植物表觀遺傳學(xué)PPT課件

1、植物表觀遺傳學(xué) Epigenetics（表觀遺傳學(xué)）:是指以不涉及到 DNA序列的改變、但可以通過有絲分裂和減數(shù)分 裂進(jìn)行遺傳的生物現(xiàn)象。 自然界中的表觀遺傳學(xué)現(xiàn)象： Paramutation 最早的例子來自果蠅的變化。 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 brow

2、n 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 現(xiàn)象 Chandler et al, PMB, 2000 Chandler et al, PMB, 2000 Chandler et al, PMB, 2000 With

3、 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 methylation, more dense chromatin str

4、ucture Low expressionStam 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 the tobacco genome via the Agrobact

5、erium-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 A

6、J. Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 2000 19: 5194-201. 植物甲基化DNADNA分子機(jī)制研究的 遺傳學(xué)方法 擬南芥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

7、-8. 1. 利用DNA甲基化敏感的酶，酶切基因組DNA，檢測甲基化程度的變化 （centromeric repetitive DNA ） WT ddm1 ddm1突變體：整體基因組DNA甲基化與野生型 相比減少了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 Methyltransferase Antisen

8、se-Met1: reduce 32-71% cytosine DNA methylation Anti-Met1 WT DNAmethylation site: CG dinucleotides 2. 轉(zhuǎn)基因法 Finnegan et al, PNAS 1996 93:8449-54 3.3.遺傳學(xué)方法 promoterMarker gene Me Me Me Mutant screening PromoterMarker gene Me Me Me MOM1 promoterMarker gene HOG1, KYP1/SUVH4, CMT3,AGO4 RTS1/ HDA6, DRD1 ,

9、2,3, NRPD1a, DDM1, MET1 - promoterMarker gene Me Me Me 外源沉默基因: 帶有標(biāo)記基因的T-DNA插入；在基因組的某處產(chǎn)生dsRNA, 沉默基因組同源序列。 內(nèi)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 ? DsRNA siRNAs DCL3 24bp TGS: RNA-directe

10、d DNA methylation: Establishing DNA methylation Inverted DNA R Pol II Me MeMe MET1? DRM2 DNA RNA pol IV (NRDP2,NRPD1A) RDR2 Me MeMe CG CNG CHH AGO4DRD1 RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3), RNA POLYMERASE D1 (RPD1) and ARGONAUTE 4 (AGO4) DRM2: DOMAINS REARRANGED METHYLASE Maintenance of CG D

11、NA methylation MET1 HDA6 (HISTONE DEACETYLASE 6) DDM1 Maintenance of CNG methylation CMT3, KYP(KRYPTONITE) /SU(VAR)3-9 HOMOLOG 4 (SUVH4) AGO1 DsRNA AGO1 KYP CMT3 CNG Me CHH Me siRNAs DCL3 24bp TGS: RNA-directed DNA methylation Inverted DNA R Pol II Me MeMe MET1? DRM2 MET1 HDA6 CG Me DDM1 DNA RNA pol

12、 IV RDR2 Me MeMe CG CNG CHH AGO4DRD1 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: hypermethyla

13、tion hypomethylation hypomethylation 23 1 2 3 I top Top, VMiddle, 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, VMiddle, I 1 F2 hypermethylated Low gen

14、e 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 ） (hyper

15、methylated ) Suppressor ago4, cmt3, kyp, 在基因組水平上，DNA甲基化多發(fā)現(xiàn)于位于著絲粒 及附近的DNA重復(fù)區(qū)、轉(zhuǎn)座子。擬南芥多于5% 的表達(dá) 基因，其啟動子區(qū)域有DNA甲基化,大約1/3以上的基因 在編碼區(qū)有DNA甲基化,但生物學(xué)意義不清楚。一般編 碼區(qū)甲基化程度高的地方，基因轉(zhuǎn)錄水平也高。但啟動 子區(qū)域甲基化高的基因，轉(zhuǎn)錄水平較低，且多表現(xiàn)基因 表達(dá)的組織特異性。 擬南芥基因組水平上的甲基化 組蛋白修飾 Histone modifications Nucleosomes are complexes of histones H2A is yellow;

16、 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

17、 - 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 present Acetyl Methyl Phosp

18、horyl Ubiquitin 常見化學(xué)修飾基團(tuán) De/Acetylation Methylation Phosphorylation Ubiquitination ADP-Rybosilation Swi/Snf complex, which, in vitro, uses the energy of ATP hydrolysis to disrupt histone-DNA interactions 組蛋白修飾 組蛋白修飾作用 Transcription Acetylation/Methylation DNA repair H2A -Phosphorylation Mitosis chro

19、mosomal arrangement Chromatin assembly DNA replication 組蛋白修飾組蛋白修飾: H3, H4 組蛋白修飾: H2A, H2B FCAT A) 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 hypoacet

20、ylated, 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 294(5543):853-8. Lau NC, Lim LP, Weinstein EG, Bart

21、el 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, Rhoa

22、des 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-asso

23、ciated 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

24、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. Conservatio n between the Arabidopsis and Oryza predicted stem-loop precursors. MicroRNAs trans-acting siRNAs nat-siRNA

25、s 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), 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

26、; 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 (Zilber

27、man 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

28、 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

29、et al., 2001); and MRE11 (meiotic recombination 11 ) Genes that regulate TGS without changing DNA methylation RD29A-LUC Before stressAfter stress(B) dsRNA siRNA RD29A Endogenous RD29A 24 bp LUCRD29ANPTIILUCRD29ANPTII ColdABA NaCl ros1-1ros1-2 WT ColdABA NaCl ros1-1ros1-2 WT Repressor Of Silencing 1

30、ROS1: LUC NPTII RD29A COR47 WTros1-1 rDNA ros1ros1突變體基因沉默發(fā)生突變體基因沉默發(fā)生 在轉(zhuǎn)錄水平在轉(zhuǎn)錄水平 Cell. 2002, 111:803-14. BstUI LUCRD29A 5 kb 8 kb 12 kb WT ros1-1 MluI WT ros1-1 6 kb 8 kb BstUI LUCRD29A 5 kb 8 kb 12 kb WT ros1-1 MluI 5 kb 8 kb 12 kb WT ros1-1 MluI WT ros1-1 6 kb 8 kb WT ros1-1 6 kb 8 kb WT ros1-1 6 k

31、b 8 kb RD29A hypermethylation in ros1 mutant LUCRD29ANPTIILUCRD29ANPTII CH3 CH3CH3 CH3 RD29A Endogenous RD29A CH3 CH3 BstUI LUCRD29A 5 kb 8 kb 12 kb WT ros1-1 MluI WT ros1-1 6 kb 8 kb BstUI LUCRD29A 5 kb 8 kb 12 kb WT ros1-1 MluI 5 kb 8 kb 12 kb WT ros1-1 MluI WT ros1-1 6 kb 8 kb WT ros1-1 6 kb 8 kb

32、 WT ros1-1 6 kb 8 kb Cell. 2002, 111:803-14. WT ros1-1 (plus RD29A-LUC) ros1-1 (minus RD29A-LUC) 23 bp tRNA and 5S rRNA B WT ros1-1 (plus RD29A-LUC) ros1-1 (minus RD29A-LUC) 23 bp tRNA and 5S rRNA WT ros1-1 (plus RD29A-LUC) ros1-1 (minus RD29A-LUC) 23 bp tRNA and 5S rRNA WT (minus RD29A-LUC) ros1-1

33、(minus RD29A-LUC) RD29A C 6 kb6 kb 8 kb8 kb 23 bp WT ros1-1 23 bp WT ros1-1 tRNA and 5S rRNA A 23 bp WT ros1-1 23 bp WT ros1-1 tRNA and 5S rRNA T-DNAT-DNA產(chǎn)生的小產(chǎn)生的小RNARNA可能是引起可能是引起 DNADNA甲基化的信號分子甲基化的信號分子 Cell. 2002, 111:803-14. C FES motif HhH-GPD domain MUTYH 157 QDLASASLEEVNQLWAGLGYYS-RGRRLQEGARKVVE

34、ELGG-HMPRTAETLQ MUTY 32 TDLANAPLDEVLHLWTGLGYYA-RARNLHKAAQQVATLHGG-KFPETFEEVA 2ABK 31 AAMLELGVEGVKTYIKTIGLYNSKAENIIKTCRILLEQHNG-EVPEDRAALE D75275 81 DAITLAPTDAVAHAIRRSNYPESKAPRIQETLRRIKAAPGGYDLDFLRDLPVKDALKWLT ROS1 886 KAIRAADVKEVAETIKSRGMNHKLAERIQGFLDRLVNDHGSIDLEWLRDVPPDKAKEYLL NTG2 159 DGLLKIDEPVLA

35、NLIRCVSFYTRKANFIKRTAQLLVDNFDS-DIPYDIEGIL MUTYH 207 QLLPGVGRYTAGAIASIAFGQAT-GVVDGNVARVLCRVRAIGADPSSTLVSQQLWGLA- MUTY 82 ALP-GVGRSTAGAILSLSLGKHF-PILDGNVKRVLARCYAVSGWPGKKEVENKLWSLS- 2ABK 82 ALP-GVGRKTANVVLNTAFGWPT-IAVDTHIFRVCNRTQFAPGKNVEQVEEKLLK- D75275 141 DLP-GVGVKTASLVLLFNYARPV-FPVDTHVHRVSTRVGVIPRMG

36、EQAAHRALLALLP- ROS1 946 SFN-GLGLKSVECVRLLTLHHLA-FPVDTNVGRIAVRLGWVPLQPLPESLQLHLLEMYPM NTG2 210 SLP-GVGPKMGYLTLQKGWGLIAGICVDVHVHRLCKMWNWVDPIKCKTAEHTRKELQV- MUTYH 264 -QQLVDPARPGDFNQAAMELGATVCTPQRPLCSQCPVESLCRAR MUTY 138 -EQVTPAVGVERFNQAMMDLGAMICTRSKPKCSLCPLQNGCIAA 2ABK 135 -VVPAEFKVDCHHWLILHGRYTCIARKP

37、RCGSCIIEDLCEYK D75275 197 -PDPPYLYELHINFLSHGRQVCTWTRPKCGKCILRERCD- ROS1 1004 LESIQKYLWPRLCKLDQKTLYELHYQMITFGKVFCTKSKPNCNACPMKGECRHF NTG2 267 -WLPHSLWYEINTVLVGFGQLICMARGKRCDLCLANDVCNAR ROS1ROS1基因編碼一個具雙重活性基因編碼一個具雙重活性 的的DNADNA修復(fù)酶修復(fù)酶 Cell. 2002, 111:803-14. 12 200 116 97 66 45 31 21 34 Average Nicks/Mol

38、ecule Protein (pmol) 0.00 0.05 0.10 0.15 0.20 0510152025 Unmethylated Sss I Msp I Unmethylated mCCGG GGCCm Msp I mCG GCm Sss I CC OC A B C ROS1 was able to introduce strand breaks to an MspI-methylated DNA template Cell. 2002, 111:803-14. Kapoor et al., FEBS Lett. 2005 Sep 12; 3 5 ROS1 Working model dsRNA siRNA RD29A Endogenous RD29A RD29A CH3 CH3 CH3 CH3CH3CH3 RD29A demethylation ROS1 LUCRD29ANPTIILUCRD29ANPTII