The Roles of Enhancer RNAs in the Regulation of Gene Expression

项目总结/摘要 我们的长期目标是了解正常细胞中转录调控的基本原理， 发展，压力反应和人类疾病。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， 产生大量的非编码增强子RNA（eRNA），其表达方式 与响应促炎信号的基因表达变化相关。我们还确定 一些转录因子和表观遗传修饰因子被募集到这些增强子中以控制eRNA 通过我们的目标是阐明在我们提出的研究计划的机制合成。值得注意的是，我的实验室也 发现几种鉴定的eRNA在基因表达的调节中表现出直接作用， 这一发现是为数不多的证明eRNA功能的研究之一。这些重要 调查结果向我们揭示了实施新技术方法的必要性，以便采取下一步措施， 研究eRNA的功能机制，因为目前的方法无法解偶联 eRNA的功能来自增强子转录的行为。为了满足这一需求，并推动我们的 了解eRNA的精确功能后，我们开始实施我们强大的无细胞检测 用克隆的增强子和基因，从细胞提取物中纯化的因子，以及合成转录的eRNA。 利用这个系统，我们最近发现了一种令人兴奋的机制，通过这种机制，eRNA增强了 通过降低解离动力学，从而增加转录激活的停留时间， 关键的表观遗传调节因子。 在未来5年，我们的目标是通过拟议的研究计划，释放新的前沿基因 通过揭示控制eRNA合成及其随后的 行动机制。我们将特别关注（i）增强子- 特定的表观遗传书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 近十年来，对组蛋白标记的新见解已经表明了增强子，但它们的功能仍然存在 未知和（ii）新的eRNA结合伴侣及其在增强子依赖性转录调控中的功能 调控重要的是，我们的诱导型细胞系统与我们的无细胞测定一起将被用作 范例来理解控制eRNA合成和功能的事件。重要的是， 从拟议的工作将很容易推进我们的分析eRNA在真核转录在多个新的 方向鉴于功能性eRNA的新兴领域以及 使用eRNA作为诊断标记物和治疗靶点来改变增强子活性的潜力， 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.