Functional assessment of enhancer-gene interactions in vivo

体内增强子-基因相互作用的功能评估

基本信息

批准号：
10331859
负责人：
Evgeny Kvon
金额：
$ 24.17万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-07 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10331859
关键词：
3-Dimensional Address Adopted Affect Architecture Area Biological Models Biology CRISPR/Cas technology Categories Cells Chromatin Structure Code DNA Development Disease Distal Distant Educational workshop Elements Embryo Enhancers Ensure Environment Evolution Faculty Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Transcription Genome Genomics Goals Heart Diseases Human Human Genome Human Genome Project Knock-in Knock-in Mouse Knock-out Leadership Limb Development Limb structure Link Location Malignant Neoplasms Maps Mediating Mentors Mentorship Methods Mus Mutation Organism Pattern Phase Phenotype Play Positioning Attribute Proteins Regulation Research Research Proposals Research Training Resolution Rodent Role SHH gene Secure Series Specificity Technology Testing Tissues Training Untranslated RNA Work career development cohort embryo tissue experimental study gene interaction genome analysis genome editing genome-wide genome-wide analysis genomic locus human disease in vivo insight mammalian genome novel promoter response skills technology development transcriptome sequencing

项目摘要

PROJECT SUMMARY Transcriptional enhancers are a predominant category of functional elements in the non-coding portion of the human genome, far outnumbering the ~20,000 protein-coding genes. Mutations affecting enhancers have been implicated in human disease, and comprehensively understanding the genome-wide architecture and function of enhancers remains a major unsolved challenge arising from The Human Genome Project. Despite substantial progress in mapping of these elements (e.g., by the ENCODE consortium), the in vivo target genes of enhancers are generally unknown, and the mechanisms of their long range regulation during development are not well explored. Recently, I developed a novel method that allows manipulation of enhancers at their endogenous genomic location in mice using CRISPR/Cas9 genome editing (Kvon et al., Cell, 2016). In this application, I propose to better understand the mechanisms of gene regulation by distant-acting enhancers through in vivo mouse studies, exploiting this highly efficient CRISPR/Cas9 genome editing technology to create enhancer knock-out and knock-in mice and employing novel methods to map enhancer-promoter interactions. I will address the following questions regarding distal enhancer function in the genome: 1) Which genes do different classes of enhancers regulate? 2) Is there enhancer-promoter specificity for distant-acting enhancers? and 3) What are the consequences of enhancer loss or replacement on an organism's function? Mentored phase: First, I propose to adapt CRISPR/Cas9 genome editing for studying long-range enhancer- gene interactions in vivo using mouse embryonic limb as a model system. I will create a series of enhancer knock-outs to identify their target gene(s) and a series of enhancer knock-ins to study enhancer-promoter specificity. Second, I will adopt and optimize the Capture-C technology to identify interaction partners of enhancers directly in mouse tissues. Independent phase: I will use methods developed in the mentored phase to systematically map target genes for important developmental enhancers in vivo and to gain a detailed understanding of the mechanisms governing long-range enhancer-promoter interactions on a genomic scale. I will also use elucidated enhancer-promoter interactions to study basic principles of long-range enhancer regulation in the mammalian genome using CRISPR/Cas9 technology. This will enable me to develop several long-term research directions, focused on the role of enhancer-gene interactions in human evolution, disease, and development. The main areas of research training will include: 1) Further advancing the use of CRISPR/Cas9 in mice, 2) Capture-C technology development in mouse tissues, and 3) computational genome analysis. My mentor (Dr. Len Pennacchio) and co-mentor (Dr. Axel Visel) are leaders in these fields. My career development activities will focus on skills in key areas of my research, attending courses and workshops, developing leadership and mentorship skills, and securing a faculty position. To ensure progress in my goals I have also established a scientific advisory board consisting of my mentors, and Drs. D. Dickel, and E. Rubin.

项目摘要转录增强剂是在非编码部分中功能元素的主要类别人类基因组，远远超过约20,000个蛋白质编码基因。影响增强子的突变具有与人类疾病有关，并全面了解全基因组的结构和增强子的功能仍然是人类基因组项目引起的主要未解决的挑战。尽管这些元素的映射（例如，通过编码财团），体内靶基因的映射取得了长足进展增强剂通常是未知的，并且在开发过程中的远程调节机制探索不好。最近，我开发了一种新颖的方法，该方法允许在他们的使用CRISPR/CAS9基因组编辑小鼠中的内源基因组位置（Kvon等，Cell，2016）。在这个应用，我建议通过远距离作用增强子更好地理解基因调节的机制通过体内小鼠研究，利用这种高效的CRISPR/CAS9基因组编辑技术创建增强剂的敲除和敲门鼠标，并采用新颖的方法来绘制增强器促进剂互动。我将解决有关基因组远端增强子功能的以下问题：1）基因是否会调节不同类别的增强子？ 2）是否有增强剂启动器特异性的特异性增强剂？ 3）增强子丢失或替换对生物体功能的后果是什么？指导阶段：首先，我建议适应CRISPR/CAS9基因组编辑，以研究远程增强子 - 使用小鼠胚胎肢体作为模型系统的体内基因相互作用。我将创建一系列增强器确定其靶基因和一系列增强子敲击以研究增强剂促进剂的敲除特异性。其次，我将采用并优化捕获-C技术来确定的交互合作伙伴直接在小鼠组织中的增强子。独立阶段：我将使用指导中开发的方法阶段到系统地绘制目标基因的体内重要发育增强子的阶段，并获得详细的理解有关基因组量表上远程增强子促销相互作用的机制。我还将使用阐明的增强器促销相互作用来研究远程增强器的基本原理使用CRISPR/CAS9技术在哺乳动物基因组中进行调节。这将使我能够发展几个长期研究方向，重点是增强子 - 基因相互作用在人类进化，疾病，和发展。研究培训的主要领域将包括：1）进一步前进小鼠的CRISPR/CAS9，2）捕获C中的C技术开发和3）计算基因组分析。我的导师（Len Pennacchio博士）和同事（Axel Visel博士）是这些领域的领导者。我的职业开发活动将集中于我研究的关键领域的技能，参加课程和讲习班，发展领导能力和指导能力，并确保教师职位。为了确保我的目标进展还建立了一个由我的导师和博士组成的科学顾问委员会。 D. Dickel和E. Rubin。