植物表观遗传学--课件

植物表观遗传学植物表观遗传学植物表观遗传学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。Epigenetics（表观遗传学）:是指以不涉及到DNA序自然界中的表观遗传学现象：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).Types of visible variations induced by x-rays in Drosophila.J Genet.22,299334.Hinton,T.,and Goodsmith,W.(1950).An analysis of phenotypic reversions at the brown locus in Drosophila.J.Exp.Zool.114,103114.自然界中的表观遗传学现象：Paramutation has been extensively characterized at three maize loci:r1,b1（helixloop-helix(bHLH)factors）,and pl1(myb)Brink(1956,1958)首先描述了r1基因的 Paramutation 现象Paramutation has been extensivChandler et al,PMB,2000Chandler et al,Chandler et al,PMB,2000Chandler et al,Chandler et al,PMB,2000With activating mutatorChandler et al,With activatinParamutation 是由基因控制的Dorweiler et al,Plant cell,2000Alleman et al.,Nature,2006,442:295-8.mop1:mediator of paramutation1，RNA dependent RNA Pol IVParamutation 是由基因控制的Dorweiler In B-I:more methylation,but more open chromatin structure High expression In B-P:less methylation,more dense chromatin structureLow expressionStam et al.,2002,Gene&Dev853-bp repeats In B-I:more methylation,but Henderson IR,Jacobsen SE.Nature,2007447:418-24 Henderson IR,Jacobsen SE.Nat目前表观遗传学研究概况目前表观遗传学研究概况目前表观遗传学研究概况拟南芥作为模式植物的优点:1.个体小，易于管理2.生长周期短，种子量大3.易于转化，进行基因功能研究4.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: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：植物植物DNADNA甲基化的分子机制甲基化的分子机制植物DNA甲基化的分子机制A DNA molecule consists of two strands,each strand=polynucleotide.Strands held together by hydrogen bonds between complementary nucleotide pairs:Adenine with Thymine,Cytosine with Guanine.double-helixstructureCH3A DNA molecule consists of two植物植物DNADNA甲基化的形式甲基化的形式CGCH3CNGCH3CHH(C/A/T)CH3动植物共有植物DNA甲基化的形式CGCH3CNGCH3CHH(C/ADNA甲基化的生物学意义：1.调控转座子的活性，保护基因组DNA2.调控基因的表达DNA甲基化的生物学意义：如何研究植物的如何研究植物的DNADNA甲基化甲基化1.DNA甲基化敏感的限制性内切酶2.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 3.HPLC：整体基因组甲基化水平,4.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RNA介导的DNA甲基化最初发现Wassenegger M,Heimes S,Riedel L,Snger HL.RNA-directed de novo methylation of genomic sequences in plants Cell.1994 Feb 11;76(3):567-76 Max-Planck-Institut fr Biochemie,Abteilung Viroidforschung,Martinsried,Federal Republic of Germany.One monomeric and three oligomeric potato spindle tuber viroid(PSTVd)cDNA units were introduced into the tobacco genome via the Agrobacterium-mediated leaf-disc transformation.RNA介导的DNA甲基化最初发现Wassenegger M,Mette MF,van der Winden J,Matzke MA,Matzke AJ.Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans.EMBO J.1999 18:241-8.Mette MF,Aufsatz W,van der Winden J,Matzke MA,Matzke AJ.Transcriptional silencing and promoter methylation triggered by double-stranded RNA.EMBO J.2000 19:5194-201.Mette MF,van der Winden J,Ma植物甲基化植物甲基化DNADNA分子机制研究的分子机制研究的遗传学方法遗传学方法植物甲基化DNA分子机制研究的遗传学方法拟南芥DDM1（decrease in DNA methylation 1)基因Vongs A,Kakutani T,Martienssen RA,Richards EJ.Arabidopsis thaliana DNA methylation mutants.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（centromeric repetitive DNA）拟南芥DDM1（decrease in DNA methylWTddm1ddm1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Kakutani et al PNAS,1996,93:12406-12411Kakutani et al PNAS,1996,93:DDM1 encodes a SWI2/SNF2-like proteinJeddeloh et al.Nature Genetics 22:94-971999Mouse:LshDDM1 encodes a SWI2/SNF2-like MET1/DDM2:Cytosine MethyltransferaseAntisense-Met1:reduce 32-71%cytosine DNA methylation Anti-Met1 WTDNAmethylation site:CG dinucleotides 2.转基因法MET1/DDM2:Cytosine MethyltranFinnegan et al,PNAS 1996 93:8449-54 Finnegan et al,PNAS 1996 93:83.3.遗传学方法遗传学方法promoterMarker geneMe Me MeMutant screeningPromoterMarker geneMe Me MeMOM1promoterMarker geneHOG1,KYP1/SUVH4,CMT3,AGO4RTS1/HDA6,DRD1,2,3,NRPD1a,DDM1,MET1-3.遗传学方法promoterMarker geneMeMepromoterMarker geneMe Me Me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,SupermanpromoterMarker geneMeMeMe外源沉默基Chan et al.,Nat Rev Genet.2005 6:351-60DOMAINS REARRANGED METHYLASE Four classes of DNA methyltransferase in Arabidopsis thaliana?Chan et al.,Nat Rev Genet.20DsRNAsiRNAsDCL324bpTGS:RNA-directed DNA methylation:Establishing DNA methylationInverted DNA RPol IIMe MeMeMET1?DRM2DNARNA pol IV(NRDP2,NRPD1A)RDR2Me MeMeCG CNG CHHAGO4DRD1RNA POLYMERASE 2(RDR2),DICER-LIKE 3(DCL3),RNA POLYMERASE D1(RPD1)and ARGONAUTE 4(AGO4)DRM2:DOMAINS REARRANGED METHYLASEDsRNAsiRNAsDCL324bpTGS:RNA-dMaintenance of CG DNA methylationMET1HDA6(HISTONE DEACETYLASE 6)DDM1Maintenance of CNG methylationCMT3,KYP(KRYPTONITE)/SU(VAR)3-9 HOMOLOG 4(SUVH4)AGO1Maintenance of CG DNA methylatDsRNAAGO1KYPCMT3CNGMeCHHMesiRNAsDCL324bpTGS:RNA-directed DNA methylationInverted DNA RPol IIMe MeMeMET1?DRM2MET1HDA6CGMeDDM1DNARNA pol IVRDR2Me MeMeCG CNG CHHAGO4DRD1DRM2DsRNAAGO1KYPCMT3CNGMeCHHMesiRNSpecific DNA methylation loci in Arabidopsis Chan et al.,Nat Rev Genet.2005 6:351-60.(pathogen related)Specific DNA methylation loci Wassilewskija strainArabidopsis tryptophan enzyme phosphoribosylanthranilate isomerase(PAI)S15a promoter+first exonPAI1-4:350bp+ORF:hypermethylationhypomethylationhypomethylationWassilewskija strainArabidopsi23123I topTop,VMiddle,ICol hypomethylationF1PAI2 gene is silencedXPAI3,no activityMelquist S,Bender J.Genetics.2004 166:437-48.Genes Dev.2003 17:2036-47.ATG350 bpTAGWS,hypermethylation23123I topTop,VMiddle,ICol h23I topTop,VMiddle,I1F2hypermethylatedLow gene expressionSome plants revert to hypomethylation status23I topTop,VMiddle,I1F2hypersuvh4/hda6 cmt3WS pai1pai1 strain accumulates fluorescent tryptophan pathway intermediates,as well as displaying yellow-green leaf pigmentation,reduced size,increased bushiness,and reduced fertility.suvh4/hda6 WS pai1pai1 strain Superman（clark kent alleles）(hypermethylated)Suppressorago4,cmt3,kyp,Superman（clark kent alleles）植物表观遗传学-课件 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组蛋白修饰Histone modifications组蛋白修饰Overall plant DNA is highly compacted by DNA-protein complexes to 10,000 to 50,000-fold Even in interphase,DNA is highly structuredOverall plant DNA is highlyNucleosomes are complexes of histonesH2A is yellow;H2B is red;H3 is blue;H4 is greenNucleosomes are complexes of hThe solenoid model of condensed chromatin146bp DNA wraps the histones2nm2 of H2A,H2B,H3 and H4 40-70 bp About 200bp for each bead 700 fold compacted180 to 300 nucleosomes Each chromatid would account for 1.2 million bp of DNA chromatin fiberThe solenoid model of condenseHeterochromatin-Telomeres-Centromeres-Repetitive DNA-genes-N-termini of histones are not(=hypo)acetylated-DNA is methylated(mammals and plants)Euchromatin-active and inactive genes-in transcribed regions,histones are(hyper)acetylated and DNase I sensitive sites are presentHeterochromatinAcetylMethylPhosphorylUbiquitin常见化学修饰基团Acetyl常见化学修饰基团De/AcetylationMethylationPhosphorylationUbiquitinationADP-RybosilationSwi/Snf complex,which,in vitro,uses the energy of ATP hydrolysis to disrupt histone-DNA interactions 组蛋白修饰De/Acetylation组蛋白修饰组蛋白修饰作用Transcription Acetylation/MethylationDNA repair H2A-PhosphorylationMitosis chromosomal arrangementChromatin assembly DNA replication组蛋白修饰作用Transcription Acetyla植物表观遗传学-课件组蛋白修饰组蛋白修饰:H3,H4组蛋白修饰:H3,H4组蛋白修饰:H2A,H2B组蛋白修饰:H2A,H2BFCATFCATA)Methyl-CpG-binding proteins recruit HDAC complex to deacetylate histone so that the histone tails will be suitable for subsequent methylation by HMTs.B)In chromatin domains where histones are hypoacetylated,the MBD domain-containing HMTs may bind directly and methylate the histones.C)Methylated histone tails may recruit DNMTs to methylate DNA for long-term gene silencing.DNA methylation,histone deacetylation,and histone methylation Genes&Dev 15,2343-2360 Methyl-CpG-binding proteins reChromatin influences nuclear processes from replication,recombination and repair to transcriptional control.Regulation of the organization of DNA and the histone octamers into nucleosomes,as well as regulation of the higher-order condensation of chromatin,not only plays a role in transcriptional activation and repression but is also required for stable silencing and differentiation.Chromatin influences nuclear p植物表观遗传学-课件植物表观遗传学-课件Histone Modifications and HeterochromatinMeARTARTK KQTARQTARK KSTGGSTGGK KAPRAPRK KQLATQLATK KAARAARK K-K KMe9H3Heterochromatin27MeMe36141823SDG8:SET Domain Group 8MeMeVRN5VIN3VRN1VRN2SDG 8LHP1FLCLHP1:LIKE HETEROCHROMATIN PROTEIN 1VRN1:VERNALIZATION1,VRN2:A Polycomb group proteinVIN3:VERNALIZATION INSENSITIVE 3,A PHD Finger Protein VRN5:Homolog of VIN3,A PHD Finger Protein Histone Modifications and HetesiRNAsiRNAsiRNAs:produced by cleaving dsRNAs(double-stranded RNA)that are resulted from transposons,viruses,or endogenous genes that express long dsRNA or when dsRNA is introduced experimentally into plant or animal cells.miRNAs:the products of endogenous,noncoding genes whose precursor RNA transcripts can form small stem loops from which mature miRNAs are cleaved by Dicer.miRNAs are encoded in genes distinct from the mRNAs whose expression they control.The expression of some miRNAs is developmentally controlled.siRNAs:produced by cleaving dMicroRNA(miRNA):“dont ignore the little guys”-noncoding RNAs-first example of miRNAs described in C.elegans(lin-4)in 1993(Lee et al.,Cell,1993,75:843-54)21 nt RNAs(let-7,Nature.2000,403:901-6.)that control developmental timing by binding to mRNA targets and possibly attenuate translation-Later found to be subgroup of large class of miRNAs:21-24-nt long,noncoding,found throughout metazoans and also recently in plantsMicroRNA(miRNA):“dont ignorLim et al.,Genes Dev.2003,17(8):991-1008.miRNA(red)and miRNA*(blue)sequences within the context of their predicted fold-back precursors.Lim et al.,Genes Dev.2003,Lagos-Quintana M,Rauhut R,Lendeckel W,Tuschl T.Identification of novel genes coding for small expressed RNAs.Science.2001 Oct 26;294(5543):853-8.Lau NC,Lim LP,Weinstein EG,Bartel DP.An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.Science.2001 Oct 26;294(5543):858-62.Lee RC,Ambros V.An extensive class of small RNAs in Caenorhabditis elegans.Science.2001 Oct 26;294(5543):862-4.Cloning of smRNAsLagos-Quintana M,Rauhut R,LeReinhart BJ,Weinstein EG,Rhoades MW,Bartel B,Bartel DP.MicroRNAs in plants.Genes Dev.2002 Jul 1;16(13):1616-26.Rhoades MW,Reinhart BJ,Lim LP,Burge CB,Bartel B,Bartel DP.Prediction of plant microRNA targets.Cell.2002 Aug 23;110(4):513-20.Llave C,Kasschau KD,Rector MA,Carrington JC.Endogenous and silencing-associated small RNAs in plants.Plant Cell.2002 Jul;14(7):1605-19 Park W,Li J,Song R,Messing J,Chen X.CARPEL FACTORY,a Dicer homolog,and HEN1,a novel protein,act in microRNA metabolism in Arabidopsis thaliana.Curr Biol.2002 Sep 3;12(17):1484-95.Tang G,Reinhart BJ,Bartel DP,Zamore PD.A biochemical framework for RNA silencing in plants.Genes Dev.2003 Jan 1;17(1):49-63.Reinhart BJ,Weinstein EG,RhosmRNAs from different animals show high homologueLim et al.,Genes Dev.2003,17(8):991-1008.smRNAs from different animals Conservation between the Arabidopsis and Oryza predicted stem-loop precursors.Conservation between the ArabiMicroRNAstrans-acting siRNAsnat-siRNAsRepeat-associated siRNAsMicroRNAsTrends Plant Sci.2006 11:460-8.Trends Plant Sci.2006 11:460-DDM1(Vongs et al.,1993;Jeddeloh et al.,1999;Morel et al.,2000;Scheid et al.,2002),MET1(Vongs et al.,1993;Morel et al.,2000),CMT3(Bartee et al.,2001;Lindroth et al.,2001;Tompa et al.,2002),KYP1/SUVH4(Jackson et al.,2002;Malagnac et al.,2002),SUVH2(Naumann et al.,2005)and HOG1(Rocha et al.,2005)(our unpublished results),Gene that affects DNA methylation at the whole genome level.DDM1(Vongs et al.,1993;JeddDRM1 and DRM2(Cao and Jacobsen,2002),HDA6(Aufsatz et al.,2002a;ProBst et al.,2004),DCL3(Xie et al.,2004),AGO4(Zilberman et al.,2003;Zilberman et al.,2004),DRD1(Kanno et al.,2004),NRPD1b/DRD3 and NRPD2a/DRD2(Herr et al.,2005;Kanno et al.,2005;Onodera et al.,2005),Genes that affect DNA methylation only in some specific regions of the genome.DRM1 and DRM2(Cao and JacobseMOM1,which encodes a protein with limited similarity to the SWI2/SNF2 family of proteins,affects TGS probably through chromatin remodeling(Amedeo et al.,2000;Scheid et al.,2002;Tariq et al.,2002).BRU1(a DNA repair-related protein)(Takeda et al.,2004);FAS1 and FAS2(subunits of chromatin assembly factor CAF-1 complex the condensing complex(Kaya et al.,2001);and MRE11(meiotic recombination 11)Genes that regulate TGS without changing DNA methylation MOM1,which encodes a protein RD29A-LUCBefore stressAfter stress(B)dsRNAsiRNARD29AEndogenous RD29A24 bpLUCRD29ANPTIILUCRD29ANPTIIRD29A-LUCBefore stressAfter st植物表观遗传学-课件Repressor Of Silencing 1ROS1:Repressor Of Silencing 1ROS1:LUCNPTIIRD29ACOR47WTros1-1rDNAros1ros1突变体基因沉默发生突变体基因沉默发生在转录水平在转录水平Cell.2002,111:803-14.LUCNPTIIRD29ACOR47WTros1-1rDNARD29A hypermethylation in ros1 mutantLUCRD29ANPTIILUCRD29ANPTIICH3 CH3CH3 CH3RD29AEndogenous RD29ACH3 CH3Cell.2002,111:803-14.RD29A hypermethylation LUCRD29WTros1-1(plus RD29A-LUC)ros1-1(minus RD29A-LUC)23 bptRNA and 5S rRNABWTros1-1(plus RD29A-LUC)ros1-1(minus RD29A-LUC)23 bptRNA and 5S rRNAWTros1-1(plus RD29A-LUC)ros1-1(minus RD29A-LUC)23 bptRNA and 5S rRNAWT(minus RD29A-LUC)ros1-1(minus RD29A-LUC)RD29AC6 kb6 kb8 kb8 kb23 bpWTros1-123 bpWTros1-1tRNA and 5S rRNAA23 bpWTros1-123 bpWTros1-1tRNA and 5S rRNAT-DNAT-DNA产生的小产生的小RNARNA可能是引起可能是引起DNADNA甲基化的信号分子甲基化的信号分子Cell.2002,111:803-14.WTros1-1(plus RD29A-LUC)ros1-ROS1ROS1基因编码一个具双重活性基因编码一个具双重活性的的DNADNA修复酶修复酶Cell.2002,111:803-14.ROS1基因编码一个具双重活性Cell.2002,11112200116976645312134Average Nicks/MoleculeProtein(pmol)0.000.050.100.150.200510152025UnmethylatedSss IMsp IUnmethylatedmCCGG GGCCmMsp ImCG GCmSss ICCOCABCROS1 was able to introduce strand breaks to anMspI-methylated DNA templateCell.2002,111:803-14.12200116976645312134Average NiKapoor et al.,FEBS Lett.2005 Sep 12;35Kapoor et al.,FEBS Lett.2005ROS1 Working modeldsRNAsiRNARD29AEndogenous RD29ARD29ACH3CH3CH3CH3CH3CH3RD29AdemethylationROS1LUCRD29ANPTIILUCRD29ANPTIILUCRD29ANPTIILUCRD29ANPTIILUCRD29ANPTIILUCRD29ANPTIIROS1 Working modeldsRNAsiRNARD