Characterizing the mechanism of the INO80 family chromatin remodeling machine

表征 INO80 系列染色质重塑机的机制

• 批准号: 9911921
• 负责人: Muryam A.A. Gourdet
• 金额: $ 3.73万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911921
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Affect; Biochemical; Biological; Biological Assay; Biological Process; Cell physiology; Cells; Chromatin; Chromatin Structure; Complex; Cues; DNA; DNA Packaging; DNA Repair; Dependence; Eukaryota; Family; Foundations; Gene Expression; Gene Expression Regulation; Genetic; Genetic Code; Genetic Materials; Genetic Transcription; Global Change; Histone H2B; Histones; Human; Hydroxyl Radical; In Vitro; Individual; Kinetics; Laboratories; Length; Maintenance; Measures; Mediating; Methods; Molecular Conformation; Molecular Motors; Nucleosomes; Play; Positioning Attribute; Process; Reaction; Regulation; Research; Role; Saccharomyces cerevisiae; Site; Slide; Structure; Testing; Work; Yeasts; base; biological systems; chromatin remodeling; crosslink; dimer; in vivo; response

Project Summary DNA encodes the genetic material to instruct all cellular process and to establish cellular identity. Cellular identity is established by both genetic content and regulation of gene expression. Gene expression is regulated by many factors including chromatin structure in eukaryotes. Chromatin structure consists of nucleosomes, comprised of ~150 bp DNA wrapped around a histone octamer. This structure regulates several DNA dependent processes including transcription and DNA damage repair. Understanding the mechanisms that regulate chromatin structure is key to understanding how biological systems are controlled in the cell. DNA dependent cellular process are known to be regulated by chromatin remodelers. Chromatin remodelers couple the energy of ATP hydrolysis to slide nucleosomes, transfer histones, and/or distort the octamer— activities that are essential to regulate the chromatin state. A unique chromatin remodeler, INO80, requires both the DEAD family ATPase and accessory subunits to slide mononucleosomes in vitro. In vivo, INO80 plays a role in both transcription and DNA damage repairs. However, it is unclear how INO80 sliding activity contributes to its diverse biological roles. Recent work from our lab revealed the following about the INO80 mechanism: (1) INO80 is regulated by two nucleosome cues: flanking DNA length and an acidic patch on histones; (2) INO80 remodeling reaction has at least one intermediate state and transition between this intermediate to a sliding product is regulated by DNA length; (3) the Nhp10 accessory module regulates the transition between intermediate state and sliding activity. With these findings, I hypothesize that INO80 senses various nucleosome cues through the accessory subunits and that these interactions contribute to the kinetic steps of a INO80 remodeling reaction. I also hypothesize that distributing the regulation of INO80 across different nucleosome cues and accessory subunits allows INO80 to respond accordingly to nucleosome cues specific to various biological contexts. This research will first investigate the intermediate nucleosome states generated by INO80 remodeling and investigate the coordination between nucleosome substrate cues and accessory modules in regulating the kinetic steps in INO80 reaction. I specifically aim to (1) determine the intermediate nucleosome structures generated by INO80; (2) determine how the intermediate step of flanking DNA unpeeling regulates the INO80 reaction; (3) determine how INO80 accessory modules regulate the ability of INO80 to sense and respond to flanking DNA length; and (4) determine how the INO80 accessory modules regulate the ability of INO80 to sense and respond to histone cues.

项目摘要 DNA编码遗传物质以指导所有细胞过程并建立细胞身份。细胞 身份是通过遗传含量和基因表达的调节确定的。基因表达受调节 通过许多因素，包括真核生物中的染色质结构。染色质结构由核小体组成， 围绕组蛋白八聚体包裹的约150 bp DNA完成。该结构调节几个DNA 依赖过程，包括转录和DNA损伤修复。了解该机制 调节染色质结构是了解细胞中生物系统如何控制的关键。 已知DNA依赖性细胞过程受染色质重塑剂调节。 将ATP水解的能量与滑动核小体，转移组蛋白和/或扭曲八聚体的能量 - 调节染色质状态至关重要的活动。独特的染色质重塑，Ino80需要 死去的家族ATPase和附件亚基在体外滑动单核小体。在体内，Ino80播放 在转录和DNA损伤维修中的作用。但是，目前尚不清楚如何ino80滑动活动 有助于其潜水员生物学作用。我们实验室的最新工作揭示了以下有关INO80的工作 机理：（1）INO80受两个核小体线索调节：侧翼DNA长度和酸性贴片 希腊（2）INO80重塑反应至少具有一个中间状态，并且在此之间过渡 中间到滑动产物受DNA长度调节。 （3）NHP10附件模块调节 中间状态和滑动活动之间的过渡。通过这些发现，我假设Ino80感官 通过附件亚基各种核小体提示，这些相互作用有助于动力学 INO80重塑反应的步骤。我还假设在跨越INO80的调节 不同的核小体提示和附件亚基允许INO80对核小体提示做出相应的响应 特定于各种生物环境。这项研究将首先研究中间核小体状态 由INO80重塑产生并研究核体基材线索和 辅助模块确定INO80反应中的动力学步骤。我专门针对（1）确定 INO80产生的中间核探针结构； （2）确定中间步骤 侧面DNA脱离抗管可调节INO80反应； （3）确定INO80配件模块的方式 调节INO80感知和响应侧翼DNA长度的能力； （4）确定如何 INO80附件模块调节INO80感知和响应组蛋白提示的能力。