植物表观遗传学ppt课件.ppt

植物表观遗传学,巩志忠中国农业大学生物学院Tel: 6273-3733Email: ,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。,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.,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,Chandler et al, PMB, 2000,Chandler et al, PMB, 2000,Chandler et al, PMB, 2000,With activating mutator,Paramutation 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 IV,In B-I: more methylation, but more open chromatin structure,High expression,In B-P: less methylation, more dense chromatin structure,Low expression,Stam et al., 2002, Gene & Dev,853-bp repeats,Henderson IR, Jacobsen SE. Nature, 2007447:418-24,目前表观遗传学研究概况,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,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,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-helix structure,CH3,植物DNA甲基化的形式,CG,CH3,动植物共有,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,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,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.,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.,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 ）,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,Kakutani et al PNAS, 1996, 93: 12406-12411,DDM1 encodes a SWI2/SNF2-like protein,Jeddeloh et al.Nature Genetics 22 :94-971999,Mouse: Lsh,MET1/DDM2: Cytosine MethyltransferaseAntisense-Met1: reduce 32-71% cytosine DNA methylation,Anti-Met1,WT,DNAmethylation site:CG dinucleotides,2. 转基因法,Finnegan et al, PNAS 1996 93:8449-54,3.遗传学方法,Mutant screening,Promoter,Marker gene,Me,Me,Me,MOM1,promoter,Marker gene,HOG1, KYP1/SUVH4, CMT3,AGO4RTS1/ HDA6, DRD1 ,2,3, NRPD1a, DDM1, MET1 -,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,Chan et al., Nat Rev Genet. 2005 6:351-60,DOMAINS REARRANGED METHYLASE,Four classes of DNA methyltransferase in Arabidopsis thaliana,?,TGS: RNA-directed DNA methylation:Establishing DNA methylation,Inverted DNA R,Pol II,DNA,RNA pol IV (NRDP2,NRPD1A)RDR2,Me,Me,Me,RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3),RNA POLYMERASE D1 (RPD1) and ARGONAUTE 4 (AGO4)DRM2: DOMAINS REARRANGED METHYLASE,Maintenance of CG DNA methylation,MET1HDA6 (HISTONE DEACETYLASE 6)DDM1,Maintenance of CNG methylation,CMT3,KYP(KRYPTONITE) /SU(VAR)3-9 HOMOLOG 4(SUVH4)AGO1,CHH,Me,TGS: RNA-directed DNA methylation,DRM2,Specific DNA methylation loci in Arabidopsis,Chan et al., Nat Rev Genet. 2005 6:351-60.,(pathogen related),Wassilewskija strain,Arabidopsis,tryptophan enzyme phosphoribosylanthranilate isomerase (PAI),S15a promoter+ first exon,PAI1-4: 350bp+ORF: hypermethylation,hypomethylation,hypomethylation,2,3,1,2,3,I top,Top, V,Middle, I,Col hypomethylation,F1,PAI2 gene is silenced,X,PAI3, no activity,Melquist S, Bender J. Genetics. 2004 166:437-48.,Genes Dev. 2003 17:2036-47.,ATG,350 bp,TAG,WS, hypermethylation,2,3,I top,Top, V,Middle, I,1,F2,hypermethylated,Low gene expression,Some plants revert to hypomethylation status,suvh4/hda6 cmt3,WS pai1,pai1 strain accumulates fluorescent tryptophan pathway intermediates, as well as displaying yellow-green leaf pigmentation, reduced size, increased bushiness, and reduced fertility.,Superman（clark kent alleles ） (hypermethylated ),Suppressor,ago4, cmt3, kyp,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,组蛋白修饰Histone modifications,Overall plant DNA is highly compacted by DNA-protein complexes to 10,000 to 50,000 -fold Even in interphase, DNA is highly structured,Nucleosomes are complexes of histones,H2A is yellow; H2B is red; H3 is blue; H4 is green,The solenoid model of condensed chromatin,146bp DNA wraps the histones,2nm,2 of H2A, H2B, H3 and H4,40-70 bp,About 200bp for each bead,700 fold compacted,180 to 300 nucleosomes,Each chromatid would account for 1.2 million bp of DNA,chromatin fiber,Heterochromatin Telomeres Centromeres Repetitive DNAgenes 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 present,AcetylMethylPhosphorylUbiquitin,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,组蛋白修饰作用,Transcription Acetylation/MethylationDNA repair H2A -PhosphorylationMitosis chromosomal arrangementChromatin assembly DNA replication,组蛋白修饰: H3, H4,组蛋白修饰: H2A, H2B,FCAT,Methyl-CpG-binding proteins recruit HDAC complex to deacetylate histone so that the histone tails will be suitable for subsequent methylation by HMTs. In chromatin domains where histones are hypoacetylated, the MBD domain-containing HMTs may bind directly and methylate the histones. 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,Chromatin 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.,Histone Modifications and Heterochromatin,ARTKQTARKSTGGKAPRKQLATKAARK-K,9,H3,Heterochromatin,27,36,14,18,23,SDG8: SET Domain Group 8,Me,Me,VRN5,VIN3,VRN1,VRN2,SDG 8,LHP1,FLC,LHP1: 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,siRNA,siRNAs: 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.,MicroRNA (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 plants,Lim et al., Genes Dev. 2003, 17(8):991-1008.,miRNA (red) and miRNA* (blue) sequences within the context of their predicted fold-back precursors.,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 smRNAs,Reinhart 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.,smRNAs from different animals show high homologue,Lim et al., Genes Dev. 2003, 17(8):991-1008.,Conservation between the Arabidopsis and Oryza predicted stem-loop precursors.,MicroRNAstrans-acting siRNAsnat-siRNAsRepeat-associated siRNAs,Trends Plant Sci. 2006 11:460-8.,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.,DRM1 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.,MOM1, 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,dsRNA,siRNA,RD29A,Endogenous RD29A,24 bp,Repressor Of Silencing 1,ROS1:,LUC,NPTII,RD29A,COR47,WT,ros1-1,rDNA,ros1突变体基因沉默发生在转录水平,Cell. 2002, 111:803-14.,Cell. 2002, 111:803-14.,T-DNA产生的小RNA可能是引起DNA甲基化的信号分子,Cell. 2002, 111:803-14.,ROS1基因编码一个具双重活性的DNA修复酶,Cell. 2002, 111:803-14.,A,B,C,ROS1 was able to introduce strand breaks to anMspI-methylated DNA template,Cell. 2002, 111:803-14.,Kapoor et al., FEBS Lett. 2005 Sep 12;,3,5,ROS1 Working model,dsRNA,siRNA,Endogenous RD29A,CH3,CH3,CH3,CH3,CH3,CH3,demethylation,ROS1,