Characterizing a mechanism of enhancer-promoter interaction in vivo

表征体内增强子-启动子相互作用的机制

• 批准号: 10680165
• 负责人: Grace Cabot Bower
• 金额: $ 4.35万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2026-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680165
• 关键词: 3-Dimensional; Address; Affect; Architecture; Area; Automobile Driving; Base Pairing; Binding; Bioinformatics; Biological Assay; Cell Nucleus; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Colon Carcinoma; Communication; Complex; Congenital Disorders; DNA; DNA Sequence; DNA Structure; Data; Data Set; Development; Developmental Gene; Disease; Distal; Elements; Embryo; Enhancers; Environment; Fluorescent in Situ Hybridization; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Transfer Techniques; Genes; Genetic Enhancer Element; Genomics; Goals; Homeobox; Investigation; Knock-out; Limb structure; Link; Malignant Neoplasms; Mediating; Mentors; Methods; Modeling; Multiomic Data; Mus; Mutagenesis; Mutagens; Mutation; Nuclear; Other Genetics; Pathogenesis; Pathology; Play; Property; Protein Family; Regulatory Element; Reporter; Research; Resolution; Role; System; Technical Expertise; Techniques; Testing; Training; Transgenes; Transplantation; Untranslated RNA; Visualization; Work; career development; chromosome conformation capture; craniofacial disorder; design; developmental genetics; genomic locus; homeodomain; in vivo; insight; novel; promoter; remote control; response; superresolution microscopy; tool; transcription factor

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.

项目概要/摘要 增强子是短DNA序列，在发育过程中调节复杂的基因表达模式。 增强子活性的失调与多种病理有关，从先天性疾病到 癌症。然而，增强子的许多特性尚不清楚，特别是增强子如何作用于 基因组距离长。虽然一些增强子位于其靶基因附近，但其他增强子位于 在远端，激活基因组距离数千甚至数百万碱基对的基因表达。大多数在 评估增强子活性的体内技术利用直接放置增强子的转基因系统 记者的上游。虽然转基因是一个强大的工具，但它无法评估基因组距离在 增强子活性。为了解决这个问题，我们的小组开发了一种新型的体内小鼠增强子报告基因测定法 表征增强子的远端活性。使用这种方法，我们鉴定了一个新的顺式调控序列， RC 元件，对于远端增强子活性是必要且充分的。该提案的总体目标是 表征这个新发现的 RC 元件并剖析其在介导远端增强子活性中的作用。到 为了实现这一目标，该提案概述了一个计划，以可视化 RC 元素对增强子-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子（目标 2）。对于 前者，将采用荧光原位杂交和基于染色体捕获的技术 仔细分析增强子-启动子共定位和整体核组织的存在和 缺少 RC 元件。对于后者，RC 元件的诱变将用于识别关键基序。 同时，候选转录因子调节因子的敲除将用于评估它们在 RC 中的重要性 元素依赖性增强子活性。该提案的结果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）和共同发起人（Ken Cho 博士）是发育遗传学领域的专家，并且 在他们的指导下，我设计了一个培训计划来帮助指导我向独立研究的过渡。我的计划 专注于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。