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

项目摘要/摘要 本课题研究的总主题是了解染色质结构的形成机制 以及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)并研究其机制 2)在染色体水平上，我们将研究核小体移位和染色质开放 高位三维染色体组织对基因调控机制的研究。在过去五年中， 在两笔NIGMS R01赠款的支持下，我们在两个方向上都取得了重大进展。新的 我们从这些研究中获得的观察和见解，以及我们开发的几种新方法， 形成了这项提议的基础。 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的错误调控与癌症密切相关 和发育疾病。尽管它们具有基本的功能，但只确定了少数几个PFS 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 系统地表征PFS及其在健康和疾病细胞中的先驱活性，并开发完整的 了解这些活动的机制。在我们最近的PF研究中，我们创新了一个 集成合成寡聚(ISO)分析，以高通量方式研究PF功能。在接下来的五年里 几年来，我们计划1)将ISO分析方法应用到人类细胞系和多能干细胞中，以剖析基因 Pf结合和核小体移位的基本规则，以及2)进一步理解先驱 通过研究PFS的辅助因素，核小体置换过程中的事件顺序，以及 这些事件的动态率。 主题2：成像和基于3C的方法揭示了3D染色体的广泛组织 染色体的远距离相互作用。这一主题的长期目标是理解 这些高阶染色体结构的机制及其在基因调控中的作用。我们最近的研究 表明远距离染色体相互作用有助于酵母中的基因调控。更确切地说， 一些等位基因或共调控基因在3D空间中聚集，这种聚集与更高的基因相关 活动。前一种情况类似于果蝇的“横行”现象。这些小说 观察揭示了酵母中新的一层基因调控，并打开了利用 强大的遗传系统，用于研究3D基因组功能。目前我们还不太了解 调节簇的形成以及簇如何增强基因表达。在未来五年，我们计划 1)使用无偏见的基因筛查来识别负面调节转基因的因素，并调查 潜在的机制，以及2)检验一些激活剂通过液-液缩合的假设 相分离，导致共调控基因的聚集和增强表达。

Project Summary/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.