DYNAMIC BOTTOM-UP DISSECTION OF CHROMATIN LOOPING AND GENE REGULATION

染色质环和基因调控的动态自下而上解剖

• 批准号: 10000531
• 负责人: Anders Sejr Hansen
• 金额: $ 231.83万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10000531
• 关键词: Cells; Chemicals; Chromatin Loop; Complement; Complex; Computing Methodologies; DNA; Development; Developmental Gene; Disease; Dissection; Distal; Elements; Enhancers; Etiology; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomic approach; Genomics; Goals; Human; Malignant Neoplasms; Methods; Mutate; Neurodevelopmental Disorder; Probability; Regulator Genes; Research; Resolution; Role; Testing; Time; Transcriptional Regulation; Work; cohesin; computerized tools; developmental disease; experimental study; genomic locus; mammalian genome; molecular imaging; nanometer resolution; nervous system disorder; new technology; promoter; single molecule; synthetic biology; tool

Project Summary/Abstract Mammalian genomes are folded into spatial domains termed Topologically Associating Domains (TADs) spanning hundreds of kilobases. By increasing the contact probability of DNA loci inside the same TAD, TADs are thought to regulate gene expression by regulating enhancer-promoter contact. Consistently, several recent studies have demonstrated that TAD disruption can cause disease, including cancer and human neurodevelopmental disorders. Additionally, the causal regulators of TADs, CTCF and the cohesin complex, are among the most frequently mutated genes in cancer. At the same time, however, other recent studies have found little to no role for CTCF and TADs in regulating specific genes and loci. The premise of this proposal is that the current disagreements in the field are partly due to at least two limitations. First, genome organization and chromatin looping is likely inherently dynamic, but we lack the tools to capture this (Part A). Second, developmental gene regulation is selected for robustness, and therefore characterized by redundancy and complexity. Here, a bottom-up strategy is proposed to overcome the redundancy of natural developmental gene regulatory circuits (Part B). First, in Part A of this proposal, the development of an integrated set of new experimental and computational tools is proposed. Previous approaches, although powerful, relied on static snapshot approaches that are limited to dead and chemically fixed cells. Here, live-cell single-molecule imaging is proposed as a method to overcome this limitation to follow chromatin looping in live cells with nanometer resolution in space and second resolution in time. By complementing this approach with the development of new computational tools and a complementary genomics approach, this will allow the dissection of how genome folding regulates transcription in both time and space. Second, in Part B of this proposal, these tools will be applied in a bottom-up synthetic biology approach. The goal will be to build TADs, enhancers, and promoters de novo in a simple genomic context. This will make it possible to escape the complexity of natural developmental gene regulation, where redundancy makes establishing causality highly challenging. Through a bottom up approach, this proposal aims to distill out the general mechanistic principles, test a wide range of mechanistic hypotheses through perturbation experiments, and to establish causality. The overarching goal of this proposal is to elucidate how and to what extent genome organization contributes to transcriptional regulation and to develop a quantitatively predictive understanding. Ultimately, this may allow us to correct genome misfolding in disease.

项目概要/摘要 哺乳动物基因组折叠成空间域，称为拓扑关联域 （TAD）跨越数百个碱基。通过增加同一DNA位点内的接触概率 TAD，TAD被认为通过调节增强子-启动子接触来调节基因表达。一贯地， 最近的几项研究表明，TAD 破坏可能导致疾病，包括癌症和人类疾病 神经发育障碍。此外，TAD、CTCF 和粘连蛋白复合物的因果调节因子， 是癌症中最常见的突变基因之一。 然而，与此同时，最近的其他研究发现 CTCF 和 TAD 在治疗中几乎没有作用。 调节特定基因和位点。该提案提出的前提是当前该领域的分歧 部分原因是至少有两个限制。首先，基因组组织和染色质循环可能本质上是 动态的，但我们缺乏捕捉这一点的工具（A 部分）。其次，选择发育基因调控 鲁棒性，因此具有冗余性和复杂性的特点。这里，自下而上的策略是 提出克服自然发育基因调控电路的冗余（B 部分）。 首先，在本提案的 A 部分中，开发一套集成的新实验和 提出了计算工具。以前的方法虽然功能强大，但依赖于静态快照 仅限于死细胞和化学固定细胞的方法。在这里，活细胞单分子成像是 提出了一种克服这种限制的方法，即用纳米技术跟踪活细胞中的染色质循环 空间分辨率和时间第二分辨率。通过补充这种方法与发展 新的计算工具和互补的基因组学方法，这将允许剖析如何 基因组折叠在时间和空间上调节转录。 其次，在该提案的B部分中，这些工具将应用于自下而上的合成生物学 方法。目标是在简单的基因组环境中从头构建 TAD、增强子和启动子。这 将使摆脱自然发育基因调控的复杂性成为可能，其中冗余 使得建立因果关系变得极具挑战性。该提案旨在通过自下而上的方法提炼出 一般机械原理，通过扰动测试各种机械假设 实验，并建立因果关系。 该提案的总体目标是阐明基因组组织的方式和程度 有助于转录调控并形成定量预测的理解。最终， 这可能使我们能够纠正疾病中的基因组错误折叠。