RNA polymerase II is required for spatial chromatin reorganization following exit from mitosis

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces marked chromosome condensation, but following reentry into the G₁ phase of the cell cycle, chromosomes reestablish their interphase organization. Here, we tested the role of RNA polymerase II (RNAPII) in this transition using a cell line that allows its auxin-mediated degradation. In situ Hi-C showed that RNAPII is required for both compartment and loop establishment following mitosis. RNAPs often counteract loop extrusion, and in their absence, longer and more prominent loops arose. Evidence from chromatin binding, super-resolution imaging, and in silico modeling allude to these effects being a result of RNAPII-mediated cohesin loading upon G₁ reentry. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.