Interplay Between Chromatin and Co-Activator Complexes

摘要 增强子和启动子如何相互作用是真核生物研究领域的重要问题之一 基因调控尽管教科书和评论文章中有挑衅性的模型， 了解该机制。我们是第一个展示TFIID和Mediator之间直接相互作用的实验室 共活化剂，并证明这种相互作用是Pol II preinitiation复合物组装所必需的 体外基于这些研究，我们假设驱动Pol II预启动的关键相互作用 复杂的组装和功能是激活剂调节的增强子处的介体和 TFIID在启动器。我们将利用生物化学/全基因组技术 从过去三个资助周期的研究中获得，以全面鉴定形成蛋白质的蛋白质。 体外TFIID-介体界面，并确定这些相互作用是否是基因表达的核心， 启动子-增强子循环。 我们的研究将采用生物化学和生物学检测方法来分析小鼠 胚胎干细胞激活剂Esrrb激活小鼠胚胎干细胞体内转录 （mESC）和体外提取物中。这一系统将作为一个模型和焦点， TFIID-Mediator接口。 在目标1中，我们将使用交联质量来识别Esrrb、Mediator和TFIID之间的界面 质谱法（XL-MS）。我们将验证和进一步研究这些表面使用蛋白质-蛋白质相互作用 测定和诱变。在目标2中，我们将通过以下方法在体内鉴定TFIID-介体共激活剂表面： 系统RNAi敲低共激活子亚基，单独地，然后成对组合， 随后进行RNA-seq以鉴定重叠效应。我们将进行TFIID的全基因组ChIP-seq， 模拟和敲低条件下的介体，以探测效应并与Aim的结果相关 1.目的3检验TFIID-Mediator界面对于启动子-增强子是否是必需的 使用4C在体内成环。我们的研究结果将提供一个详细的真核基因激活机制 使用强大的最先进的生化和生物方法。

Abstract How enhancers and promoters communicate is one of the important questions in the field of eukaryotic gene regulation. Despite provocative models from textbooks and review articles, very little is genuinely known of the mechanism. We were the first lab to show a direct interaction between TFIID and Mediator co-activators, and demonstrate that this interaction was required for Pol II preinitiation complex assembly in vitro. Based on these studies, we hypothesize that the key interaction driving Pol II preinitiation complex assembly and function is activator-regulated interaction between Mediator at the enhancer and TFIID at the promoter. We will leverage the knowledge and biochemical/genomewide technologies acquired from studies over the last three funding cycles to comprehensively identify proteins forming the TFIID-Mediator interface in vitro and determine if these interactions are central to gene expression and promoter-enhancer looping in cells. Our studies will employ biochemical and biological assays developed to analyze how the murine embryonic stem cell activator Esrrb activates transcription in vivo in murine embryonic stem cells (mESCs) and in vitro in extracts. This system will serve as a model and focal point for understanding the TFIID-Mediator interface. In Aim 1, we will identify the interface between Esrrb, Mediator and TFIID using crosslinking mass spectrometry (XL-MS). We will validate and further study these surfaces using protein-protein interaction assays and mutagenesis. In Aim 2, we will identify the TFIID-Mediator co-activator surface in vivo by systematic RNAi knockdown of co-activator subunits, individually and then in pairwise combinations, followed by RNA-seq to identify overlapping effects. We will perform genomewide ChIP-seq of TFIID and Mediator under mock and knockdown conditions to probe effects and correlate with the results from Aim 1. Aim 3 examines whether the TFIID-Mediator interface is necessary or not for promoter-enhancer looping in vivo using 4C. Our results will provide a detailed mechanism of eukaryotic gene activation using powerful state-of-the-art biochemical and biological approaches.