Mechanisms for Histone H2B Deubiquitination

 描述（由申请人提供）：组蛋白的翻译后修饰调节所有需要进入DNA的过程。小蛋白泛素与组蛋白H2 B的连接是活跃转录基因的普遍特征，并被认为在染色质结构的动态变化中发挥作用。然而，缺乏对酶如何调节这种修饰以及泛素的附着和去除如何与核小体动力学耦合的机制理解。在阐明H2 B泛素化如何调节转录的许多障碍中，缺乏对调节这种修饰的酶与核小体底物结合的方式的分子见解。酵母中的H2 B泛素化水平由两种去泛素化酶调节：四蛋白佐贺DUB模块，它是1.9 MDa佐贺转录辅激活因子复合物的一部分，和Ubp 10，一种单体酶。虽然DUB模块和Ubp 10表面上靶向相同的底物进行泛素化，但缺失任一酶在体内具有不同的效果，表明这些酶靶向不同的基因组位置或与染色质不同地相互作用。在过去的资助期间，我们成功地结晶了佐贺DUB模块-Ubp 8/Sgf 11/Sus 1/Sgf 73-结合到泛素化的核小体上。我们将建立在这一成就，以解决H2 B泛素化是如何调节和H2 B去泛素化是如何耦合到核小体动力学。在目标1中，我们将通过确定与泛素化核小体结合的Ubp 8/Sgf 11/Sus 1/Sgf 73的原子分辨率结构来解决佐贺DUB模块如何识别泛素化核小体。这些结果也将为探索组蛋白H2 A-Y 58磷酸化的串扰如何调节酵母和人类细胞中DUB模块的活性提供基础。在目标2中，我们将确定Ubp 10特异性参与染色质的机制，以及佐贺DUB模块和Ubp 10与H2 B结合之间的差异可能是其独特生物学功能的基础。在目标3中，我们将探讨组蛋白伴侣在控制Ubp 10和DUB模块对组蛋白底物的特异性方面所起的作用。

 DESCRIPTION (provided by applicant): Post-translational modification of histone proteins regulates all processes requiring access to DNA. Attachment of the small protein, ubiquitin, to histone H2B is a universal feature of actively transcribed genes and has been proposed to play a role in dynamic changes in chromatin structure. However, a mechanistic understanding of how enzymes regulate this modification, and how attachment and removal of ubiquitin may be coupled to nucleosome dynamics, is lacking. Among many barriers to elucidating how H2B ubiquitination regulates transcription has been the lack of molecular insights into the way that enzymes that regulate this modification engage their nucleosome substrates. Levels of H2B ubiquitination in yeast are regulated by two deubiquitinating enzymes: the four-protein SAGA DUB module, which is part of the 1.9 MDa SAGA transcriptional coactivator complex, and Ubp10, a monomeric enzyme. Although both the DUB module and Ubp10 ostensibly target the same substrate for ubiquitination, deletion of either enyzme has a distinct effect in vivo, suggesting that these enzyme are either targeted to different genomic locations or interact differently with chromatin. In the past funding period, we succeeded in crystallizing the SAGA DUB module - Ubp8/Sgf11/Sus1/Sgf73 - bound to ubiquitinated nucleosomes. We will build upon this accomplishment to address how H2B ubiquitination is regulated and how H2B deubiquitination is coupled to nucleosome dynamics. In Aim 1, we will address how the SAGA DUB module recognizes ubiquitinated nucleosomes by determining the atomic resolution structure of Ubp8/Sgf11/Sus1/Sgf73 bound to ubiquitinated nucleosomes. These results will also provide a foundation for probing how cross-talk with phosphorylation of histone H2A-Y58 regulates DUB module activity in yeast and human cells. In Aim 2, we will determine the mechanism by which Ubp10 specifically engages chromatin and how differences between SAGA DUB module and Ubp10 binding to H2B might underlie their distinct biological functions. In Aim 3, we will explore the role that histone chaperones play in governing the specificity of both Ubp10 and the DUB module for their histone substrates.