UC San Diego

The three-dimensional (3D) organization of the genome played a crucial role in gene regulation, yet the precise mechanisms linking chromatin architecture to transcriptional regulation remained incompletely understood. In mammals, the CCCTC-binding factor (CTCF) functioned as a key architectural protein that mediated transcriptional insulation and facilitated genome organization into topologically associating domains (TADs). In Chapter 1, I contributed to a project in which we used a sensitive insulator reporter in mouse embryonic stem cells to demonstrate that the potency of CTCF-mediated insulation depended on both the number of binding sites in tandem and the sequence context flanking its binding motif. Furthermore, we showed that CTCF binding sites at TAD boundaries were more likely to function as insulators than those outside boundaries, independent of binding strength. Additionally, we observed that disrupting insulation weakened enhancer-promoter interactions and altered chromatin domain architecture, reinforcing the functional link between genome topology and gene regulation.

In Chapter 2, we built on these findings by systematically investigating the role of loop extrusion machinery—comprised of CTCF and cohesin—in maintaining chromatin organization and transcriptional control. We found that acute depletion of cohesin or CTCF disrupted TADs but had gene-specific and context-dependent effects on transcription, with loop extrusion being largely dispensable for transcription in steady-state conditions but critical during cellular transitions. A genome-wide CRISPR screen identified chromatin regulators, including the MORF acetyltransferase complex, that modulated the transcriptional effects of loop extrusion. Notably, inhibition of Kat6b, a MORF complex member, partially rescued insulator defects in Nipbl-deficient cells, revealing a functional interplay between histone modifications and 3D genome organization. Together, these studies provided mechanistic insights into how chromatin topology and epigenetic factors coordinated to regulate gene expression, with implications for developmental disorders linked to genome misfolding.