MASSIVELY PARALLEL CIS-REGULATORY ELEMENT ANALYSIS IN MAMMALIAN CELLS

用于1 R 01 HG 006790 -01 在此输入文本，它是应用程序的新摘要信息。此部分不得超过30行文本。 下一代测序（NGS）技术如ChIP-seq正在推动推定的顺式调节元件（克雷斯;即，增强子/启动子）以惊人的速度。测试这些预测的调控潜力是我们理解人类基因组中基因调控逻辑的一个严重瓶颈。目前还没有可行的技术来功能性地测试甚至可以从单个ChIP-seq实验中生成的数千个预测，更不用说ENCODE和其他基于联盟的努力生成的数百万个预测了。打破CRE预测的僵局需要一种新技术，可以在哺乳动物细胞中进行大规模并行顺式调控分析。在这项提案中，我们介绍了CRE-seq（顺式调控元件测序分析），一种新的技术，用于在哺乳动物细胞中的一个实验中测定数千个克雷斯。我们的策略是将克雷斯文库与条形码报告基因融合，将这些文库转染到细胞中并通过NGS定量其输出。我们将开发CRE-seq作为一种高效，强大和高度并行的技术，用于在单个实验中评估数千个ChIP-seq峰的顺式调节活性。如果成功的话，这种策略应该可以量化整个“顺式调节组”的启动子活性（即，在给定细胞类型中控制基因表达的顺式调节区的整个互补序列）。我们将通过研究两种不同的哺乳动物细胞类型，视网膜光感受器（一种分化的神经元细胞类型）和未分化的胚胎干细胞中的顺式调节来证明该测定的实用性。CRE-seq有望促进我们对人类基因调控的理解，并将成为诊断和治疗疾病的个性化基因组信息的新来源。

for 1R01HG006790-01 Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Next-generation sequencing (NGS) technologies such as ChIP-seq are driving the discovery of putative cis-regulatory elements (CREs; i.e., enhancers/promoters) at an astonishing pace. Testing the regulatory potential of these predictions is a serious bottleneck in our efforts to understand the logic of gene regulation in the human genome. Currently there are no viable technologies for functionally testing the thousands of predictions that can be generated from even a single ChIP-seq experiment, let alone the millions of predictions being generated by ENCODE and other consortium-based efforts. Breaking the logjam of CRE predictions requires a new technology that enables massively parallel cis-regulatory analysis in mammalian cells. In this proposal, we introduce CRE-seq (Cis-Regulatory Element analysis by sequencing), a novel technique for assaying thousands of CREs in a single experiment in mammalian cells. Our strategy is to fuse libraries of CREs to barcoded reporter genes, transfect these libraries into cells and quantify their output by NGS. We will develop CRE-seq as an efficient, robust and highly parallel technology for assessing the cis-regulatory activity of thousands of ChIP-seq peaks in a single experiment. If successful, this strategy should make it possible to quantify the promoter activity of entire 'cis-regulomes' (i.e., the entire complement of cis-regulatory regions controlling gene expression in a given cell type). We will demonstrate the utility of this assay by studying cis-regulation in two different mammalian cell types, retinal photoreceptors (a differentiated neuronal cell type) and undifferentiated embryonic stem cells. CRE-seq promises to advance our understanding of human gene regulation and will serve as a novel source of personalized genomic information available for diagnosis and treatment of disease.