In situ evaluation of combinatorial gene regulation in the human genome

项目摘要 基因表达增强子对于在许多情况下建立转录程序至关重要 整个后生动物。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， 增强子和最近的联盟的努力已经确定了数百万候选增强子在整个人类 基因组不幸的是，很难在候选增强子的内源性基因座处用电流检测增强子。 需要大量努力来评估单个增强子的活性的方法。增强剂通常 组合工作以调节单个基因，并且目前的方法不适合于研究组合 大规模使用增强剂。为了解决研究增强子的高通量方法的需求， 他们的天然基因组背景下，我们已经调整了CRISPR/Cas9系统的目标增强子，并计划测试 数百个基因组位点在驱动转录反应中的必要性和充分性。通过靶向 多个远端调控区同时，我们也有能力评估增强子如何在 演唱会在这个项目中，我们将开发原位高通量测试增强子的方法，使用 雌激素受体α（ER）和子宫内膜癌细胞中的雌激素信号传导作为模型系统。有 建议项目的三个目标：（1）确定候选增强剂的组合， （2）确定适当转录调控所必需的调控区的组合， 足以概括ER驱动的转录应答;（3）比较ER基因的遗传缺失， 表观遗传工程的增强剂。该项目的成功完成将导致发现 调控区域，对转录调控很重要，并提供了如何理解 增强子的组合协同工作以调节转录。该项目还将建立方法 用于增强子内源位点的高通量检测，这将有助于研究增强子 在许多其他的生物系统中。

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.