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

基因组的包装和拆包：染色质的单分子操作

• 批准号: 1305509
• 负责人: Simon Mochrie
• 金额: $ 42万
• 依托单位: Yale University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1305509&HistoricalAwards=false
• 关键词: Packing; Unpacking; Genome; Single; Molecule

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.

在这个项目中，PI将使用单分子光镊来阐明生物相关的翻译后修饰（PTM）对单个核小体和染色质凝聚行为的结构，机械和热力学后果。拟议研究的基本生物学意义在于，DNA包装成核小体和染色质，以及组蛋白修饰和组蛋白H1对其的调节，是所有真核生物中基因调控的重要方面。本项目的具体目标包括：（1）使用具有指定的单位点和多位点特异性乙酰化的组蛋白H4组装核小体和染色质;（2）比较存在镁离子或镁离子和连接体组蛋白H1的染色质的行为，这是调节凝聚的关键因素，以及没有这些因素的染色质的行为;（3）使用智能力步进协议确定遇到的不同分子状态之间的准确自由能差异;（4）比较含有乙酰化尾部的组蛋白H4的核小体与含有突变尾部的H4的核小体的行为，模拟乙酰化，揭示电荷的作用。因此，该项目将提供创建和验证真核基因表达物理模型所需的观察和测量。该项目更广泛的影响包括：（1）这些研究具有潜在的人类健康益处，因为包括哮喘和许多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课程（IGPPEB），并在一个新颖的物理学入门课程中担任助教，该课程旨在吸引生命科学本科生，参与这一项目的研究生将加入新一代研究人员/教师的行列，他们擅长定量方法，具有生物学方面的复杂性，能够确定并将其技能应用于尖端生物学问题，并乐于教育后代;（3）为了鼓励下一代对科学的兴趣，PI将与Breakthrough New Haven合作，这是一个针对7年级和8年级社会经济弱势学生的高中预备计划，为Breakthrough的暑期项目实施实践科学模块，并将在他们的实验室举办高中学生的暑期研究项目。该项目由物理学系的生命系统物理学项目和分子和细胞生物科学系的分子生物物理学项目共同支持。