分子实验高级-组蛋白修饰课件

1、DNA携带的信息是如何读取的？DNA本身是有生命的吗？基因组的可塑性（与环境相互作用）是如何形成的？2022/7/24FUDAN EPIGENETICS LAB1Parabiosis experiment (共生试验)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Villeda S.A. 2011, Nature基因组的组成染色质 = DNA + 蛋白质 + 化学修饰 + RNA2022/7/24FUDAN EPIGENETICS LAB5Nucleosome and Histones2022/7/24FUDAN EPIGENE

2、TICS 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-acetylglucosaminationBBiotinylationRADP ribosylationAAAAAAAAA

3、APPPMMMMMMMMMMPMMPPAGBAAMMRRRMMAAH2BH2AWritersErasersReaders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，H4Amino acids and PositionsK LysineK9 Lysine 9R ArginineModificationsMe Methylation （1mono、2di、3tri）Ac AcetylationP Phosphorylation2022/7/24FUDAN EPIGENETICS LAB8AAAAAAAAAAPPPMMMMMMMMMMPMM

4、PPAGBAAMMRRRMMAAH2BH2AH3K27Ac染色质功能域的多样性Epigenetics animation2022/7/24FUDAN EPIGENETICS LAB10很多重要生命现象有表观遗传的参与表观遗传: 不涉及到DNA序列变化的可遗传变异; 在基因组和环境的相互作用中发挥重要作用生物的进化个体对环境的适应胚胎发育、器官形成记忆同卵双生子的后天获得2022/7/24FUDAN EPIGENETICS LAB11生命的选择很多时候不是简单的Yes or No精细调节 多样化本日大纲2022/7/24FUDAN EPIGENETICS LAB121.组蛋白修饰与组蛋白密码 2

5、.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2022/7/24FUDAN EPIGENETICS LAB13I. 组蛋白修饰与组蛋白密码Histone Code Hypothesis2000年Histone Acetylation,HAT （GCN5、CBP） and HDACTranscription activationHistone methylationHMT (SuvH3-9)Heterochromatin and silenceHistone phosphorylationDNA methylation2022

6、/7/24FUDAN EPIGENETICS LAB142022/7/24FUDAN EPIGENETICS LAB15What do these mean?2022/7/24FUDAN EPIGENETICS LAB162022/7/24FUDAN 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）2022/7/24FUDAN E

7、PIGENETICS 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？2022/7/24FUDAN EPIGENETICS LAB19Developmental Cell, Volume 21, Issue 6, 993-10042022/7/24FUDAN EPIGENETICS LAB20Matthew G. Guenther , Stuart S. Levine , Laurie A. Boyer , Rudolf Jaenisch , Richard A. Youn

8、gA 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？2022/7/24FUDAN EPIGENETICS LAB21H3K4me3H3K27me3MLLPRC2Two possible ways to inherit histone methylation2022/7/24FUDAN E

9、PIGENETICS LAB22I. Histone marker semiconservative replicationII. Enzymesemiconservative replication2022/7/24FUDAN EPIGENETICS LAB232022/7/24FUDAN EPIGENETICS LAB242022/7/24FUDAN EPIGENETICS LAB25II.组蛋白修饰与基因表达调控已知的组蛋白赖氨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2

10、022/7/24FUDAN EPIGENETICS LAB27HDACsHATs and HDACs催化位点功能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 Tat interactionHBO1H4 (K5, K8, K12)ORC interactionSIRTsH4 K16Metabo

11、lism, agingHDACsH3 (K9, K14, K18) and many other sitesRepressionGCN5的发现2022/7/24FUDAN EPIGENETICS LAB28Material: Macronuclei (Enriched for Histone acetylation)Assay: Enzyme + Histone + 3H-Acteyl-coA = SignalAssay done in GelBrownell JE 1995 PNASBrownell JE 1996 Cell四膜虫 TetrahymenaDavid AllisMolecula

12、r Characterization of GCN52022/7/24FUDAN EPIGENETICS LAB29Brownell JE 1996 CellGCN5的功能2022/7/24FUDAN 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）2022/7/24FUDAN EPIGENETICS LAB31Trapoxin 微生物来源的天然

13、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）2022/7/24FUDAN EPIGENETICS LAB32Taunton J, 2000, ScienceStuart Schreiber酰基化和转录的关系2022/7/24FUDAN EPIGENETICS LAB33Struhl K 1998 Genes&DevNon-histone 乙酰化与代谢2022/7/24FUDAN EPIGENETICS LA

14、B34K. 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35MLysine methylationMMMMMMH2BH2A2022/7/24FUDAN EPIGENETICS LAB362022/7/24FUDAN EPIGENETICS LAB37主要的组蛋白甲基化修饰的功能2022/7/24FUDAN EPIGENETICS LAB38甲基化位点分布特点功能甲基化

15、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Suv4-20H1-22022/7/24FUDAN EPIGENETICS LAB39III. 重要组蛋白修饰相关的Complex介绍Suv39H1-2 HP1Trithorax (MLL) and Polycomb (EZH2)LSD1 complexPosition-effect varieg

16、ation2022/7/24FUDAN EPIGENETICS LAB40Useful tools in studying epigenetics - wm4(speak white-mottled-4) - An inversion places white gene close to centromere Gunter Reuter2022/7/24FUDAN EPIGENETICS LAB41H3K9 methylationSu(var)3-9 and HP1Su(var)3-9 suppresses white2022/7/24FUDAN EPIGENETICS LAB42Su(var

17、)3-9 encodes a H3K9 methyltransferase2022/7/24FUDAN EPIGENETICS LAB43Su(var)3-9H3K9me3H3K9me3 loss in Su(Var)3-9 mutant Su(var)3-9 and Su(var)2-5/HP12022/7/24FUDAN EPIGENETICS LAB44ChromoSETSu(var)3-9Su(var)2-5/HP1ChromoChromo-ShadowHingeinteractionH3K9me3Enzymes and readers often work together2022/

18、7/24FUDAN EPIGENETICS LAB45Thomas Jenuwen2022/7/24FUDAN EPIGENETICS LAB46H3K27 and H3K4 methylationPcG (EZH2) and TrxG (MLL)Polycomb and Trithorax groupsPolycomb group （PcG）多聚梳类Trithorax group （TrxG) 三胸类2022/7/24FUDAN EPIGENETICS LAB47WTPcG MutantPcG and TrxG control Hox expression2022/7/24FUDAN EPI

19、GENETICS LAB48PcGTrxGFly Hox genessuppresses transcription by H3K27me3 and H2AK119ub2022/7/24FUDAN EPIGENETICS LAB49PcG催化H3K27me3甲基化识别H3K27me3甲基化催化H2AK119泛素化activates transcription by H3K4me3 and H3K27 demethylation2022/7/24FUDAN EPIGENETICS LAB50TrxG2022/7/24FUDAN EPIGENETICS LAB51H3K4 demethylatio

20、nLSD1(KDM1) complexIt all started from a virusAdenovirus E1A protein is an oncogene2022/7/24FUDAN EPIGENETICS LAB52E1A C-term binding protein 1 (CtBP1) complex2022/7/24FUDAN EPIGENETICS LAB53Yujiang Shi Nature 2003Yang ShiYujiang ShiWe call it LSD1 (Lysine Specific Demethylase 1)NPAO catalyzes H3K4m

21、e2 demethylationLSD1 was lately found as the homolog of fly Su(var)3-3 and is required for proper H3K9me2/3 heterochromatin boundary in fly.2022/7/24FUDAN EPIGENETICS LAB54Yujiang Shi Cell 2004No LSD1+LSD1LSD1 alone is not enough - A PHD finger in LSD1 complex2022/7/24FUDAN EPIGENETICS LAB5511697312

22、536665520014(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 nucleosomesBHC80HDAC1/2CorestLSD1Histone deacetylaseLysine 4 demeth

23、ylase?BHC80Again, enzyme needs a reader2022/7/24FUDAN EPIGENETICS LAB5620% InputH3H3K4me2 No peptideHIS-BHC80Biotinylated PeptidesH3K9me2 H3H3CorestBHC80LSD1HDAC1/2BHC80HDAC1/2CorestH3H3LSD1CorestBHC80HDAC1/2CorestH3H3PropagationBHC80LSD1Methyl Zero can carry epigenetic information!F. Lan 2007, Natu

24、re2022/7/24FUDAN EPIGENETICS LAB57IV.组蛋白修饰与疾病和衰老Epigenetics and diseaseCancerEZH2, NSD2, MLL2, SETD2, UTX, SNF5, ARID1ANeuronal diseasesKDM5C, PHF8, PHF21A/BHC80, MeCP2, ATRXAutoimmuneAIRE2022/7/24FUDAN EPIGENETICS LAB58Anna Portela & Manel Esteller Nature Biotechnology 2010EZH2 Y641 mutation in DLB

25、CL59DLBCL cell linesA gain of function mutation!EZH2 inhibitor reactivates tumor suppressor gene p16Ink4A2022/7/24FUDAN EPIGENETICS LAB60Novartis Shanghai Qi W, PNAS 2012Targeted therapy by EZH2 inhibitor2022/7/24FUDAN EPIGENETICS LAB61EZH2 mutated cellHyper H3K27me3Sensitive to EZH2iEZH2 WT cellNormal H3K27me3Insensitive to EZH2iColony formation assayProliferation assayKDM6A/UTX (H3K27me3 demethylase) inactivation mutations in cancer62基因型酶活野生型100%F642L40%L731F37%Y751C51%A388P14%Contro