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 人类基因组含有大约100万增强剂，可以调节其特定目标 基因从远处到巨蛋，并以与方向无关的方式走开。广泛的目标 该项目是要以深入的方式定义人类增强剂的基本架构和功能 并更好地了解使用不同类别的启动子的相互作用的特异性 转录因子和/或在转录的不同步骤中受到调节。我们已经证明增强剂可以 使用我们的gro/pro-cap测定法识别并精确映射映射转录开始位点 所有可用方法的RNA具有最高灵敏度。该测定表明增强剂和 发起人共享一个共同的体系结构，通过两个不同的核心启动子（CPS）对两者进行了界定 和转录因子（TF）结合基序的中央簇。角色和要求的组织 多个构成增强子和两个CP的多个序列基序需要完全剖析以了解 主动增强剂的功能。此外，增强子可以与特定启动子有效互动，并且 这种特异性的基础，尤其是在远距离上的基础。最后，我们知道那个发起人和 增强子元素的序列基序可以在转录周期的不同调节步骤中起作用，但是 这些活动如何协调基因调节尚未检查。在AIM 1中，我们将测试所有人的活动 Pro-CAP从代表性的人类染色体8和11中鉴定出K562中的增强剂候选者， 精心选择的一组具有不同监管特征的启动子。一组主动增强器启动器 然后，组合将经过TF结合的中央簇的全面基序诱变 主题和两个核心启动子中的每个。这些研究检验了我们的基本增强子单位假设和 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 增强器功能的功能。在AIM 2中，我们检查了定量增强子和关键突变体 AIM 1通过条形码和整合WT和突变体增强剂5 KB的正常响应速度上游 AAVS1安全港基因座的染色体环境中的发起人。这些测定将严格测试功能 增强子图案，核心启动子和恒定染色体中增强剂的整体结构 背景。此外，我们还将测试发起人和 评估突变体TF基序对使用靶向基因组的TF结合和新生转录的影响 测定。最后，在AIM 3中，我们探讨了增强剂在长距离行动的能力，使用NMU 增强子（ENMU），它驻留于NMU启动子上游的94 KB，并通过 10,000倍。我们将利用这种强大的模型增强器，它不会被冗余/阴影混淆 天然基因座的增强剂，建立一个新型的远程，染色体，大规模并行记者 系统使用ENMU基因座的降落垫，并通过诱变表征ENMU的功能基序。