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

项目摘要 我的研究计划的总主题是了解染色质结构和 它在基因调控中的作用。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(PFS)，并研究核小体的机制 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。在过去的五年中，由两个NIGMS R01支持 在赠款方面，我们在两个方向上都取得了重大进展。我们从以下方面获得了新的观察和见解 这些研究以及我们开发的几种新方法构成了这一提议的基础。 主题1：先锋因子(PFS)可侵入核小体，并增加其结合附近染色质的可及性 因此在基因调控中起着关键作用。PFS的错误调控与癌症和 发育性疾病。尽管它们具有基本的功能，但只确定了少数几个私营部门，而且 先锋活动背后的机制尚不清楚。这一主题的长期目标是系统地 描述PFS及其在健康和疾病细胞中的先驱活动，并充分了解 这些活动的机制。在我们最近的PF研究中，我们创新了一种集成的合成寡聚 (ISO)法，以高通量方式研究PF功能。在接下来的五年里，我们计划适应 对人类细胞系和多能干细胞进行ISO检测，以剖析PF结合的遗传规则 和核小体置换，以及2)通过研究 PFS的共同因素，核小体置换过程中的事件顺序，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的长染色体的3D染色体组织 染色体间的距离相互作用。这一主题的长远目标是了解其形成机制 这些高阶染色体结构及其在基因调控中的作用。我们最近的研究表明，长期- 在酵母中，远距离染色体的相互作用有助于基因调控。更具体地说，一些等位基因或共- 受调控的基因在3D空间中聚集，这种聚集与更高的基因活性相关。前者 CASE类似于果蝇的“横行”现象。这些新奇的观察揭示了一种新的 酵母基因调控层的构建，为利用强大的遗传系统进行研究提供了机会 3D基因组的功能。目前，我们对星系团形成的媒介以及如何形成知之甚少 这些簇增强了基因的表达。在接下来的五年里，我们计划1)使用无偏见的基因筛查 找出负面调节转运的因素，并调查其潜在的机制，以及2)测试 假设一些活化剂通过液-液相分离进行缩合，导致Co-C 调控基因和增强表达。

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.