Disentangling the architectural and non-architectural functions of CTCF and cohesin in global gene regulation

Cohesin- and CTCF-mediated chromatin loops shape enhancer-promoter interactions, but their global impact on gene regulation remains unclear. We show that cohesin and CTCF regulate hundreds of genes in mouse cells, though the magnitude of expression changes is modest. Acute loss of cohesin loops mainly downregulates CBP/p300-dependent enhancer targets, while CTCF depletion can both up- and downregulate putative enhancer targets. Interestingly, beyond regulating enhancer-dependent transcription via loop anchoring, CTCF acts as a transcriptional activator or repressor of sense and antisense transcripts, depending on its binding position and orientation in promoters. Mechanistically, promoter-bound CTCF enhances DNA accessibility and RNA polymerase II recruitment, thereby activating housekeeping genes essential for mammalian cell proliferation. CTCF’s transcriptional activation function—but not its loop-anchoring role—is shared with its vertebrate-specific paralog, CTCFL. These findings resolve cohesin and CTCF’s roles in global gene regulation, offering a unified model that integrates their enhancer-dependent and -independent functions in transcription control.