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1、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 revers
2、ions 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 ac
3、tivating 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 expr
4、ession,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 to
5、gether 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
6、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
7、 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
8、 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 D
9、NA 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
10、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
11、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,
12、?,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 D
13、EACETYLASE 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 enzy
14、me 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-
15、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,redu
16、ced 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
17、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 th
18、e 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 me
19、thylated(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
20、,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
21、 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-t
22、erm 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
23、 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,S
24、DG8: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
25、 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 c
26、leaved 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,199
27、3,75:843-54)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.
28、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.A
29、n 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,B
30、artel 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 Ce
31、ll.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.Gene
32、s 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
33、(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 unpub
34、lished 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
35、;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;Tari
36、q 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 R
37、D29A,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,
