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.

项目概要 DNA 编码遗传物质来指导所有细胞过程并建立细胞身份。 同一性是通过遗传内容和基因表达的调节来确定的。基因表达受到调节。 受许多因素影响，包括真核生物的染色质结构，染色质结构由核小体组成， 由包裹在组蛋白八聚体周围的约 150 bp DNA 组成，该结构调节多个 DNA。 依赖过程，包括转录和 DNA 损伤修复。 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 依赖性细胞过程受到染色质重塑剂的调节。 将 ATP 水解的能量耦合到滑动核小体、转移组蛋白和/或扭曲八聚体 — 调节染色质状态所必需的活性需要一种独特的染色质重塑剂 INO80。 INO80 在体外发挥 DEAD 家族 ATP 酶和辅助亚基滑动单核小体的作用。 然而，目前还不清楚 INO80 的滑动活性如何。 我们实验室最近的工作揭示了有关 INO80 的以下内容。 机制：(1) INO80 受到两个核小体信号的调节：侧翼 DNA 长度和侧翼 DNA 上的酸性片段 (2) INO80重塑反应至少有一个中间态以及该中间态之间的过渡 滑动产品的中间体受DNA长度调节；（3）Nhp10附件模块调节 有了这些发现，我追寻了 INO80 的感觉。 通过辅助亚基的各种核小体线索，这些相互作用有助于动力学 INO80 重塑反应的步骤我还采取了分布 INO80 的调节。 不同的核小体线索和辅助亚基使 INO80 能够对核小体线索做出相应的反应 这项研究将首先研究中间核小体状态。 由 INO80 重塑产生并研究核小体底物线索和 我的具体目标是 (1) 确定 INO80 反应动力学步骤的辅助模块。 INO80生成的中间核小体结构；(2)确定中间步骤如何 侧翼 DNA 脱皮调节 INO80 反应 (3) 确定 INO80 辅助模块的作用； 调节 INO80 感知和响应侧翼 DNA 长度的能力；以及 (4) 确定如何 INO80 辅助模块调节 INO80 感知和响应组蛋白线索的能力。