Analysis of conserved eukaryotic transcription elongation factors

保守的真核转录延伸因子分析

• 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
• 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

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.

项目摘要/摘要 该项目的长期目标是增加我们对真核转录的理解 伸长，重点是组蛋白伴侣。组蛋白伴侣控制组装和拆卸 转录，复制和修复过程中的核小组。提出的实验解决了保守的 组蛋白伴侣SPT6，使用酿酒酵母作为模型系统。 SPT6是保守的 它的人类对应物与发育控制和癌症有关。先前的分析 SPT6已证明，在酵母和 哺乳动物细胞。虽然确定SPT6与组蛋白相互作用，RNA聚合酶II和其他 蛋白质，其功能的机制尚不清楚。特定目标1中的拟议实验将 解决SPT6与其他两个必需和保守的组蛋白伴侣的相互作用，SPN1/IWS1和 事实。初步研究表明，SPT6突变体SPT6-YW会损害其物理相互作用 SPT6带有SPN1，具有生长，转录和染色质结构的变化。确定的其他研究 补偿SPT6-YW突变缺陷的抑制突变。这些抑制突变中的几个 导致事实的保守表面中的聚类变化。 AIM 1.1测试SPT6-YW及其的模型 抑制器通过改变转录伸长复合物控制转录和染色质结构 和组蛋白的修饰。这将通过一组CHIP-seq实验来测定。 AIM 1.2研究 实际上，弥补SPT6缺陷的变化。这些结果将提供对 组蛋白伴侣之间的功能关系，并提供有关为什么这么多至关重要的见解 在转录期间。特定的目标2重点是SPT6与组蛋白的结合，这是所有组蛋白的重要功能 伴侣。该目标测试了SPT6在其高度酸性和 n末端域无序。 AIM 2.1将在N末端区域分离出SPT6突变体，这些突变体有缺陷 功能。 AIM 2.2将使用这些突变体在体外定义SPT6-固定结合。 AIM 2.3将解决关键问题 关于转录过程中的SPT6-酮相互作用。这些实验将共同阐明 SPT6的基本功能。特定目标3在控制中解决了SPT6的相关但独特的作用 基因组完整性，因为SPT6突变体显示基因组不稳定性表型。提出的实验将测试 是否需要通过控制染色质结构，转录或解决基因组稳定性SPT6 转录复制冲突。实验将测定RNA：DNA杂交，这有助于基因组 不稳定性，双链DNA断裂，并将测试SPT6也需要DNA复制的模型 作为转录。结果将为控制基因组稳定性的控制提供新的理解，这是一个基本的 保守的过程对人类健康很重要。