Functional assessment of enhancer-gene interactions in vivo

项目摘要 转录增强子是转录因子非编码部分的主要功能元件类别。 人类基因组，数量远远超过约2万个蛋白质编码基因。影响增强子的突变 与人类疾病有关，全面了解全基因组结构， 增强子的功能仍然是由人类基因组计划引起的主要未解决的挑战。尽管 在绘制这些要素方面取得了实质性进展（例如，由ENCODE联盟），体内靶基因 的增强子通常是未知的，其在发育过程中的远程调节机制 还没有很好地探索。最近，我开发了一种新的方法，允许在其 使用CRISPR/Cas9基因组编辑在小鼠中的内源基因组定位（Kvon等人，Cell，2016）。在这 应用，我建议更好地了解基因调控的机制，远距离作用增强子 通过小鼠体内研究，利用这种高效的CRISPR/Cas9基因组编辑技术， 创建增强子敲除和敲入小鼠，并采用新的方法来定位增强子-启动子 交互.我将解决以下有关基因组中远端增强子功能的问题：1） 不同类型的增强子调节的基因呢？2)是否存在增强子-启动子特异性， 增强剂增强子丢失或替换对生物体功能的影响是什么？ 指导阶段：首先，我建议调整CRISPR/Cas9基因组编辑以研究远程增强子- 以小鼠胚胎肢体为模型系统进行体内基因相互作用的研究。我会制造一系列的增强剂 敲除以鉴定它们的靶基因和一系列增强子敲入以研究增强子-启动子 的特异性第二，采用并优化Capture-C技术， 增强子直接在小鼠组织中。独立阶段：我将使用在指导下开发的方法 阶段，系统地绘制体内重要发育增强子的靶基因，并获得详细的 在基因组规模上对控制长距离增强子-启动子相互作用的机制的理解。我 还将使用阐明的增强子-启动子相互作用来研究远程增强子的基本原理， 使用CRISPR/Cas9技术在哺乳动物基因组中进行调控。这将使我能够开发几个 长期的研究方向，重点是增强子-基因相互作用在人类进化，疾病， 发展先行者的要求研究培训的主要领域将包括：1）进一步推进使用 小鼠中的CRISPR/Cas9，2）小鼠组织中的Capture-C技术开发，以及3）计算基因组 分析.我的导师（Len Pennacchio博士）和共同导师（阿克塞尔Visel博士）是这些领域的领导者。我的职业生涯 发展活动将集中在我的研究，参加课程和研讨会的关键领域的技能， 发展领导和指导技能，并确保教师职位。为了确保我的目标取得进展， 我还成立了一个科学顾问委员会，由我的导师和D.迪克尔和E.鲁宾

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.