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/抽象事实是在包括人类在内的所有真核生物中发现的两种蛋白质的复合物，对于所有测试的生物体的生存力至关重要。核小体是染色质的基本亚基，这是真核生物中的DNA包装。它们的特性控制了来自基因组的信息流。该项目的目标是确定事实是如何导致核小体采用替代结构，并了解形成和解决此类结构的能力何时何地对活细胞有用。事实在调节染色质的因素之间是独一无二的，因为它没有改变组成核小体的组蛋白，因此不使用能源来移动组蛋白核心沿DNA，并且它通过未与任何其他组蛋白伴侣观察到的机制起作用。在广泛的系统生物学研究中已经确定了事实，以寻求调节细胞生理学，促进正常胚胎发育并允许干细胞保持多能性的因素。因此，事实行动的机制及其使用的环境对于理解基本真核生理学既重要。为这些研究提出了使用酵母模型系统的集成方法，以利用这些生物中可用的工具范围。 该提案的第一个目的是分析核小体的特性，这些核小体的特性已被事实改变，以产生所谓的“重组”形式。该结构与规范核小体有显着不同，但其性质的特征仍然很差。用荧光染料标记的纯化的组蛋白和DNA片段将用于探测重组核小体的特性，其中突变组蛋白和事实蛋白将在这些测定中使用，以允许将结果与活细胞中事实的特性进行比较。第二个目的是通过识别突变来表征事实的作用，这些突变使使用全基因组测序耐受事实缺陷变得更加容易或难以忍受事实缺陷，并询问实际上以及其他组蛋白伴侣的不同突变如何影响抑制隐性启动子的抑制能力，并使用定量的CRCR测量转录蛋白免疫蛋白（CHIPS）（CHIPS）（CHIPS）（CHIPS）（CHIPS）（CHIPS）（CHIPS）（CHIPS）（CHIPS）（CHIPS）促进稳定性的稳定性。第三个目的是询问事实如何影响DNA复制的起源附近的染色质结构，这如何有助于调节起源的触发，并测试事实在复制过程中重复基因组后沉积新染色质的假设作用。该目标使用一些已建立的芯片技术，但还需要开发一些新颖的方法。