High-throughput functional characterization of human enhancers

项目摘要/摘要： 高等生物的基因组及其表达受到高度调控，以应对各种 发育、环境和营养方面的线索。未能执行适当的基因调控可能会导致 发育缺陷和疾病状态。这项研究的首要目标是了解基本的 转录水平上的基因调控机制，即RNA聚合酶II(POL II)阶段 将基因转录成信使核糖核酸。这一调控是通过DNA元素的共同努力来进行的 (启动子和增强剂)和许多蛋白质机制，如POL II、TF和染色质/组蛋白修饰 复合体。最近对果蝇和哺乳动物细胞的研究表明，转录主要是 以两个限速步骤进行调控；1)Pol II招募到启动子，Pol II在20-50个基点暂停 在转录起始点下游，以及2)释放暂停的Pol II以产生延伸。一组 互补的方法(一些新的)被设计来识别人类的TF和染色质重构体 在这些步骤中，然后严格阐述它们实施转录调控的机制。由TFS提供 以及在不同的细胞系中具有最高时空分辨率的染色质修饰物， 包括K562、HCT116、HEK293和未分化和分化WTC-11。现有的财富 这些细胞系中的信息将与连续分析(RO-SEQ)以及必要时的其他分析相补充， 鉴定主要作用于转录中限速调控步骤中的一个或两个的转录因子。这些 观察性分析包括多个高灵敏的RO-SEQ分析，用于绘制 跨基因和增强子全基因组转录，用于检测转铁蛋白结合和占位的芯片外显子， STARR-SEQ法检测增强子和启动子的转录调控活性，以及退化子 系统，以快速降解因子，以评估其主要影响，通过这些‘-序贯’分析。总而言之，这些系统 将测试我们的监管模式，并评估其普遍性。在目标1中，使用一种新的适体为基础的下拉和 通过质谱学方法，我们将识别通过重塑复合体工作的开创性TF，例如 PBAP(SWI/SNF)打开染色质，用于随后的TF和POL II招募。在目标2中，我们试图识别细胞- 特定于类型的转录因子，作用于转录的主要调控步骤，即步骤1、2或两者。这将通过一个 细胞系特异性新生转录图谱的比较分析。目标3将测试特遣部队候选人的角色， 它们与转录周期中的一个或两个调控步骤有关，在活细胞中使用 Degron方法和多个‘-seq’分析。在目标4中，我们将评估信托基金在以下相互作用中的作用 增强子和启动子的相互作用使用高通量的异构体和染色体增强子分析。这个 提案将确定在转录调控的特定步骤中起作用的转录因子，并为 他们的行动机制。

PROJECT SUMMARY/ABSTRACT: The genomes of higher organisms and their expression are highly regulated in response to a variety of developmental, environmental, and nutritional cues. The failure to execute proper gene regulation can lead to developmental defects and disease states. The overarching goal of this research is to understand basic regulatory mechanisms of genes at the level of transcription, the stage where RNA polymerase II (Pol II) transcribes the genes into mRNA. This regulation is conducted by the concerted effort of DNA elements (promoters and enhancers) and many protein machineries such as Pol II, TFs, and chromatin/histone modifying complexes. Recent studies in Drosophila and mammalian cells have revealed that transcription is primarily regulated at two rate-limiting steps; 1) Recruitment of Pol II to promoter and Pol II pausing at 20-50bp downstream of transcription initiation site, and 2) Release of paused Pol II to productive elongation. A battery of complementary approaches (some novel) are designed to identify human TFs and chromatin remodelers that act at these steps and then rigorously address the mechanism by which they exert transcriptional regulation. by TFs and chromatin modifiers with the highest possible spatiotemporal resolution genome-wide in distinct cell lines, including K562, HCT116, HEK293, and undifferentiated and differentiated WTC-11. The wealth of existing information in these cell lines will be complemented with Run-On assays (RO-seq), and others when necessary, to identify TFs that act primarily on one or both of the rate-limiting regulated steps in transcription. These observational assays include multiple highly-sensitive RO-seq assays for mapping the position and amount of transcription across genes and enhancers genome-wide, ChIP-exo for detecting TF binding and occupancy, STARR-seq assays to measure transcription regulatory activity of enhancers and promoters, and a degron system to rapidly degrade TFs to assess their primary effects by these ‘-seq’ assays. Together, these systems will test our regulatory models and assess their generality. In Aim 1, using a novel aptamer-based pulldown and mass spectrometry approach, we will identify pioneering TFs that work through remodeling complexes such as PBAP (SWI/SNF) to open chromatin for subsequent TF and Pol II recruitment. In Aim 2, we seek to identify cell- type-specific TFs that act at major regulated steps of transcription, Steps 1, 2, or both. This will be achieved by a comparative analysis of cell line specific nascent transcription profiles. Aim 3 will test the role of TF candidates, which are associated with one or both of the regulatory steps in the transcription cycle, in living cells using a Degron approach and multiple ‘-seq’ assays. In Aim 4, we will assess the roles of TFs in the interplay of enhancers and promoters interactions using high-throughput episomal and chromosomal enhancer assays. The proposal will identify TFs that act at specific steps in transcription regulation and provide insights to the mechanics of their action.