CAREER: Dynamic dissection of how transcription and loop extrusion regulate 3D genome structure

职业：动态剖析转录和环挤出如何调节 3D 基因组结构

• 批准号: 2337728
• 负责人: Anders Hansen
• 金额: $ 99.96万
• 依托单位: Massachusetts Institute of Technology
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-03-01 至 2029-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2337728&HistoricalAwards=false
• 关键词: CAREER; Dynamic; dissection; how; transcription

Understanding how a single genome can give rise to a fully formed animal composed of diverse cell types remains one of biology’s great unsolved mysteries. This process requires exquisite control of gene expression. Enhancers are the primary units of gene control in mammals and often activate genes across vast genomic distances, thus, an understanding of 3D genome structure is needed to understand the action of enhancers. This project will investigate the regulation of 3D genome structure through the study of protein-mediated loops and domains termed Topologically Associating Domains (TADs). A major focus will be to integrate the proposed research into a class that the investigator teaches, in which students will build their own microscopes and study DNA looping. Outreach efforts to high school students and undergraduates with a focus on promoting the full participation of women, persons with disabilities, and underrepresented minorities will also be conducted. Additionally, the technological developments pioneered by this proposal will be openly shared with the scientific community. An understanding of dynamic genome topology is needed for understanding the regulation of gene expression. At the 3D genome scale relevant to enhancers, the proteins CTCF and cohesin fold mammalian genomes into loops and domains termed Topologically Associating Domains (TADs). Cohesin extrudes loops until it is blocked by CTCF, which then holds together a TAD. To better understand TADs, the proposed work will visualize TADs and CTCF loops directly in living cells and quantify their dynamics, loop lifetime, and looped fraction. An integrated approach that combines genome-editing, perturbation experiments, super-resolution live-cell imaging of CTCF/cohesin loops, 3D genomics, and integrative 3D polymer modeling will be used to address three key mechanistic questions related to the formation and regulation of TADs and loops. The investigators will explore how adjacent TADs form nested structures and higher-order interactions; how cohesin abundance and residence time controls TAD and loop formation and whether active transcription regulates TADs and loops positively or negatively. These measurements will provide quantitative and mechanistic information on TAD and loop formation in living cells, which will improve current understanding of 3D genome architecture and associated modulation during gene expression.This award is funded by the Molecular Biophysics Program in the Molecular and Cellular Biosciences Division of the Biological Sciences Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

了解单个基因组如何产生由不同细胞类型组成的完全形成的动物仍然是生物学未解之谜之一。这一过程需要对基因表达的精确控制。增强子是哺乳动物中基因控制的主要单位，并且经常跨越巨大的基因组距离激活基因，因此，需要理解3D基因组结构以理解增强子的作用。 本计画将借由蛋白质介导的环与拓扑关联结构域（Topologically Associating Domains，TADs）来探讨基因组三维结构的调控。 一个主要的重点将是将拟议的研究整合到一个类，研究人员教，学生将建立自己的显微镜和研究DNA循环。 还将开展面向高中生和大学生的外联工作，重点是促进妇女、残疾人和代表性不足的少数群体的充分参与。此外，该提案开创的技术发展将与科学界公开分享。了解动态基因组拓扑结构是理解基因表达调控的必要条件。 在与增强子相关的3D基因组尺度上，蛋白质CTCF和粘附素将哺乳动物基因组折叠成称为拓扑关联结构域（TADs）的环和结构域。黏连蛋白挤压成环，直到被CTCF阻断，然后CTCF将一个环结合在一起。为了更好地理解TADs，拟议的工作将直接在活细胞中可视化TADs和CTCF循环，并量化它们的动力学，循环寿命和循环分数。一个综合的方法，结合基因组编辑，扰动实验，超分辨率活细胞成像的CTCF/cohesin循环，3D基因组学和集成的3D聚合物建模将被用来解决三个关键的机制问题有关的形成和调节的TAD和循环。研究人员将探索相邻的TADs如何形成嵌套结构和高阶相互作用;粘附素丰度和停留时间如何控制转录和环的形成，以及活性转录是否积极或消极地调节TADs和环。 这些测量将提供关于活细胞中的微循环和环形成的定量和机制信息，这将提高目前对3D基因组结构和基因表达过程中相关调节的理解。该奖项由生物科学理事会分子和细胞生物科学部的分子生物物理学计划资助。该奖项反映了NSF的法定使命，并被认为值得通过以下方式获得支持：使用基金会的知识价值和更广泛的影响审查标准进行评估。