Interplay Between Chromatin and Co-Activator Complexes

项目总结/摘要 增强子-启动子（E-P）通讯使真核生物中的基因激活成为可能，但尽管如此， 尽管在该领域的许多其他领域取得了进展，但这种通信的性质还没有得到很好的理解 在细胞的天然染色质环境中，尤其是在细胞的天然染色质环境中。我们已经开发 在小鼠胚胎干细胞（mESC）中研究E-P通讯的新系统， 物理水平，E-P循环，和功能水平，Pol II预启动复合物（PIC）组装 和基因激活。我们将利用一种称为雌激素相关受体的关键转录因子 β（ESRRB）在许多mESC增强子上大量结合。ESRRB还直接结合至 介体，控制增强子和启动子功能的主要辅激活复合物。ESRRB- 应答基因通常在称为绝缘邻近区的环状结构域中发现， 由粘着蛋白和CTCF的强峰限定。siRNA对ESRRB的消耗极大地导致了 增强子内ESRRB位点的介体结合减少，靶基因 表达仅限于隔离社区内。此外，我们还开发了一种degron 靶向TAF 12并抑制TFIID和Pol II PIC的系统，沿着基因 表情我们将使用这些系统，并在此基础上加以改进，以解决根本问题 关于E-P通信的物理和功能机制。在目标1中，我们将采用 ESRRB、粘附素和TAF 12的降解决定子，沿着一些选择的CRISPR增强子缺失， 近端启动子和核心启动子。关键因子的ChIP-seq、RNA-seq和启动子- 捕获Hi-C将用于定量每个降解决定子和遗传缺失对物理特性的影响。 E-P环和功能相互作用，包括介体结合，PIC组装，染色质 状态和基因表达。在目标2中，我们将正交地处理这个问题。巧合的是， 一些ESRRB调节子含有在发育后期表达的失活基因。CRISPR-dCAS9 激活域（VPR）、染色质修饰（p300）和重塑的靶向策略 机器（BRG 1）将被用来异位激活这些基因，并确定这是否 这一过程依赖于在调节子内绑架功能增强子。总之，我们的 一项提案试图确定物理E-P循环是否能够实现或独立于 功能性沟通，以及哪些决定因素（即，PIC，染色质状态，激活剂，近端 启动子）控制功能相互作用。结果将提供重要的新信息， 在一个定义明确的实验模型中进行E-P通信，与发展和 分化

Project Summary/Abstract Enhancer-promoter (E-P) communication enables gene activation in eukaryotes but despite advances in many other areas of the field, the nature of this communication is not well understood mechanistically, particularly within the native chromatin environment of a cell. We have developed novel systems in mouse embryonic stem cells (mESCs) to study E-P communication at both the physical level, E-P looping, and the functional level, Pol II Pre-Initiation Complex (PIC) assembly and gene activation. We will leverage a key transcription factor termed Estrogen Related Receptor Beta (ESRRB) that binds in abundance at many mESC enhancers. ESRRB also binds directly to Mediator, a major co-activator complex controlling enhancer and promoter function. ESRRB- responsive genes are typically found within looped domains termed Insulated Neighborhoods, bounded by strong peaks of Cohesin and CTCF. Depletion of ESRRB by siRNAs causes greatly diminished Mediator binding to ESRRB sites within enhancers and decreased target gene expression restricted to within Insulated Neighborhoods. In addition, we have developed a degron system that targets TAF12 and dismantles both TFIID and Pol II PICs, along with gene expression. We will use these systems, and build onto them, to address fundamental questions about the physical and functional mechanisms of E-P communication. In Aim 1, we will employ degrons of ESRRB, Cohesin and TAF12, along with a few select CRISPR deletions of enhancers, proximal promoters and core promoters. ChIP-seq of key factors, RNA-seq, and Promoter- Capture Hi-C will be used to quantitate the effects of each degron and genetic deletion on physical E-P looping and functional interactions including Mediator binding, PIC assembly, chromatin state, and gene expression. In Aim 2 we will approach the problem orthogonally. Paradoxically, some ESRRB regulons contain inactive genes expressed later in development. CRISPR-dCAS9 targeting strategies of activation domains (VPR), chromatin modification (p300), and remodeling machines (BRG1) will be used to ectopically activate these genes, and determine whether this process depends upon kidnapping the functional enhancer within the regulon. In sum, our proposal seeks to determine whether physical E-P looping enables or is independent from functional communication, and what determinants (i.e., PICs, chromatin state, activators, proximal promoters) control functional interactions. The results will provide important new information on E-P communication in a well-defined experimental model, highly relevant to development and differentiation.