Uncovering fundamentals of gene regulation by enhancers

项目摘要/摘要 一类被称为增强子的顺式调控元件在时空调控中起着核心作用。 在发育过程中精确的基因表达程序。增强子序列或调控中的扰动可能 导致疾病，包括先天畸形和癌症。此外，增强子序列差异 正在成为人类表型变异的重要介体。增强剂的一个关键特征是它们的能力 在距离它们的基因组几十甚至几百个碱基的很长的基因组距离上激活转录 目标推动者。发现，当激活时，增强剂由独特的染色质签名标记， 结合基因组学方法，如芯片序列或染色体构象捕获技术 (3C及其衍生产品)促进了跨细胞类型和物种的增强子注释，并提供了关键见解 转变为远程监管。然而，一般而言，在这些群体水平的固定细胞分析中，动力学信息 在单细胞水平上的潜在增强子激活已经丢失。 我们最近开发了一种新的成像方法，允许我们标记和跟踪单个增强剂和 活细胞中的启动子元件，在它们的天然染色体背景下，在不同的细胞和活性中 各州。我们提议的工作进一步将这项技术与新生转录的活细胞可视化相结合，以 捕捉增强子和启动子的动力学行为及其与不连续性质的关系 抄写。使用未分化或分化的干细胞作为细胞模型，我们将解决主要的 增强子生物学中的公开问题，包括增强子-启动子的实时频率和动态 接触，它们与转录突发的关联，以及染色质拓扑组织在 增强功能。我们计划引入一系列扰动来调查特定的干扰是如何 增强子的事件，如组蛋白修饰、变异体掺入或核小体重塑，会影响 远距离染色体接触的动态和单细胞水平的转录激活。在……里面 在提案的第二个主题中概述的补充研究中，我们采用了一套不同的 基因组和遗传学方法，以确定远程基因调控所需的新因子和 确定在天然染色质环境中激活增强子的必要性和充分性。两个人 主要主题将允许我们重温增强器功能的当前模型(例如增强器循环、增强器 按拓扑上相关的区域等进行划定。)并将产生新的概念和机制模型 哺乳动物的远程基因控制，对理解和治疗人类具有广泛的未来意义 疾病。

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.