics（表观遗传学）:是指以不涉及到DNA序2自然界中的表观遗传学现象：Paramutation最早的例子来自果蝇的变化。Muller,H.J.(1930).Typesofvisiblevariationsinducedbyx-raysinDrosophila.JGenet.22,299–334.Hinton,T.,andGoodsmith,W.(1950).AnanalysisofphenotypicreversionsatthebrownlocusinDrosophila.J.Exp.Zool.114,103–114.自然界中的表观遗传学现象：3Paramutationhasbeenextensivelycharacterizedatthreemaizeloci:r1,b1（helixloop-helix(bHLH)factors）,andpl1(myb)Brink(1956,1958)首先描述了r1基因的

Paramutation现象Paramutationhasbeenextensiv4Chandleretal,PMB,2000Chandleretal,5Chandleretal,PMB,2000Chandleretal,6Chandleretal,PMB,2000WithactivatingmutatorChandleretal,Withactivatin7Paramutation是由基因控制的Dorweileretal,Plantcell,2000Allemanetal.,Nature,2006,442:295-8.

mop1:mediatorofparamutation1，RNAdependentRNAPolIVParamutation是由基因控制的Dorweiler8InB-I:moremethylation,butmoreopenchromatinstructureHighexpressionInB-P:lessmethylation,moredensechromatinstructureLowexpressionStametal.,2002,Gene&Dev853-bprepeatsInB-I:moremethylation,but9HendersonIR,JacobsenSE.Nature,2007447:418-24HendersonIR,JacobsenSE.Nat10目前表观遗传学研究概况目前表观遗传学研究概况11拟南芥作为模式植物的优点:个体小，易于管理生长周期短，种子量大易于转化，进行基因功能研究基因组小，重复序列少，完成测序拟南芥作为模式植物的优点:12表观遗传学的分子生物学机制包括：DNA甲基化组蛋白修饰染色质重组RNA干扰表观遗传学的分子生物学机制包括：13植物DNA甲基化的分子机制植物DNA甲基化的分子机制14ADNAmoleculeconsistsoftwostrands,eachstrand=polynucleotide.Strandsheldtogetherbyhydrogenbondsbetweencomplementarynucleotidepairs:AdeninewithThymine,CytosinewithGuanine.double-helixstructureCH3ADNAmoleculeconsistsoftwo15植物DNA甲基化的形式CGCH3CNGCH3CHH(C/A/T)CH3动植物共有植物DNA甲基化的形式CGCH3CNGCH3CHH(C/A16DNA甲基化的生物学意义：调控转座子的活性，保护基因组DNA调控基因的表达DNA甲基化的生物学意义：17如何研究植物的DNA甲基化DNA甲基化敏感的限制性内切酶亚硫酸氢钠测序：重亚硫酸盐使DNA中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶,而甲基化的胞嘧啶保持不变，PCR扩增所需片段,则尿嘧啶全部转化成胸腺嘧啶，最后,对PCR产物进行测序HPLC：整体基因组甲基化水平,免疫化学法：利用特异的5mC抗体，结合整体基因组芯片，测定DNA甲基化区域如何研究植物的DNA甲基化DNA甲基化敏感的限制性内切酶18RNA介导的DNA甲基化最初发现WasseneggerM,HeimesS,RiedelL,SängerHL.RNA-directeddenovomethylationofgenomicsequencesinplantsCell.1994Feb11;76(3):567-76Max-Planck-InstitutfürBiochemie,AbteilungViroidforschung,Martinsried,FederalRepublicofGermany.Onemonomericandthreeoligomericpotatospindletuberviroid(PSTVd)cDNAunitswereintroducedintothetobaccogenomeviatheAgrobacterium-mediatedleaf-disctransformation.RNA介导的DNA甲基化最初发现WasseneggerM,19MetteMF,vanderWindenJ,MatzkeMA,MatzkeAJ.Productionofaberrantpromotertranscriptscontributestomethylationandsilencingofunlinkedhomologouspromotersintrans.EMBOJ.199918:241-8.MetteMF,AufsatzW,vanderWindenJ,MatzkeMA,MatzkeAJ.Transcriptionalsilencingandpromotermethylationtriggeredbydouble-strandedRNA.

EMBOJ.200019:5194-201.

MetteMF,vanderWindenJ,Ma20植物甲基化DNA分子机制研究的遗传学方法植物甲基化DNA分子机制研究的遗传学方法21拟南芥DDM1（decreaseinDNAmethylation1)基因VongsA,KakutaniT,MartienssenRA,Richards

EJ.ArabidopsisthalianaDNAmethylationmutants.Science.1993，260:1926-8.1.利用DNA甲基化敏感的酶，酶切基因组DNA，检测甲基化程度的变化（centromericrepetitiveDNA）拟南芥DDM1（decreaseinDNAmethyl22WTddm1ddm1突变体：整体基因组DNA甲基化与野生型相比减少了70%。rDNAWTddm1ddm1突变体：整体基因组DNA甲基化与野生型相23KakutanietalPNAS,1996,93:12406-12411KakutanietalPNAS,1996,93:24DDM1encodesaSWI2/SNF2-likeproteinJeddelohetal.NatureGenetics22:94-971999Mouse:LshDDM1encodesaSWI2/SNF2-like25MET1/DDM2:CytosineMethyltransferaseAntisense-Met1:reduce32-71%cytosineDNAmethylationAnti-Met1WTDNAmethylationsite:CGdinucleotides2.转基因法MET1/DDM2:CytosineMethyltran26Finneganetal,PNAS199693:8449-54Finneganetal,PNAS199693:8273.遗传学方法promoterMarkergeneMeMeMeMutantscreeningPromoterMarkergeneMeMeMeMOM1promoterMarkergeneHOG1,KYP1/SUVH4,CMT3,AGO4RTS1/HDA6,DRD1,2,3,NRPD1a,DDM1,MET1----

3.遗传学方法promoterMarkergeneMeMe28promoterMarkergeneMeMeMe外源沉默基因:带有标记基因的T-DNA插入；在基因组的某处产生dsRNA,沉默基因组同源序列。内源沉默基因：PAI,SupermanpromoterMarkergeneMeMeMe外源沉默基29Chanetal.,NatRevGenet.20056:351-60DOMAINSREARRANGEDMETHYLASEFourclassesofDNAmethyltransferaseinArabidopsisthaliana???Chanetal.,NatRevGenet.2030DsRNAsiRNAsDCL3~24bpTGS:RNA-directedDNAmethylation:EstablishingDNAmethylationInvertedDNARPolIIMeMeMeMET1?DRM2DNARNApolIV(NRDP2,NRPD1A)RDR2MeMeMeCGCNGCHHAGO4DRD1RNAPOLYMERASE2(RDR2),DICER-LIKE3(DCL3),RNAPOLYMERASED1(RPD1)andARGONAUTE4(AGO4)DRM2:DOMAINSREARRANGEDMETHYLASEDsRNAsiRNAsDCL3~24bpTGS:RNA-d31MaintenanceofCGDNAmethylationMET1HDA6(HISTONEDEACETYLASE6)DDM1MaintenanceofCNGmethylationCMT3,KYP(KRYPTONITE)/SU(VAR)3-9HOMOLOG4(SUVH4)AGO1MaintenanceofCGDNAmethylat32DsRNAAGO1KYPCMT3CNGMeCHHMesiRNAsDCL3~24bpTGS:RNA-directedDNAmethylationInvertedDNARPolIIMeMeMeMET1?DRM2MET1HDA6CGMeDDM1DNARNApolIVRDR2MeMeMeCGCNGCHHAGO4DRD1DRM2DsRNAAGO1KYPCMT3CNGMeCHHMesiRN33SpecificDNAmethylationlociinArabidopsisChanetal.,NatRevGenet.20056:351-60.(pathogenrelated)SpecificDNAmethylationloci34WassilewskijastrainArabidopsistryptophanenzymephosphoribosylanthranilateisomerase(PAI)S15apromoter+firstexonPAI1-4:350bp+ORF:hypermethylationhypomethylationhypomethylationWassilewskijastrainArabidopsi3523123ItopTop,VMiddle,IColhypomethylationF1PAI2geneissilencedXPAI3,noactivityMelquistS,BenderJ.

Genetics.2004166:437-48.GenesDev.200317:2036-47.ATG350bpTAGWS,hypermethylation23123ItopTop,VMiddle,IColh3623ItopTop,VMiddle,I1F2hypermethylatedLowgeneexpressionSomeplantsreverttohypomethylationstatus23ItopTop,VMiddle,I1F2hyper37suvh4/hda6cmt3WSpai1pai1strainaccumulatesfluorescenttryptophanpathwayintermediates,aswellasdisplayingyellow-greenleafpigmentation,reducedsize,increasedbushiness,andreducedfertility.suvh4/hda6WSpai1pai1strain38Superman（clarkkentalleles）(hypermethylated)Suppressorago4,cmt3,kyp,Superman（clarkkentalleles）39植物表观遗传学ppt课件40在基因组水平上，DNA甲基化多发现于位于着丝粒及附近的DNA重复区、转座子。拟南芥多于5%的表达基因，其启动子区域有DNA甲基化,大约1/3以上的基因在编码区有DNA甲基化,但生物学意义不清楚。一般编码区甲基化程度高的地方，基因转录水平也高。但启动子区域甲基化高的基因，转录水平较低，且多表现基因表达的组织特异性。拟南芥基因组水平上的甲基化在基因组水平上，DNA甲基化多发现于位于着丝粒及41组蛋白修饰Histonemodifications组蛋白修饰42Overall–plantDNAishighlycompactedbyDNA-proteincomplexesto10,000to50,000-fold

Evenininterphase,DNAishighlystructuredOverall–plantDNAishighly43NucleosomesarecomplexesofhistonesH2Aisyellow;H2Bisred;H3isblue;H4isgreenNucleosomesarecomplexesofh44Thesolenoidmodelofcondensedchromatin146bpDNAwrapsthehistones2nm2ofH2A,H2B,H3andH440-70bpAbout200bpforeachbead700foldcompacted180to300nucleosomesEachchromatidwouldaccountfor~1.2millionbpofDNAchromatinfiberThesolenoidmodelofcondense45HeterochromatinTelomeresCentromeresRepetitiveDNAgenesN-terminiofhistonesarenot(=hypo)acetylatedDNAismethylated(mammalsandplants)Euchromatinactiveandinactivegenesintranscribedregions,histonesare(hyper)acetylatedandDNaseIsensitivesitesarepresentHeterochromatin46AcetylMethylPhosphorylUbiquitin常见化学修饰基团Acetyl常见化学修饰基团47De/AcetylationMethylationPhosphorylationUbiquitinationADP-RybosilationSwi/Snfcomplex,which,invitro,usestheenergyofATPhydrolysistodisrupthistone-DNAinteractions 组蛋白修饰De/Acetylation组蛋白修饰48组蛋白修饰作用Transcription–Acetylation/MethylationDNArepair–H2A-PhosphorylationMitosis–chromosomalarrangementChromatinassembly–DNAreplication组蛋白修饰作用Transcription–Acetyla49植物表观遗传学ppt课件50组蛋白修饰:H3,H4组蛋白修饰:H3,H451组蛋白修饰:H2A,H2B组蛋白修饰:H2A,H2B52FCATFCAT53Methyl-CpG-bindingproteinsrecruitHDACcomplextodeacetylatehistonesothatthehistonetailswillbesuitableforsubsequentmethylationbyHMTs.Inchromatindomainswherehistonesarehypoacetylated,theMBDdomain-containingHMTsmaybinddirectlyandmethylatethehistones.MethylatedhistonetailsmayrecruitDNMTstomethylateDNAforlong-termgenesilencing.

DNAmethylation,histonedeacetylation,andhistonemethylationGenes&Dev15,2343-2360Methyl-CpG-bindingproteinsre54Chromatininfluencesnuclearprocessesfromreplication,recombinationandrepairtotranscriptionalcontrol.RegulationoftheorganizationofDNAandthehistoneoctamersintonucleosomes,aswellasregulationofthehigher-ordercondensationofchromatin,notonlyplaysaroleintranscriptionalactivationandrepressionbutisalsorequiredforstablesilencinganddifferentiation.Chromatininfluencesnuclearp55植物表观遗传学ppt课件56植物表观遗传学ppt课件57HistoneModificationsandHeterochromatinMeARTKQTARKSTGGKAPRKQLATKAARK---KMe9H3Heterochromatin27MeMe36141823SDG8:SETDomainGroup8MeMeVRN5VIN3VRN1VRN2SDG8LHP1FLCLHP1:LIKEHETEROCHROMATINPROTEIN1VRN1:VERNALIZATION1,VRN2:APolycombgroupproteinVIN3:VERNALIZATIONINSENSITIVE3,

APHDFingerProtein

VRN5:HomologofVIN3,APHDFingerProtein

HistoneModificationsandHete58siRNAsiRNA59siRNAs:producedbycleavingdsRNAs(double-strandedRNA)thatareresultedfromtransposons,viruses,orendogenousgenesthatexpresslongdsRNAorwhendsRNAisintroducedexperimentallyintoplantoranimalcells.miRNAs:theproductsofendogenous,noncodinggeneswhoseprecursorRNAtranscriptscanformsmallstemloopsfromwhichmaturemiRNAsarecleavedbyDicer.miRNAsareencodedingenesdistinctfromthemRNAswhoseexpressiontheycontrol.TheexpressionofsomemiRNAsisdevelopmentallycontrolled.siRNAs:producedbycleavingd60MicroRNA(miRNA):“don’tignorethelittleguys”-noncodingRNAs-firstexampleofmiRNAsdescribedinC.elegans(lin-4)in1993(Leeetal.,Cell,1993,75:843-54)21ntRNAs(let-7,Nature.2000,403:901-6.

)thatcontroldevelopmentaltimingbybindingtomRNAtargetsandpossiblyattenuatetranslation-LaterfoundtobesubgroupoflargeclassofmiRNAs:21-24-ntlong,noncoding,foundthroughoutmetazoansandalsorecentlyinplantsMicroRNA(miRNA):“don’tignor61Limetal.,GenesDev.2003,17(8):991-1008.miRNA(red)andmiRNA*(blue)sequenceswithinthecontextoftheirpredictedfold-backprecursors.Limetal.,GenesDev.2003,62Lagos-QuintanaM,RauhutR,LendeckelW,TuschlT.IdentificationofnovelgenescodingforsmallexpressedRNAs.

Science.2001Oct26;294(5543):853-8.

LauNC,LimLP,WeinsteinEG,BartelDP.AnabundantclassoftinyRNAswithprobableregulatoryrolesinCaenorhabditiselegans.

Science.2001Oct26;294(5543):858-62.

LeeRC,AmbrosV.AnextensiveclassofsmallRNAsinCaenorhabditiselegans.

Science.2001Oct26;294(5543):862-4.

CloningofsmRNAsLagos-QuintanaM,RauhutR,Le63ReinhartBJ,WeinsteinEG,RhoadesMW,BartelB,BartelDP.MicroRNAsinplants.

GenesDev.2002Jul1;16(13):1616-26.RhoadesMW,ReinhartBJ,LimLP,BurgeCB,BartelB,BartelDP.PredictionofplantmicroRNAtargets.

Cell.2002Aug23;110(4):513-20.LlaveC,KasschauKD,RectorMA,CarringtonJC.Endogenousandsilencing-associatedsmallRNAsinplants.

PlantCell.2002Jul;14(7):1605-19ParkW,LiJ,SongR,MessingJ,ChenX.CARPELFACTORY,aDicerhomolog,andHEN1,anovelprotein,actinmicroRNAmetabolisminArabidopsisthaliana.

CurrBiol.2002Sep3;12(17):1484-95.TangG,ReinhartBJ,BartelDP,ZamorePD.

AbiochemicalframeworkforRNAsilencinginplants.

GenesDev.2003Jan1;17(1):49-63.

ReinhartBJ,WeinsteinEG,Rho64smRNAsfromdifferentanimalsshowhighhomologueLimetal.,GenesDev.2003,17(8):991-1008.smRNAsfromdifferentanimals65ConservationbetweentheArabidopsisandOryzapredictedstem-loopprecursors.ConservationbetweentheArabi66MicroRNAstrans-actingsiRNAsnat-siRNAsRepeat-associatedsiRNAsMicroRNAs67TrendsPlantSci.200611:460-8.TrendsPlantSci.200611:460-68DDM1

(Vongsetal.,1993;Jeddelohetal.,1999;Moreletal.,2000;Scheidetal.,2002),

MET1(Vongsetal.,1993;Moreletal.,2000),

CMT3

(Barteeetal.,2001;Lindrothetal.,2001;Tompaetal.,2002),KYP1/SUVH4

(Jacksonetal.,2002;Malagnacetal.,2002),SUVH2(Naumannetal.,2005)andHOG1(Rochaetal.,2005)(ourunpublishedresults),GenethataffectsDNAmethylationatthewholegenomelevel.DDM1(Vongsetal.,1993;Jedd69DRM1andDRM2

(CaoandJacobsen,2002),HDA6(Aufsatzetal.,2002a;ProBstetal.,2004),DCL3(Xieetal.,2004),AGO4

(Zilbermanetal.,2003;Zilbermanetal.,2004),DRD1(Kannoetal.,2004),NRPD1b/DRD3andNRPD2a/DRD2(Herretal.,2005;Kannoetal.,2005;Onoderaetal.,2005),GenesthataffectDNAmethylationonlyinsomespecificregionsofthegenome.DRM1andDRM2(CaoandJacobse70MOM1,whichencodesaproteinwithlimitedsimilaritytotheSWI2/SNF2familyofproteins,affectsTGSprobablythroughchromatinremodeling(Amedeoetal.,2000;Scheidetal.,2002;Tariqetal.,2002).BRU1

(aDNArepair-relatedprotein)(Takedaetal.,2004);FAS1andFAS2(subunitsofchromatinassemblyfactor[CAF-1]complexthecondensingcomplex(Kayaetal.,2001);andMRE11(meioticrecombination11)GenesthatregulateTGSwithoutchangingDNAmethylationMOM1,whichencodesaprotein71RD29A-LUCBeforestressAfterstress(B)dsRNAsiRNARD29AEndogenousRD29A~24bpLUCRD29ANPTIILUCRD29ANPTIIRD29A-LUCBeforestressAfterst72植物表观遗传学ppt课件73RepressorOfSilencing1ROS1:RepressorOfSilencing1ROS1:74LUCNPTIIRD29ACOR47WTros1-1rDNAros1突变体基因沉默发生在转