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整动物仍然是生物学中未解的重大谜团之一。这个过程需要对基因表达进行精确控制，增强子是哺乳动物基因控制的主要单位，并且经常激活跨基因的基因。巨大的基因组距离，因此，需要了解 3D 基因组结构来了解增强子的作用，该项目将通过研究蛋白质介导的环和域（称为拓扑关联）来研究 3D 基因组结构的调节。一个主要重点是将拟议的研究整合到研究者教授的课程中，学生将在课程中建造自己的显微镜并向高中生和本科生研究 DNA 循环，重点是促进这一领域的发展。此外，该提案开创的技术发展也将与科学界公开分享，以了解基因表达的调控需要了解动态基因组拓扑结构。在 3D与增强子相关的基因组规模，蛋白质 CTCF 和粘连蛋白将哺乳动物基因组折叠成称为拓扑关联结构域 (TAD) 的环和结构域，粘连蛋白会挤出环，直到被 CTCF 阻断，然后将 TAD 结合在一起。这项工作将直接在活细胞中可视化 TAD 和 CTCF 环，并量化它们的动态、环寿命和环分数。研究人员将利用微扰实验、CTCF/粘连蛋白环的超分辨率活细胞成像、3D 基因组学和综合 3D 聚合物建模来解决与 TAD 和环的形成和调节相关的三个关键机制问题。 TAD 形成嵌套结构和高阶相互作用；粘连蛋白丰度和停留时间如何控制 TAD 和环形成，以及活性转录是否正向或负向调节 TAD 和环。提供活细胞中 TAD 和环形成的机制信息，这将提高目前对 3D 基因组结构和基因表达过程中相关定量调节的理解。该奖项由生物科学分子和细胞生物科学部的分子生物物理学项目资助该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。