Cohesin extrudes chromatin loop unidirectionally through two modes of mechanisms in human cells

Cohesin is crucial for establishing 3D genome organization and functions. However, the in vivo mechanism of cohesin-mediated extrusion remains unsettled. Here, we investigated extrusion directionality using integrated genome mapping coupled with perturbation in human cells. By defining cohesin loading at NIPBL binding loci and anchoring at CTCF sites, we systematically characterized the aggregated and single-molecule trajectories of extrusion “stripes” and contact “dots” from and between individual cohesin loading and anchoring points. Our analyses delineated two dynamic modes of in vivo mechanism in which NIPBL/cohesin initiates extrusion at its loading sites in a ‘one-sided’ manner; after encountering CTCF, CTCF/cohesin switches its extrusion direction backward and robustly extrude DNA unidirectionally until completing the convergent CTCF loop. Surprisingly, depleting CTCF resulted substantial increase of NIPBL/cohesin-mediated extrusion activity, which is further validated by in silico perturbation. These findings reveal novel mechanistic insights into how cohesin and CTCF orchestrate cohesin-mediated loop extrusion in vivo.