植物表观遗传学PPT课件.pptVIP

植物表观遗传学 巩志忠中国农业大学生物学院Tel 6273 3733Email gongzz 1 Epigenetics 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 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 3 Paramutationhasbeenextensivelycharacterizedatthreemaizeloci r1 b1 helixloop helix bHLH factors andpl1 myb Brink 1956 1958 首先描述了r1基因的Paramutation现象 4 Chandleretal PMB 2000 5 Chandleretal PMB 2000 6 Chandleretal PMB 2000 Withactivatingmutator 7 Paramutation是由基因控制的 Dorweileretal Plantcell 2000Allemanetal Nature 2006 442 295 8 mop1 mediatorofparamutation1 RNAdependentRNAPolIV 8 InB I moremethylation butmoreopenchromatinstructure Highexpression InB P lessmethylation moredensechromatinstructure Lowexpression Stametal 2002 Gene Dev 853 bprepeats 9 HendersonIR JacobsenSE Nature 2007447 418 24 10 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 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 13 植物DNA甲基化的分子机制 14 ADNAmoleculeconsistsoftwostrands eachstrand polynucleotide Strandsheldtogetherbyhydrogenbondsbetweencomplementarynucleotidepairs AdeninewithThymine CytosinewithGuanine double helixstructure CH3 15 植物DNA甲基化的形式 CG CH3 动植物共有 16 DNA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 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 18 RNA介导的DNA甲基化最初发现 WasseneggerM HeimesS RiedelL S ngerHL RNA directeddenovomethylationofgenomicsequencesinplantsCell 1994Feb11 76 3 567 76Max Planck Institutf rBiochemie AbteilungViroidforschung Martinsried FederalRepublicofGermany Onemonomericandthreeoligomericpotatospindletuberviroid PSTVd cDNAunitswereintroducedintothetobaccogenomeviatheAgrobacterium mediatedleaf disctransformation 19 MetteMF vanderWindenJ MatzkeMA MatzkeAJ Productionofaberrantpromotertranscriptscontributestomethylationandsilencingofunlinkedhomologouspromotersintrans EMBOJ 199918 241 8 MetteMF AufsatzW vanderWindenJ MatzkeMA MatzkeAJ Transcriptionalsilencingandpromotermethylationtriggeredbydouble strandedRNA EMBOJ 200019 5194 201 20 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 RichardsEJ 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 22 WT ddm1 ddm1突变体 整体基因组DNA甲基化与野生型相比减少了70 rDNA 23 KakutanietalPNAS 1996 93 12406 12411 24 DDM1encodesaSWI2 SNF2 likeprotein Jeddelohetal NatureGenetics22 94 971999 Mouse Lsh 25 MET1 DDM2 CytosineMethyltransferaseAntisense Met1 reduce32 71 cytosineDNAmethylation Anti Met1 WT DNAmethylationsite CGdinucleotides 2 转基因法 26 Finneganetal PNAS199693 8449 54 27 3 遗传学方法 Mutantscreening Promoter Markergene Me Me Me MOM1 promoter Markergene HOG1 KYP1 SUVH4 CMT3 AGO4RTS1 HDA6 DRD1 2 3 NRPD1a DDM1 MET1 28 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 Superman 29 Chanetal NatRevGenet 20056 351 60 DOMAINSREARRANGEDMETHYLASE FourclassesofDNAmethyltransferaseinArabidopsisthaliana 30 TGS RNA directedDNAmethylation EstablishingDNAmethylation InvertedDNAR PolII DNA RNApolIV NRDP2 NRPD1A RDR2 Me Me Me RNAPOLYMERASE2 RDR2 DICER LIKE3 DCL3 RNAPOLYMERASED1 RPD1 andARGONAUTE4 AGO4 DRM2 DOMAINSREARRANGEDMETHYLASE 31 MaintenanceofCGDNAmethylation MET1HDA6 HISTONEDEACETYLASE6 DDM1 MaintenanceofCNGmethylation CMT3 KYP KRYPTONITE SU VAR 3 9HOMOLOG4 SUVH4 AGO1 32 CHH Me TGS RNA directedDNAmethylation DRM2 33 SpecificDNAmethylationlociinArabidopsis Chanetal NatRevGenet 20056 351 60 pathogenrelated 34 Wassilewskijastrain Arabidopsis tryptophanenzymephosphoribosylanthranilateisomerase PAI S15apromoter firstexon PAI1 4 350bp ORF hypermethylation hypomethylation hypomethylation 35 2 3 1 2 3 Itop Top V Middle I Colhypomethylation F1 PAI2geneissilenced X PAI3 noactivity MelquistS BenderJ Genetics 2004166 437 48 GenesDev 200317 2036 47 ATG 350bp TAG WS hypermethylation 36 2 3 Itop Top V Middle I 1 F2 hypermethylated Lowgeneexpression Someplantsreverttohypomethylationstatus 37 suvh4 hda6cmt3 WSpai1 pai1strainaccumulatesfluorescenttryptophanpathwayintermediates aswellasdisplayingyellow greenleafpigmentation reducedsize increasedbushiness andreducedfertility 38 Superman clarkkentalleles hypermethylated Suppressor ago4 cmt3 kyp 39 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 41 2020 2 6 42 组蛋白修饰Histonemodifications 43 Overall plantDNAishighlycompactedbyDNA proteincomplexesto10 000to50 000 foldEvenininterphase DNAishighlystructured 44 Nucleosomesarecomplexesofhistones H2Aisyellow H2Bisred H3isblue H4isgreen 45 Thesolenoidmodelofcondensedchromatin 146bpDNAwrapsthehistones 2nm 2ofH2A H2B H3andH4 40 70bp About200bpforeachbead 700foldcompacted 180to300nucleosomes Eachchromatidwouldaccountfor 1 2millionbpofDNA chromatinfiber 46 HeterochromatinTelomeresCentromeresRepetitiveDNAgenesN terminiofhistonesarenot hypo acetylatedDNAismethylated mammalsandplants Euchromatinactiveandinactivegenesintranscribedregions histonesare hyper acetylatedandDNaseIsensitivesitesarepresent 47 AcetylMethylPhosphorylUbiquitin 常见化学修饰基团 48 De AcetylationMethylationPhosphorylationUbiquitinationADP RybosilationSwi Snfcomplex which invitro usestheenergyofATPhydrolysistodisrupthistone DNAinteractions 组蛋白修饰 49 组蛋白修饰作用 Transcription Acetylation MethylationDNArepair H2A PhosphorylationMitosis chromosomalarrangementChromatinassembly DNAreplication 50 51 组蛋白修饰 H3 H4 52 组蛋白修饰 H2A H2B 53 FCAT 54 Methyl CpG bindingproteinsrecruitHDACcomplextodeacetylatehistonesothatthehistonetailswillbesuitableforsubsequentmethylationbyHMTs Inchromatindomainswherehistonesarehypoacetylated theMBDdomain containingHMTsmaybinddirectlyandmethylatethehistones MethylatedhistonetailsmayrecruitDNMTstomethylateDNAforlong termgenesilencing DNAmethylation histonedeacetylation andhistonemethylation Genes Dev15 2343 2360 55 Chromatininfluencesnuclearprocessesfromreplication recombinationandrepairtotranscriptionalcontrol RegulationoftheorganizationofDNAandthehistoneoctamersintonucleosomes aswellasregulationofthehigher ordercondensationofchromatin notonlyplaysaroleintranscriptionalactivationandrepressionbutisalsorequiredforstablesilencinganddifferentiation 56 57 58 HistoneModificationsandHeterochromatin ARTKQTARKSTGGKAPRKQLATKAARK K 9 H3 Heterochromatin 27 36 14 18 23 SDG8 SETDomainGroup8 Me Me VRN5 VIN3 VRN1 VRN2 SDG8 LHP1 FLC LHP1 LIKEHETEROCHROMATINPROTEIN1VRN1 VERNALIZATION1 VRN2 APolycombgroupproteinVIN3 VERNALIZATIONINSENSITIVE3 APHDFingerProteinVRN5 HomologofVIN3 APHDFingerProtein 59 siRNA 60 siRNAs producedbycleavingdsRNAs double strandedRNA thatareresultedfromtransposons viruses orendogenousgenesthatexpresslongdsRNAorwhendsRNAisintroducedexperimentallyintoplantoranimalcells miRNAs theproductsofendogenous noncodinggeneswhoseprecursorRNAtranscriptscanformsmallstemloopsfromwhichmaturemiRNAsarecleavedbyDicer miRNAsareencodedingenesdistinctfromthemRNAswhoseexpressiontheycontrol TheexpressionofsomemiRNAsisdevelopmentallycontrolled 61 MicroRNA 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 foundthroughoutmetazoansandalsorecentlyinplants 62 Limetal GenesDev 2003 17 8 991 1008 miRNA red andmiRNA blue sequenceswithinthecontextoftheirpredictedfold backprecursors 63 Lagos 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 CloningofsmRNAs 64 ReinhartBJ 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 65 smRNAsfromdifferentanimalsshowhighhomologue Limetal GenesDev 2003 17 8 991 1008 66 ConservationbetweentheArabidopsisandOryzapredictedstem loopprecursors 67 MicroRNAstrans actingsiRNAsnat siRNAsRepeat associatedsiRNAs 68 TrendsPlantSci 200611 460 8 69 DDM1 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 70 DRM1andDRM2 CaoandJacobsen 2002 HDA6 Aufsatzetal 2002a ProBstetal 2004 DCL3 Xieetal 2004 AGO4 Zilbermanetal 2003 Zilbermanetal 2004 DRD1 Kannoetal 2004 NRP