CTCF binding landscape is shaped by the epigenetic state of the N-terminal nucleosome in relation to CTCF motif orientation.

CTCF binding sites serve as anchors for the 3D chromatin architecture in vertebrates. The functionality of these anchors is influenced by the residence time of CTCF on chromatin, which is determined by its binding affinity and its interactions with nucleosomes and other chromatin-associated factors. In this study, we demonstrate that CTCF occupancy is driven by CTCF motifs, strategically positioned at the entry sides of a well-positioned nucleosome, such that, upon binding, the N-terminus of CTCF is oriented towards the nucleosome. We refer to this nucleosome as the CTCF priming nucleosome (CPN). Our analyses suggest that CTCF can more easily displace the CPN if the nucleosome is not marked by CpG methylation or repressive histone modifications. Under these permissive conditions, the N-terminus of CTCF recruits SMARCA5 to reposition the CPN downstream, thereby creating nucleosome-free regions that enhance CTCF occupancy and cohesin stalling. In contrast, when CPNs carry repressive epigenetic marks, CTCF binding is weaker, with no nucleosome displacement or chromatin opening, and cohesin is retained less effectively at CTCF binding sites. We propose that the epigenetic status of CPNs shapes cell-specific CTCF binding patterns, ensuring the maintenance of chromatin architecture throughout the cell cycle.