Influence of NIPBL Knockdown-Induced Alterations in Cohesin Loop Extrusion on CTCF Loops, Enhancer-Promoter Connectivity, and PRC Domain Contacts During hESC Differentiation into Pancreatic Lineages

Cell differentiation is orchestrated by precise enhancer-promoter interactions that rely on three-dimensional (3D) chromatin organization, influenced by cohesin dynamics and architectural proteins such as CTCF and NIPBL. In this study, we investigated the effects of knocking down NIPBL, a critical cohesin loading factor, in human embryonic stem cells (hESCs) and their differentiated pancreatic lineages. Our results demonstrate that NIPBL depletion leads to a global loss of cohesin occupancy and altered chromatin architecture, particularly affecting enhancer-promoter loops and CTCF loops. Notably, while CTCF anchors maintain cohesin, enhancer-promoter interactions diminish significantly, highlighting the role of cohesive loading in transcriptional regulation. Additionally, we observed disrupted interactions within Polycomb Repressive Complex (PRC) domains and super enhancers, suggesting that NIPBL is crucial for maintaining the structural integrity of these regulatory elements. The study reveals that cohesin loading and loop extrusion processes are vital for establishing and preserving 3D chromatin interactions essential for gene expression during pancreatic differentiation. These findings underscore the intricate relationships between chromatin organization and gene regulation, with implications for understanding developmental processes and diseases associated with chromatin dysregulation.