CTCF’s loop-independent functions prevail over chromatin looping in the acute degradation system

CTCF is widely known for its critical role in mediating chromatin loops across the genome to regulate gene expression, particularly through promoting or insulating enhancer-promoter (EP) interactions. Surprisingly, acute CTCF depletion has little effect on these interactions and gene expression, leaving much of its regulatory mechanism unclear. Employing high-resolution chromatin interaction assays in an acute CTCF degradation model, we uncover a loop-independent mechanism where promoter-bound CTCF enhances gene expression by increasing chromatin accessibility through enhancing CHD4/CHD8 binding. In contrast, only a small subset of differentially expressed genes are regulated via CTCF loops, which exert a limited impact on EP or promoter-promoter (PP) interactions. Effective enhancement of EP/PP interactions requires CTCF binding at one or both anchors of these interactions and forming loops in the same orientation. And insulation on EP/PP interactions by CTCF necessitates relatively strong CTCF loop strength. Moreover, nearly half of EP/PP interactions are surrounded by multiple CTCF loops with either promoting or insulating functions, which can counterbalance each other and diminish the overall impact of CTCF loops on EP/PP interactions. Together, our findings underscore a dual role for CTCF as a loop-dependent and -independent transcription regulator, with its loop-independent functions playing a more dominant role in the acute degradation system.