Functional Architecture and Interplay of Transcription Regulatory Elements of the Human Genome

项目摘要/摘要 人类基因组中估计有一百万个增强子可以调节他们的特定靶点。 基因(S)从距离上到百万碱基之外，并且以一种与方位无关的方式。的宏伟目标 这个项目是以深入的方式定义人类增强器的基本架构和功能 为了更好地了解它们与不同类别的启动子相互作用的特殊性，这些启动子使用不同的 转录因子和/或在转录过程中的不同步骤受到调控。我们已经证明了增强剂可以 使用我们的GRO/PRO-CAP分析来识别和精确定位新生基因的转录起始点 在所有可用的方法中，RNA的灵敏度最高。这项测试表明，增强剂和 启动子共享共同的体系结构，其中两者由两个不同的核心启动子(CP)分隔 和转录因子(TF)结合基序的中心簇。的角色和所需的组织 需要充分剖析构成增强子和两个CP的多个序列基序才能理解 活性促进剂如何发挥作用。此外，增强剂可以与特定的启动子有效地相互作用，并且 这种特异性的基础仍不明确，特别是在远距离。最后，我们知道发起人和 增强子元件有序列基序，可以在转录周期的不同调控步骤中发挥作用，但 这些活动如何协调基因调控还有待研究。在目标1中，我们将测试所有 Pro-CAP从具有代表性的人类8号和11号染色体中鉴定了K562基因的增强子候选者 精心挑选的一组具有明显监管特征的推动者。一组具有活性的增强子-启动子 然后，组合将受到Tf结合中心簇的全面基序突变 以及两个核心启动子中的每一个。这些研究测试了我们的基本增强单位假说 评估增强子与目标启动子的关系，以及特定基序和序列的作用 增强剂功能的特点。在目标2中，我们定量检查了在 目标1通过条形码和整合WT和突变增强子在其正常响应的上游5kb 在AAVS1安全港基因座的染色体背景下的启动子。这些化验将严格测试功能 一个恒定染色体中的增强子基序、核心启动子和增强子的整体结构 背景资料。此外，我们还将测试启动子和增强子特异性潜在的监管代码 利用靶向基因组评价突变型转铁蛋白基序对转铁蛋白结合和新生转录的影响 化验。最后，在目标3中，我们使用NMU探索增强剂在远距离发挥作用的能力 增强子(ENMU)，位于NMU启动子上游94kb，通过以下方式刺激其转录 一万倍。我们将利用这个健壮的模型增强器，它不会受到冗余/阴影的干扰 在天然基因座上的增强子，建立一个新的长距离、染色体、大规模平行的报告 在ENMU基因座上使用着陆垫的系统，并通过突变来表征ENMU的功能基序。

PROJECT SUMMARY/ABSTRACT Human genome is peppered with an estimated one million enhancers that can regulate their specific target gene(s) from a distance up to megabases away, and in an orientation-independent manner. The broad goal of this project is to define the fundamental architecture and function of human enhancers in a in-depth manner and to better understand the specificity of their interplay with different classes of promoters that use different transcription factors and/or are regulated at distinct steps in transcription. We have shown that enhancers can be identified and precisely mapped using our GRO/PRO-cap assay that map transcription start sites of nascent RNA with highest sensitivity of all available methods. This assay has shown that both enhancers and promoters share a common architecture whereby both are delimited by two divergent core promoters (CPs) and a central cluster of transcription factor (TF) binding motifs. The roles and required organization of the multiple sequence motifs that constitute enhancers and the two CPs need to be fully dissected to understand how active enhancers function. Additionally, enhancers can interact productively with specific promoters, and the basis of this specificity especially at long range remains ill-defined. Finally, we know that promoter and enhancer elements have sequence motifs that can act at distinct regulatory steps of the transcription cycle, but how these activities coordinate gene regulation has yet to be examined. In Aim 1, we will test the activity of all PRO-cap identified enhancer candidates in K562 from representative human Chromosomes 8 and 11 with a carefully chosen set of promoters harboring distinct regulatory features. A set of active enhancer-promoter combinations will then be subjected to a comprehensive motif mutagenesis of the central clusters of TF binding motifs and each of the two core promoters. These studies test our fundamental enhancer unit hypothesis and assess the relationships of enhancers to targeted promoters, and the role of specific motifs and sequence features in enhancer function. In Aim 2, we examine quantitatively enhancers and key mutants identified in Aim 1 by barcoding and integrating WT and mutant enhancers 5 kb upstream of their normally responsive promoter in a chromosomal context at the AAVS1 safe harbor locus. These assays will rigorously test function of enhancer motifs, core promoters, and the overall architecture of enhancers in a constant chromosomal background. In addition, we will also test the regulatory code underlying enhancer specificity for promoters and evaluate effects of mutant TF motifs on TF binding and on nascent transcription using targeted genomic assays. Finally, in Aim 3, we explore the ability of enhancers to act over long distances, using the NMU enhancer (eNMU), which resides 94 kb upstream of the NMU promoter and stimulates its transcription by 10,000-fold. We will utilize this robust model enhancer, which is not confounded by redundant/shadow enhancers at the native locus, to establish a novel long-range, chromosomal, massively-parallel reporter system using a landing pad at the eNMU locus and characterize functional motifs of eNMU by mutagenesis.