Packing and Unpacking the Genome: Single-Molecule Manipulation of Chromatin

在这个项目中，pi将使用单分子光学镊子来阐明生物相关翻译后修饰（PTMs）对单个核小体行为和染色质凝聚的结构，机械和热力学后果。本研究的基本生物学意义在于，将DNA包装成核小体和染色质，并通过组蛋白修饰和组蛋白H1对其进行调节，是所有真核生物基因调控的重要方面。该项目的具体目标包括：(1)使用组蛋白H4进行规定的单位点和多位点特异性乙酰化，组装核小体和染色质；(2)比较在有镁离子或镁离子和连接体组蛋白H1的情况下染色质的行为，镁离子和连接体组蛋白H1是调节缩聚的关键因素，而没有这些因素时染色质的行为；(3)使用智能强制步进协议精确确定不同分子状态之间的自由能差；(4)比较含有组蛋白H4尾部乙酰化的核小体与含有组蛋白H4尾部突变的核小体的行为，揭示电荷的作用。因此，该项目将为创建和验证真核基因表达的物理模型提供所需的观察和测量。该项目的更广泛影响包括：(1)这些研究具有潜在的人类健康益处，因为包括哮喘和一些癌症在内的几种疾病与组蛋白ptm有关；(2)通过遵循耶鲁大学物理与工程生物学综合研究生计划（IGPPEB），并担任旨在吸引生命科学本科生的新颖入门物理序列的教学研究员，参与该项目的研究生将加入新一代的研究人员/教师，他们擅长定量方法，具有生物学的复杂性，能够识别并应用他们的技能来解决前沿的生物学问题。并且很高兴能教育下一代；(3)为了鼓励下一代对科学的兴趣，pi将与“突破纽黑文”（Breakthrough New Haven）合作，为7年级和8年级的社会经济弱势学生提供高中准备计划，为“突破”的暑期计划实施实践科学模块，并将接待高中生在他们的实验室进行暑期研究项目。该项目由物理系的生命系统物理学项目和分子与细胞生物科学系的分子生物物理学项目共同支持。

In this project, the PIs will use single-molecule optical tweezers to elucidate the structural, mechanical and thermodynamic consequences of biologically-relevant post-translational modifications (PTMs) on the behavior of individual nucleosomes and on chromatin condensation. The fundamental biological significance of the proposed research is that the packaging of DNA into nucleosomes and chromatin, and its modulation by histone modifications and histone H1, are essential aspects of gene regulation in all eukaryotes. Specific objectives of this project include: (1) assemble nucleosomes and chromatin, using histone H4 with prescribed single and multiple site-specific acetylation; (2) compare the behavior of chromatin in the presence of magnesium ions or magnesium ions and linker histone H1, which are key factors regulating condensation, to the behavior of chromatin without these factors; (3) determine accurate free energy differences between the different molecular states encountered, using an intelligent force-stepping protocol; (4) compare the behavior of nucleosomes containing histone H4 with acetylated tails to those containing H4 with mutated tails, that mimic acetylation, revealing the role of charge. Thus, this project will provide observations and measurements needed to create and validate physical models of eukaryotic gene expression. The broader impacts of this project include: (1) these studies have potential human health benefits, because several diseases, including asthma and a number of cancers, are linked to histone PTMs; (2) by following Yale's Integrated Graduate Program in Physical and Engineering Biology (IGPPEB) and serving as teaching fellows in a novel introductory physics sequence, that seeks to engage life-science undergraduates, the graduate students involved in this project will join a new generation of researcher/teachers, who excel at quantitative approaches, have the biological sophistication to identify and apply their skills to cutting-edge biological problems, and are excited to educate future generations; (3) to encourage an interest in science in the next generation, the PIs will work with Breakthrough New Haven, a high school preparation program for socio-economically disadvantaged students in Grades 7 and 8, to implement hands-on science modules for Breakthrough's summer program, and will host high-school students for summer research projects in their laboratories.This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences.