Mechanism of chromatin accessibility, 3D chromosome organization, and their functions in gene regulation

染色质可及性机制、3D 染色体组织及其在基因调控中的功能

• 批准号: 10322650
• 负责人: Lu Bai
• 金额: $ 60.04万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10322650
• 关键词: 3-Dimensional; Address; Alleles; Binding; Binding Sites; Biological Assay; Cells; Chromatin; Chromatin Structure; Chromosome Structures; Chromosomes; Disease; Drosophila genus; Event; Five-Year Plans; Foundations; Gene Cluster; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Screening; Genome; Goals; Grant; Health; Human Cell Line; Image; Invaded; Kinetics; Link; Liquid substance; Malignant Neoplasms; Mediating; Methods; National Institute of General Medical Sciences; Nucleosomes; Oligonucleotides; Phase; Play; Pluripotent Stem Cells; Regulation; Research; Role; System; Testing; Yeasts; chromosome conformation capture; developmental disease; human disease; innovation; insight; novel; programs

Project Abstract The overall theme of my research program is to understand the formation mechanism of chromatin structure and its role in gene regulation. We plan to address this problem at two different levels: 1) at the chromatin / nucleosome level, we will identify pioneer factors (PFs) and investigate the mechanism underlying nucleosome displacement and chromatin opening, and 2) at the chromosome level, we will study the mechanism of gene regulation by high-order 3D chromosome organization. In the past five years, supported by two NIGMS R01 grants, we have made significant progress in both directions. New observations and insights we gained from these studies, as well as several novel methods we developed, form the foundation of this proposal. Theme1: Pioneer factors (PFs) can invade nucleosome and increase chromatin accessibility near their binding sites and therefore play critical roles in gene regulation. Mis-regulation of PFs is highly linked to cancer and developmental disease. Despite their essential functions, only a handful of PFs have been identified and the mechanism underlying the pioneer activity is unclear. The long term goal of this theme is to systematically characterize PFs and their pioneer activities in health and disease cells, and to develop a full understanding of the mechanism of these activities. In our recent PF studies, we have innovated an Integrated Synthetic Oligo (ISO) assay to investigate PF function in a high-throughput manner. In the next five years, we plan to 1) adapt the ISO assay into human cell lines and pluripotent stem cells to dissect the genetic rules underlying PF binding and nucleosome displacement, and 2) further our understanding of the pioneering activity by investigating the co-factors of PFs, the sequence of events during nucleosome displacement, and the kinetic rates of these events. Theme2: Imaging and 3C-based methods have revealed 3D chromosome organization with extensive long- distance chromosomal interactions. The long-term goal of this theme is to understand the formation mechanism of these high-order chromosome structures and their roles in gene regulation. Our recent studies show that long- distance chromosomal interactions contribute to gene regulation in yeast. More specifically, some allelic or co- regulated genes cluster in the 3D space, and such clustering is correlated with higher gene activities. The former case is analogous to the “transvection” phenomenon in Drosophila. These novel observations revealed a new layer of gene regulation in yeast and opened the opportunity of using the powerful genetic system to investigate the 3D genome function. Currently we have little understanding of what mediates the cluster formation and how the clusters enhance gene expression. In the next five years, we plan to 1) use an unbiased genetic screen to identify factors that negatively regulate transvection, and investigate the underlying mechanism, and 2) test the hypothesis that some activators condense though liquid-liquid phase separation, leading to the clustering of co- regulated genes and enhanced expression.

项目摘要 我的研究计划的总体主题是了解染色质结构的形成机制和 我们计划在两个不同的层面上解决这个问题：1）在染色质/ 核小体水平，我们将识别先锋因子（PF）并研究核小体的机制 位移和染色质开放，2）在染色体水平上，我们将研究基因的机制 高阶3D染色体组织的调控在过去五年中得到了两个NIGMS R01的支持。 拨款，我们在两个方向上都取得了重大进展，并从中获得了新的观察和见解。 这些研究以及我们开发的几种新颖方法构成了该提案的基础。 主题1：先锋因子（PF）可以侵入核小体并增加其结合附近的染色质可及性 因此，PF 的错误调控与癌症和癌症密切相关。 尽管它们具有重要的功能，但只有少数 PF 已被识别出来，并且 先锋活动背后的机制尚不清楚，该主题的长期目标是系统地进行。 描述 PF 及其在健康和疾病细胞中的先驱活动的特征，并全面了解 在我们最近的 PF 研究中，我们创新了一种综合合成寡核苷酸。 (ISO) 检测以高通量方式研究 PF 功能 在未来五年中，我们计划 1) 进行调整。 对人类细胞系和多能干细胞进行 ISO 分析，以剖析 PF 结合的遗传规则 和核小体置换，2）通过调查进一步加深我们对开创性活动的理解 PF 的辅因子、核小体位移过程中的事件顺序以及这些事件的动力学速率。 主题 2：成像和基于 3C 的方法揭示了具有广泛长链的 3D 染色体组织。 该主题的长期目标是了解远距离染色体相互作用。 我们最近的研究表明，这些高阶染色体结构及其在基因调控中的作用。 更具体地说，一些等位基因或共染色体相互作用有助于酵母中的基因调控。 受调控的基因在3D空间中聚集，这种聚集与较高的基因活性相关。 这种情况类似于果蝇中的“平移”现象。这些新颖的观察结果揭示了一种新的现象。 酵母中的基因调控层，并开启了使用强大的遗传系统进行研究的机会 目前我们对什么介导簇形成以及如何形成知之甚少。 在接下来的五年中，我们计划1）使用公正的遗传筛选来增强基因表达。 识别负面调节横传的因素，并研究潜在的机制，2) 测试 假设一些活化剂通过液-液相分离凝结，导致共- 调节基因并增强表达。