Uncovering fundamentals of gene regulation by enhancers

项目概要/摘要 一类称为增强子的顺式调控元件在协调时空中起着核心作用， 在发育过程中精确的基因表达程序。增强子序列或调节的扰动可以 导致疾病，包括先天畸形和癌症。此外，增强子序列趋异 正在成为人类表型变异的重要媒介。增强子的一个关键特征是它们能够 为了激活与它们的基因组相距数十甚至数百个酶的长基因组距离上的转录， 靶向启动子。发现当激活时，增强子被独特的染色质标记所标记， 结合基因组方法，如ChIP-seq或染色体构象捕获技术 (3C和衍生物）促进了跨细胞类型和物种的增强子注释， 长期监管。然而，通常，在这些群体水平的固定细胞测定中， 在单细胞水平上潜在的增强子激活已经丧失。 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子， 启动子元件在活细胞中、在它们的天然染色体背景中以及在不同的细胞和活性中， states.我们提出的工作进一步将这项技术与新生转录本的活细胞可视化结合起来， 捕获增强子和启动子的动力学行为及其与不连续性质的关系， 转录。使用未分化或分化的干细胞作为细胞模型，我们将解决主要问题。 增强子生物学中的开放问题，包括增强子-启动子的实时频率和动力学 接触，它们与转录爆发的关联，以及染色质拓扑组织在 增强子功能我们计划引入一系列扰动来研究特定的 增强子上的事件，如组蛋白修饰、变体掺入或核小体重塑，影响 在单细胞水平上的长距离染色体接触和转录激活的动力学。在 在建议的第二个主题中概述的补充研究中，我们采用了一套多样化的 基因组和遗传学方法，以确定长期基因调控所需的新因子， 定义天然染色质背景下增强子激活的必要性和充分性。两 主要主题将使我们能够重新审视增强子功能的当前模型（例如，增强子循环，增强子 通过拓扑关联域等来定界）并将产生新的概念和机械模型， 哺乳动物的远程基因控制，对理解和治疗人类疾病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.