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

项目摘要 DNA-组蛋白相互作用至少在复制和转录过程中暂时丢失，这 导致染色质结构的深刻变化，并伴随着衰老。因此，高效 已知在复制和转录过程中保持染色质结构对于 细胞存活和正常衰老。最近，H3.3组蛋白变异体引起了特别的兴趣 在衰老的背景下，因为H3.3变异随着年龄的增长而积累并影响基因 表情。在后生动物中，组蛋白变异体H3.3和H4一起沉积在整个 由Huc(Hira/UBN1/CABIN1)复合体转录的基因，而在子囊菌中，如 酵母，只是组蛋白H3的一种形式，它与H3.3有着相同的进化祖先 后生动物，由Hir(Hir1/Hir2/Hir3/Hpc2)杂岩沉积。了解员工如何 酵母中复杂的功能最终将促进治疗途径的发展 治疗人类年龄相关疾病的发病。作为我的论文项目，我寻求一种可能的 酵母菌全细胞抽提物中HIR复合体的辅因子，并鉴定 Spt4/5，一种高度保守的PolII伸长因子，几乎所有mRNA的转录都需要它 基因，这为PolII和组蛋白之间的物理相互作用提供了第一个直接证据 组蛋白沉积的伴侣。这项提议旨在揭示Spt4/5的分子基础 通过招募HIR复合体来促进转录PolII通过后组蛋白的沉积。 我希望这项研究能提供第一个组蛋白相互作用的分子证据。 沉积和转录，以及其结构基础的首个结构视图， 以下目标： (1)探讨Spt4/5在体外转录偶联H3/H4沉积中的作用 转录系统。 (2)确定转录延伸复合体中HIR复合体的分子基础 通过交联质谱联用和冷冻-EM分析。

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.