Reconstitution and structural dissection of transcription-coupled histone H3.3 deposition

转录偶联组蛋白 H3.3 沉积的重建和结构剖析

• 批准号: 10323529
• 负责人: Hee Jong Kim
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10323529
• 关键词: Address; Affect; Age; Age of Onset; Aging; Ascomycota; Binding; Biochemical; Cell Extracts; Cell Survival; Chromatin; Chromatin Structure; Complex; Coupled; Crosslinker; Cryoelectron Microscopy; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Data Analyses; Dependence; Deposition; Disease; Dissection; Electron Microscopy; Elongation Factor; Gene Expression; Genes; Genetic Transcription; Goals; Histone H3; Histones; Human; In Vitro; Length; Light; Location; Longevity; Maintenance; Maps; Mass Spectrum Analysis; Messenger RNA; Modeling; Molecular; Molecular Chaperones; Nucleosomes; Pathway interactions; RNA; Role; Sampling; Structure; System; Transcription Elongation; Ursidae Family; Variant; Yeasts; analysis pipeline; cofactor; crosslink; cryogenics; hereditary prostate cancer protein 2; in vivo; interest; knockout gene; large datasets; normal aging; particle; reconstitution; recruit; restraint; therapeutic development; tool

Project Summary DNA-histone interactions are at least transiently lost during replication and transcription, which causes profound change in chromatin structure, and accompanies aging. Thus, efficient maintenance of chromatin structure during replication and transcription is known to be critical for cell survival and normal aging. Recently, the H3.3 histone variant has garnered particular interest in the context of aging because the H3.3 variant accumulates with age and impacts gene expression. In metazoans, the histone variant H3.3 along with H4 is deposited throughout transcribed genes by the HUC (HIRA/UBN1/CABIN1) complex, while in ascomycetes, such as yeast, only one form of histone H3, which shares the same evolutionary ancestry with H3.3 in metazoans, is deposited by the Hir (Hir1/Hir2/Hir3/Hpc2) complex. Understanding how the Hir complex functions in yeast will ultimately facilitate the development of therapeutic pathways to treat the onset of age-associated disease in humans. As my thesis project, I sought a possible cofactor of the Hir complex in yeast whole cell extract using mass spectrometry, and identified Spt4/5, a highly-conserved pol II elongation factor required for transcription of nearly all mRNA genes, which provided first direct evidence of physical interaction between pol II and histone chaperones for histone deposition. This proposal aims to uncover the molecular basis of Spt4/5 in facilitating histone deposition after passage of transcribing pol II by recruiting the Hir complex. I expect this study to provide the first molecular evidence of the interplay between histone deposition and transcription, as well as a first-of-its-kind structural view of its structural basis with following aims: (1) Investigate the functional roles of Spt4/5 in transcription coupled H3/H4 deposition in vitro transcription system. (2) Determine the molecular basis of the Hir complex in the transcription elongation complex via crosslinking mass spectrometry and cryo-EM.

项目概要 DNA-组蛋白相互作用在复制和转录过程中至少会暂时丢失，这 导致染色质结构发生深刻变化，并伴随衰老。因此，高效 众所周知，复制和转录过程中染色质结构的维持对于 细胞存活和正常衰老。最近，H3.3 组蛋白变体引起了特别关注 在衰老的背景下，因为 H3.3 变体随着年龄的增长而积累并影响基因 表达。在后生动物中，组蛋白变体 H3.3 和 H4 沉积在整个细胞中。 由 HUC (HIRA/UBN1/CABIN1) 复合体转录的基因，而在子囊菌中，例如 酵母，组蛋白 H3 的唯一一种形式，与酵母中的 H3.3 具有相同的进化祖先 后生动物由 Hir (Hir1/Hir2/Hir3/Hpc2) 复合体沉积。了解 Hir 如何 酵母中的复杂功能最终将促进治疗途径的开发 治疗人类与年龄相关的疾病的发作。作为我的论文项目，我寻求一种可能的 使用质谱法分析酵母全细胞提取物中 Hir 复合物的辅因子，并鉴定 Spt4/5，几乎所有 mRNA 转录所需的高度保守的 pol II 延伸因子 基因，首次提供了 pol II 和组蛋白之间物理相互作用的直接证据 组蛋白沉积的伴侣。该提案旨在揭示Spt4/5的分子基础 通过招募 Hir 复合物，在转录 pol II 通过后促进组蛋白沉积。 我希望这项研究能够提供组蛋白之间相互作用的第一个分子证据 沉积和转录，以及其结构基础的第一个结构视图 以下目标： (1) 体外研究Spt4/5在转录偶联H3/H4沉积中的功能作用 转录系统。 (2)确定转录延伸复合物中Hir复合物的分子基础 通过交联质谱和冷冻电镜。