Analysis of conserved eukaryotic transcription elongation factors

保守的真核转录延伸因子分析

• 批准号: 10321279
• 负责人: FRED M. WINSTON
• 金额: $ 43.61万
• 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/10321279
• 关键词: Address; Alanine; Amino Acids; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Cancer Etiology; Cells; ChIP-seq; Chromatin; Chromatin Structure; Complex; Conflict (Psychology); 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; Malignant Neoplasms; Mammalian Cell; Modeling; Molecular Chaperones; Mutation; N-terminal; Nature; Nucleosomes; Organism; Phenotype; Play; Positioning Attribute; Process; Protein Analysis; Proteins; Publishing; RNA; RNA Polymerase II; Regulation; Role; Saccharomyces cerevisiae; Series; 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.

项目总结/摘要 这个项目的长期目标是增加我们对真核生物转录的理解 延伸，重点是组蛋白伴侣。组蛋白分子伴侣控制蛋白质的组装和分解。 在转录、复制和修复过程中的核小体。拟议的实验解决了保守的 组蛋白伴侣Spt 6，使用酵母酿酒酵母，作为模型系统。Spt 6是保守的 人类的对应物与发育控制和癌症有关。的先前分析 Spt 6已经证明它在酵母和酵母中的转录和染色质结构中广泛需要。 哺乳动物细胞虽然已经确定Spt 6与组蛋白、RNA聚合酶II和其他蛋白相互作用， 蛋白质，它的功能机制是未知的。具体目标1中拟议的实验将 解决Spt 6与其他两个必需的和保守的组蛋白伴侣Spn 1/Iws 1和 事实初步研究表明，一种spt 6突变体spt 6-YW，它损害了 Spt 6与Spn 1在生长、转录和染色质结构上都有变化。确定的其他研究 抑制基因突变补偿spt 6-YW突变缺陷。其中几种抑制基因突变 引起FACT保守表面的聚集性变化。目的1.1检验spt 6-YW及其 抑制子通过改变转录延伸复合体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研究了 FACT的变化，弥补了Spt 6的缺陷。这些结果将提供新的认识， 组蛋白伴侣之间的功能关系，并提供了为什么这么多的人是至关重要的见解 在转录过程中。具体目标2关注Spt 6与组蛋白的结合，这是所有组蛋白的基本功能， 监护人这一目的测试了Spt 6在其高酸性和低pH环境中具有多个组蛋白结合位点的模型。 无序的N端结构域。目的2.1将分离N-末端区域中的spt 6突变体，这些突变体在以下方面有缺陷： 功能目的2.2将使用这些突变体来定义Spt 6-组蛋白体外结合。目标2.3将解决关键问题 关于Spt 6-组蛋白在转录过程中的相互作用。总之，这些实验将阐明 Spt 6的基本功能。具体目标3解决了Spt 6的一个相关但独特的作用，即控制 基因组完整性，因为SPT 6突变体显示基因组不稳定表型。拟议的实验将测试 Spt 6是否是通过控制染色质结构、转录或解析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，这有助于基因组 不稳定性，双链DNA断裂，并将测试DNA复制所需的Spt 6模型 作为转录。研究结果将为控制基因组稳定性提供新的认识，这是一个基本问题 和保存过程对人类健康很重要。