Biochemical, Genetic, and Genomic Analysis of Nucleosome Reorganization by FACT

通过 FACT 进行核小体重组的生化、遗传学和基因组分析

• 批准号: 9908077
• 负责人: Timothy G Formosa
• 金额: $ 34.31万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908077
• 关键词: ATP Hydrolysis; Access to Information; Address; Affect; Animal Model; Architecture; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Cells; Chromatin; Chromatin Structure; Collaborations; Complex; DNA; DNA Binding; DNA Binding Domain; DNA biosynthesis; Dissociation; Embryo; Eukaryota; Fibroblasts; Gene Expression; Genes; Genetic; Genetic Transcription; Genomic approach; Genomics; Goals; Grant; HMGB Proteins; Health; Histones; Housekeeping; Human; In Vitro; Individual; Intercistronic Region; Mediating; Methods; Modeling; Molecular Chaperones; Mus; Mutation; N-terminal; Nucleosomes; Organism; Outcome; Physiological; Plants; Polymerase; Process; Property; Publications; RNA Polymerase II; RNA Recognition Motif; Role; Saccharomyces cerevisiae; Structure; Surface; Testing; Time; Transcriptional Elongation Factors; Transcriptional Regulation; Variant; Work; Yeasts; biochemical tools; chromatin remodeling; collaborative approach; genome-wide; genomic tools; human tissue; in vivo; preservation; promoter; repaired; single molecule; tool

Project Summary/Abstract This proposal addresses mechanisms used by the histone chaperones FACT and Spt6 to promote assembly, disassembly, and repair of chromatin. Understanding how chromatin is formed and maintained is central to understanding regulation of transcription, DNA replication, and repair. The yeast Saccharomyces cerevisiae is used as a model system that has powerful genetic, biochemical, and genomic tools available to study fundamental processes common to all eukaryotes. The highly collaborative approach proposed here takes full advantage of these tools by using a broad range of methods simultaneously. FACT uses multiple histone- binding domains to convert nucleosomes into an alternative structural form (the reorganized nucleosome) in which both the DNA and the histones are more accessible. It does this without ATP hydrolysis, distinguishing it from chromatin remodelers. Reorganization is reversible, so FACT can also assemble nucleosomes out of loosely associated DNA and histones to construct chromatin. Spt6 also binds histones, DNA, and nucleosomes, but does not induce reorganization. Comparing the activities and functions of these two chaperones will help us understand how each of these factors can both block histone interactions and promote them at the appropriate times, yet each has unique physiological roles that the other cannot perform. FACT has been viewed as a housekeeping factor that promotes transcription and DNA replication by loosening each nucleosome encountered to allow polymerases to progress through chromatin. This view has been challenged and we are in the process of replacing it with a model in which FACT acts to establish and maintain chromatin in a precisely optimized architecture, and to restore this form after disturbances like transcription or replication. A key goal of this proposal is to continue challenging the existing model and to contribute to proposing and testing the new one. Aim 1 addresses questions about what drives the differential localization of FACT and Spt6, and how they promote or inhibit nucleosome turnover locally to sculpt chromatin structure genome-wide. Reorganization can promote assembly or disassembly of nucleosomes, but we do not know what the reorganized form looks like or what influences how it is resolved. Aim 2 examines this using two distinct single molecule methods in collaborations with the Ha and Studitsky labs, as well as our established and emerging biochemical assays examining the features of nucleosomes, histones, and chaperones that affect reorganization. Aim 3 examines the role of the DNA binding module that is common to all FACT complexes, but whose architecture varies among species. Together these approaches will provide answers to persistent questions about the mechanisms used by histone chaperones, as well as new questions about their physiological roles.

项目摘要/摘要 该建议介绍了组蛋白伴侣事实和SPT6使用的机制，以促进组装， 拆卸和染色质修复。了解染色质的形成和维护是至关重要的 了解转录，DNA复制和修复的调节。酿酒酵母的酵母菌是 用作具有强大遗传，生化和基因组工具可用于研究的模型系统 所有真核生物共有的基本过程。这里提出的高度协作方法可以满足 通过同时使用广泛的方法来优势。事实使用多个组蛋白 - 结合结构域将核小体转化为替代结构形式（重组核小体） DNA和组蛋白都更容易获得。它在没有ATP水解的情况下这样做，将其区分 来自染色质重塑。重组是可逆的，因此事实也可以从核素中汇出。 松散相关的DNA和组蛋白以构建染色质。 SPT6还结合组蛋白，DNA和核小体， 但不会引起重组。比较这两个伴侣的活动和功能将有助于 我们了解这些因素中的每一个都可以阻止组蛋白的相互作用并在 适当的时间，但是每个人都有对方无法执行的独特生理角色。事实一直是 被视为一个管家因素，通过松动每个 遇到的核小体是为了使聚合酶通过染色质进展。这种观点受到挑战 我们正在用一个模型来代替它，在该模型中，事实是建立和维护染色质的模型 在精确优化的体系结构中，并在诸如转录或复制之类的干扰之后恢复此形式。 该提案的关键目标是继续挑战现有模型，并为提议和 测试新的。 AIM 1解决了有关驱动事实差异定位的问题的问题 SPT6，以及它们如何促进或抑制局部核小体离职率，以塑造染色质结构范围。 重组可以促进核小体的组装或拆卸，但我们不知道什么 重组形式看起来像是什么影响它的解决方案。 AIM 2使用两个不同的单曲来检查这一点 分子方法与HA和Studitsky实验室合作，以及我们已建立和新兴的 生化分析检查影响核小体，组蛋白和伴侣的特征 重组。 AIM 3检查了所有事实复合物共有的DNA结合模块的作用， 但是其建筑在物种之间有所不同。这些方法共同为持久提供答案 有关组蛋白伴侣使用的机制的问题，以及有关其的新问题 生理角色。