PDS5 proteins control genome architecture by limiting the lifetime of cohesin-NIPBL complexes

Cohesin-NIPBL complexes extrude genomic DNA into loops that are constrained by CTCF boundaries. This process has important regulatory functions and weakens the separation between euchromatic and heterochromatic compartments. Cohesin can also bind PDS5 proteins, which do not support loop extrusion but are required for the formation of CTCF boundaries. How PDS5 proteins perform this function is unknown. Here we show, by in vitro single-molecule imaging, that human PDS5 proteins stop loop extrusion by facilitating the dissociation of NIPBL from cohesin. Hi-C experiments suggest that this function is required for the establishment of CTCF boundaries in cells. In silico modeling indicates that PDS5 proteins enable the separation between compartments by limiting cohesin’s velocity and chromatin residence time. The degree of this compartmentalization depends on the frequency with which chromatin is extruded relative to the time it takes for compartments to form. These results identify PDS5 proteins as key regulators of genome organization. • PDS5 proteins stop loop extrusion by facilitating dissociation of NIPBL from cohesin • PDS5 proteins strengthen CTCF boundaries by limiting the lifetime of cohesin-NIPBL • PDS5 proteins regulate compartmentalization • Compartmentalization is governed by polymer relaxation and loop extrusion dynamics PDS5 proteins regulate genome organization by halting cohesin-mediated loop extrusion. Wutz et al. show that PDS5 promotes NIPBL dissociation from cohesin, enabling CTCF boundary formation and proper separation of chromatin compartments. These findings establish PDS5 as a key regulator of three-dimensional genome architecture.