Interplay Between Chromatin and Co-Activator Complexes

项目摘要/摘要 增强子-启动子(E-P)通讯使真核细胞中的基因激活，但尽管 尽管在该领域的许多其他领域取得了进展，但这种交流的性质还没有得到很好的理解 机械上，特别是在细胞的天然染色质环境中。我们已经开发出 在小鼠胚胎干细胞中研究E-P通讯的新系统 物理层，E-P环，和功能层，POL II预引发复合体(PIC)组装 和基因激活。我们将利用一种名为雌激素相关受体的关键转录因子 在许多mESC增强剂中大量结合的β(Esrrb)。Esrrb还直接绑定到 介体是一种控制增强子和启动子功能的主要共激活复合体。Esrrb- 响应基因通常被发现在被称为绝缘邻域的环状结构域中， 以粘附素和CTCF的强峰为界。由siRNAs耗尽Esrrb导致的 增强子内与Esrrb位点的介体结合减少和靶基因减少 仅限于在隔热社区内表达。此外，我们还发展了一种退化 以TAF12为靶点并拆除TFIID和POLII PICS以及基因的系统 表情。我们将使用这些系统，并在它们的基础上，解决基本问题 关于E-P通信的物理和功能机制。在目标1中，我们将使用 Esrrb、Coherin和TAF12的退化，以及CRISPR中一些增强子的精选缺失， 近端启动子和核心启动子。关键因素的芯片序列，RNA序列，和启动子- 捕获Hi-C将被用来量化每个退化和基因缺失对物理 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，我们将以正交化的方式处理这个问题。矛盾的是， 一些Esrrb调节子含有在发育后期表达的非活性基因。CRISPR-dCAS9 激活结构域(VPR)、染色质修饰(P300)和重塑的靶向策略 机器(BRG1)将被用来异位激活这些基因，并确定这是否 这一过程取决于绑架调节子内的功能增强子。总而言之，我们的 该提案寻求确定物理E-P环路是否启用或独立于 功能性沟通，以及哪些决定因素(即PIC、染色质状态、激活物、近端 启动子)控制功能相互作用。这一结果将提供有关 在定义明确的实验模型中进行E-P通信，与开发和 差异化。

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.