分子實驗高級-組蛋白修飾課件

1、DNA攜帶的信息是如何讀取的？DNA本身是有生命的嗎？基因組的可塑性（與環(huán)境相互作用）是如何形成的？2022/7/24FUDAN EPIGENETICS LAB1Parabiosis experiment (共生試驗)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(rèn)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 + RNA2022/7/24FUDAN EPIGENETICS LAB5Nucleosome and Histones2022/7/24FUDAN EPIGENE

2、TICS LAB6H3H2A/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7MAPUbAcetylationMethylationPhosphorylationMono-ubiquitinationG-N-acetylglucosaminationBBiotinylationRADP ribosylationAAAAAAAAA

3、APPPMMMMMMMMMMPMMPPAGBAAMMRRRMMAAH2BH2AWritersErasersReaders組蛋白密碼學(xué)說龐大的組合數(shù)對DNA信息精細(xì)調(diào)節(jié)組蛋白修飾的命名法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染色質(zhì)功能域的多樣性Epigenetics animation2022/7/24FUDAN EPIGENETICS LAB10很多重要生命現(xiàn)象有表觀遺傳的參與表觀遺傳: 不涉及到DNA序列變化的可遺傳變異; 在基因組和環(huán)境的相互作用中發(fā)揮重要作用生物的進(jìn)化個體對環(huán)境的適應(yīng)胚胎發(fā)育、器官形成記憶同卵雙生子的后天獲得2022/7/24FUDAN EPIGENETICS LAB11生命的選擇很多時候不是簡單的Yes or No精細(xì)調(diào)節(jié) 多樣化本日大綱2022/7/24FUDAN EPIGENETICS LAB121.組蛋白修飾與組蛋白密碼 2

5、.組蛋白修飾與基因表達(dá)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2022/7/24FUDAN EPIGENETICS LAB13I. 組蛋白修飾與組蛋白密碼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(biāo)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）2022/7/24FUDAN E

7、PIGENETICS LAB18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？2022/7/24FUDAN 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？2022/7/24FUDAN 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.組蛋白修飾與基因表達(dá)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)26MLysine acetylationAAAAAAAAAAAAAH2BH2AA酰基化調(diào)控體系2

10、022/7/24FUDAN EPIGENETICS LAB27HDACsHATs and HDACs催化位點功能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的發(fā)現(xiàn)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四膜蟲 TetrahymenaDavid AllisMolecula

12、r Characterization of GCN52022/7/24FUDAN EPIGENETICS LAB29Brownell JE 1996 CellGCN5的功能2022/7/24FUDAN EPIGENETICS LAB30GCN5 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M(jìn)nucleosome 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）2022/7/24FUDAN EPIGENETICS LAB31Trapoxin 微生物來源的天然

13、化合物可抑制哺乳細(xì)胞去乙酰化并殺傷細(xì)胞HeLa cell whole cell extractA: untreatedB: Treated with TrapoxinWB: anti-H3AcHDAC/Rdp3的發(fā)現(xiàn) （化學(xué)生物學(xué)法）2022/7/24FUDAN EPIGENETICS LAB32Taunton J, 2000, ScienceStuart Schreiber?；娃D(zhuǎn)錄的關(guān)系2022/7/24FUDAN EPIGENETICS LAB33Struhl K 1998 Genes&DevNon-histone 乙酰化與代謝2022/7/24FUDAN EPIGENETICS LA

14、B34K. 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、H3K27、H3K36、H3K79和H4K20甲基化有單、雙和三甲基化三種水平35MLysine methylationMMMMMMH2BH2A2022/7/24FUDAN EPIGENETICS LAB362022/7/24FUDAN EPIGENETICS LAB37主要的組蛋白甲基化修飾的功能2022/7/24FUDAN EPIGENETICS LAB38甲基化位點分布特點功能甲基化

15、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Suv4-20H1-22022/7/24FUDAN EPIGENETICS LAB39III. 重要組蛋白修飾相關(guān)的Complex介紹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）多聚梳類Trithorax group （TrxG) 三胸類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甲基化識別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.組蛋白修飾與疾病和衰老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