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）： 该提案涉及组蛋白伴侣 FACT 和 Spt6 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的转录、复制和 DNA 修复，因此了解染色质的形成和维持方式对于理解这些核心过程至关重要。酿酒酵母被用作强大的模型系统，具有可用于研究所有真核生物共有的基本过程的遗传、生化和分子工具。这里提出的高度协作方法通过同时使用多种方法来充分利用这些工具。 FACT 可以将核小体转化为另一种结构形式（重组核小体），其中 DNA 和组蛋白都比正常情况更容易接近，这对于打开染色质以从 DNA 读取信息的过程非常重要。这种重组活动是可逆的，因此 FACT 还可以从松散关联的 DNA 和组蛋白中组装核小体来构建染色质，这对于限制 DNA 的接触以保护其物理完整性以及在适当的水平上优化基因表达非常重要。目前尚不清楚如何决定制造核小体或将其分开，如何将 FACT 招募到特定底物上，或者如何调节它在正确的时间执行正确的功能。目标 1 在体外用纯化成分检查这些问题，使用不同版本的 FACT 和组蛋白来表征重组核小体，以确定 FACT 如何影响这种状态的形成和解析，以及如何选择 FACT 底物。 Spt6 是另一种重要的组蛋白伴侣，具有许多与 FACT 相同的生化特性，但 Spt6 不能重组核小体，FACT 不能执行 Spt6 独特的生理功能。因此，比较这两种伴侣的活性和功能将有助于深入了解如何利用相似的生化活性来执行不同的功能。组蛋白伴侣在调节染色质的局部特性方面发挥着重要作用，但所使用的机制却知之甚少。目标 2 使用 MNase-Seq 和 RNA-Seq 来解决这一问题，以评估染色质质量并在具有 FACT、Spt6 和组蛋白突变的菌株中建立适当的抑制。还测试了其他人提出的具体模型，以及探索 curaxins 对 FACT 的影响，探索报告表明此类潜在化疗药物通过抑制 FACT 活性发挥作用。人们发现了一种意想不到的、之前未曾描述过的系统，可以防止组蛋白 mRNA 积累超出正常水平，揭示了一种新的调节机制。目标 3 建议对该系统以及组蛋白伴侣在调节组蛋白和染色质周转率中的作用进行初步研究。