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.

项目摘要/摘要 增强子是简短的DNA序列，可调节发育过程中基因表达的复杂模式。 增强剂活性的正调与多种病理有关，从先天性疾病到 癌症。但是，增强剂的许多特性尚不清楚，尤其是增强剂如何行动 长基因组距离。虽然某些增强剂位于其靶基因近端，而另一些则位于 远端，激活数千甚至数百万碱基对基因组距离的基因表达。大多数 评估增强剂活性的体内技术利用了直接放置增强剂的转基因系统 记者的上游。尽管转基因是一种强大的工具，但它无法评估基因组距离的作用 增强剂活动。为了解决这个问题，我们的小组开发了一种小说中的体内鼠标增强器记者测定法 表征增强子的远端活性。使用这种方法，我们确定了一种新型的顺式调节序列， RC元素，对于远端增强剂活性是必要且足够的。该提议的总体目标是 为了表征这种新确定的RC元素，并剖析了其在介导远端增强剂活性中的作用。到 解决这个目标，该提案概述了一个计划，以可视化RC元素对增强器促销的影响 相互作用（AIM 1）并确定哪些因素是RC元素函数的关键调节因子（AIM 2）。为了 以前，将采用荧光原位杂交和基于染色体捕获的技术 仔细分析增强剂促销的共定位和整体核组织，并在存在下 没有RC元素。对于后者，RC元素的诱变将用于识别关键基序。 同时，将使用候选转录因子调节剂的敲除来评估其在RC中的重要性 元素依赖性增强子活性。该提案产生的发现将有助于阐明新颖 协调增强剂活性的机制，增加了我们对基因调节的整体理解。我的赞助商 （Evgeny Kvon博士）和共同赞助商（Ken Cho博士）是发展遗传学领域的专家，并且与 他们的指导，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 专注于在五个关键领域发展我的能力：技术技能，科学沟通，指导和 职业发展。