In situ evaluation of combinatorial gene regulation in the human genome

项目总结 基因表达增强剂对于在多种环境中建立转录程序至关重要 在后生动物中。在过去的十年里，我们的预测能力有了令人难以置信的进步 增强剂和最近联盟的努力已经确定了人类体内数百万个候选增强剂 基因组。不幸的是，很难用电流在候选增强子的内源位点上测试它们 需要大量工作来评估单个增强子的活性的方法。增强剂通常 组合调控单个基因的工作和目前的方法不适用于组合研究 增强剂的大规模使用。为了满足对研究增强剂的高通量方法的需求， 他们的天然基因组背景下，我们已经调整CRISPR/Cas9系统来靶向增强子，并计划测试 数百个基因组基因座在驱动转录反应中的必要性和充分性。通过锁定目标 多个远端调控区域同时，我们也有能力评估增强剂如何在 演唱会。在这个项目中，我们将开发高通量现场测试增强剂的方法，使用 雌激素受体α(ER)和子宫内膜癌细胞中的雌激素信号作为模型系统。确实有 拟议项目的三个目标：(1)确定符合以下条件的候选增强剂组合 对适当的转录调控是必要的；(2)鉴定 足以概括内质网驱动的转录反应；(3)比较 表观遗传工程的增强剂。该项目的成功完成将导致发现 对转录调控很重要的调控区域，并提供了对如何 增强剂的组合协同作用来调节转录。该项目还将建立方法 用于高通量测试其内源基因座中的增强剂，这将有助于研究增强剂 在一些额外的生物系统中。

PROJECT SUMMARY Gene expression enhancers are critical for establishing transcriptional programs in numerous contexts throughout metazoans. There have been incredible advancements over the last decade in our ability to predict enhancers and recent consortium efforts have identified millions of candidate enhancers across the human genome. Unfortunately, it is difficult to test candidate enhancers at their endogenous locus with current approaches requiring a large amount of effort to assess the activity of a single enhancer. Enhancers often work in combination to regulate a single gene and current methods are not amenable to studying combinatorial enhancer usage on a large-scale. To address the need for high-throughput methods for studying enhancers in their native genomic context, we have adapted the CRISPR/Cas9 system to target enhancers and plan to test the necessity and sufficiency of hundreds of genomic loci in driving transcriptional responses. By targeting multiple distal regulatory regions simultaneously, we also have the ability to assess how enhancers work in concert. During this project we will develop methods for high-throughput testing of enhancers in situ, using estrogen receptor α (ER) and estrogen signaling in endometrial cancer cells as a model system. There are three aims of the proposed project: (1) Determination of combinations of candidate enhancers that are necessary for proper transcription regulation; (2) Identification of combinations of regulatory regions that are sufficient to recapitulate an ER-driven transcriptional response; (3) Comparison of genetic deletion of enhancers to epigenetic engineering. The successful completion of this project will lead to the discovery of regulatory regions that are important for transcription regulation and provide an understanding of how combinations of enhancers work in concert to regulate transcription. This project will also establish methods for high-throughput testing of enhancers in their endogenous locus, which will be useful for studying enhancers in a number of additional biological systems.