Structural biology of chromatin in vitro and in cells

项目摘要 由PiGalia DebelouChina领导的实验室有以下目标：1)发展结构 生物学方法论，研究复杂和动态的体外生物组装，以及2)扩展 这些方法用于细胞环境中的结构生物学研究。我们的方法论 发展结合了固体核磁共振波谱和最先进的技术 用于全面描述体外和细胞内生物系统的化学生物学工具。我们 使用这些技术来研究染色质，一种复杂的蛋白质-DNA聚合物，负责 将遗传信息打包到细胞核中。未来五年，我们计划重点做好以下工作 项目：1)蛋白质翻译后修饰对染色质结构和动力学的影响。 甲基化和乙酰化等修饰对染色质的紧凑有深远的影响 聚合物和包装的DNA的可及性。我们计划研究其分子机制。 使用固态在原子分辨率下由这些修饰带来的染色质压缩和去压缩 核磁共振波谱。我们期望这项研究将为人类的生物物理特性提供独特的见解 染色质聚合物以及如何将其转化为功能性生物产物。2)分子基础 异染色质形成。异染色质隔间与基因沉默和重复有关 DNA序列及其形成是细胞分化和发育过程中至关重要的一步。最近的假设 表明它们是通过液-液相分离形成的。我们计划研究这种相互作用 促进相分离并提供这一过程的第一个分子描述。3)发展 基于核磁共振的细胞结构生物学工具。在这里，我们计划重点设计和实现一个 针对细胞环境中的特定蛋白质并选择性地增加其核磁共振信号的策略 手机背景。最终，我们的目标是开发一种选择性和灵敏的核磁共振方法，使 美国记录内源性细胞蛋白量的相关结构数据。这些工具将提供 史无前例的机会构建细胞环境的原子分辨率图像并研究 当我们对细胞施加压力、老化或用治疗剂处理细胞时，蛋白质结构和动力学的变化。

Project Summary The laboratory led by PI Galia Debelouchina has the following objectives: 1) The development of structural biology methodology to study complex and dynamic biological assemblies in vitro, and 2) The extension of these methodologies for structural biology investigations in the cellular environment. Our methodology development combines solid-state nuclear magnetic resonance (NMR) spectroscopy with state-of-the-art chemical biology tools for the comprehensive description of biological systems both in vitro and in cells. We use these technologies to investigate chromatin, a complex protein-DNA polymer responsible for the packaging of genetic information in the nucleus. Over the next five years, we plan to focus on the following projects: 1) The influence of protein post-translational modifications on chromatin structure and dynamics. Modifications such as methylation and acetylation have profound effects on the compaction of the chromatin polymer and the accessibility of the packaged DNA. We plan to investigate the molecular mechanisms of chromatin compaction and decompaction imparted by these modifications at atomic resolution using solid-state NMR spectroscopy. We expect that this study will provide unique insights into the biophysical properties of the chromatin polymer and how they can be translated into functional biological outputs. 2) The molecular basis of heterochromatin formation. Heterochromatin compartments are associated with gene silencing and repetitive DNA sequences, and their formation is a vital step in cell differentiation and development. Recent hypotheses indicate that they are formed through liquid-liquid phase separation. We plan to investigate the interactions that promote phase separation and to provide the first molecular description of this process. 3) Development of NMR-based tools for structural biology in cells. Here, we plan to focus on the design and implementation of a strategy to target a specific protein in the cellular milieu and to increase its NMR signals selectively over the cellular background. Ultimately, we aim to develop a selective and sensitive NMR methodology that will allow us to record relevant structural data of endogenous amounts of cellular proteins. Such tools will provide the unprecedented opportunity to build an atomic resolution picture of the cellular milieu and to investigate changes in protein structure and dynamics as we stress, age or treat cells with therapeutic agents.