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

项目总结/摘要 核小体是真核生物染色质分层结构的基本单位， 基因组DNA的碱基对包裹着核心组蛋白的八聚体。通常，核小体 已被视为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，我们和其他人已经表明，核小体也可以作为有效的 募集、调节和刺激各种必需染色质调节因子活性的热点，表明 核小体在为基因组提供大量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 调节染色质调节剂的活性，构成了一个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交易。这些参数 包括核小体核心颗粒的形状和组成以及核小体核心颗粒的间隔和几何形状。 阵列中的连续核小体。为了验证这一假设，我建议使用单分子荧光 在我的实验室里建立的探测和力操纵技术， 瞬时和异质分子相互作用，研究核小体的物理特性 拓扑结构，决定了它的能力，以调整几个重要类别的染色质调节剂的活动， 多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 必需的先锋转录因子（Aim 1）的活性和合作。然后我们将探讨几何学 局部核小体的排列调节染色质的参与、募集和增殖， 染色质修饰酶（Aim 2）。最后，我们将探讨整体核小体的生物物理基础 定位和功能化的能量消耗分子机器（目标3）。这些研究将 放大核小体的拓扑结构，核小体包括染色质内编码的生物物理参数层 调节细胞核中基因组交易的结构。他们将为一个新的 这种观点将核小体视为基因组调节器，利用其独特的物理特征， 主动调节、募集和刺激染色质相关因子的活性，而不是被动的DNA 包装单位。拟议的调查将揭示一个核小体为重点的角度来解决几个 关于染色质及其调节因子之间相互作用的长期问题，并有望从机制上 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，其可以在治疗上用于治疗人类疾病。

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.