Analysis of conserved eukaryotic transcription elongation factors

保守的真核转录延伸因子分析

基本信息

批准号：
10531245
负责人：
FRED M. WINSTON
金额：
$ 43.22万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10531245
关键词：
Address Alanine Amino Acids Binding Binding Sites Biochemical Biological Assay Biological Models Cancer Etiology Cells ChIP-seq Chromatin Chromatin Structure Compensation Complex DNA DNA Damage DNA Double Strand Break DNA biosynthesis Defect Development Disease Double Strand Break Repair Elongation Factor Gene Expression Genetic Transcription Genome Stability Genomic Instability Genomic approach Genomics Goals Growth Growth and Development function Health Histones Human Human Biology Hybrids Impairment In Vitro Knowledge Learning Malignant Neoplasms Mammalian Cell Modeling Molecular Chaperones Mutation N-terminal Nature Nucleosomes Organism Phenotype Play Positioning Attribute Process Protein Analysis Protein Dynamics Proteins Publishing RNA RNA Polymerase II Regulation Role Saccharomyces cerevisiae Series Suppressor Genes Suppressor Mutations Surface Testing Transcription Alteration Transcription Elongation Transcriptional Elongation Factors Transcriptional Regulation Yeasts experimental study genetic information genome integrity histone modification human disease improved in vivo insight mutant mutation screening repaired

项目摘要

PROJECT SUMMARY/ABSTRACT The long-term objectives of this project are to increase our understanding of eukaryotic transcription elongation, with a focus on histone chaperones. Histone chaperones control the assembly and disassembly of nucleosomes during transcription, replication, and repair. The proposed experiments address the conserved histone chaperone Spt6, using the yeast Saccharomyces cerevisiae, as a model system. Spt6 is conserved and its human counterpart has been implicated in developmental control and in cancer. Previous analysis of Spt6 has demonstrated that it is broadly required for transcription and chromatin structure in both yeast and mammalian cells. While it is established that Spt6 interacts with histones, RNA polymerase II, and other proteins, the mechanisms by which it functions are unknown. The proposed experiments in Specific Aim 1 will address the interactions of Spt6 with two other essential and conserved histone chaperones, Spn1/Iws1 and FACT. Preliminary studies have shown that an spt6 mutant, spt6-YW, that impairs the physical interaction of Spt6 with Spn1, has changes in growth, transcription and chromatin structure. Additional studies identified suppressor mutations that compensate for spt6-YW mutant defects. Several of these suppressor mutations cause clustered changes in a conserved surface of FACT. Aim 1.1 tests the model that spt6-YW and its suppressors control transcription and chromatin structure via alterations of the transcription elongation complex and histone modifications. This will be assayed by a set of ChIP-seq experiments. Aim 1.2 studies the changes in FACT that compensate for the Spt6 defect. These results will provide new understanding of the functional relationships among histone chaperones and provide insights into why so many of them are vital during transcription. Specific Aim 2 focuses on Spt6 binding to histones, an essential function for all of histone chaperones. This aim tests the model that Spt6 has multiple histone binding sites in its highly acidic and disordered N-terminal domain. Aim 2.1 will isolate spt6 mutants in the N-terminal region that are defective for function. Aim 2.2 will use these mutants to define Spt6-histone binding in vitro. Aim 2.3 will address key issues regarding Spt6-histone interactions during transcription. Together, these experiments will elucidate an essential function of Spt6. Specific Aim 3 addresses a related but distinct role for Spt6, in the control of genome integrity, as spt6 mutants display genome instability phenotypes. The proposed experiments will test whether Spt6 is required for genome stability by the control of chromatin structure, transcription, or resolving transcription-replication conflicts. Experiments will assay RNA:DNA hybrids, which contribute to genome instability, double-strand DNA breaks, and will test the model that Spt6 is required for DNA replication as well as transcription. The results will provide new understanding of the control of genome stability, a fundamental and conserved process important for human health.

项目概要/摘要该项目的长期目标是增加我们对真核转录的理解延伸，重点关注组蛋白伴侣。组蛋白伴侣控制组蛋白的组装和拆卸转录、复制和修复过程中的核小体。所提出的实验解决了保守的问题组蛋白伴侣 Spt6，使用酿酒酵母作为模型系统。 Spt6 是保守的其人类对应物与发育控制和癌症有关。之前的分析 Spt6 已证明它是酵母菌和酵母菌中转录和染色质结构所广泛需要的。哺乳动物细胞。虽然已确定 Spt6 与组蛋白、RNA 聚合酶 II 和其他蛋白相互作用蛋白质，其发挥作用的机制尚不清楚。具体目标 1 中提出的实验将解决 Spt6 与另外两个重要且保守的组蛋白伴侣 Spn1/Iws1 和事实。初步研究表明，spt6 突变体 spt6-YW 会损害 Spt6 与 Spn1 一样，在生长、转录和染色质结构方面都有变化。确定的其他研究补偿 spt6-YW 突变体缺陷的抑制突变。其中一些抑制突变引起 FACT 保守表面的聚集变化。目标1.1测试spt6-YW及其模型抑制因子通过改变转录延伸复合物来控制转录和染色质结构和组蛋白修饰。这将通过一组 ChIP-seq 实验进行测定。目标 1.2 研究 FACT 的变化弥补了 Spt6 的缺陷。这些结果将为我们提供新的认识组蛋白伴侣之间的功能关系，并深入了解为什么其中如此多的蛋白伴侣如此重要在转录过程中。具体目标 2 重点关注 Spt6 与组蛋白的结合，这是所有组蛋白的基本功能伴侣。该目的测试了Spt6在其高酸性和高酸性中具有多个组蛋白结合位点的模型。无序的 N 端结构域。目标 2.1 将分离 N 末端区域有缺陷的 spt6 突变体功能。目标 2.2 将使用这些突变体来定义 Spt6-组蛋白的体外结合。目标 2.3 将解决关键问题关于转录过程中 Spt6-组蛋白相互作用。这些实验将共同阐明 Spt6 的基本功能。具体目标 3 解决了 Spt6 的一个相关但独特的角色，即控制基因组完整性，因为 spt6 突变体表现出基因组不稳定表型。拟议的实验将测试通过控制染色质结构、转录或解析，Spt6 是否是基因组稳定性所必需的转录复制冲突。实验将分析 RNA:DNA 杂交体，这对基因组有贡献不稳定性、双链 DNA 断裂，并将测试 DNA 复制所需的 Spt6 模型作为转录。这些结果将为基因组稳定性的控制提供新的认识，这是一个基本的和保守的过程对人类健康很重要。