1、植物表观遗传学植物表观遗传学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：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
2、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, 2000Chandler et al, PMB, 2000Chandler et al, PMB, 2000
3、With activating mutatorParamutation 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 IVIn B-I: more methylation, but more open chromatin structure High expression In B-P: less methylation, more dense chromatin struc
4、tureLow expressionStam et al., 2002, Gene & Dev853-bp repeats Henderson IR, Jacobsen SE. Nature, 2007447:418-24 目前表观遗传学研究概况目前表观遗传学研究概况拟南芥作为模式植物的优点:1.个体小，易于管理2.生长周期短，种子量大3.易于转化，进行基因功能研究4.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干扰植物植物DNADNA甲基化的分子机制甲基化的分子机制A DNA molecule consists of two stran
5、ds, each strand = polynucleotide.Strands held together by hydrogen bonds between complementary nucleotide pairs: Adenine with Thymine, Cytosine with Guanine.double-helix structureCH3植物植物DNADNA甲基化的形式甲基化的形式CGCH3CNGCH3CHH (C/A/T)CH3动植物共有DNA甲基化的生物学意义：1.调控转座子的活性，保护基因组DNA2.调控基因的表达如何研究植物的如何研究植物的DNADNA甲基化甲基
6、化1.DNA甲基化敏感的限制性内切酶2.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 3.HPLC：整体基因组甲基化水平, 4.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. 19
7、94 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 transformat
8、ion. 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 pro
9、moter methylation triggered by double-stranded RNA.EMBO J. 2000 19: 5194-201. 植物甲基化植物甲基化DNADNA分子机制研究的分子机制研究的遗传学方法遗传学方法拟南芥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
10、因组DNA，检测甲基化程度的变化（centromeric repetitive DNA ）WTddm1ddm1突变体：整体基因组DNA甲基化与野生型相比减少了70%。rDNAKakutani et al PNAS, 1996, 93: 12406-12411DDM1 encodes a SWI2/SNF2-like proteinJeddeloh et al.Nature Genetics 22 :94-971999Mouse: LshMET1/DDM2: Cytosine MethyltransferaseAntisense-Met1: reduce 32-71% cytosine DNA
11、methylation Anti-Met1 WTDNAmethylation site:CG dinucleotides 2. 转基因法Finnegan et al, PNAS 1996 93:8449-54 3.3.遗传学方法遗传学方法promoterMarker geneMe Me MeMutant screeningPromoterMarker geneMe Me MeMOM1promoterMarker geneHOG1, KYP1/SUVH4, CMT3,AGO4RTS1/ HDA6, DRD1 ,2,3, NRPD1a, DDM1, MET1 - promoterMarker ge
12、neMe 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, SupermanChan et al., Nat Rev Genet. 2005 6:351-60DOMAINS REARRANGED METHYLASE Four classes of DNA methyltransferase in Arabidopsis thaliana?DsRNAsiRNAsDCL324bpTGS: RNA-directed DNA methylation:Establishing DNA methylationInverted
13、 DNA RPol IIMe MeMeMET1? DRM2DNARNA pol IV (NRDP2,NRPD1A)RDR2Me MeMeCG CNG CHHAGO4DRD1RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3),RNA POLYMERASE D1 (RPD1) and ARGONAUTE 4 (AGO4)DRM2: DOMAINS REARRANGED METHYLASEMaintenance of CG DNA methylationMET1HDA6 (HISTONE DEACETYLASE 6)DDM1Maintenance of CNG
14、methylationCMT3,KYP(KRYPTONITE) /SU(VAR)3-9 HOMOLOG 4(SUVH4)AGO1DsRNAAGO1KYPCMT3CNGMeCHHMesiRNAsDCL324bpTGS: RNA-directed DNA methylationInverted DNA RPol IIMe MeMeMET1? DRM2MET1HDA6CGMeDDM1DNARNA pol IVRDR2Me MeMeCG CNG CHHAGO4DRD1DRM2Specific DNA methylation loci in Arabidopsis Chan et al., Nat Re
15、v Genet. 2005 6:351-60. (pathogen related)Wassilewskija strainArabidopsis tryptophan enzyme phosphoribosylanthranilate isomerase (PAI) S15a promoter+ first exonPAI1-4: 350bp+ORF: hypermethylationhypomethylationhypomethylation23123I topTop, VMiddle, ICol hypomethylationF1PAI2 gene is silencedXPAI3, n
16、o activityMelquist S, Bender J. Genetics. 2004 166:437-48. Genes Dev. 2003 17:2036-47. ATG350 bpTAGWS, hypermethylation23I topTop, VMiddle, I1F2hypermethylatedLow gene expressionSome plants revert to hypomethylation statussuvh4/hda6 cmt3WS pai1pai1 strain accumulates fluorescent tryptophan pathway i
17、ntermediates, as well as displaying yellow-green leaf pigmentation, reduced size, increased bushiness, and reduced fertility. Superman（clark kent alleles ） (hypermethylated )Suppressorago4, 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
18、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 Nucleosomes are complexes of histonesH2A is yellow; H2B is red; H3 is blue; H4 is greenThe solenoid model of condensed chromatin146bp DNA wraps the histones2nm2 of H2A, H2B, H3 and H4 40-70 bp About 20
19、0bp for each bead 700 fold compacted180 to 300 nucleosomes Each chromatid would account for 1.2 million bp of DNA chromatin fiberHeterochromatin- Telomeres- Centromeres- Repetitive DNA-genes- N-termini of histones are not (=hypo) acetylated- DNA is methylated (mammals and plants)Euchromatin- active
20、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 hydrolysi
21、s 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组蛋白修饰组蛋白修饰: H3, H4组蛋白修饰: H2A, H2BFCATA) Methyl-CpG-binding proteins recruit HDAC complex to deacetylate histone s
22、o 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 silen
23、cing. 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 re
24、gulation 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 HeterochromatinMeARTKQTARKSTGGKAPRKQLATKAARK-KMe9H3Heterochromatin27MeMe36141823SDG8: SET
25、 Domain Group 8MeMeVRN5VIN3VRN1VRN2SDG 8LHP1FLCLHP1: 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 siRNAsiRNAs: produced by cleaving dsRNAs (double-stranded RNA) tha
26、t 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 cleav
27、ed 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., Ce
28、ll, 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 plantsLi
29、m 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
30、, 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 smRNAsReinha
31、rt 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. Endoge
32、nous 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, Bar
33、tel 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 homologueLim et al., Genes Dev. 2003, 17(8):991-1008. Conservation between the Arabidopsis and Oryza predicted stem-loop precursors. MicroRNAstrans-acting
34、 siRNAsnat-siRNAsRepeat-associated siRNAsTrends 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 (Jackso
35、n 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)
36、, 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 sim
37、ilarity 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
38、complex (Kaya et al., 2001); and MRE11 (meiotic recombination 11 ) Genes that regulate TGS without changing DNA methylation RD29A-LUCBefore stressAfter stress(B)dsRNAsiRNARD29AEndogenous RD29A24 bpLUCRD29ANPTIILUCRD29ANPTIIColdABANaClros1-1ros1-2WTColdABANaClros1-1ros1-2WTRepressor Of Silencing 1ROS
39、1: LUCNPTIIRD29ACOR47WTros1-1rDNAros1ros1突变体基因沉默发生突变体基因沉默发生在转录水平在转录水平Cell. 2002, 111:803-14. BstUILUCRD29A5 kb8 kb12 kbWTros1-1MluIWTros1-16 kb8 kbBstUILUCRD29A5 kb8 kb12 kbWTros1-1MluI5 kb8 kb12 kbWTros1-1MluIWTros1-16 kb8 kbWTros1-16 kb8 kbWTros1-16 kb8 kbRD29A hypermethylation in ros1 mutantLUCRD
40、29ANPTIILUCRD29ANPTIICH3 CH3CH3 CH3RD29AEndogenous RD29ACH3 CH3BstUILUCRD29A5 kb8 kb12 kbWTros1-1MluIWTros1-16 kb8 kbBstUILUCRD29A5 kb8 kb12 kbWTros1-1MluI5 kb8 kb12 kbWTros1-1MluIWTros1-16 kb8 kbWTros1-16 kb8 kbWTros1-16 kb8 kbCell. 2002, 111:803-14. WTros1-1 (plus RD29A-LUC)ros1-1 (minus RD29A-LUC
41、)23 bptRNA and 5S rRNABWTros1-1 (plus RD29A-LUC)ros1-1 (minus RD29A-LUC)23 bptRNA and 5S rRNAWTros1-1 (plus RD29A-LUC)ros1-1 (minus RD29A-LUC)23 bptRNA and 5S rRNAWT (minus RD29A-LUC)ros1-1 (minus RD29A-LUC)RD29AC6 kb6 kb8 kb8 kb23 bpWTros1-123 bpWTros1-1tRNA and 5S rRNAA23 bpWTros1-123 bpWTros1-1tR
42、NA and 5S rRNAT-DNAT-DNA产生的小产生的小RNARNA可能是引起可能是引起DNADNA甲基化的信号分子甲基化的信号分子Cell. 2002, 111:803-14. CFES motifHhH-GPD domainMUTYH 157 QDLASASLEEVNQLWAGLGYYS-RGRRLQEGARKVVEELGG-HMPRTAETLQ MUTY 32 TDLANAPLDEVLHLWTGLGYYA-RARNLHKAAQQVATLHGG-KFPETFEEVA 2ABK 31 AAMLELGVEGVKTYIKTIGLYNSKAENIIKTCRILLEQHNG-EVPEDRAA
43、LE D75275 81 DAITLAPTDAVAHAIRRSNYPESKAPRIQETLRRIKAAPGGYDLDFLRDLPVKDALKWLT ROS1 886 KAIRAADVKEVAETIKSRGMNHKLAERIQGFLDRLVNDHGSIDLEWLRDVPPDKAKEYLL NTG2 159 DGLLKIDEPVLANLIRCVSFYTRKANFIKRTAQLLVDNFDS-DIPYDIEGIL MUTYH 207 QLLPGVGRYTAGAIASIAFGQAT-GVVDGNVARVLCRVRAIGADPSSTLVSQQLWGLA- MUTY 82 ALP-GVGRSTAGAILS
44、LSLGKHF-PILDGNVKRVLARCYAVSGWPGKKEVENKLWSLS- 2ABK 82 ALP-GVGRKTANVVLNTAFGWPT-IAVDTHIFRVCNRTQFAPGKNVEQVEEKLLK- D75275 141 DLP-GVGVKTASLVLLFNYARPV-FPVDTHVHRVSTRVGVIPRMGEQAAHRALLALLP- ROS1 946 SFN-GLGLKSVECVRLLTLHHLA-FPVDTNVGRIAVRLGWVPLQPLPESLQLHLLEMYPM NTG2 210 SLP-GVGPKMGYLTLQKGWGLIAGICVDVHVHRLCKMWNWV
45、DPIKCKTAEHTRKELQV- MUTYH 264 -QQLVDPARPGDFNQAAMELGATVCTPQRPLCSQCPVESLCRAR MUTY 138 -EQVTPAVGVERFNQAMMDLGAMICTRSKPKCSLCPLQNGCIAA 2ABK 135 -VVPAEFKVDCHHWLILHGRYTCIARKPRCGSCIIEDLCEYK D75275 197 -PDPPYLYELHINFLSHGRQVCTWTRPKCGKCILRERCD- ROS1 1004 LESIQKYLWPRLCKLDQKTLYELHYQMITFGKVFCTKSKPNCNACPMKGECRHF NTG
46、2 267 -WLPHSLWYEINTVLVGFGQLICMARGKRCDLCLANDVCNAR ROS1ROS1基因编码一个具双重活性基因编码一个具双重活性的的DNADNA修复酶修复酶Cell. 2002, 111:803-14. 12200116976645312134Average Nicks/MoleculeProtein (pmol)0.000.050.100.150.200510152025UnmethylatedSss IMsp IUnmethylatedmCCGG GGCCmMsp ImCG GCmSss ICCOCABCROS1 was able to introduce strand breaks to anMspI-methylated DNA templateCell. 2002, 111:803-14. Kapoor et al., FEBS Lett. 2005 Sep 12; 35ROS1 Working modeldsRNAsiRNARD29AEndogenous RD29ARD29ACH3CH3CH3CH3CH3CH3RD29AdemethylationROS1LUCRD29ANPTIILUCRD29ANPTIILUCRD29ANPTIILUCRD29ANPTIILUCRD29ANPTIILUCRD29ANPTII
