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

了解单个基因组如何产生由多种细胞类型组成的完全成形的动物，仍然是生物学中未解之谜之一。这个过程需要对基因表达进行精确的控制。增强子是哺乳动物基因控制的主要单位，经常激活跨基因组距离的基因，因此，需要了解三维基因组结构才能理解增强子的作用。该项目将通过研究蛋白质介导的环和称为拓扑相关结构域（TADs）的结构域来研究三维基因组结构的调控。一个主要的重点将是将拟议的研究整合到研究者所教授的课程中，在这门课程中，学生将建立自己的显微镜并研究DNA环。还将开展面向高中生和本科生的外联工作，重点是促进妇女、残疾人和代表性不足的少数民族的充分参与。此外，这项提案所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。在与增强子相关的三维基因组尺度上，蛋白质CTCF和内聚蛋白将哺乳动物基因组折叠成称为拓扑相关结构域（TADs）的环和结构域。内聚蛋白挤出环状物，直到它被CTCF阻断，然后CTCF将TAD结合在一起。为了更好地理解TADs，建议的工作将直接在活细胞中可视化TADs和CTCF环，并量化它们的动力学、环寿命和环分数。结合基因组编辑、微扰实验、CTCF/内聚蛋白环的超分辨率活细胞成像、3D基因组学和综合3D聚合物建模的综合方法将用于解决与TADs和环的形成和调控相关的三个关键机制问题。研究人员将探索相邻tad如何形成嵌套结构和高阶相互作用；内聚丰度和停留时间如何控制TAD和环的形成，以及主动转录是正调控还是负调控TAD和环。这些测量将提供活细胞中TAD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项目资助。该奖项反映了美国国家科学基金会的法定使命，并通过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，被认为值得支持。

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.