Biochemical and Genetic Analysis of yFACT, A Novel Nucleosome Reorganizing Factor

新型核小体重组因子 yFACT 的生化和遗传分析

• 批准号: 8450938
• 负责人: Timothy G Formosa
• 金额: $ 29.98万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8450938
• 关键词: Adopted; Affect; Attention; Biochemical Genetics; Biological Assay; Cell physiology; Cells; Characteristics; Chromatin; Chromatin Modeling; Chromatin Structure; Complex; Crosslinker; Crystallography; DNA; DNA Packaging; DNA biosynthesis; DNA replication origin; Defect; Deposition; Development; Embryonic Development; Energy-Generating Resources; Enhancers; Eukaryota; Eukaryotic Cell; Fluorescent Dyes; Gene Expression; Genetic; Genetic Screening; Genetic Transcription; Genome; Goals; Grant; Health; Histone H2A; Histones; Human; In Vitro; Label; Lead; Learning; Life; Maps; Measurement; Methods; Modeling; Molecular Chaperones; Mutation; Nature; Nucleosomes; Organism; Outcome; Phase; Physiological; Physiology; Play; Polymerase; Process; Property; Proteins; RNA; Reactive Oxygen Species; Repression; Research; Role; Stem cells; Structure; Systems Biology; Techniques; Technology; Testing; Transcript; Work; Yeast Model System; Yeasts; abstracting; chromatin immunoprecipitation; dimer; genetic analysis; genetic manipulation; genome sequencing; histone-binding proteins; in vivo; insight; mutant; novel; pluripotency; prevent; promoter; protein complex; public health relevance; research study; tool

DESCRIPTION (provided by applicant): Project Summary/Abstract FACT is a complex of two proteins that are found in all eukaryotes, including humans, and is essential for viability in all organisms tested. Nucleosomes are the fundamental subunit of chromatin, which is the DNA packaging in eukaryotes. Their properties govern the flow of information from the genome. The goals of this project are to determine how FACT causes nucleosomes to adopt an alternative structure and to learn when and where the ability to form and resolve such structures is useful in living cells. FACT is unique among factors that regulate chromatin because it does not modify the histones that make up nucleosomes, it does not use an energy source to move the histone cores along the DNA, and it works through a mechanism that has not been observed with any other histone chaperone. FACT has been identified in a wide range of systems biology studies seeking factors that regulate cellular physiology, promote normal embryonic development, and allow stem cells to maintain pluripotency. Both the mechanism of FACT action and the circumstances in which it is used are therefore of high importance for understanding basic eukaryotic physiology. An integrated approach using a yeast model system is proposed for these studies to take advantage of the range of tools available in these organisms. The first aim of the proposal is to analyze the properties of nucleosomes that have been altered by FACT to produce what is called a "reorganized" form. This structure is significantly different from canonical nucleosomes, but its nature remains poorly characterized. Purified histones and DNA fragments labeled with fluorescent dyes will be used to probe the properties of reorganized nucleosomes, and mutant histone and FACT proteins will be used in these assays to allow the results to be compared with the properties of FACT in living cells. The second aim is to characterize the roles of FACT by identifying mutations that make it easier or harder to tolerate FACT defects using whole-genome sequencing, and by asking how distinct mutations in FACT and other histone chaperones affect the ability to maintain repression of cryptic promoters and promote stability of distinct chromatin states using quantitative PCR measurements of transcripts and chromatin immunoprecipitations (ChIPs). The third aim is to ask how FACT affects chromatin structure near origins of DNA replication, how this contributes to regulating origin firing, and to test a hypothetical role for FACT in depositing new chromatin after the genome is duplicated during replication. This aim uses some established ChIP technology but also requires development of some novel methods.

描述（由申请人提供）： 项目摘要/摘要 FACT 是一种由两种蛋白质组成的复合物，存在于包括人类在内的所有真核生物中，对于所有测试的生物体的生存能力至关重要。核小体是染色质的基本亚基，染色质是真核生物中的 DNA 包装。它们的特性控制着来自基因组的信息流。该项目的目标是确定 FACT 如何导致核小体采用替代结构，并了解形成和解析此类结构的能力何时何地在活细胞中有用。 FACT 在调节染色质的因素中是独一无二的，因为它不会修改构成核小体的组蛋白，它不使用能源来沿着 DNA 移动组蛋白核心，并且它通过任何其他组蛋白伴侣尚未观察到的机制发挥作用。 FACT 已在广泛的系统生物学研究中得到证实，这些研究旨在寻找调节细胞生理学、促进正常胚胎发育和允许干细胞保持多能性的因素。因此，FACT 作用机制及其使用环境对于理解基本真核生理学非常重要。为这些研究提出了一种使用酵母模型系统的综合方法，以利用这些生物体中可用的一系列工具。 该提案的第一个目的是分析被 FACT 改变的核小体的特性，以产生所谓的“重组”形式。这种结构与典型核小体显着不同，但其性质仍知之甚少。纯化的组蛋白和荧光染料标记的 DNA 片段将用于探测重组核小体的特性，突变组蛋白和 FACT 蛋白将用于这些测定中，以便将结果与活细胞中 FACT 的特性进行比较。第二个目标是通过使用全基因组测序识别使 FACT 缺陷更容易或更难耐受的突变来表征 FACT 的作用，并通过使用转录本和染色质免疫沉淀 (ChIP) 的定量 PCR 测量来探究 FACT 和其他组蛋白伴侣中的不同突变如何影响维持隐性启动子的抑制和促进不同染色质状态的稳定性的能力。第三个目标是询问 FACT 如何影响 DNA 复制起点附近的染色质结构，这如何有助于调节起点激发，并测试 FACT 在基因组复制过程中复制后沉积新染色质的假设作用。这一目标使用了一些成熟的 ChIP 技术，但也需要开发一些新方法。