1、FIST-Epi3: 組蛋白修飾藍斐復(fù)旦楓林IBS研究所DNA攜帶的信息是如何讀取的？nDNA本身是有生命的嗎？n基因組的可塑性（與環(huán)境相互作用）是如何形成的？2022-5-10FUDAN EPIGENETICS LAB2Parabiosis experiment (共生試驗共生試驗)血液中有調(diào)控肌肉再生能力的因子血液中有調(diào)控肌肉再生能力的因子Conboy I.M., 2005, Nature神經(jīng)系統(tǒng)再生和認知的衰老是可控的神經(jīng)系統(tǒng)再生和認知的衰老是可控的Villeda S.A. 2011, Nature基因組的組成 染色質(zhì) = DNA + 蛋白質(zhì) + 化學(xué)修飾 + RNA2022-5-10F

2、UDAN EPIGENETICS LAB6Nucleosome and Histones2022-5-10FUDAN EPIGENETICS LAB7H3H2A/H2BH4Whole Cell ExtractPurified HistonesOligonucleosome (H3.1)15KD10KD25KDH3.1H2BH2AH435KD40KD組蛋白上存在大量表觀遺傳修飾組蛋白修飾可發(fā)生在多個賴氨酸和精氨酸位點上組蛋白修飾酶 100種識別因子(Reader) 300多種8MAPUbAcetylationMethylationPhosphorylationMono-ubiquitinatio

3、nG -N-acetylglucosaminationB BiotinylationR ADP ribosylationAAAAAAAAAAPPPMMMMMMMMMMPMMPPAGBAAMMRRRMMAAH2BH2AWritersErasersReaders組蛋白密碼學(xué)說組蛋白密碼學(xué)說龐大的組合數(shù)對DNA信息精細調(diào)節(jié)組蛋白修飾的命名法 Histones：H2A，H2B，H3，H4 Amino acids and Positions K Lysine K9 Lysine 9 R Arginine Modifications Me Methylation （1mono、2di、3tri） Ac A

4、cetylation P Phosphorylation2022-5-10FUDAN EPIGENETICS LAB9AAAAAAAAAAPPPMMMMMMMMMMPMMPPAGBAAMMRRRMMAAH2BH2AH3K27Ac染色質(zhì)功能域的多樣性染色質(zhì)功能域的多樣性 Epigenetics animation2022-5-10FUDAN EPIGENETICS LAB11很多重要生命現(xiàn)象有表觀遺傳的參與表觀遺傳表觀遺傳: 不涉及到不涉及到DNA序列變化的可遺傳變異序列變化的可遺傳變異; 在基在基因組和環(huán)境的相互作用中發(fā)揮重要作用因組和環(huán)境的相互作用中發(fā)揮重要作用n生物的進化n個體對環(huán)境的適應(yīng)

5、n胚胎發(fā)育、器官形成n記憶n同卵雙生子的后天獲得n2022-5-10FUDAN EPIGENETICS LAB12生命的選擇很多時候不是簡單的生命的選擇很多時候不是簡單的Yes or No精細調(diào)節(jié)精細調(diào)節(jié) 多樣化多樣化本日大綱2022-5-10FUDAN EPIGENETICS LAB13 1. 組蛋白修飾與組蛋白密碼 2. 組蛋白修飾與基因表達調(diào)控 3. 重要組蛋白修飾相關(guān)的Complex介紹 4. 組蛋白修飾與疾病和衰老 5. 組蛋白修飾的檢測2022-5-10FUDAN EPIGENETICS LAB14I. 組蛋白修飾與組蛋白密碼Histone Code Hypothesis 2000

6、年 Histone Acetylation, HAT （GCN5、CBP） and HDAC Transcription activation Histone methylation HMT (SuvH3-9) Heterochromatin and silence Histone phosphorylation DNA methylation2022-5-10FUDAN EPIGENETICS LAB152022-5-10FUDAN EPIGENETICS LAB16What do these mean?2022-5-10FUDAN EPIGENETICS LAB172022-5-10FUD

7、AN EPIGENETICS LAB18組蛋白密碼假說 單一的和組合的組蛋白修飾攜帶有特定的調(diào)控信號 組蛋白修飾的特征可以通過招募相關(guān)效應(yīng)蛋白從而決定局部染色質(zhì)的活性和結(jié)構(gòu)例如：H3K4me3 標記在活化基因的啟示位點（TSS）H3K9Ac, H3K14Ac 轉(zhuǎn)錄活性H3K9me3 異染色質(zhì)（Heterochromatin）2022-5-10FUDAN EPIGENETICS LAB19關(guān)于Histone Code的爭論 問題一：Histone modification 真的可以預(yù)測染色質(zhì)活性嗎？ 有轉(zhuǎn)錄和無轉(zhuǎn)錄的基因都有H3K4me3，如何解釋？2022-5-10FUDAN EPIGENET

8、ICS LAB20Developmental Cell, Volume 21, Issue 6, 993-10042022-5-10FUDAN EPIGENETICS LAB21Matthew 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 - 8

9、8關(guān)于Histone Code的爭論 問題二：Histone code 可以自我復(fù)制嗎？ 如果不能自我復(fù)制，可以被稱為code嗎？2022-5-10FUDAN EPIGENETICS LAB22H3K4me3H3K27me3MLLPRC2Two possible ways to inherit histone methylation2022-5-10FUDAN EPIGENETICS LAB23I. Histone marker semiconservative replicationII. Enzymesemiconservative replication2022-5-10FUDAN EP

10、IGENETICS LAB242022-5-10FUDAN EPIGENETICS LAB252022-5-10FUDAN EPIGENETICS LAB26II.組蛋白修飾與基因表達調(diào)控已知的組蛋白賴氨酸酰基化位點大部分賴氨酸位點均可以被?；；c轉(zhuǎn)錄激活正相關(guān)27MLysine acetylationAAAAAAAAAAAAAH2BH2AA?；{(diào)控體系2022-5-10FUDAN EPIGENETICS LAB28HDACsHATs and HDACs催化位點催化位點功能功能HAT1H4 (K5, K12)Nucleosome assemblyCBP/P300H3 (K14, K18

11、) 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 K16Metabolism, agingHDACsH3 (K9, K14, K18) and many other sitesRepressionGCN5的發(fā)現(xiàn)2022-5-10FUDAN EPIGENETICS LAB29Material

12、: Macronuclei (Enriched for Histone acetylation)Assay: Enzyme + Histone + 3H-Acteyl-coA = SignalAssay done in GelBrownell JE 1995 PNASBrownell JE 1996 Cell四膜蟲 TetrahymenaDavid AllisMolecular Characterization of GCN52022-5-10FUDAN EPIGENETICS LAB30Brownell JE 1996 CellGCN5的功能2022-5-10FUDAN EPIGENETIC

13、S LAB31GCN5 是SAGA復(fù)合體的酶活組分，并與activator結(jié)合GCN5 在酵母內(nèi)?；谢騿幼悠浠钚钥纱龠Mnucleosome remodeling人的同源物是 p300 和 PCAFSterner 2000 MMBRHDAC/Rdp3的發(fā)現(xiàn) （化學(xué)生物學(xué)法）2022-5-10FUDAN EPIGENETICS LAB32Trapoxin 微生物來源的天然化合物可抑制哺乳細胞去乙?；毎鸋eLa cell whole cell extractA: untreatedB: Treated with TrapoxinWB: anti-H3AcHDAC/Rdp3的發(fā)現(xiàn) （化

14、學(xué)生物學(xué)法）2022-5-10FUDAN EPIGENETICS LAB33Taunton J, 2000, ScienceStuart Schreiber?；娃D(zhuǎn)錄的關(guān)系2022-5-10FUDAN EPIGENETICS LAB34Struhl K 1998 Genes&DevNon-histone 乙?；c代謝2022-5-10FUDAN EPIGENETICS LAB35K. Guan and Y. Xiong Trend in Biochemical Sciences 2010Yue XiongKunliang Guan已知的組蛋白賴氨酸甲基化位點H3K4、H3K9、H3K

15、27、H3K36、H3K79和H4K20甲基化有單、雙和三甲基化三種水平36M Lysine methylationMMMMMMH2BH2A2022-5-10FUDAN EPIGENETICS LAB372022-5-10FUDAN EPIGENETICS LAB38主要的組蛋白甲基化修飾的功能2022-5-10FUDAN EPIGENETICS LAB39甲基化位點甲基化位點分布特點分布特點功能功能甲基化酶甲基化酶H3K4me3轉(zhuǎn)錄起始點轉(zhuǎn)錄活化MLL1-5、SETD1A-BH3K9me3大區(qū)域、異染色質(zhì)基因沉默Suv39H1-2H3K27me3大區(qū)域轉(zhuǎn)錄抑制EZH1-2H3K36me3轉(zhuǎn)錄

16、活性區(qū)、外顯子轉(zhuǎn)錄延伸SETD2H3K79me3轉(zhuǎn)錄活性區(qū)轉(zhuǎn)錄活化DOT1LH4K20me3異染色質(zhì)、基因沙漠基因組穩(wěn)定Suv4-20H1-22022-5-10FUDAN EPIGENETICS LAB40III. 重要組蛋白修飾相關(guān)的Complex介紹Suv39H1-2 HP1Trithorax (MLL) and Polycomb (EZH2)LSD1 complexPosition-effect variegation2022-5-10FUDAN EPIGENETICS LAB41Useful tools in studying epigenetics - wm4(speak white

17、-mottled-4) - An inversion places white gene close to centromere Gunter Reuter2022-5-10FUDAN EPIGENETICS LAB42H3K9 methylationSu(var)3-9 and HP1Su(var)3-9 suppresses white2022-5-10FUDAN EPIGENETICS LAB43Su(var)3-9 encodes a H3K9 methyltransferase2022-5-10FUDAN EPIGENETICS LAB44Su(var)3-9H3K9me3H3K9m

18、e3 loss in Su(Var)3-9 mutant Su(var)3-9 and Su(var)2-5/HP12022-5-10FUDAN EPIGENETICS LAB45ChromoSETSu(var)3-9Su(var)2-5/HP1ChromoChromo-ShadowHingeinteractionH3K9me3Enzymes and readers often work together2022-5-10FUDAN EPIGENETICS LAB46Thomas Jenuwen2022-5-10FUDAN EPIGENETICS LAB47H3K27 and H3K4 met

19、hylationPcG (EZH2) and TrxG (MLL)Polycomb and Trithorax groups Polycomb group （PcG）多聚梳類 Trithorax group （TrxG) 三胸類2022-5-10FUDAN EPIGENETICS LAB48WTPcG MutantPcG and TrxG control Hox expression2022-5-10FUDAN EPIGENETICS LAB49PcGTrxGFly Hox genessuppresses transcription by H3K27me3 and H2AK119ub2022-

20、5-10FUDAN EPIGENETICS LAB50PcG催化催化H3K27me3甲基化甲基化識別識別H3K27me3甲基化甲基化催化催化H2AK119泛素化泛素化activates transcription by H3K4me3 and H3K27 demethylation2022-5-10FUDAN EPIGENETICS LAB51TrxG2022-5-10FUDAN EPIGENETICS LAB52H3K4 demethylationLSD1(KDM1) complexIt all started from a virus Adenovirus E1A protein is a

21、n oncogene2022-5-10FUDAN EPIGENETICS LAB53 E1A C-term binding protein 1 (CtBP1) complex2022-5-10FUDAN EPIGENETICS LAB54Yujiang Shi Nature 2003Yang ShiYujiang ShiWe call it LSD1 (Lysine Specific Demethylase 1) NPAO catalyzes H3K4me2 demethylation LSD1 was lately found as the homolog of fly Su(var)3-3

22、 and is required for proper H3K9me2/3 heterochromatin boundary in fly.2022-5-10FUDAN EPIGENETICS LAB55Yujiang Shi Cell 2004No LSD1+LSD1LSD1 alone is not enough - A PHD finger in LSD1 complex2022-5-10FUDAN EPIGENETICS LAB5611697312536665520014(Kd)Fg:HA:LSD1hLSD1 comlepxCtBP1MockHDAC1CoRESTRREB-1Z198Z

23、NF217BHC80BRAF35HMG20AY182KIAA1343Hsp70.1Tubulin-1*Hsp70.21 33-54 427-437 486-532 586-607 660 LeuZip AT-Hook PHD LeuzipFacilitates demethylation on hypoacetylated nucleosomesBHC80HDAC1/2CorestLSD1Histone deacetylaseLysine 4 demethylase?BHC80Again, enzyme needs a reader2022-5-10FUDAN EPIGENETICS LAB5

24、720% InputH3H3K4me2 No peptideHIS-BHC80Biotinylated PeptidesH3K9me2 H3H3CorestBHC80LSD1HDAC1/2BHC80HDAC1/2CorestH3H3LSD1CorestBHC80HDAC1/2CorestH3H3PropagationBHC80LSD1Methyl Zero can carry epigenetic information!F. Lan 2007, Nature2022-5-10FUDAN EPIGENETICS LAB58IV.組蛋白修飾與疾病和衰老Epigenetics and diseas

25、e Cancer EZH2, NSD2, MLL2, SETD2, UTX, SNF5, ARID1A Neuronal diseases KDM5C, PHF8, PHF21A/BHC80, MeCP2, ATRX Autoimmune AIRE2022-5-10FUDAN EPIGENETICS LAB59Anna Portela & Manel Esteller Nature Biotechnology 2010EZH2 Y641 mutation in DLBCL60DLBCL cell linesA gain of function mutation!EZH2 inhibit

26、or reactivates tumor suppressor gene p16Ink4A2022-5-10FUDAN EPIGENETICS LAB61Novartis Shanghai Qi W, PNAS 2012Targeted therapy by EZH2 inhibitor2022-5-10FUDAN EPIGENETICS LAB62EZH2 mutated cellHyper H3K27me3Sensitive to EZH2iEZH2 WT cellNormal H3K27me3Insensitive to EZH2iColony formation assayProliferation assayKDM6A/UTX (H3K27me3 demethylase) inactivation mutations in cancer63基因型基因型酶活酶活野生型100%F642L40%L731F37%Y751C51%A388P14%Control MOdSMCX MO 抑制魚SMCX導(dǎo)致神經(jīng)分化后