Deciphering the mechanism of long-range gene regulation in vivo

项目摘要 由转录因子（TF）结合的远距离作用（“远程”）转录增强子驱动基因表达 空间和时间的模式。越来越多的证据表明， 影响人类疾病远程增强子的突变。目前尚不清楚的是这些远程增强子 在很长的基因组距离上调节它们的靶基因，通常跳过干预基因。远程增强子 通常在高级染色质组织的支持下与靶启动子相互作用，但破坏 这种染色质组织不能消除增强子-启动子相互作用。哪些额外的因素调节 这些长距离的调节相互作用在很大程度上仍未被探索。为了解决这个核心问题， 一个拟议的项目将使用一种新的功能获得方法来鉴定介导 哺乳动物发育过程中的长距离增强子-启动子通讯。传统方法 评估体内增强子活性依赖于增强子-报道基因转基因;然而，技术限制 排除了这种方法评估长程增强子活性。拟议的工作克服了这一点 在小鼠中利用有效增强子置换技术系统地表征体内特性的局限性 长距离增强子活性，并鉴定对于这种长距离活性至关重要的顺式调节序列。到 通过这些远程控制元件确定远程调节的机制，并识别调节元件， 结合它们的蛋白质，这项工作将使用多学科的方法来映射和可视化增强子-启动子 接触在大分子分辨率。远程控制元件的功能特性将扩展 已知顺式调节元件库，为长距离增强子- 启动子通信，并大大推进了3D基因组组织在基因组中的作用的知识。 发展与疾病的关系。

PROJECT SUMMARY Distant-acting (“remote”) transcriptional enhancers bound by transcription factors (TFs) drive gene expression patterns in space and time. The importance of this function is underscored by mounting evidence linking mutations affecting remote enhancers to human disease. What remains unclear is how these remote enhancers regulate their target genes over long genomic distances, often skipping intervening genes. Remote enhancers typically interact with target promoters with the support of higher-order chromatin organization, but disruption of this chromatin organization does not abolish enhancer–promoter interactions. What additional factors regulate these long-range regulatory interactions remains largely unexplored. To address this central question, the proposed project will use a novel gain-of-function approach to identify cis-regulatory sequences that mediate long-range enhancer–promoter communication in the context of mammalian development. Traditional methods that assess in vivo enhancer activity rely on enhancer-reporter transgenesis; however, technical limitations preclude this approach from assessing long-range enhancer activity. The proposed work overcomes this limitation by utilizing efficient enhancer replacement technology in mice to systematically characterize in vivo long-range enhancer activity and identify cis-regulatory sequences that are critical for this long-range activity. To determine the mechanism of long-range regulation by these remote control elements and identify regulatory proteins that bind them, this work will use a multidisciplinary approach to map and visualize enhancer–promoter contacts at macromolecular resolution. Functional characterization of remote control elements will expand the repertoire of known cis-regulatory elements, provide a novel general mechanism for long-range enhancer– promoter communication, and substantially advance knowledge of the role of 3D-genome organization in the context of development and disease.