MULTIPLEX CIS-REGULATORY ANALYSIS IN MAMMALIAN CELLS

描述（由申请人提供）：98%的哺乳动物基因组是非编码的，其中很大一部分具有顺式调控功能。如果我们要解释人类群体中大量非编码多态性的影响，我们必须对基因表达的顺式调控语法有更全面的了解。在基因组规模技术的推动下，潜在顺式调控元件（cre）的鉴定正在迅速进行。不幸的是，发现假定的cre的速度远远超过了我们在体内对它们进行功能分析的能力。显然，迫切需要新的高通量技术来阐明哺乳动物细胞中cre的功能结构。报告基因分析是解剖哺乳动物cre功能的标准分析方法，但该分析的时间和劳动密集型性质使得测试大量cre不切实际。我们建议利用下一代测序（NGS）的力量来创建一个系统，用于哺乳动物细胞中报告基因的多路分析。我们的策略是将cre文库与条形码报告基因融合，并通过NGS量化其输出。我们将通过研究哺乳动物神经元细胞类型（视网膜感光细胞）的顺式调节来证明这种测定方法的实用性。在目标1中，我们将在单核苷酸分辨率下解剖光受体特异性视紫红质（Rho）启动子。超过15000个突变启动子将在活体视网膜中进行单次、多重分析，这是任何现有方法都无法实现的实验。这个实验的数据将允许我们第一次在核苷酸分辨率上量化进化序列守恒和顺式调控活性之间的关系。在Aim 2中，我们将部署该分析来测试1,000个Chip-seq“峰”，这些峰富含与关键光受体转录因子Crx结合。在一个单一的多路复用实验中，我们将测试所有1000个基因组序列在体内驱动光感受器特异性表达的能力。此外，在Aim 2中，我们将检测由Crx和另外两个转录因子Nrl和Nr2e3结合位点组成的合成启动子组合文库，这两个转录因子已知在控制光受体特异性基因表达中起关键作用。所有这些实验的输出将使用组合顺式调节的正式热力学模型进行分析。我们的目的是证明，在相对较短的时间内，有可能在体内解开一种重要哺乳动物细胞类型的顺式调节语法。这项研究将作为一个概念证明，CRE分析可以做得像CRE发现一样高通量。

DESCRIPTION (provided by applicant): 98% of the mammalian genome is non-coding, a significant fraction of which has cis- regulatory function. If we are ever to interpret the effects of the vast number of non-coding polymorphisms in the human population, we must gain a more complete understanding of the cis-regulatory grammar of gene expression. Fueled by genomic-scale technologies, the identification of potential cis-regulatory elements (CREs) is proceeding rapidly. Unfortunately, the rate of discovery of putative CREs far outpaces our capacity to functionally analyze them in vivo. Clearly, there is an urgent need for new high-throughput technologies to elucidate the functional architecture of CREs in mammalian cells. Reporter gene analysis is the standard assay for dissecting the function of mammalian CREs, but the time- and labor-intensive nature of the assay makes it impractical for testing large numbers of CREs. We propose to harness the power of Next Generation Sequencing (NGS) to create a system for the multiplexed analysis of reporter genes in mammalian cells. Our strategy is to fuse libraries of CREs to barcoded reporter genes and quantify their output by NGS. We will demonstrate the utility of this assay by studying cis-regulation in a mammalian neuronal cell type, the retinal photoreceptor cell. In Aim 1 we will dissect, at single nucleotide resolution, the photoreceptor- specific Rhodopsin (Rho) promoter. More than 15,000 mutant promoters will be assayed in a single, multiplexed assay in living retinas, an experiment that is not possible with any existing methodologies. The data from this experiment will allow us, for the first time, to quantify the relationship between evolutionary sequence conservation and cis-regulatory activity at nucleotide resolution. In Aim 2 we will deploy the assay to test 1,000 Chip-seq "peaks" that are enriched for binding to the key photoreceptor transcription factor, Crx. In a single, multiplexed experiment we will test all 1,000 genomic sequences for their ability to drive photoreceptor- specific expression in vivo. In addition, in Aim 2 we will assay combinatorial libraries of synthetic promoters composed of binding sites for Crx and two other transcription factors, Nrl and Nr2e3, known to play a key role in controlling photoreceptor-specific gene expression. The output of all these experiments will be analyzed using a formal thermodynamic model of combinatorial cis-regulation. Our intention is to demonstrate, in a relatively short time frame, that it is possible to unravel the cis-regulatory grammar of an important mammalian cell type in vivo. This study will serve as a proof-of-concept that CRE analysis can be made as high- throughput as CRE discovery. PUBLIC HEALTH RELEVANCE: Differences in the DNA sequence of the human genome account for variation between individuals in their susceptibilities to many diseases. Many of these genetic differences occur in regions of the genome containing stretches of DNA that control which genes are turned on and off. The goal of the proposed research is to develop a new methodology for determining the function of these control regions so that we can better predict the effects of human genetic variation on disease susceptibility.