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

基本信息

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
项目类别：
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

项目摘要

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）：该提案阐述了组蛋白伴侣 FACT 和 Spt6 用于促进染色质组装、分解和重建的机制。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，具有可用于研究常见基本过程的遗传、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充分利用这些工具，可以将核小体转化为更容易接近 DNA 和组蛋白的替代结构形式（重组核小体）。比正常情况下，这对于打开染色质以读取 DNA 信息的过程非常重要，因此 FACT 还可以将松散关联的 DNA 组装成核小体。组蛋白构建染色质，这对于限制 DNA 的接触以保护其物理完整性以及在适当水平上优化基因表达非常重要。目前尚不清楚如何做出制造核小体或将其分开的决定，FACT 是如何做出的。目标 1 使用不同版本的 FACT 和组蛋白来表征重组，在体外使用纯化成分检查这些问题。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，其许多生化特性与 FACT 相同，但 Spt6 不能重组核小体，并且 FACT 不能执行独特的生理功能。因此，比较这两个伴侣的活性和功能将有助于了解相似的生化活性如何用于执行不同的功能。在调节染色质的局部特性方面发挥着重要作用，但目标 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 发挥作用发现了一种意想不到的、先前未描述的系统，用于防止组蛋白 mRNA 积累超出正常水平，揭示了一种新的调节机制，建议对该系统以及组蛋白伴侣在调节周转率中的作用进行初步研究。组蛋白和染色质。