Characterizing a mechanism of enhancer-promoter interaction in vivo

项目摘要/摘要 增强子是短DNA序列，在发育过程中调节基因表达的复杂模式。 增强子活性的错误调节与一系列的病理有关，从先天性疾病到 癌症。然而，增强剂的许多性质还不是很清楚，特别是增强剂是如何起作用的 基因组距离很远。虽然一些增强子位于其目标基因附近，但其他增强子位于 在远端，激活超过数千甚至数百万个碱基对的基因组距离的基因表达。最受欢迎 评估增强子活性的体内技术利用的是直接放置增强子的转基因系统 一名记者的上游。虽然转基因是一个强大的工具，但它不能评估基因组距离在 增强剂活性。为了解决这个问题，我们小组开发了一种新的体内小鼠增强子报告试验来 表征增强剂的远端活性。利用这种方法，我们鉴定了一个新的顺式调控序列， RC元件，这是远端增强子活性所必需和充分的。这项提案的总体目标是 鉴定这个新发现的RC元件，并分析其在调节远端增强子活性中的作用。至 为了实现这一目标，这项提案概述了一项计划，以可视化RC元件对增强子-启动子的影响 相互作用(目标1)，并确定哪些因素是RC元件功能的关键调节因素(目标2)。对于 前者，荧光原位杂交和基于染色体捕获的技术将被用于 仔细分析增强子-启动子共定位和整体核组织 缺少RC元素。对于后者，将使用RC元件的突变来确定关键基序。 同时，候选转录因子调控因子的敲除将被用于评估它们在RC中的重要性 依赖元素的增强子活性。由这一提议产生的发现将有助于阐明一部小说 协调增强子活性的机制，增加了我们对基因调控的整体理解。我的赞助商 Evgeny Kvon博士和共同赞助人Ken Cho博士是发育遗传学领域的专家，并与 在他们的指导下，我设计了一个培训计划，以帮助指导我向独立研究的过渡。我的计划 重点发展我在五个关键领域的能力：技术技能、科学沟通、指导和 职业发展。

PROJECT SUMMARY/ABSTRACT Enhancers are short DNA sequences that regulate complex patterns of gene expression during development. Misregulation of enhancer activity is associated with a wide range of pathologies, from congenital disorders to cancer. However, many properties of enhancers are not well understood, particularly how enhancers act over long genomic distances. While some enhancers are located proximally to their target genes, others are located distally, activating gene expression over thousands or even millions of base pairs of genomic distance. Most in vivo techniques that assess enhancer activity make use of a transgene system that places the enhancer directly upstream of a reporter. While transgenesis is a powerful tool, it cannot assess the role of genomic distance in enhancer activity. To address this, our group developed a novel in vivo mouse enhancer reporter assay to characterize the distal activity of enhancers. Using this method, we identified a novel cis-regulatory sequence, the RC element, that is necessary and sufficient for distal enhancer activity. The overall goal of this proposal is to characterize this newly identified RC element and dissect its role in mediating distal enhancer activity. To address this goal, this proposal outlines a plan to visualize the impact of the RC element on enhancer-promoter interaction (Aim 1) and to determine what factors are critical regulators of RC element function (Aim 2). For the former, both fluorescence in situ hybridization and chromosome capture based techniques will be employed to carefully analyze enhancer-promoter co-localization and overall nuclear organization in the presence and absence of the RC element. For the latter, mutagenesis of the RC element will be used to identify critical motifs. In parallel, knockout of candidate transcription factor regulators will be used to evaluate their importance in RC element-dependent enhancer activity. Findings resulting from this proposal will help elucidate a novel mechanism coordinating enhancer activity, adding to our overall understanding of gene regulation. My sponsor (Dr. Evgeny Kvon) and co-sponsor (Dr. Ken Cho) are experts in the field of developmental genetics, and, with their guidance, I have designed a training plan to help guide my transition to independent research. My plan focuses on developing my abilities in five key areas: technical skills, scientific communication, mentoring, and career development.