Uncovering fundamentals of gene regulation by enhancers

揭示增强子基因调控的基础

• 批准号: 10176537
• 负责人: Joanna Wysocka
• 金额: $ 32.87万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10176537
• 关键词: Address; Affect; Behavior; Biology; Cell model; Cells; Cellular Assay; ChIP-seq; Chromatin; Communication; Congenital Abnormality; Couples; Development; Disease; Elements; Enhancers; Event; Frequencies; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Enhancer Element; Genetic Transcription; Genomic approach; Genomics; Goals; Human; Individual; Kinetics; Label; Lead; Malignant Neoplasms; Mammals; Mediator of activation protein; Modeling; Molecular Conformation; Nature; Nucleosomes; Phenotype; Play; Population; Process; Regulation; Regulatory Element; Research; Role; Series; Technology; Therapeutic; Time; Transcript; Transcriptional Activation; Undifferentiated; Variant; Visualization; Work; cell type; genetic approach; histone modification; human disease; imaging approach; insight; novel; programs; promoter; spatiotemporal; stem cell differentiation

PROJECT SUMMARY/ABSTRACT A class of cis-regulatory elements, called enhancers, play a central role in orchestrating spatiotemporally precise gene expression programs during development. Perturbations in enhancer sequence or regulation can lead to disease, including congenital malformations and cancer. Furthermore, enhancer sequence divergence is emerging as an important mediator of human phenotypic variation. A key feature of enhancers is their ability to activate transcription over long genomic distances of tens or even hundreds of kilobases away from their target promoters. Discovery that, when active, enhancers are marked by unique chromatin signatures, combined with genomic approaches such as ChIP-seq or Chromosomal Conformation Capture technologies (3C and derivatives) facilitated enhancer annotation across cell types and species and provided key insights into long-range regulation. Generally, however, in these population-level, fixed-cell assays, kinetic information underlying enhancer activation at a single-cell level has been lost. We recently developed a new imaging approach that allows us to label and track individual enhancer and promoter elements in living cells, in their native chromosomal context and in different cellular and activity states. Our proposed work further couples this technology with live-cell visualization of nascent transcripts to capture the kinetic behavior of enhancers and promoters and its relationship with the discontinuous nature of transcription. Using undifferentiated or differentiating stem cells as a cellular model, we will address major open questions in enhancer biology, including the real-time frequency and dynamics of enhancer-promoter contacts, their association with transcriptional bursts, and the role of chromatin topological organization in enhancer function. We plan to introduce a series of perturbations to investigate how disruption of specific events at enhancers, such as histone modification, variant incorporation or nucleosome remodeling, affects dynamics of long-range chromosomal contacts and transcriptional activation at the single-cell level. In complementary studies outlined in the second theme of the proposal, we are employing a diverse set of genomic and genetic approaches to identify novel factors that are required for long-range gene regulation and to define necessities and sufficiencies for enhancer activation within the native chromatin context. The two main themes will allow us to revisit current models of enhancer function (e.g. enhancer looping, enhancer delimitation by topologically associated domains, etc.) and will yield new concepts and mechanistic models of long-range gene control in mammals, with broad future implications for understanding and treatment of human disease.

项目摘要/摘要 一类称为增强剂的顺式调节元素在编排时空中起着核心作用 开发过程中精确的基因表达程序。增强子序列或调节中的扰动可以 导致疾病，包括先天性畸形和癌症。此外，增强子序列差异 正在成为人类表型变异的重要介体。增强剂的关键特征是他们的能力 激活在远离其远离其远距离的长度基因组距离上的转录 目标启动子。发现当活动时，增强剂的标志是独特的染色质特征， 结合基因组方法，例如芯片序列或染色体构象捕获技术 （3C和衍生物）促进细胞类型和物种之间的增强剂注释，并提供了关键的见解 进入远程调节。但是，通常在这些人群级别的固定细胞测定中，动力学信息 在单细胞水平上的基本增强子激活已经丢失。 我们最近开发了一种新的成像方法，使我们能够标记和跟踪单个增强器和 活细胞中的启动子元素在其天然染色体环境以及不同的细胞和活性中 国家。我们提出的工作进一步伴侣，通过对新生成绩单的实时可视化这项技术 捕获增强子和启动子的动力学行为及其与不连续性的关系 转录。使用未分化或区分干细胞作为细胞模型，我们将解决主要 增强剂生物学中的开放问题，包括增强器促进剂的实时频率和动态 接触，它们与转录爆发的关联以及染色质拓扑组织在 增强器功能。我们计划引入一系列扰动，以调查特定的破坏 增强剂的事件，例如组蛋白修饰，变体掺入或核小体重塑，会影响 远程染色体接触和单细胞水平上转录激活的动力学。在 提案的第二个主题中概述了互补研究，我们正在采用各种各样的 基因组和遗传方法，以鉴定远程基因调节所需的新因素和 定义在天然染色质环境中增强剂激活的必需品和充分性。两个 主要主题将使我们能够重新访问当前的增强器功能模型（例如增强器循环，增强器 按拓扑相关的域等划界），并将产生新的概念和机械模型 哺乳动物中的远程基因控制，对理解和治疗人类具有广泛的未来影响 疾病。