CTCF depletion uncouples the role of enhancer-promoter interactions and higher-order chromatin hubs in gene regulation during cellular differentiation [ChIPmentation]

Enhancers and promoters interact in 3D chromatin structures to regulate gene expression. The mechanisms that drive the formation of these structures and their function during cellular differentiation are incompletely understood. Here, we study the structure-function relationship of the genome in a lymphoid-to-myeloid differentiation system at very high resolution. We demonstrate a close correlation between binding of regulatory proteins, formation of chromatin interactions, and gene expression. By integrating analysis of single-allele topologies and computational modeling, we show that tissue-specific gene loci are organized into chromatin hubs, characterized by cooperative interactions between multiple enhancers, promoters, and CTCF-binding sites. Depletion of CTCF leads to a near-complete loss of these structures, which indicates that CTCF-mediated interactions provide a scaffold for chromatin hub formation. In contrast, the effects of CTCF depletion on gene expression are relatively mild and can be explained by rewired enhancer-promoter interactions. Together, our results demonstrate an instructive role for enhancer-promoter interactions in gene regulation during cellular differentiation, which does not depend on cooperative interactions in chromatin hubs.