CAREER: The structural basis of the multivalent readout of histone PTMs and PTM/interaction mediated modulation of nucleosome dynamics

每个真核细胞的细胞核中都有一个基因组的拷贝，但在任何给定的细胞中，只有一个基因子集在任何给定的时间被激活。适当的基因调控是发育和基本的生物功能从细胞到有机体水平的关键。真核生物的基因组与称为组蛋白的蛋白质复合物存在。组蛋白/DNA复合体称为染色质，由称为核小体的重复亚基组成。每个核小体看起来像一个用绳子包裹着的线轴，组蛋白组成线轴，基因组DNA的一段是线轴。正是DNA包裹组蛋白核心（称为核小体结构）的方式以及核小体相互之间的组织方式，允许或限制了对潜在基因组的访问，从而决定了基因表达的模式。该项目解决了被称为翻译后修饰（PTM）的小化学标签模式如何被与染色质相关的其他蛋白质（称为辅助因子）读出的基本问题，以及这种关联如何改变核小体结构以影响下游事件的精确调节。核磁共振（NMR）光谱学将用于研究各种核小体结构，以确定特定的模型转录因子如何与含有不同模式PTMs的核小体相关联，以及这种关联如何在原子分辨率上改变核小体结构及其动态行为。本项目支持本科生、预科和博士后的教育和培训，既在课堂上通过PI指导的课程，也在实验室中，他们将实现项目目标。此外，研究小组将与爱荷华州的非营利组织“工作场所学习连接”合作，在实验室为高中生建立一个实习项目。来自爱荷华市和周边县的学生将获得45-90小时的实验室经验，在项目的各个方面工作，他们将获得高中学分。该项目将为学生提供在基础科学方面的实际研究经验，并将允许PI与他们建立指导关系，预计这种关系将远远超出他们在实验室的时间。研究者将继续这些活动，为提高STEM领域的参与度做出贡献，这对美国经济的增长和稳定以及我们的全球竞争力至关重要。技术：提出的研究的主要目的是了解组蛋白翻译后修饰（PTMs）调节染色质结构的基本和生物学重要机制。研究者和她的学生将利用核磁共振波谱和其他生化和生物物理方法来研究一个模型系统，即BPTF博士-手指-溴结构域基序与修饰核小体的相互作用。研究人员希望详细了解多结构域基序如何在核小体水平上识别PTMs，特别是如何特异性读取多个组蛋白尾部的PTMs的不同模式。此外，通过测量核磁共振弛豫参数，他们将解决组蛋白修饰和效应结构域结合如何调节核小体的构象动力学的重要问题，可能稳定或破坏构象状态，这可能是下游功能所必需的。这项研究将与一项教育推广计划充分结合，重点是激发高中生对STEM领域的兴趣。

Every eukaryotic cell contains a copy of the genome in its nucleus, but only a subset of genes is turned on in any given cell at any given time. Proper regulation of genes is critical in development and basic biological function from the cellular to organismal level. The eukaryotic genome exists in complex with proteins called histones. The histone/DNA complex is called chromatin and is composed of repeating subunits called nucleosomes. Each nucleosome looks like a spool wrapped with string in which the histones form the spool and a segment of genomic DNA is the string. It is the way in which the DNA wraps the histone core (called nucleosome structure) and the organization of the nucleosomes with respect to each other that allows or restricts access to the underlying genome, determining patterns of gene expression. This project addresses the fundamental questions of how patterns of small chemical tags, termed post-translational modification (PTM), are read-out by other proteins associated with chromatin (called co-factors) and how this association may alter nucleosome structure to affect precise regulation of downstream events. Nuclear magnetic resonance (NMR) spectroscopy will be used to study a variety of nucleosome constructs to determine how a specific model transcription factor associates with a nucleosome containing different patterns of PTMs and how this association alters the nucleosome structure and its dynamic behavior at atomic resolution.This project supports the education and training of undergraduate and pre-and post-doctoral students both in the classroom via courses instructed by the PI, as well as in the laboratory in which they will carry out the project goals. In addition, the research team will partner with the Iowa-based non-profit organization, the Workplace Learning Connection, to establish an internship program in the laboratory for high school students. Students in from Iowa City and surrounding counties will gain 45-90 hours of laboratory experience, working on various aspects of the project for which they will receive high school credit. This program will provide students with a tangible research experience in the basic sciences, and will also allow the PI to establish mentoring relationships with them that are expected to last well beyond their time in the laboratory. The investigator will continue these activities to contribute towards increased participation in STEM areas, which is imperative for the growth and stability of the United States economy as well as our global competitiveness.Technical: The main objective of the proposed research is to understand the fundamental and biologically important mechanisms by which histone post-translational modifications (PTMs) regulate chromatin structure. The investigator and her students will utilize NMR spectroscopy and other biochemical and biophysical methods to investigate the interaction of a model system, the BPTF PHD-finger-Bromodomain motif with modified nucleosomes. The investigators hope to understand, in detail, how a multi-domain motif recognizes PTMs at the level of the nucleosome, especially how distinct patterns of PTMs across multiple histone tails are specifically read out. In addition, through measurement of NMR relaxation parameters, they will address the important questions of how histone modifications and effector domain binding regulate the conformational dynamics of the nucleosome, likely stabilizing or destabilizing conformational states that may be essential for downstream function. This research will be fully integrated with a plan for education outreach, focused on sparking interest of high school students in STEM fields.