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

工程摘要 我的研究计划的总体主题是了解染色质结构的形成机制， 在基因调控中的作用。我们计划在两个不同的水平上解决这个问题：1）在染色质/ 在核小体水平上，我们将鉴定先驱因子（PFs）并研究核小体的机制 在染色体水平上，我们将研究基因表达的机制， 通过高阶3D染色体组织进行调控。在过去的五年里，在两个NIGMS R 01的支持下， 我们在这两个方面都取得了重大进展。我们从以下方面获得的新观察和见解： 这些研究以及我们开发的几种新方法构成了这项建议的基础。 主题1：先锋因子（Pioneer factors，PFs）可以侵入核小体，并增加其结合附近的染色质可及性 位点，因此在基因调控中发挥关键作用。PFs的错误调节与癌症高度相关， 发育性疾病尽管它们具有重要的功能，但只有少数几个PF被确定， 先驱活动的潜在机制尚不清楚。本主题的长期目标是系统地 表征PF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合成寡核苷酸， (ISO)测定以高通量方式研究PF功能。在接下来的五年里，我们计划1）适应 ISO试验用于人类细胞系和多能干细胞，以剖析PF结合的遗传规律 和核小体置换，以及2）通过调查 PF的辅因子，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的方法已经揭示了具有广泛的长染色体的3D染色体组织。 距离染色体相互作用。本主题的长期目标是了解形成机制 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- 染色体间的相互作用有助于酵母的基因调控。更具体地说，一些等位基因或共- 受调控的基因在三维空间中聚集，并且这种聚集与更高的基因活性相关。前 这种情况类似于果蝇中的“transvection”现象。这些新的观察揭示了一个新的 酵母基因调控层的研究，为利用强大的遗传系统研究 3D基因组功能。目前，我们对是什么介导了集群的形成以及如何介导集群的形成知之甚少 簇增强基因表达。在接下来的五年里，我们计划1）使用无偏见的基因筛选， 确定负调节transvection的因素，并调查潜在的机制，和2）测试 假设一些活化剂通过液-液相分离冷凝，导致共- 调节基因并增强表达。

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.