Effects of histone ubiquitylation on nucleosome dynamics

组蛋白泛素化对核小体动力学的影响

• 批准号: 9816424
• 负责人: Tae-Hee Lee
• 金额: $ 30.15万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9816424
• 关键词: Acetylation; Affect; Amino Acids; Autoimmune Diseases; Biochemical; Biological Process; Biophysics; Chromatin; Chromatin Fiber; Chromosomes; Collaborations; Complex; Conflict (Psychology); Core Protein; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Disease; Dissociation; Gene Expression; Gene Expression Regulation; Generations; Genes; Genome; Goals; Health; Histone H2A; Histone H2B; Histones; Human; Impairment; Inherited; Kinetics; Knowledge; Light; Location; Malignant Neoplasms; Measures; Mediating; Metabolic Diseases; Methods; Molecular; Molecular Chaperones; Monitor; Motion; Nucleosomes; Play; Process; Recruitment Activity; Regulation; Regulator Genes; Repression; Resolution; Role; Site; Stress; Structure; System; Testing; Thermodynamics; Time; Torsion; Transcription Elongation; Transcriptional Regulation; Ubiquitin; Yeasts; base; dimer; flexibility; gene repression; mechanical force; single molecule; spatiotemporal

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.

项目摘要 本研究的长期目标是阐明组蛋白泛素化的分子机制， 调节染色质活性，这在很大程度上仍然未知。去理解人类 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， 由~147 bp DNA，两个组蛋白H2 A-H2 B二聚体和一个（H3-H4）2四聚体组成的染色质，以及（2）它是如何 通过组蛋白伴侣调节六酶体产生的效率和通过RNA调节转录延伸的效率 聚合酶II（Pol II）。这些研究利用了位点特异性泛素化组蛋白和尖端的 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 改变核小体内动力学，从而物理调节基因可及性。初步 生物化学和单分子研究表明，组蛋白H2 BK 34和H2 BK 120泛素化 伴随核小体结构的不稳定，诱导H2 A-H2 B二聚体的不稳定。在 与组蛋白受体如组蛋白伴侣Nap 1、H2 BK 34和H2 BK 120泛素化协作 促进两种H2 A-H2 B二聚体之一的解离，产生六酶体， 处于活性染色质状态的亚核小体中间体。本提案的目的是阐明如何 组蛋白泛素化改变了核小体的动力学，并最终改变了这些变化与 调节六酶体产生和转录延伸的效率。具体目标是：（1） 定义H2 BK 34/120泛素化对核小体结构动力学的影响， 开发了单分子结构柔性测试方法;（2）评估H2 BK 34/K120 ub的效果 （i）通过组蛋白伴侣Nap 1和FACT产生六酶体的动力学，以及（ii） 在Nap 1和FACT介导的核小体分解和重组的背景下的转录延伸， 两者都是基于最近开发的转录延伸监测系统的单分子水平。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。