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不同细胞类型组成的完全形成的动物，仍然是生物学尚未解开的重大谜团之一。这一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，经常激活跨越基因组距离的基因，因此，需要了解三维基因组结构才能理解增强子的作用。这个项目将通过研究蛋白质介导的环和结构域来研究3D基因组结构的调节，这些环和结构域被称为拓扑相关结构域(TADS)。一个主要的重点将是将拟议的研究整合到调查员教授的课程中，在课堂上，学生们将建造自己的显微镜，并研究DNA环路。还将向高中生和本科生开展外联工作，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，这项提议开创的技术发展将与科学界公开分享。为了理解基因表达的调节，需要了解动态基因组拓扑结构。在与增强子相关的3D基因组尺度上，CTCF和粘附素蛋白将哺乳动物基因组折叠成环和结构域，称为拓扑关联结构域(TADS)。粘附素挤压循环，直到它被CTCF阻止，然后CTCF将其稍微结合在一起。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环，并量化它们的动力学、环寿命和环比例。结合基因组编辑、扰动实验、CTCF/粘附素环的超分辨率活细胞成像、3D基因组学和一体化3D聚合物建模的综合方法将被用于解决与TADS和环的形成和调节相关的三个关键机制问题。研究人员将探索相邻的TADS是如何形成嵌套结构和高阶相互作用的；粘附素的丰度和停留时间如何控制TAD和环的形成，以及活跃的转录是积极还是消极地调节TADS和环的形成。这些测量将提供关于活细胞中TAD和环形成的定量和机械信息，这将改善目前对3D基因组结构和基因表达过程中相关调控的理解。该奖项由生物科学总监分子和细胞生物科学部门的分子生物学计划资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.