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

描述（由申请人提供）： FACT是两种蛋白质的复合物，在所有真核生物中发现，包括人类，并且对所有测试生物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DNA的包装体。它们的特性控制着基因组的信息流。该项目的目标是确定FACT如何导致核小体采用替代结构，并了解何时何地形成和解析这种结构的能力在活细胞中有用。FACT在调节染色质的因子中是独特的，因为它不修饰组成核小体的组蛋白，它不使用能量源来使组蛋白核心沿着DNA移动，并且它通过一种在任何其他组蛋白伴侣中未观察到的机制起作用。FACT已在广泛的系统生物学研究中被鉴定，这些研究寻求调节细胞生理学、促进正常胚胎发育和允许干细胞维持多能性的因子。因此，FACT的作用机制和使用它的环境对于理解基本的真核生物生理学具有重要意义。一个集成的方法，使用酵母模型系统，提出了这些研究，以利用这些生物体中可用的工具的范围。 该提案的第一个目的是分析被FACT改变的核小体的性质，以产生所谓的“重组”形式。这种结构与典型的核小体明显不同，但其性质仍不清楚。用荧光染料标记的纯化组蛋白和DNA片段将用于探测重组核小体的性质，并且突变组蛋白和FACT蛋白将用于这些测定中，以允许将结果与活细胞中FACT的性质进行比较。第二个目的是通过使用全基因组测序鉴定使其更容易或更难耐受FACT缺陷的突变，并通过询问FACT和其他组蛋白伴侣中的不同突变如何影响维持隐蔽启动子抑制的能力，并使用转录本和染色质免疫沉淀（ChIP）的定量PCR测量促进不同染色质状态的稳定性，来表征FACT的作用。第三个目的是询问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发射，并测试FACT在复制过程中基因组复制后沉积新染色质的假设作用。这一目标使用一些已建立的ChIP技术，但也需要开发一些新的方法。

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.