植物表观遗传学

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, 299 334. Hinton, T., and Goodsmith, W. (1950). An analysis of phenotypic reversions at the brown locus in Drosophila. J. Exp. Zool. 114, 103 114. 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, 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 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 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 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 promoter Marker gene Me Me Me Mutant screening Promoter Marker gene Me Me Me MOM1 promoter Marker gene HOG1, KYP1/SUVH4, CMT3,AGO4 RTS1/ HDA6, DRD1 ,2,3, NRPD1a, DDM1, MET1 - promoter Marker gene Me 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, 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-directed DNA methylation: Establishing DNA methylation Inverted DNA R Pol II Me Me Me MET1? DRM2 DNA RNA pol IV (NRDP2,NRPD1A) RDR2 Me Me Me CG CNG CHH AGO4 DRD1 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, 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 Me Me MET1? DRM2 MET1 HDA6 CG Me DDM1 DNA RNA pol IV RDR2 Me Me Me CG CNG CHH AGO4 DRD1 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 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 Phosphoryl Ubiquitin 常见化学修饰基团 De/Acetylation Methylation Phosphorylation Ubiquitination ADP-Rybosilation Swi/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/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 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 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 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. Conservatio n 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), 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 RD29A-LUC Before stress After stress (B) dsRNA siRNA RD29A Endogenous RD29A 24 bp LUC RD29A NPTII LUC RD29A NPTII Cold ABA NaC l ros1 - 1 ros1 - 2 WT Cold ABA NaC l ros1 - 1 ros1 - 2 WT 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. B st U I LUC R D 2 9 A 5 k b 8 k b 1 2 k b WT ros1 - 1 Ml u I WT ros1 - 1 6 k b 8 k b WT ros1 - 1 WT ros1 - 1 WT ros1 - 1 WT ros1 - 1 WT ros1 - 1 RD29A hypermethylation in ros1 mutant LUC RD29A NPTII LUC RD29A NPTII CH3 CH3 CH3 CH3 RD29A Endogenous RD29A CH3 CH3 B st U I LUC R D 2 9 A 5 k b 8 k b 1 2 k b WT ros1 - 1 Ml u I WT ros1 - 1 6 k b 8 k b WT ros1 - 1 WT ros1 - 1 WT ros1 - 1 WT ros1 - 1 WT ros1 - 1 Cell. 2002, 111:803-14. 23 bp tRNA and 5S rRNA B RD29A C 6 kb 8 kb 23 bp tRNA and 5 S rRNA A 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. C F ES mo t if H h H - GPD d o m a in MUTYH 157 QD L AS A S LE EV NQ L WAG L GYY S - RG R R L Q EG AR KV V E EL GG - H M P RTA E T L Q MUTY 32 TD L AN A P LD EV LH L WTG L GYY A - R A RN L H K A A QQ V ATL HGG - KF P E TF E E V A 2ABK 31 A A M LELG VE G V KTY I K T I G L Y NS KA EN I I K T C R I L LE Q H N G - E V P E DRAA L E D75275 81 D A I TL A PT D A VA HA I R RSN Y PES KA P RIQ E T L R RI KAAP GG YDLDFLRDLP V K D ALKW L T ROS1 886 K A I RA A D V K EVA ET I K SR G MNHKL A E RIQ GFLD R LV ND HG SIDLEWLRDVPPDKAK E Y L L NTG2 159 D G L LKIDEPV L A N LI R C V S F Y TR KA NF I K R TA QL LV D NFDS - D I P YDI E G I L MUTYH 207 Q L LP GVGR Y TA GA I AS IAF G QAT - GV VD GN V A RV LC R V RA I G A D P SSTL V SQQ L WG L A - MUTY 82 A LP - GVGR S TA GA I L S L SL G K HF - PI L D GN V K RV LA R CYA V S G W P GKKE V EN K L WS L S - 2ABK 82 A LP - GVGRKTA N V VL NT AF G WPT - IA VDT H I F RV CN R TQ F A P G KNVEQVEEKL L K - D75275 141 D LP - GVG V KTA S L VL LFN YAR PV - FP VDT H V H RV ST R V GV I P RMGEQAAHRAL L AL L P - ROS1 946 SFN - G L G L K S VEC V RL L TLH H LA - FP VDT N V G R I AV R L G W VP LQ P LPES L QL H L LE M YPM NTG2 210 S LP - GVG P K M G Y L T L QK GW G LIAGIC VD VH V H R L CKMWN W V DPIKCKTAEHT R KELQV - MUTYH 264 - QQ LV DPARPG D FNQAA M EL G AT V CT PQ R P L C SQ CP V ES L C R A R MUTY 138 - EQ V TPAVG V ERFNQA MM DL G A MI CT R S KP K C SL CP L QNG C I A A 2ABK 135 - VV PAEFKV D CHHW LI LH G R YT C IAR KP R C GS C I I E DL C EY K D75275 197 - PDPPY L YE L HINF L SH G R Q V CT W TR P K C GK C I L R E R C D - ROS1 1004 LESIQKYLWPRLCK L DQKT L YE L HYQ MI TF G KV F CT K S KP N C NA CP M KGE C RHF NTG2 267 - W L PHSLW YE I NTV LV GF G Q LI C MARGK R C DL C LAN DV C N A R ROS1基因编码一个具双重活性 的 DNA修复酶 Cell. 2002, 111:803-14. 1 2 200 116 97 66 45 31 21 3 4 Aver age Nicks/Mole cul e Protein (pmol) 0.00 0.05 0.10 0.15 0.20 0 5 10 15 20 25 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 CH3 CH3 CH3 RD29A demethylation ROS1 LUC RD29A NPTII LUC RD29A NPTII LUC RD29A NPTII LUC RD29A NPTII LUC RD29A NPTII LUC RD29A NPTII