Cohesin collisions maintain ordered nucleosome architecture at boundaries and promoters.

Cohesin is best known for its role in loop extrusion, while nucleosome phasing at regulatory elements is usually attributed to local DNA-bound factors and remodelers. Here we identify a previously unrecognized role for cohesin-mediated extrusion in maintaining local nucleosome architecture at CTCF sites and transcription start sites. Using single-molecule nano-NOMe-seq during SCC1 depletion, cell-cycle progression and Sororin perturbation, we show that CTCF-bound sites contain distinct nucleosome architectures ranging from ordered CTCF-footprinted arrays to footprint-free nucleosomal and inaccessible configurations.. In unperturbed cells, ordered CTCF-footprinted nucleosome arrays were strongest at a boundary-enriched class of CTCF sites without regulatory elements. By contrast, CTCF sites overlapping regulatory elements showed stronger aggregate CTCF ChIP-seq signal despite weaker footprinting and less regular nucleosome phasing, indicating that boundary-like nucleosome architecture is not predicted by CTCF occupancy alone. At TSSs, promoter-proximal CTCF defined a distinct state balance: CTCF-positive promoters were enriched for accessible and footprinted configurations, whereas CTCF-negative promoters showed proportionately fewer footprinted states and were dominated by footprint-free phased arrays. Acute SCC1 depletion disrupted nucleosome organization at CTCF sites without regulatory elements and at promoters with promoter-proximal CTCF, despite retention of aggregate CTCF ChIP-seq signal at CTCF-bound sites. SCC1 depletion also altered nucleosome organization at promoters lacking promoter-proximal CTCF, highlighting that cohesin-dependent nucleosome patterning is not simply a CTCF-barrier phenomenon. Cell-cycle and Sororin analyses further separated extrusion-associated states from Sororin-stabilized post-replicative cohesin, highlighting that nucleosome order depends on effective cohesin-barrier encounters rather than cohesin occupancy alone. Together, these findings establish cohesin collisions as an active local mechanism that patterns nucleosomes at boundaries and promoters.