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.

项目摘要/摘要 哺乳动物基因组被折叠成称为拓扑关联结构域的空间域 (TADS)跨越数百个千碱基。通过增加同一DNA基因座内的接触概率 TAD、TADS被认为通过调节增强子-启动子的接触来调节基因的表达。始终如一， 最近的几项研究表明，TAD中断可以导致疾病，包括癌症和人类 神经发育障碍。此外，TADS、CTCF和粘附素复合体的因果调节因子， 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TADS在 调控特定的基因和位点。这项提议的前提是，目前该领域的分歧 这在一定程度上是由于至少两个限制。首先，基因组组织和染色质循环很可能是天生的 动态的，但我们缺乏捕捉这一点的工具(A部分)。第二，选择发育基因调控 健壮性，因此具有冗余性和复杂性的特点。在这里，自下而上的策略是 建议克服自然发育基因调控回路的冗余性(B部分)。 首先，在本提案的A部分中，开发了一套综合的新的实验和 提出了计算工具的概念。以前的方法虽然功能强大，但依赖于静态快照 仅限于死亡和化学固定细胞的方法。在这里，活细胞单分子成像是 提出了一种克服这一限制的方法，以跟随染色质在纳米级活细胞中的循环 空间分辨率和时间分辨率。通过将此方法与开发 新的计算工具和互补的基因组学方法，这将允许解剖如何 基因组折叠在时间和空间上调节转录。 其次，在本提案的B部分，这些工具将应用于自下而上的合成生物学 接近。目标将是在简单的基因组环境中构建TADS、增强子和启动子。这 将有可能逃脱自然发育基因调控的复杂性，在那里冗余 这使得建立因果关系具有极大的挑战性。通过自下而上的方法，这项建议旨在提炼出 一般力学原理通过微扰检验各种力学假说 实验，并建立因果关系。 这项提案的首要目标是阐明基因组组织的方式和程度 有助于转录调控和发展定量预测性理解。最终， 这可能会让我们纠正疾病中的基因组错误折叠。