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

人类基因组转录调控元件的功能结构和相互作用

• 批准号: 10639574
• 负责人: JOHN T LIS
• 金额: $ 69.2万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10639574
• 关键词: Architecture; Bar Codes; Base Pairing; Binding; Biological Assay; CCRL2 gene; Capsicum; Chromatin Conformation Capture and Sequencing; Chromosomes; Code; Communication; Complex; DNA; DNA Polymerase II; Disease; Dissection; Elements; Enhancers; Gene Expression; Gene Expression Regulation; Genes; Genetic Enhancer Element; Genetic Transcription; Genomics; Goals; Histones; Human; Human Chromosomes; Human Genome; Hypersensitivity; K-562; K562 Cells; Maps; Measures; Messenger RNA; Methods; Modeling; Molecular; Mutagenesis; Mutate; NMU gene; Normal Cell; Nucleic Acid Regulatory Sequences; Productivity; Property; RNA; Regulation; Regulator Genes; Reporter; Reporting; Role; Site; Specificity; System; Testing; Transcript; Transcription Initiation; Transcription Initiation Site; Upstream Enhancer; Work; global run on sequencing; human study; insight; mRNA Stability; mutant; novel; promoter; transcription factor

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.

项目摘要/摘要 人类基因组中散布着大约一百万个增强子，它们可以调节特定的靶点。 基因从一个距离到兆位数的距离，并以方向独立的方式。的广泛目标 本项目旨在深入研究人类增强子的基本结构和功能 为了更好地理解它们与不同类型的启动子相互作用的特异性， 转录因子和/或在转录的不同步骤被调节。我们已经证明，增强剂可以 使用我们的GRO/PRO-cap测定法进行鉴定和精确定位， 在所有可用方法中灵敏度最高的RNA。该测定显示增强子和 启动子共享一个共同的结构，其中两者都由两个不同的核心启动子（CP）限定， 和转录因子（TF）结合基序的中心簇。的作用和所需的组织 构成增强子和两个CP的多个序列基序需要被充分解剖以理解 活性增强子如何发挥作用。此外，增强子可以与特异性启动子有效地相互作用， 这种特异性的基础，尤其是在远距离上，仍然不清楚。最后，我们知道发起人和 增强子元件具有可以在转录周期的不同调节步骤起作用的序列基序，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鉴定了来自代表性人染色体8和11的K562中的增强子候选物， 精心选择的一组具有不同调控特征的启动子。一组活性增强子-启动子 然后将组合进行TF结合的中心簇的综合基序诱变， 基序和两个核心启动子。这些研究验证了我们的基本增强子单位假设， 评估增强子与靶向启动子的关系，以及特定基序和序列的作用 增强子功能中的特征。在目标2中，我们定量地研究了增强子和关键突变体， 目的1通过条形码化和整合WT和突变增强子上游5kb的正常响应 在AAVS1安全港基因座处的染色体背景中的启动子。这些分析将严格测试功能 增强子基序，核心启动子和增强子在恒定染色体中的整体结构 背景此外，我们还将测试启动子的增强子特异性背后的调控密码， 使用靶向基因组技术评估突变TF基序对TF结合和新生转录的影响， 分析。最后，在目标3中，我们使用NMU探索增强子在长距离上起作用的能力。 增强子（eNMU），其位于NMU启动子上游94 kb处，并通过以下方式刺激其转录： 一万倍我们将利用这个强大的模型增强器，它不会被冗余/阴影混淆 在天然基因座的增强子，以建立一个新的长距离，染色体，平行报道 系统，并通过诱变表征eNMU的功能基序。