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）： 该建议介绍了组蛋白链纳斯事实和SPT6使用的机制，以促进染色质的组装，拆卸和重建。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，因此了解如何形成和维持染色质对于理解这些核心过程中的每一个至关重要。酿酒酵母的酵母菌被用作具有遗传，生化和分子工具的强大模型系统，可用于研究所有真核生物共有的基本过程。这里提出的高度协作方法通过简单地使用广泛的方法来充分利用这些工具。事实可以将核小体转化为一种替代结构形式（重组的核小体），其中DNA和组蛋白都比正常人更容易获取，这对于打开从DNA读取信息的过程的染色质很重要。这种重组活性是可逆的，因此，事实还可以从松散相关的DNA和组蛋白中组装出核体，以构建染色质，这对于限制访问DNA以保护其物理完整性并在适当水平上优化基因表达非常重要。目前尚不清楚如何做出核病组或将其拆开的决定，如何将事实招募到特定的底物，或者如何调节其在正确的时间执行正确功能。 AIM 1使用不同版本的事实和组蛋白在体外进行纯化的组件来检查这些问题，以表征重组的核组组，以确定事实如何影响该状态的形成和解决方案，以及如何选择事实底物。 SPT6是另一个必不可少的组蛋白链酮，具有许多与事实共同的生化特性，但是SPT6无法重新组织核体组，事实无法执行SPT6的不同物理功能。因此，比较这两个链条的活动和功能将提供有关如何使用类似的生化活动来执行不同功能的洞察力。组蛋白链酮在调整染色质的局部特性方面具有重要作用，但是所使用的机制知之甚少。 AIM 2使用MNase-Seq和RNA-SEQ解决了这一问题，以评估具有突变，SPT6和组蛋白的菌株中适当表达的染色质质量并建立适当的表达。还测试了其他人提出的特定模型，以及探索库拉辛对事实的影响，探测报告表明，这类潜在的化学治疗药物通过抑制事实活动来起作用。已经发现了一种出乎意料的和以前未描述的系统，用于防止组蛋白mRNA超过其正常水平的积累，从而揭示了一种新型调节机制。 AIM 3建议该系统的初始投资以及组蛋白链酮在确定组蛋白和染色质的营业率速率中的作用。