Effects of histone ubiquitylation on nucleosome dynamics

项目摘要 这项研究的长期目标是阐明组蛋白泛素化的分子机制。 调节染色质的活性，这在很大程度上仍然是未知的。要了解以下基本方式 组蛋白泛素化影响染色质介导的生物学过程，本项目重点揭示(1) 组蛋白泛素化如何影响核小体的结构动力学，核小体是组蛋白的基本包装单位 染色质由约147bp的DNA、两个组蛋白H_2A-H_2B二聚体和一个(H_3-H_4)_2四聚体组成，以及(2)它是如何 组蛋白伴侣调节六倍体的生成效率和RNA调节转录延长 聚合酶II(POL II)。这些研究利用了特定部位泛素化的组蛋白和尖端技术 生物化学和单分子方法。这个项目的一个新主题是组蛋白泛素化 改变核小体内的动态，从而在物理上调节基因的可及性。预赛 生化和单分子研究表明，组蛋白H_2BK34和H_2BK120泛素化 伴随着核小体结构的不稳定，导致H_2A-H_2B二聚体的不稳定。在……里面 与组蛋白受体如组蛋白伴侣NAP1、H_2BK34和H_2BK120泛素化的协作 促进两个H_2A-H_2B二聚体中的一个解离，产生关键的六体 处于活跃染色质状态的亚核小体中间体。这项提议的目的是阐明如何 组蛋白泛素化改变核小体动态，并最终改变如何牵涉到 调节六倍体的产生和转录延伸的效率。具体目标是(1) 用最近的一项研究确定了H_2BK34/120泛素化对核小体结构动力学的影响 发展了单分子结构柔韧性测试方法和(2)评价H_2BK34/K120ub的效果 关于(I)组蛋白伴侣NAP1和FACT产生六倍体的动力学 在NAP1和FACT介导的核小体拆解和重组的背景下的转录延长， 两者都是基于最近开发的转录延伸监测系统在单分子水平上的。这 该项目将导致对组蛋白泛素化的直接物理作用的基本理解 调节染色质动力学。

Project Summary The long-term goal of this study is to elucidate the molecular mechanisms of how histone ubiquitylation regulates chromatin activity, which remains largely unknown. To understand the fundamental ways in which histone ubiquitylation influences chromatin-mediated biological processes, this project focuses on revealing (1) how histone ubiquitylation affects the structural dynamics of the nucleosome, the fundamental packing unit of chromatin made of ~147 bp DNA, two histone H2A-H2B dimers and one (H3-H4)2 tetramer, and (2) how it regulates the efficiency of hexasome generation by histone chaperone and transcription elongation by RNA polymerase II (Pol II). These studies take advantage of site-specifically ubiquitylated histones and cutting-edge biochemical and single-molecule approaches. An emerging theme in this project is that histone ubiquitylation alters intra-nucleosomal dynamics and thus physically regulates gene accessibility. The preliminary biochemical and single-molecule studies have shown that histone H2BK34 and H2BK120 ubiquitylation accompanies destabilization of the nucleosome structure, inducing labilization of H2A-H2B dimers. In collaboration with a histone acceptor such as histone chaperone Nap1, H2BK34 and H2BK120 ubiquitylation promote dissociation of one of the two H2A-H2B dimers, producing hexasomes that are emerging as critical sub-nucleosomal intermediates in an active chromatin state. The goal of this proposal is to elucidate how histone ubiquitylation alters the nucleosome dynamics and eventually how the changes are implicated in regulating the efficiencies of hexasome generation and transcription elongation. The specific aims are (1) to define the effects of H2BK34/120 ubiquitylation on the structural dynamics of the nucleosome with a recently developed single-molecule structural flexibility test method and (2) to evaluate the effects of H2BK34/ K120ub on (i) the kinetics of hexasome generation by histone chaperone Nap1 and FACT and (ii) the kinetics of transcription elongation in the context of Nap1- and FACT-mediated nucleosome disassembly and reassembly, both at a single-molecule level based on a recently developed transcription elongation monitoring system. This project will result in fundamental understanding on the direct physical roles of histone ubiquitylation in regulating chromatin dynamics.