Dynamics of DNA-histone interactions

项目摘要 我的研究计划的首要目标是阐明动态DNA-组蛋白的作用 单分子方法调控基因可及性中核小体的相互作用。这个 核小体是真核生物基因的基本包装单位，在基因中起着重要的作用 监管。基因调控的错误往往会导致发育障碍和致命性疾病，如各种 癌症的类型。核小体由147bpds-DNA和一个八聚体组蛋白核心组成。DNA和 组蛋白是各种染色质修饰的靶标，这些修饰通常与基因调控密切相关 机械装置。我们报告了在结构和结构动力学方面的几个重要变化 核小体由各种染色质修饰和组蛋白变体诱导，如DNA甲基化， 组蛋白乙酰化、SUMO化、CENP-A和H_2A.Z在基因调控中的作用。AS 这些更改是异类的、不同步的和/或复杂的，通常无法解决 集合-平均生化、遗传和静态结构生物学工具。我们开发和改进单项- 分子荧光方法和系统，主要基于FRET、FCS和光学共定位 解决这些问题的超级解决方案。我们将继续推动这些实验的界限 支持我们未来研究的系统和方法，这些研究将围绕三个协同主题：如何(1) 染色质修饰和组蛋白变体，(2)自发的分子运动和相互作用，以及(3) 主动和被动分子机器和酶影响核小体DNA-组蛋白动态和 核小体阵列及其在基因调控机制中的作用。 我们最近开始发展我们的主要以核小体为重点的实验系统，通过将 作用于核小体并将其扩展到包括核小体阵列的各种酶。基于 对于这些系统，我们将在未来五年继续在三个主要领域进行研究：(1)核小体 与RNA聚合酶II和DOT1L等复杂酶相互作用的动力学 H3K79甲基化依赖白血病的发病机制，(2)染色质修饰对H3K79甲基化依赖白血病的影响 核小体的结构和动力学及其在核小体-酶相互作用中的意义(3) 核小体阵列的构象和动力学以及染色质修饰对核小体的影响 核小体阵列中的动力学。对这些领域的研究将有助于理解基本的分子 核小体-酶相互作用中调节核小体动力学和基因可及性的过程 在核小体和核小体阵列的背景下，以及在高度精炼的 易于处理的单分子系统。

Project Summary The overarching goal of my research program is to elucidate the roles of dynamic DNA-histone interactions in the nucleosome in regulating gene accessibility from single-molecule approaches. The nucleosome is the fundamental packing unit of genes in eukaryotes and plays important roles in gene regulation. Errors in gene regulation often lead to developmental failure and lethal diseases such as various types of cancer. The nucleosome is made of ~147 bp ds-DNA with an octameric histone protein core. DNA and histones are targets for various chromatin modifications that are often critically implicated in gene regulation mechanisms. We reported several important changes in the structure and structural dynamics of the nucleosome induced by various chromatin modifications and histone variants such as DNA methylation, histone acetylations, SUMOylation, CENP-A, and H2A.Z in the context of their roles in gene regulation. As these changes are heterogeneous, unsynchronized, and/or complex, they are often impossible to resolve with ensemble-averaging biochemical, genetic, and static structural biology tools. We develop and improve single- molecule fluorescence methods and systems, mainly based on FRET, FCS, and colocalization at an optical super-resolution to address these problems. We will continue pushing the boundaries of these experimental systems and methods to support our future research that will center around three synergistic themes: how (1) chromatin modifications and histone variants, (2) spontaneous molecular motions and interactions, and (3) active and passive molecular machines and enzymes affect DNA-histone dynamics in nucleosomes and nucleosome arrays and how the effects are implicated in gene regulation mechanisms. We recently started evolving our mostly nucleosome-focused experimental systems by combining various enzymes that act on the nucleosome and expanding them to include nucleosome arrays. Based on these systems, our research will continue largely in three topical areas in the next five years: (1) nucleosome dynamics during its interaction with complex enzymes such as RNA polymerase II and Dot1L, a key player in H3K79 methylation-dependent leukemia pathogenesis, (2) the effects of chromatin modifications on the structure and dynamics of the nucleosome and their implications in nucleosome-enzyme interactions, (3) conformations and dynamics of nucleosome arrays and the effects of chromatin modifications on nucleosome dynamics in nucleosome arrays. Investigations in these areas will help understand the fundamental molecular processes that regulate nucleosome dynamics and gene accessibility during nucleosome-enzyme interactions in a nucleosome and a nucleosome-array context and at depth and clarity afforded by our highly-refined tractable single-molecule systems.