MASSIVELY PARALLEL CIS-REGULATORY ELEMENT ANALYSIS IN MAMMALIAN CELLS

适用于1R01HG006790-01 在此输入文本，它是您的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，正在以惊人的速度推动顺式调控元件(CRE；即增强子/启动子)的发现。测试这些预测的调控潜力是我们努力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对即使是一个芯片序列实验也能产生的数千个预测进行功能测试，更不用说由encode和其他基于联盟的努力产生的数百万个预测了。打破Cre预测的僵局需要一种新的技术，能够在哺乳动物细胞中进行大规模平行的顺式调控分析。在这项建议中，我们引入了CRE-SEQ(顺式测序调节元件分析)，这是一种在哺乳动物细胞中一次实验中分析数千种Cre的新技术。我们的策略是将Cres文库与条形码报告基因融合，将这些文库导入细胞，并通过NGS量化它们的输出。我们将开发CRE-SEQ作为一种高效、健壮和高度并行的技术，用于在单个实验中评估数千个芯片序列峰的顺式调节活性。如果成功，这一策略应该可以量化整个顺式调节体(即控制给定细胞类型中基因表达的整个顺式调节区)的启动子活性。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(一种分化的神经细胞类型)和未分化的胚胎干细胞--的顺式调节来证明该方法的实用性。Cre-seq有望促进我们对人类基因调控的理解，并将成为可用于疾病诊断和治疗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.