Pioneer transcription factors direct tissue-specific cohesin chromatin entry and three-dimensional genome organization.

Cohesin organizes three-dimensional genome architecture by extruding DNA loops, but the mechanisms specifying its chromatin entry sites remain unclear. Here we show that the cohesin loader NIPBL is prepositioned at highly tissue-specific sites, largely distinct from the broadly conserved binding profiles of CTCF and cohesin. Motif and chromatin immunoprecipitation-sequencing analyses identify pioneer transcription factors, particularly FOXA1, as key mediators of the tissue-specific NIPBL recruitment. FOXA1 directs NIPBL to intratopologically associating domain (TAD) regions, enabling symmetric loop extrusion, whereas ETS factors guide NIPBL to TAD boundaries, supporting asymmetric extrusion. FOXA1 depletion disrupts NIPBL binding and impairs intra-TAD loops selectively, whereas NIPBL loss reduces both intra-TAD and boundary-to-boundary interactions. In prostate cancer, the recurrent FOXA1 R219S mutation redirects FOXA1-NIPBL to TAD boundaries, fostering a more insulated and aggressive genome. These findings reveal how pioneer factors orchestrate cohesin loading to shape tissue-specific three-dimensional genome organization and how this mechanism is co-opted in cancer.