The Roles of Enhancer RNAs in the Regulation of Gene Expression

项目摘要/摘要 我们的长期目标是理解正常转录调控的基本原理。 发展、应激反应和人类疾病。在过去的几年里，我的实验室一直专注于 研究细胞类型特异性增强子影响基因表达程序的机制。 使用高通量测序，我们最近发现了一类新的增强子，支持 产生大量非编码增强子RNA(ERNA)，其表达方式为 与反应促炎信号的基因表达的变化有关。我们还确认了 被招募到这些增强子以控制Erna的几个转录因子和表观遗传修饰物 通过我们在拟议的研究计划中旨在阐明的机制进行合成。值得注意的是，我的实验室还 发现一些已识别的eRNA在基因表达的调节中显示出直接的作用，而这 该发现是为数不多的证明eRNAs功能的研究之一。这些都很重要 调查结果向我们揭示了需要实施新的技术方法来采取下一步 研究eRNAs的作用机制，因为目前的方法无法解偶联 ERNAs在增强转录过程中的作用。为了满足这一需求并推进我们的 了解eRNAs的精确功能，我们已经开始实施强大的无细胞检测 利用克隆的增强子和基因，从细胞提取液中提纯因子，并合成转录的eRNAs。 利用这个系统，我们最近发现了一种令人兴奋的机制，eRNAs通过这种机制增强了 通过降低解离动力学从而增加A蛋白的停留时间来激活转录 增强子中的关键表观遗传调控因子。 在接下来的5年里，我们的目标是通过拟议的研究计划，开拓基因的新领域 通过揭示支配Erna合成及其后续的机械原理来进行监管 行动机制。我们将特别集中于确定(I)增强剂- 特定的表观遗传编写者和组蛋白标记调节Erna的产生。实现这一目标的进展将提供 对组蛋白标记的新见解，这些标记近十年来一直是增强剂，但它们的功能仍然存在 未知和(Ii)新的Erna结合伙伴及其在增强子依赖转录中的功能 监管。重要的是，我们的可诱导细胞系统和我们的无细胞检测将被用作 范式，以了解控制Erna合成和功能的事件。重要的是，结果是 将很容易地推进我们对多个新的真核转录中的eRNA的分析 方向。鉴于新兴的功能性eRNAs领域和 将eRNA用作诊断标记物和治疗靶点以改变以下增强子活性的可能性 与人类疾病有关。

Project Summary/Abstract Our long-term goal is to understand the fundamental principles of transcriptional regulation in normal development, stress responses, and human disease. Over the past several years, my lab has focused on an investigation of the mechanisms by which cell type-specific enhancers influence gene expression programs. Using high throughput sequencing, we recently uncovered a new class of enhancers that support the production of a vast number of noncoding enhancer RNAs (eRNAs) that are expressed in a manner that correlates with changes in gene expression in response to proinflammatory signaling. We also identified several transcription factors and epigenetic modifiers that are recruited to these enhancers to control eRNA synthesis through mechanisms that we aim to elucidate in our proposed research plan. Notably, my lab also found that several of the identified eRNAs exhibit direct roles in the regulation of gene expression, and this finding is among the few number of studies that have demonstrated a function for eRNAs. These important findings revealed to us the need of implementing new technical approaches to take the next step of investigating the mechanisms by which eRNAs function since current methodologies are unable to uncouple the functions of eRNAs from the act of enhancer transcription. To address this need and to advance our understanding of the precise functions of eRNAs, we have started implementing our powerful cell free assays with cloned enhancers and genes, factors purified from cellular extracts, and synthetically transcribed eRNAs. Using this system, we have very recently uncovered an exciting mechanism by which eRNAs enhance transcriptional activation by decreasing the dissociation kinetics and thereby increasing the residence time of a key epigenetic regulator at enhancers. In the next 5 years, we aim, through the proposed research plan to unleash new frontiers of gene regulation by uncovering mechanistic principles that govern eRNA synthesis and their subsequent mechanisms of action. We will focus specifically on the identification of (i) mechanisms by which enhancer- specific epigenetic writers and histone marks regulate eRNA production. Progress toward this goal will provide new insights into histone marks that have denoted enhancers for almost a decade but their functions remain unknown and (ii) new eRNA binding partners and their functions in enhancer-dependent transcriptional regulation. Importantly, our inducible cellular system together with our cell-free assays will be employed as a paradigm to understand the events controlling eRNA synthesis and function. Importantly, the results stemming from the proposed work will readily advance our analysis of eRNAs in eukaryotic transcription in multiple new directions. This is an issue of great significance in light of the emerging field of functional eRNAs and the potential of using eRNAs as diagnostic markers and therapeutic targets for altering enhancer activities that are linked to human disease.