Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
Eric F. Joyce
Inside of the nucleus, chromosomes are intricately folded to regulate expression of their genes. Early models of how chromosomes are organized were driven by genomics-based methods that average chromatin contact frequencies across cell populations. These studies revealed that the genome folds into megabase-sized topologically associating domains (TADs), reproducible clusters of self-interacting chromatin. TADs are proposed to form via the loop extrusion model, in which cohesin, a ring-shaped protein complex, extrudes chromatin until paused by the insulator protein CTCF. TADs typically encompass a gene and its regulatory elements, leading to a model in which TADs spatially insulate enhancer-promoter communication to regulate transcription. However, subsequent studies revealed that TAD boundaries were variable in single cells, suggesting TAD formation is dynamic. How such a mercurial system could reliably regulate gene expression was unclear. Here, we unpack the role of cohesin and its regulators in TAD folding and gene expression. We developed a novel imaging-based system to visualize TAD insulation in single cells. Using this system, we find that cohesin promotes chromatin interactions across domain boundaries, contributing to variability in boundary positions. We propose that cohesin dynamically extrudes chromatin past population-defined boundaries in a process we call asymmetric boundary bypass. We find that depleting NIPBL, which is required for cohesin loading and extrusion, decreased inter-domain intermingling, while knocking down WAPL, which opens the cohesin ring to dissociate it from chromatin, increases domain interactions. These knockdowns are accompanied by dysregulation of largely different sets of genes, which were enriched at loop anchors, indicating they are a direct effect of altering levels of cohesin on chromatin. We propose that boundary bypass allows promoters to sample neighboring chromatin for potential regulatory elements, building resiliency into the system. Interestingly, we find this process is regulated by the relative, rather than absolute, amounts of NIPBL and WAPL, as co-depletion of both proteins rescues the effect of either single knockdown on both gene expression and chromatin organization. Our data suggest that gene expression phenotypes associated with reduced cohesin levels could be corrected for by enhancing its processivity on chromosomes, which has wide implications for targeted therapies in cohesinopathies.
Luppino, Jennifer Mary, "Investigating The Role Of Cohesin In Dynamic Genome Folding And Gene Regulation" (2022). Publicly Accessible Penn Dissertations. 4823.