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

Cohesin- and CTCF-mediated chromatin loops facilitate enhancer-promoter and promoter-promoter interactions, but their impact on global gene regulation remains debated. Here we show that acute removal of cohesin or CTCF in mouse cells dysregulates hundreds of genes. Cohesin depletion primarily downregulates CBP/p300-dependent putative enhancer targets, whereas CTCF loss both up- and downregulates enhancer targets. Beyond loop anchoring, CTCF directly modulates transcription, acting as an activator or repressor depending on its binding position and orientation at promoters. Mechanistically, when activating, CTCF increases DNA accessibility and promotes RNA polymerase II recruitment; when repressing, it prevents RNA polymerase II binding without altering chromatin accessibility. Promoter-bound CTCF activates housekeeping genes essential for cell proliferation. CTCF's transcriptional activation function-but not its loop anchoring role-is shared with its vertebrate-specific paralog, CTCFL. These findings reconcile architectural and non-architectural roles of cohesin and CTCF, offering a unified model for their functions in enhancer-dependent and enhancer-independent transcription control.