分子实验高级-组蛋白修饰课件.pptx

1、DNA携带的信息是如何读取的？nDNA本身是有生命的吗？n基因组的可塑性（与环境相互作用）是如何形成的？2022-8-1FUDAN EPIGENETICS LAB1Parabiosis experiment(共生试验共生试验)血液中有调控肌肉再生能力的因子血液中有调控肌肉再生能力的因子Conboy I.M.,2005,Nature神经系统再生和认知的衰老是可控的神经系统再生和认知的衰老是可控的Villeda S.A.2011,Nature基因组的组成 染色质=DNA+蛋白质+化学修饰+RNA2022-8-1FUDAN EPIGENETICS LAB5Nucleosome and Histone

2、s2022-8-1FUDAN EPIGENETICS LAB6H3H2A/H2BH4Whole Cell ExtractPurified HistonesOligonucleosome(H3.1)15KD10KD25KDH3.1H2BH2AH435KD40KD组蛋白上存在大量表观遗传修饰组蛋白修饰可发生在多个赖氨酸和精氨酸位点上组蛋白修饰酶 100种识别因子(Reader)300多种7MAPUbAcetylationMethylationPhosphorylationMono-ubiquitinationG-N-acetylglucosaminationB BiotinylationR ADP

3、 ribosylationAAAAAAAAAAPPPMMMMMMMMMMPMMPPAGBAAMMRRRMMAAH2BH2AWritersErasersReaders组蛋白密码学说组蛋白密码学说庞大的组合数对DNA信息精细调节组蛋白修饰的命名法 Histones：H2A，H2B，H3，H4 Amino acids and Positions K Lysine K9 Lysine 9 R Arginine Modifications Me Methylation（1mono、2di、3tri）Ac Acetylation P Phosphorylation2022-8-1FUDAN EPIGENE

4、TICS LAB8AAAAAAAAAAPPPMMMMMMMMMMPMMPPAGBAAMMRRRMMAAH2BH2AH3K27Ac染色质功能域的多样性染色质功能域的多样性 Epigenetics animation2022-8-1FUDAN EPIGENETICS LAB10很多重要生命现象有表观遗传的参与表观遗传表观遗传:不涉及到不涉及到DNA序列变化的可遗传变异序列变化的可遗传变异;在基在基因组和环境的相互作用中发挥重要作用因组和环境的相互作用中发挥重要作用n生物的进化n个体对环境的适应n胚胎发育、器官形成n记忆n同卵双生子的后天获得n2022-8-1FUDAN EPIGENETICS LA

5、B11生命的选择很多时候不是简单的生命的选择很多时候不是简单的Yes or No精细调节精细调节 多样化多样化本日大纲2022-8-1FUDAN EPIGENETICS LAB12 1.组蛋白修饰与组蛋白密码 2.组蛋白修饰与基因表达调控 3.重要组蛋白修饰相关的Complex介绍 4.组蛋白修饰与疾病和衰老 5.组蛋白修饰的检测2022-8-1FUDAN EPIGENETICS LAB13I.组蛋白修饰与组蛋白密码Histone Code Hypothesis 2000年 Histone Acetylation,HAT（GCN5、CBP）and HDAC Transcription acti

6、vation Histone methylation HMT(SuvH3-9)Heterochromatin and silence Histone phosphorylation DNA methylation2022-8-1FUDAN EPIGENETICS LAB142022-8-1FUDAN EPIGENETICS LAB15What do these mean?2022-8-1FUDAN EPIGENETICS LAB162022-8-1FUDAN EPIGENETICS LAB17组蛋白密码假说 单一的和组合的组蛋白修饰携带有特定的调控信号 组蛋白修饰的特征可以通过招募相关效应蛋白

7、从而决定局部染色质的活性和结构例如：H3K4me3 标记在活化基因的启示位点（TSS）H3K9Ac,H3K14Ac 转录活性H3K9me3 异染色质（Heterochromatin）2022-8-1FUDAN EPIGENETICS LAB18关于Histone Code的争论 问题一：Histone modification 真的可以预测染色质活性吗？有转录和无转录的基因都有H3K4me3，如何解释？2022-8-1FUDAN EPIGENETICS LAB19Developmental Cell,Volume 21,Issue 6,993-10042022-8-1FUDAN EPIGENE

8、TICS LAB20Matthew G.Guenther,Stuart S.Levine,Laurie A.Boyer,Rudolf Jaenisch,Richard A.YoungA Chromatin Landmark and Transcription Initiation at Most Promoters in Human CellsCell Volume 130,Issue 1 2007 77-88关于Histone Code的争论 问题二：Histone code 可以自我复制吗？如果不能自我复制，可以被称为code吗？2022-8-1FUDAN EPIGENETICS LAB2

9、1H3K4me3H3K27me3MLLPRC2Two possible ways to inherit histone methylation2022-8-1FUDAN EPIGENETICS LAB22I.Histone marker semiconservative replicationII.Enzymesemiconservative replication2022-8-1FUDAN EPIGENETICS LAB232022-8-1FUDAN EPIGENETICS LAB242022-8-1FUDAN EPIGENETICS LAB25II.组蛋白修饰与基因表达调控已知的组蛋白赖氨

10、酸酰基化位点大部分赖氨酸位点均可以被酰基化酰基化与转录激活正相关26MLysine acetylationAAAAAAAAAAAAAH2BH2AA酰基化调控体系2022-8-1FUDAN EPIGENETICS LAB27HDACsHATs and HDACs催化位点催化位点功能功能HAT1H4(K5,K12)Nucleosome assemblyCBP/P300H3(K14,K18)H4(K5,K8)H2A(K5)H2B(K12,K15)CoactivatorPCAF/GCN5H3(K9,K14,K18)CoactivatorTIP60H4(K5,K8,K12,K16)H3 K14HIV T

11、at interactionHBO1H4(K5,K8,K12)ORC interactionSIRTsH4 K16Metabolism,agingHDACsH3(K9,K14,K18)and many other sitesRepressionGCN5的发现2022-8-1FUDAN EPIGENETICS LAB28Material:Macronuclei(Enriched for Histone acetylation)Assay:Enzyme+Histone+3H-Acteyl-coA=SignalAssay done in GelBrownell JE 1995 PNASBrownel

12、l JE 1996 Cell四膜虫 TetrahymenaDavid AllisMolecular Characterization of GCN52022-8-1FUDAN EPIGENETICS LAB29Brownell JE 1996 CellGCN5的功能2022-8-1FUDAN EPIGENETICS LAB30GCN5 是SAGA复合体的酶活组分，并与activator结合GCN5 在酵母内酰基化靶基因启动子其活性可促进nucleosome remodeling人的同源物是 p300 和 PCAFSterner 2000 MMBRHDAC/Rdp3的发现（化学生物学法）2022

13、-8-1FUDAN EPIGENETICS LAB31Trapoxin 微生物来源的天然化合物可抑制哺乳细胞去乙酰化并杀伤细胞HeLa cell whole cell extractA:untreatedB:Treated with TrapoxinWB:anti-H3AcHDAC/Rdp3的发现（化学生物学法）2022-8-1FUDAN EPIGENETICS LAB32Taunton J,2000,ScienceStuart Schreiber酰基化和转录的关系2022-8-1FUDAN EPIGENETICS LAB33Struhl K 1998 Genes&DevNon-histone

14、 乙酰化与代谢2022-8-1FUDAN EPIGENETICS LAB34K.Guan and Y.Xiong Trend in Biochemical Sciences 2010Yue XiongKunliang Guan已知的组蛋白赖氨酸甲基化位点H3K4、H3K9、H3K27、H3K36、H3K79和H4K20甲基化有单、双和三甲基化三种水平35M Lysine methylationMMMMMMH2BH2A2022-8-1FUDAN EPIGENETICS LAB362022-8-1FUDAN EPIGENETICS LAB37主要的组蛋白甲基化修饰的功能2022-8-1FUDAN

15、EPIGENETICS LAB38甲基化位点甲基化位点分布特点分布特点功能功能甲基化酶甲基化酶H3K4me3转录起始点转录活化MLL1-5、SETD1A-BH3K9me3大区域、异染色质基因沉默Suv39H1-2H3K27me3大区域转录抑制EZH1-2H3K36me3转录活性区、外显子转录延伸SETD2H3K79me3转录活性区转录活化DOT1LH4K20me3异染色质、基因沙漠基因组稳定Suv4-20H1-22022-8-1FUDAN EPIGENETICS LAB39III.重要组蛋白修饰相关的Complex介绍Suv39H1-2 HP1Trithorax(MLL)and Polycom

16、b(EZH2)LSD1 complexPosition-effect variegation2022-8-1FUDAN EPIGENETICS LAB40Useful tools in studying epigenetics -wm4(speak white-mottled-4)-An inversion places white gene close to centromere Gunter Reuter2022-8-1FUDAN EPIGENETICS LAB41H3K9 methylationSu(var)3-9 and HP1Su(var)3-9 suppresses white20

17、22-8-1FUDAN EPIGENETICS LAB42Su(var)3-9 encodes a H3K9 methyltransferase2022-8-1FUDAN EPIGENETICS LAB43Su(var)3-9H3K9me3H3K9me3 loss in Su(Var)3-9 mutant Su(var)3-9 and Su(var)2-5/HP12022-8-1FUDAN EPIGENETICS LAB44ChromoSETSu(var)3-9Su(var)2-5/HP1ChromoChromo-ShadowHingeinteractionH3K9me3Enzymes and

18、 readers often work together2022-8-1FUDAN EPIGENETICS LAB45Thomas Jenuwen2022-8-1FUDAN EPIGENETICS LAB46H3K27 and H3K4 methylationPcG(EZH2)and TrxG(MLL)Polycomb and Trithorax groups Polycomb group（PcG）多聚梳类 Trithorax group（TrxG)三胸类2022-8-1FUDAN EPIGENETICS LAB47WTPcG MutantPcG and TrxG control Hox ex

19、pression2022-8-1FUDAN EPIGENETICS LAB48PcGTrxGFly Hox genessuppresses transcription by H3K27me3 and H2AK119ub2022-8-1FUDAN EPIGENETICS LAB49PcG催化催化H3K27me3甲基化甲基化识别识别H3K27me3甲基化甲基化催化催化H2AK119泛素化泛素化activates transcription by H3K4me3 and H3K27 demethylation2022-8-1FUDAN EPIGENETICS LAB50TrxG2022-8-1FUD

20、AN EPIGENETICS LAB51H3K4 demethylationLSD1(KDM1)complexIt all started from a virus Adenovirus E1A protein is an oncogene2022-8-1FUDAN EPIGENETICS LAB52 E1A C-term binding protein 1(CtBP1)complex2022-8-1FUDAN EPIGENETICS LAB53Yujiang Shi Nature 2003Yang ShiYujiang ShiWe call it LSD1(Lysine Specific D

21、emethylase 1)NPAO catalyzes H3K4me2 demethylation LSD1 was lately found as the homolog of fly Su(var)3-3 and is required for proper H3K9me2/3 heterochromatin boundary in fly.2022-8-1FUDAN EPIGENETICS LAB54Yujiang Shi Cell 2004No LSD1+LSD1LSD1 alone is not enough -A PHD finger in LSD1 complex2022-8-1

22、FUDAN EPIGENETICS LAB5511697312536665520014(Kd)Fg:HA:LSD1hLSD1 comlepxCtBP1MockHDAC1CoRESTRREB-1Z198ZNF217BHC80BRAF35HMG20AY182KIAA1343Hsp70.1Tubulin-1*Hsp70.21 33-54 427-437 486-532 586-607 660 LeuZip AT-Hook PHD LeuzipFacilitates demethylation on hypoacetylated nucleosomesBHC80HDAC1/2CorestLSD1His

23、tone deacetylaseLysine 4 demethylase?BHC80Again,enzyme needs a reader2022-8-1FUDAN EPIGENETICS LAB5620%InputH3H3K4me2 No peptideHIS-BHC80Biotinylated PeptidesH3K9me2 H3H3CorestBHC80LSD1HDAC1/2BHC80HDAC1/2CorestH3H3LSD1CorestBHC80HDAC1/2CorestH3H3PropagationBHC80LSD1Methyl Zero can carry epigenetic i

24、nformation!F.Lan 2007,Nature2022-8-1FUDAN EPIGENETICS LAB57IV.组蛋白修饰与疾病和衰老Epigenetics and disease Cancer EZH2,NSD2,MLL2,SETD2,UTX,SNF5,ARID1A Neuronal diseases KDM5C,PHF8,PHF21A/BHC80,MeCP2,ATRX Autoimmune AIRE2022-8-1FUDAN EPIGENETICS LAB58Anna Portela&Manel Esteller Nature Biotechnology 2010EZH2 Y6

25、41 mutation in DLBCL59DLBCL cell linesA gain of function mutation!EZH2 inhibitor reactivates tumor suppressor gene p16Ink4A2022-8-1FUDAN EPIGENETICS LAB60Novartis Shanghai Qi W,PNAS 2012Targeted therapy by EZH2 inhibitor2022-8-1FUDAN EPIGENETICS LAB61EZH2 mutated cellHyper H3K27me3Sensitive to EZH2i

26、EZH2 WT cellNormal H3K27me3Insensitive to EZH2iColony formation assayProliferation assayKDM6A/UTX(H3K27me3 demethylase)inactivation mutations in cancer62基因型基因型酶活酶活野生型100%F642L40%L731F37%Y751C51%A388P14%Control MOdSMCX MO 抑制鱼SMCX导致神经分化后的细胞大量凋亡 抑制鱼SMCX导致神经系统发育缺陷自闭症和智障病患中发现大量SMCX突变63Iwase S.,Lan F.2007

27、 CellKDM5C/SMCX-H3K4me3去甲基化酶去甲基化酶-对神经对神经功能至关重要功能至关重要Tom Rando,2013Stanford U.肌干细胞的活化伴随着肌干细胞的活化伴随着H3K27me3的提高的提高QSC 静态肌干细胞ASC 活化肌干细胞QSC特异因子的下调伴随着特异因子的下调伴随着H3K27me3的上升的上升H3K4me3和和H3K36me3与与ASC1中基中基因活化因活化H3K4me3和和H3K27me3与衰老状态的相关性与衰老状态的相关性 2022-8-1FUDAN EPIGENETICS LAB69V.组蛋白修饰的检测Global and locus speci

28、fic conceptIHC、IF、WBChIP and ChIP-seqDetect histone modification at global level2022-8-1FUDAN EPIGENETICS LAB70IHC H3K23AcIF H3K4me3WB H3K27meDetect locus specific modification2022-8-1FUDAN EPIGENETICS LAB71ChIP:Chromatin ImmunoprecipitationEnriched DNA can be quantitatively analyzed by q-PCR,Hi-seq(ChIP-seq)or array hybridization(ChIP-chip)A ChIP-chip example of Hox gene locus2022-8-1FUDAN EPIGENETICS LAB72Sperm epigenome Words to your kids?2022-8-1FUDAN EPIGENETICS LAB73Radford EJ,2014 Science