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: 9038371
• 负责人: Timothy G Formosa
• 金额: $ 32.52万
• 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 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9038371
• 关键词: Address; Affect; Alleles; Animal Model; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Cell Cycle; Cells; Chromatin; Chromatin Structure; Confusion; DNA; DNA Repair; DNA biosynthesis; DNA-Binding Proteins; Data; Development; Eukaryota; Figs - dietary; Flavoring; Gene Expression; Gene Expression Profile; Generic Drugs; Genes; Genetic; Genetic Suppression; Genetic Transcription; Grant; Growth; Health; Histones; Human; In Vitro; Investigation; Lead; Malignant Neoplasms; Measurement; Measures; Messenger RNA; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Profiling; Mutate; Mutation; Nucleosomes; Outcome; Pathway interactions; Pattern; Pharmaceutical Preparations; Physiological; Positioning Attribute; Process; Property; Proteins; Publishing; Reading; Recruitment Activity; Regulation; Reporting; Repression; Resolution; Role; Saccharomyces cerevisiae; Site; System; Testing; Time; Toxin; Transcript; Work; Yeasts; cancer therapy; chromatin protein; flexibility; gene repression; genetic analysis; improved; in vitro Model; in vivo; insight; mutant; novel; prevent; reconstruction; repaired; research study; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): This proposal addresses mechanisms used by the histone chaperones FACT and Spt6 to promote assembly, disassembly, and reconstruction of chromatin. The structure of chromatin strongly influences transcription, replication, and DNA repair in all eukaryotes, so understanding how chromatin is formed and maintained is central to understanding each of these core processes. The yeast Saccharomyces cerevisiae is used as a powerful model system that has genetic, biochemical, and molecular 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 can convert nucleosomes into an alternative structural form (the reorganized nucleosome) in which both the DNA and the histones are more accessible than normal, which is important for opening chromatin for processes that read information from the DNA. This reorganization activity is reversible, so FACT can also assemble nucleosomes out of loosely associated DNA and histones to construct chromatin, which is important for limiting access to the DNA to protect its physical integrity and also to optimize expression of genes at the appropriate level. It is not clear how the decision to make nucleosomes or take them apart is made, how FACT is recruited to specific substrates, or how it is regulated to perform the correct function at the correct time. Aim 1 examines these issues with purified components in vitro, using different versions of FACT and histones to characterize reorganized nucleosomes, to determine how FACT influences the formation and resolution of this state, and how FACT substrates are chosen. Spt6 is another essential histone chaperone with many biochemical properties in common with FACT, but Spt6 cannot reorganize nucleosomes and FACT cannot perform the distinct physiological functions of Spt6. Comparing the activities and functions of these two chaperones will therefore provide insight into how similar biochemical activities are used to perform distinct functions. Histone chaperones have an important role in tuning the local properties of chromatin, but mechanisms used are poorly understood. Aim 2 addresses this using MNase-Seq and RNA- Seq to assess chromatin quality and establishment of appropriate repression in strains with mutations in FACT, Spt6, and histones. Specific models that have been proposed by others are also tested, as well as exploration of the effects of curaxins on FACT, probing reports suggesting this class of potential chemotherapeutic drugs act by inhibiting FACT activity. An unexpected and previously undescribed system for preventing the accumulation of histone mRNAs beyond their normal levels has been discovered, revealing a novel mechanism for regulation. Aim 3 proposes initial investigation of this system and the roles of histone chaperones in regulating the rate of turnover of histone proteins and chromatin.

 描述（由申请人提供）： 这一提议涉及组蛋白伴侣FACT和Spt 6用于促进染色质组装、拆卸和重建的机制。染色质的结构强烈影响所有真核生物中的转录，复制和DNA修复，因此了解染色质如何形成和维持对于理解这些核心过程中的每一个都至关重要。酿酒酵母是一个强大的模型系统，具有遗传、生物化学和分子工具，可用于研究所有真核生物共同的基本过程。这里提出的高度协作的方法充分利用这些工具，同时使用广泛的方法。FACT可以将核小体转化为另一种结构形式（重组的核小体），其中DNA和组蛋白都比正常情况下更容易接近，这对于打开染色质以从DNA读取信息的过程非常重要。这种重组活性是可逆的，因此FACT还可以从松散缔合的DNA和组蛋白中组装核小体以构建染色质，这对于限制DNA的访问以保护其物理完整性以及优化适当水平的基因表达非常重要。目前尚不清楚如何决定制造核小体或将其拆开，FACT如何被招募到特定底物，或如何调节其在正确的时间执行正确的功能。目的1研究这些问题与纯化的成分在体外，使用不同版本的FACT和组蛋白来表征重组的核小体，以确定FACT如何影响这种状态的形成和解决，以及如何选择FACT底物。Spt 6是另一种必需的组蛋白伴侣，具有与FACT共同的许多生化特性，但Spt 6不能重组核小体，FACT不能执行Spt 6的独特生理功能。因此，比较这两种分子伴侣的活性和功能将提供对相似的生化活性如何用于执行不同功能的深入了解。组蛋白分子伴侣在调节染色质的局部性质中具有重要作用，但所使用的机制知之甚少。目的2使用MNase-Seq和RNA-Seq来解决这个问题，以评估染色质质量和在具有FACT、Spt 6和组蛋白突变的菌株中建立适当的阻遏。还测试了其他人提出的特定模型，以及探索curaxins对FACT的影响，探索表明这类潜在化疗药物通过抑制FACT活性起作用的报告。一个意想不到的和以前未描述的系统，用于防止组蛋白mRNA的积累超过其正常水平已被发现，揭示了一种新的调节机制。目的3：初步探讨组蛋白伴侣在调控组蛋白和染色质更新速率中的作用。