《植物表观遗传学》PPT课件.ppt

1、植物表观遗传学,巩志忠 中国农业大学生物学院 Tel: 6273-3733 Email: ,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

2、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,

3、2000,Chandler et al, PMB, 2000,With activating mutator,Paramutation 是由基因控制的,Dorweiler et al, Plant cell, 2000 Alleman 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 met

4、hylation, more dense chromatin structure,Low expression,Stam et al., 2002, Gene 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 t

5、he 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

6、 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 mut

7、ants. 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: Cytosin

8、e Methyltransferase Antisense-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,AGO4 RTS1/ HDA6, DRD1 ,2,

9、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,Invert

10、ed 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,MET1 HDA6 (HISTONE DEACETYLASE 6) DDM1,Maintenance of CNG methylation,CMT3, K

11、YP(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),S15

12、a 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, hypermethylatio

13、n,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, an

14、d 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,Ov

15、erall 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

16、 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 DNA genes N-termini of histones are not (=hypo) acetylated DNA is methylated (mamm

17、als and plants) Euchromatin active and inactive genes in transcribed regions, histones are (hyper) acetylated and DNase I sensitive sites are present,Acetyl Methyl Phosphoryl Ubiquitin,常见化学修饰基团,De/Acetylation Methylation Phosphorylation Ubiquitination ADP-Rybosilation Swi/Snf complex, which, in vitr

18、o, 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/Methylation DNA repair H2A -Phosphorylation Mitosis chromosomal arrangement Chromatin assembly DNA replication,组蛋白修饰: H3, H4,组蛋白修饰: H2A, H2B,FCAT,Methyl-CpG-binding proteins recruit HDAC

19、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 D

20、NA for long-term gene silencing.,DNA methylation, histone deacetylation, and histone methylation,Genes 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, Ambr

21、os 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 D

22、P. 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 n

23、ovel 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

24、 Dev. 2003, 17(8):991-1008.,Conservation between the Arabidopsis and Oryza predicted stem-loop precursors.,MicroRNAs trans-acting siRNAs nat-siRNAs Repeat-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), MET

25、1(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 wh

26、ole 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