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

Massachusetts Institute Of Technology, Cambridge MA

Investigators

Abstract

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 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.