Biophysical Determinants of the Nucleosome as an Activity Center for Chromatin Regulators

核小体作为染色质调节剂活动中心的生物物理决定因素

• 批准号: 10638494
• 负责人: Shixin Liu
• 金额: $ 33.9万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-22 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638494
• 关键词: ATP phosphohydrolase; Architecture; Atomic Force Microscopy; Base Pairing; Binding; Biological Assay; Biophysics; Cell Nucleus; Characteristics; Chemicals; Chromatin; Complex; DNA; DNA Binding; DNA Packaging; Data; Detection; Disease; Energy consumption; Enzymes; Exhibits; Fluorescence; Fluorescence Microscopy; Gene Expression; Genetic Transcription; Genome; Genomic DNA; Genomics; Geometry; Heterochromatin; Histone H1; Histone H2A; Histone H3; Histones; Human; Individual; Intervention; Investigation; Kinetics; Laboratories; Length; Licensing; Link; Linker DNA; Lysine; Measures; Methods; Methylation; Modification; Molecular Machines; Multienzyme Complexes; Mutation; Nucleosome Core Particle; Nucleosomes; ORC1L gene; Oncogenic; Pathogenesis; Polycomb; Positioning Attribute; Preparation; Proteins; Replication Initiation; Replication Origin; Reporting; Resolution; Role; Shapes; Site; Spectrum Analysis; Technology; Testing; Therapeutic; Time; Variant; Visualization; biophysical properties; chromatin protein; helicase; human disease; mutant; origin recognition complex; protein function; recruit; single molecule; transcription factor

PROJECT SUMMARY/ABSTRACT The fundamental unit of hierarchically organized eukaryotic chromatin is the nucleosome, which contains 147 base pairs of genomic DNA wrapped around an octamer of core histone proteins. Conventionally, nucleosomes have been viewed as DNA packaging units that inhibit gene expression by obstructing the accessibility of DNA to the transcriptional machinery. However, we and others have shown that nucleosomes also serve as potent hotspots which recruit, modulate, and stimulate the activity of various essential chromatin regulators, indicating a new role for nucleosomes in furnishing the genome with a multitude of physical features and interactions which direct protein function. As such, I hypothesize that the physical characteristics and topology of nucleosomes modulate the activity of chromatin regulators, constituting an underappreciated layer of physical parameters encoded within chromatin architecture that govern genomic transactions in the nucleus. These parameters include the shape and composition of the nucleosome core particle as well as the spacing and geometry of contiguous nucleosomes in an array. To test this hypothesis, I propose to use single-molecule fluorescence detection and force manipulation technologies established in my laboratory, which uniquely track real-time transient and heterogeneous molecular interactions, to investigate the physical characteristics of nucleosome topology that determine its capacity to tune the activity of several important classes of chromatin regulators at multiple scales. We will first investigate how the topology of individual nucleosomes directs the DNA targeting activity and cooperation of essential pioneer transcription factors (Aim 1). We will then probe how the geometry of local nucleosomes in an array modulates the engagement, recruitment, and propagation of chromatin- modifying enzymes on chromatin (Aim 2). Finally, we will investigate the biophysical basis of global nucleosome localization and functionalization by energy-consuming molecular machines (Aim 3). Together, these studies will zoom in on the topology of nucleosomes comprising a layer of biophysical parameters encoded within chromatin architecture that regulate genomic transactions in the nucleus. They will contribute evidence towards a new perspective that views nucleosomes as genomic regulators which harness their unique physical features to actively modulate, recruit, and stimulate the activity of chromatin-associated factors, rather than passive DNA packaging units. The proposed investigations will shed light on a nucleosome-focused angle for tackling several long-standing questions about the interplay between chromatin and its regulators and promise to mechanistically inform how disease-associated mutations perturb essential genomic activities, potentially revealing mutation- selective protein-chromatin interfaces that may be therapeutically exploited to treat human disease.

项目概要/摘要 分层组织的真核染色质的基本单位是核小体，它包含 147 个 包裹着核心组蛋白八聚体的基因组 DNA 碱基对。传统上，核小体 被视为 DNA 包装单位，通过阻碍 DNA 的可及性来抑制基因表达 到转录机器。然而，我们和其他人已经证明核小体也可以作为有效的 招募、调节和刺激各种重要染色质调节因子活性的热点，表明 核小体在为基因组提供多种物理特征和相互作用方面的新作用 直接蛋白质功能。因此，我假设核小体的物理特征和拓扑结构 调节染色质调节因子的活性，构成未被充分认识的物理参数层 在染色质结构中编码，控制细胞核中的基因组事务。这些参数 包括核小体核心颗粒的形状和组成以及间隔和几何形状 阵列中连续的核小体。为了验证这个假设，我建议使用单分子荧光 我的实验室建立的检测和力操纵技术，具有独特的实时跟踪能力 瞬时和异质分子相互作用，研究核小体的物理特征 拓扑结构决定了其调节几类重要染色质调节因子活性的能力 多重尺度。我们将首先研究单个核小体的拓扑结构如何指导 DNA 靶向 重要先锋转录因子的活性和合作（目标 1）。然后我们将探讨几何 阵列中的局部核小体调节染色质的参与、招募和传播 修饰染色质上的酶（目标 2）。最后，我们将研究整体核小体的生物物理基础 通过耗能分子机器进行定位和功能化（目标 3）。这些研究共同将 放大核小体的拓扑结构，包括染色质内编码的一层生物物理参数 调节细胞核内基因组事务的架构。他们将为新的研究提供证据 将核小体视为基因组调节剂的观点，利用其独特的物理特征 主动调节、招募和刺激染色质相关因子的活性，而不是被动的 DNA 包装单位。拟议的研究将揭示以核小体为中心的角度来解决几个问题 关于染色质与其调节因子之间相互作用的长期存在的问题，并有望从机制上解决 告知疾病相关突变如何扰乱基本基因组活动，可能揭示突变- 选择性蛋白质-染色质界面可用于治疗人类疾病。