Molecular mechanisms of the ACF chromatin remodeling complex

ACF染色质重塑复合物的分子机制

• 批准号: 10377574
• 负责人: Un Seng Chio
• 金额: $ 6.98万
• 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: 2020-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377574
• 关键词: ATP phosphohydrolase; Address; Air; Binding; Biochemical; Biochemistry; Biological Assay; Biological Models; Biophysics; California; Cell Differentiation process; Cell Nucleus; Cells; Chemicals; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Complex; Conflict (Psychology); Cryoelectron Microscopy; DNA; DNA biosynthesis; Data; Defect; Development; Disease; Drug Targeting; Electron Microscopy; Enzymes; Equipment; Eukaryotic Cell; Fixatives; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Gene Silencing; Generations; Genetic Transcription; Heterochromatin; Higher Order Chromatin Structure; Histone H1; Histone H4; Histones; Human; Institution; Lead; Link; Location; Malignant Neoplasms; Mediating; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Mutation; Nature; Nucleosomes; Physiological; Play; Principal Investigator; Process; Protein Denaturation; Protein Subunits; Proteins; Reporting; Research; Research Personnel; Resolution; Resources; Role; Sampling; San Francisco; Side; Slide; Specificity; Structure; Tail; Technology; Transact; Universities; Variant; Water; base; career; chromatin remodeling; cryogenics; developmental disease; experimental study; improved; infancy; insight; interdisciplinary approach; particle; post-doctoral training; repaired; single molecule; therapeutic development

PROJECT SUMMARY/ABSTRACT ATP-dependent chromatin remodelers catalyze nucleosome changes to regulate essential DNA-related processes, such as replication, transcription, and repair. Mutations of protein subunits within these remodeling complexes have been linked to various diseases including cancer and developmental disorders; however, the precise underlying mechanisms remain unclear. The ACF remodeling complex has served as a model system for understanding molecular mechanisms used in nucleosome remodeling. ACF is composed of the SNF2h ATPase and the Acf1 accessory subunit and generates evenly spaced nucleosome arrays that are important for forming silenced chromatin. Recent NMR and cryo-EM studies indicate SNF2h alone is able to deform histones within a nucleosome to carry out its functions, suggesting remarkable nucleosome plasticity. However, the exact molecular nature of the histone conformational changes remains unresolved. Additionally, the structure and function of the Acf1 subunit of ACF remains poorly characterized, and how ACF remodels nucleosomes in the presence of other heterochromatin-associated proteins remains unclear. The ability to address these questions using single-particle cryo-EM has faltered due to the difficulty in preparing cryo-EM sample grids of remodeling complexes bound to nucleosome substrate. We have now developed a new method to routinely prepare cryo-EM grids with intact remodeler-nucleosome complexes without the use of chemical fixatives. This technology will facilitate our ability to use an interdisciplinary approach combining single-particle cryo-EM, biochemical assays, and single-molecule fluorescence spectroscopy to elucidate mechanisms of ACF function as delineated in the research strategy. Specifically, I will: 1. determine the structural basis for ACF-mediated chromatin remodeling using single-particle cryo-EM; 2. define the interplay between ACF and linker histone H1 variants using biochemical assays, fluorescence-based assays, and single- particle cryo-EM; and 3. elucidate the connection between ACF and heterochromatin protein 1α using interaction assays and single-particle cryo-EM. In the long-term, we envision that the methods applied here will be generally applicable to study the molecular mechanisms of other chromatin-related enzymes and to understand why mutations of these enzymes lead to disease. The Principal Investigator (Un Seng Chio) will carry out the proposed experiments under the guidance of both Dr. Yifan Cheng (Sponsor), an expert in cryo-EM methodology, and Dr. Geeta Narlikar (Co- Sponsor), an expert in chromatin biochemistry, at the University of California, San Francisco (UCSF). UCSF is a well-established research institution with abundant resources for single-particle cryoEM and chromatin biochemistry both intellectually and equipment-wise, making it an ideal location for the PI to receive postdoctoral training while performing the proposed research. UCSF also offers many resources for the PI to develop career- wise as he prepares for the next stage of his career as an independent investigator.

项目总结/摘要 ATP依赖的染色质重塑催化核小体变化以调节必需的DNA相关的 过程，如复制，转录和修复。这些重塑中的蛋白质亚基突变 复合物与包括癌症和发育障碍在内的各种疾病有关;然而， 确切的潜在机制仍不清楚。ACF重塑复合体作为一个模型系统 用于理解核小体重塑的分子机制。ACF由SNF 2 h组成 ATP酶和Acf 1辅助亚基，并产生均匀间隔的核小体阵列，这对于 形成沉默的染色质。最近的NMR和cryo-EM研究表明SNF 2 h单独能够使组蛋白变形 在核小体内执行其功能，表明核小体具有显着的可塑性。但具体 组蛋白构象变化的分子本质仍然没有解决。此外，结构和 ACF的Acf 1亚基的功能仍然知之甚少，以及ACF如何重塑细胞核小体 其它异染色质相关蛋白的存在仍不清楚。 使用单粒子冷冻EM解决这些问题的能力由于以下困难而动摇： 制备与核小体基质结合的重塑复合物的冷冻-EM样品网格。我们现在已经 开发了一种新的方法来常规制备具有完整重塑核小体复合物的冷冻EM网格 而不使用化学固定剂。这项技术将促进我们使用跨学科方法的能力 结合单粒子冷冻EM、生物化学测定和单分子荧光光谱， 阐明研究策略中描述的ACF功能机制。具体来说，我会：1。确定 使用单颗粒冷冻EM的ACF介导的染色质重塑的结构基础; 2.定义相互作用 ACF和接头组蛋白H1变体之间的关系，使用生物化学测定、基于荧光的测定和单- 粒子冷冻EM;以及3.阐明ACF与异染色质蛋白1α之间的相互作用 测定和单颗粒冷冻EM。 从长远来看，我们设想这里应用的方法将普遍适用于研究 其他染色质相关酶的分子机制，并了解为什么这些酶的突变 导致疾病。主要研究者（Un Seng Chio）将根据 Yifan Cheng博士（申办者），冷冻EM方法学专家，和Geeta Narlikar博士（合作者）的指导。 赞助商），染色质生物化学专家，在加州大学弗朗西斯科分校（UCSF）。UCSF是 拥有丰富的单粒子cryoEM和染色质资源的成熟研究机构 生物化学的智力和设备明智的，使其成为PI接受博士后的理想地点 在进行拟议研究的同时进行培训。加州大学旧金山分校还为PI提供许多资源来发展职业生涯- 在他准备作为一名独立调查员进入职业生涯的下一阶段时，他是明智的。