【作者】 李静；

【导师】 翁杰敏；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 在真核细胞中,DNA与组蛋白包装形成染色质。在现有的多种组蛋白修饰中,组蛋白赖氨酸的甲基化实际上参与了所有的以染色质为基础的生物进程,包括转录调控和DNA损伤修复。每一个赖氨酸残基能够被一甲基,二甲基或三甲基化修饰。而这些组蛋白甲基化修饰的生物效应不仅仅由特异位点的甲基化修饰决定,还受甲基化修饰的阶段影响。目前认为组蛋白甲基化修饰能供提供位点来招募(或排斥)一些被称为“阅读因子”或“效应因子”的特异的染色质相关蛋白。这些效应蛋白因此对解释甲基化密码,以及组蛋白甲基化修饰介导的生物学效应是非常关键的。因此,在本文中,我们鉴定了两个H3K4二甲基化修饰的效应因子：NRDc和PAF1复合体。nardilysin(NRDc),是一个M16家族的金属肽酶。在本研究中,我们鉴定到了NRDc能作为一个新的H3K4二甲基化修饰的结合蛋白。生化实验表明NRDc与转录共抑制因子NCoR复合体相关。通过microarray我们鉴定到了受NRDc抑制的下游靶基因。发现NRDc能直接与这些靶基因结合,并且与靶基因的直接结合以及对NCoR复合体的招募都是依赖于H3K4me2的结合活性。因此,我们的研究鉴定了一个新的H3K4me2结合蛋白并揭示了NRDc在转录调控的作用。PAF1复合体是一个RNA pol Ⅱ的转录延伸因子,并能在转录过程中能够介导多种组蛋白的修饰。在本研究中,我们发现了PAF1复合体一个新的功能——与组蛋白H3和H3K4me2特异结合,并鉴定了介导这一结合功能的关键亚基——PAF1。通过构建PAF1的缺失体蛋白进行pulldown分析,发现了PAF1介导组蛋白特异结合功能的功能区域位于C端。因此,我们的研究表明PAF1复合体能通过PAF1亚基介导H3和H3K4me2特异结合。更多还原

【Abstract】 In eukaryotic cells, DNA is packaged with histones in the form of chromatin. Among various histone modifications identified, histone lysine methylation functions in virtually all chromatin based biological processes including transcriptional regulation and DNA damage repair. Each lysine residue can be mono-, di-, or tri-methylated. As a result, the biological effects of histone methylation are not only determined by the specific sites of methylation but also influenced by the methylation states. Mechanistically, histone methylation is believed to function at least in part as docking sites to recruit (or repel) specific chromatin-associated proteins called "reader" or "effector" proteins. The effector proteins are therefore critical for interpreting the methylation codes and thus mediate the biological effect of histone methylation. In this study, we identified two di-methylated H3K4effectors:NRDc and PAF1complex.nardilysin (NRDc), is a member of M16family metalloendopeptidases. In this study, we identified NRDc as a novel dimethyl-H3K4(H3K4me2) binding protein. Biochemical purification demonstrates that NRDc associates with the corepressor NCoR complex. We identified target genes repressed by NRDc through microarray. We showed that NRDc is physically associated with and recruits the NCoR complex to the target genes tested and this association requires its H3K4me2binding activity. Thus, our study has identified a novel H3K4me2binding protein and revealed a role of NRDc in transcriptional regulation.PAF1complex is a RNA pol Ⅱ associtated elongation factor and mediates several histone modifications in gene transcription. In this study, we identified PAF1complex as H3and H3K4me2specific binding proteins. We also found the PAF1subunit of the PAF1complex is likely responsible for H3and H3K4me2. We mapped the function domain responsible for H3and H3K4me2specific binding to the Oterminus of PAF1. Our study provides evidence that PAF1complex specificly binds H3and H3K4me2through PAF1subunit.更多还原