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1、Histone ubquitination&deubquitination1组蛋白的泛素化与去泛素化The structure of nucleosome组蛋白是一种11-15KDa的碱性蛋白，与DNA相互作用构成核小体。核心组蛋白的尾巴伸出来，受到广泛的修饰。2组蛋白的泛素化与去泛素化Ubquitinl泛素(ubiquitin，Ub)是高度保守的、含76个氨基酸的蛋白质，分子量为8.5 kDa，在真核生物体内广泛存在。l泛素分子氨基端1-72位点的氨基酸残基形成一个紧密球状结构紧密球状结构，紧靠羧基端的4个氨基酸残基是随机盘绕的（随机盘绕的（random-coiled）。3组蛋白的泛素化与去
2、泛素化Ubiquitination&Deubiquitination泛素化修饰泛素化修饰就是底物的赖氨酸残基位点与泛素分子的羧基末端相互结合的过程。由于泛素本身的7个赖氨酸位点位点, 也可与另一个泛素相结合, 因此单泛素化单泛素化的底物蛋白, 可作为Seed，结合多个泛素，形成了多泛素化修饰。多泛素化修饰。4组蛋白的泛素化与去泛素化GENES & DEVELOPMENT 17:27332740 . 2003 by Cold Spring Harbor Laboratory PressHistone Ubiquitination5组蛋白的泛素化与去泛素化Histone ubiquitinatio
3、nPrimary monoubiquitinationreversible，regulated by ubiquitinase and deubquitinaseHistone modifications crosstalkInvolved in multiple cellular events-gene expression regulation、DNA damage response and repair、female X chromosome inactive and so on6组蛋白的泛素化与去泛素化除了单泛素化，除了单泛素化，H2A/H2AX在在DNA损伤时可发生损伤时可发生K63
4、位的多泛素化位的多泛素化7组蛋白的泛素化与去泛素化负责H2A/H2B泛素化与去泛素化的酶H2A-specific Common to H2A/H2B H2B-specific8组蛋白的泛素化与去泛素化Histone ubiquitinase One E1(activating), mutiple E2(conjugating),substrate-specific E3(ligase)Schematic depiction of histone ubiquitinationMethods. 2011 Jul; 54(3): 315325. E3主要有两大类: pHECT(homologous
5、to the E6-AP carboxyl terminus) 结构域家族，通过与泛素形成催化作用所必需的硫酯键硫酯键发挥作用pRING(really interesting new gene)结构域家族，为E2和底物提供居留位点居留位点从而使 E2 催化泛素转移到底物上9组蛋白的泛素化与去泛素化Histone deubiquitinating enzyme（DUBs）H2A deubiquitination USP 16： HOX gene silencing, DNA damage repair X chromosome inactivation, cell cycle progressi
6、on2A-DUB ：interacts with PCAFUSP21： regulator of liver regenerationBAP1： C-terminal hydrolase,repression of HOX genesH2B deubiquitination Ubp8 ：reside within SAGA，Transcription Ubp10 （Dot4）：Sir-mediated telomeric and rDNA silencing10组蛋白的泛素化与去泛素化Function of Histone UbiquitinationTranscription regulat
7、ionDNA damage responseinactive X chromosome：H2Aubchromatin boundary integrity：H2Bubstem cell maintenance and differentiationHistone Ubiquitination and Deubiquitination in Transcription, DNA Damage Response, and Cancer. Front Oncol, 2(26),Cao J & Yan Q,201211组蛋白的泛素化与去泛素化Regulation of cell cycle progr
8、ession chromosome segregation during mitosisUbp-M (USP16) can deubiqutinate H2A.During the cell cycle, Ubp-M is sequentially phosphorylated and dephosphorylated, potentially by the cdc-2/cyclin B complex.Ubp-M is essential for Ser-10 H3 phosphorylation mediated by the Aurora B kinase and is required
9、 for chromosome segregation during mitosis.12组蛋白的泛素化与去泛素化Cell Reports, Volume 10, Issue 2, 2015, 226 - 238http:/dx.doi.org/10.1016/j.celrep.2014.12.021DNA damage repairATM:ataxia telangiectasia mutatedDSB:DNA double-strand breakRNF:Ring finger ligase（E3）DDR:DNA damage response53BPI：p53 binding prote
10、in 113组蛋白的泛素化与去泛素化Transcription regulationH2A与H2B的单泛素化与转录调节有关: H2Aub与基因沉默有关,H2Bub与基因活化有关【CHIP-on-chip实验证明 ubH2A在卫星区，ubH2B在转录活化基因的主体】H2A/H2B泛素化机制：co-transcription mechanismH2B的泛素化与去泛素化的循环与转录起始及延伸有密切关系14组蛋白的泛素化与去泛素化ubH2A&silenceH2A-specific E3：Ring1B，2A-HUB，与转录沉默有关Ring1B存在于3种不同的转录抑制复合物PRC1, BCoR及E2F-1
11、中2A-HUB与N-CoR/HDAC1/3 complex联系，定位于chemokine genes的启动子处，阻断FACT的招募15组蛋白的泛素化与去泛素化a.Histone H2A Monoubiquitination Represses Transcription byH3K4 methylation is essential for preinitiation complex(PIC) formation.Ubiquitylation of H2A does inhibitpreinitiation complex formation, not in a direct way buti
12、ndirectly by preventing H3K4 methylation. Deubiquitylation ofhistone H2A by USP21 activates transcriptional initiation viatrans-histone crosstalk with H3K4 di- and tri-methylation.Fig. 1 Model of transcriptional initiation from chromatin template.Inhibiting PIC formation16组蛋白的泛素化与去泛素化b.Histone H2A M
13、onoubiquitination Represses Transcription by 2A-HUB:a H2Aspecific ubiquitin ligase, catalyzes monoubiquitination of H2A at K119H2A monoubiquitination acts to prevent FACT recruitment at the transcriptional promoter region, blocking RNA polymerase II release at the early stage of elongation.Molecular
14、 Cell, MG Rosenfeld, 1(29), 2008Inhibiting RNAP II Transcriptional ElongationFACT:facilitates chromatin transcription二聚体，包括SPT16和SSRP1，能从核小体中置换出H2A/H2B二聚体，进而促使染色体介导的转录延伸抑制得到释放17组蛋白的泛素化与去泛素化Fig. 1. The diverse activities of H2Bub1. H2B monoubiquitylation and deubiquitylation can directly modulate the
15、 chromatin state by altering nucleosome stability, promoting partial nucleosome disassembly and reassembly, and regulating chromatin higher-order structure. modulate chromatin indirectly through the binding or repulsion of specific readers, which call into action a plethora of proteins and protein c
16、omplexes with diverse biochemical activities. The direct and indirect mechanisms are not mutually exclusiveE.g.relaxation of higher-order chromatin structure is expected to facilitate the access of H2Bub1 readers注：注：H2Bub1即对即对K120进行泛素化进行泛素化G. Fuchs, M. Oren / Biochimica et Biophysica Acta 1839 (2014
17、) 694701H2B18组蛋白的泛素化与去泛素化H2B Ubiquitination Requires Early Steps in Transcription Elongation(Co-Transcription)1.酸性激活蛋白GAL4招募H2B特异的泛素酶复合物到启动子区2.在与PAF、BUR（对Rad6的Ser120磷酸化）、延伸形式的RNAP2（其CTD的Ser5被Kin28磷酸化）的作用下，泛素酶复合物对H2B的K120位进行泛素化（co-transcription）19组蛋白的泛素化与去泛素化Model for the regulation of chromatin dyna
18、mics by H2B ubiquitination and deubiquitination during transcription elongation.Rad6 and Bre1 associate with the Paf1 complex and travel with the elongating form of RNA Pol (i and ii) Rad6/Bre1-mediated H2Bub1 stabilizes the nucleosome in front of the polymerase to counteract any torsional stress an
19、d might acts as a“checkpoint” to coordinate transcription-coupled events, such as, allowing the binding of Spt16/FACT and restricting Ctk1 binding to chromatin. 泛素化泛素化 稳定稳定(B) In (i), Ubp8 ,a component of the SAGA sub-complex that travels with RNA Pol II, removes the conjugated ubiquitin to destabil
20、ize the nucleosome. (ii) This facilitates nucleosome disassembly by Spt16/FACT. (iii) Deubiquitination by Ubp8 allows association of Ctk1 with chromatin for the phosphorylation of serine 2 in RNA Pol II CTD. 去泛素化去泛素化 不稳定不稳定(C) In (i), nucleosomes are reassembled behind the elongating RNA Pol II like
21、ly by the step-wise initial addition of H3-H4 by Spt6 followed by the addition of H2A-H2B by Spt16/FACT. (ii) Rad6/Bre1-mediated H2Bub1 stabilizes the nucleosome to facilitate nucleosome reassembly and to prevent any backtracking by RNA Pol II. (iii) Stable nucleosome also prevents any promiscuous t
22、ranscription that might occur by the binding of TBP to cryptic TATA-like sequences within the coding region.20组蛋白的泛素化与去泛素化A.RNAP2前方的H2B泛素化，检查转录偶联事件B.H2B去泛素化，核小体解体，允许RNAP2继续前行C.RNAP2通过后，核小体的H2B泛素化，发生重组，防止未知位点的转录起始regulation of chromatin dynamics by H2B ubiquitination and deubiquitination during trans
23、cription elongation.21组蛋白的泛素化与去泛素化Histone modifications crosstalk 组蛋白的修饰间是相关的，它们联合合或者顺序序地地发挥作用来调控转录。22组蛋白的泛素化与去泛素化Figure 7.The trans-histone crosstalk between histone H2B ubiquitination and H3K4/K79 methylation in budding yeast. COMPASS:complex associated with Set1Epigenetics 5:6, 460-468; August 16
24、, 2010; 2010 Landes BioscienceH2B Monoubiquitination Is a Prerequisite for Lys-4 H3 and Lys-79 H3 Methylation23组蛋白的泛素化与去泛素化 crosstalk between H2B Ubiquitination&H3K4 methylation 1.酸性激活蛋白GAL4招募H2B特异的泛素酶复合物到启动子区2.在与PAF、BUR（对Rad6的Ser120磷酸化）、延伸形式的RNAP2（其CTD的Ser5被Kin28磷酸化）的作用下，泛素酶复合物对H2B的K120位进行泛素化（co-tr
25、anscription）3.H2B的泛素化是招募CPS35必须的，Cps35是COMPASS甲基化酶复合体的一个组分，COMPASS能介导H3K4的二甲基化或三甲基化24组蛋白的泛素化与去泛素化参考文献1Higashi M, Inoue S, Ito T. Core histone H2A ubiquitylation and transcriptional regulationJ. Experimental cell research, 2010, 316(17): 2707-2712.2 Gatti M, Pinato S, Maiolica A, et al. RNF168 Promot
26、es Noncanonical K27 Ubiquitination to Signal DNA DamageJ. Cell reports, 2015, 10(2): 226-238.3Weake V M, Workman J L. Histone ubiquitination: triggering gene activityJ. Molecular cell, 2008, 29(6): 653-663.4 Chandrasekharan M B, Huang F, Sun Z W. Histone H2B ubiquitination and beyond: Regulation of
27、nucleosome stability, chromatin dynamics and the trans-histone H3 methylationJ. Epigenetics, 2010, 5(6): 460-468.5 Fuchs G, Oren M. Writing and reading H2B monoubiquitylationJ. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 2014, 1839(8): 694-701.6 Osley M A. Regulation of histone H
28、2A and H2B ubiquitylationJ. Briefings in functional genomics & proteomics, 2006, 5(3): 179-189.25组蛋白的泛素化与去泛素化Thank you for listen!26组蛋白的泛素化与去泛素化Histone Ub&Cancer利用利用H2Bub（K120）-specic antibodies进行行IHC染色染色Either too little or too much H2Bub may equally hinder proper chromatin function, distort gene e
29、xpression patterns and perhaps increase genome instability, paving the road to cancer.Explantation:H2Bub can suppress the expression of a subgroup of genes that includes several proto-oncogenes, while positively regulating the expression of the p53 tumor suppressor reducing H2Bub levels in cells by partial depletion of RNF20 or RNF40 results in impaired DNA double-strand break repair 3638, elevated replication stress and chromosomal instability 73, estrogen-independentgrowth of breast cancer cells and increased cell survival signaling27组蛋白的泛素化与去泛素化
