Supplement Characterizing the mechanism of the INO80 family chromatin remodeling machine

补充描述 INO80 系列染色质重塑机的机制

• 批准号: 10558103
• 负责人: Muryam A.A. Gourdet
• 金额: $ 2.36万
• 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 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10558103
• 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。蜂窝 身份是通过遗传内容和基因表达调节来确定的。基因表达受调控 包括真核生物的染色质结构。染色质结构由核小体组成， 由围绕组蛋白八聚体的~150 bp DNA组成。这种结构调节几种DNA 依赖过程包括转录和DNA损伤修复。了解这些机制， 调节染色质结构是理解生物系统在细胞中是如何被控制的关键。 已知DNA依赖性细胞过程受染色质重塑物调节。染色质重塑剂 耦合ATP水解的能量以滑动核小体、转移组蛋白和/或扭曲八聚体- 对调节染色质状态至关重要的活性。一种独特的染色质重塑剂INO 80需要 DEAD家族ATP酶和辅助亚基在体外滑动单核细胞。在体内，INO 80发挥 在转录和DNA损伤修复中的作用。然而，目前还不清楚INO 80滑动活动如何 有助于其多样化的生物学作用。我们实验室最近的工作揭示了以下关于INO 80的信息 INO 80的调节机制：（1）INO 80受两个核小体信号的调控：侧翼DNA长度和侧翼酸性补丁。 （2）INO 80重塑反应至少有一个中间状态，并在此之间转换。 滑动产物的中间体受DNA长度调节;（3）Nhp 10辅助模块调节滑动产物的中间体。 中间状态和滑动活动之间的过渡。根据这些发现，我假设INO 80感觉到 各种核小体线索通过辅助亚基，这些相互作用有助于动力学 INO 80重塑反应的步骤。我还假设，将INO 80的调节分布在 不同的核小体提示和辅助亚基允许INO 80对核小体提示作出相应的反应 特定于各种生物学背景。本研究将首先研究中间核小体状态 通过INO 80重塑产生，并研究核小体底物线索与 INO 80反应动力学步骤调控的辅助模块。我的主要目的是（1）确定 INO 80产生的中间核小体结构;（2）确定INO 80产生的中间步骤是如何产生的。 侧翼DNA去剥离调控INO 80反应;（3）确定INO 80辅助模块如何 调节INO 80感知和响应侧翼DNA长度的能力;以及（4）确定INO 80如何与DNA结合。 INO 80附属模块调节INO 80感知和响应组蛋白线索的能力。