Molecular mechanisms of the ACF chromatin remodeling complex

项目摘要/摘要 依赖于ATP的染色质重构体催化核小体改变以调节基本的DNA相关 复制、转录和修复等过程。这些重塑过程中蛋白质亚单位的突变 复合体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改建综合体被作为一个模型系统 以了解核小体重塑的分子机制。ACF是由SnF2H组成的 ATPase和Acf1辅助亚基，并产生均匀分布的核小体阵列，这对 形成沉默的染色质。最近的核磁共振和冷冻-EM研究表明，仅Snf2小时就能使组蛋白变形 在核小体内执行其功能，表明核小体具有显著的可塑性。然而，准确的 组蛋白构象变化的分子性质仍未解决。此外，该结构和 ACF的Acf1亚单位的功能仍未得到很好的描述，以及ACF如何重塑核小体。 其他异染色质相关蛋白的存在尚不清楚。 使用单粒子低温电磁解决这些问题的能力已经动摇，因为 制备与核小体底物结合的重塑复合体的低温EM样品网格。我们现在有了 开发了一种新的方法来常规制备具有完整重构体-核小体复合体的冷冻-EM网格 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、生化分析和单分子荧光光谱 阐明ACF的作用机制，如研究策略所示。具体地说，我将：1.确定 ACF介导的单粒子冷冻-EM染色质重塑的结构基础；2.确定相互作用 ACF和连接物组蛋白H1变异体之间使用生化分析，基于荧光的分析，和单一的 3.利用相互作用阐明acf与异染色质蛋白1α的关系。 化验和单粒子冷冻-EM。 长远来说，我们预期所采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以及为什么这些酶的突变 会导致疾病。首席调查员(赵恩生)将根据 由低温EM方法学专家郑一凡博士(发起人)和Geeta Narlikar博士(合作伙伴)共同指导。 赞助商)，加州大学旧金山分校染色质生物化学专家。加州大学旧金山分校是 一个成熟的研究机构，拥有丰富的单粒子低温EM和染色质资源 在智力和设备方面的生物化学，使它成为PI获得博士后的理想地点 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提供了许多发展事业的资源- 当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。

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.