Partial depletion of NIPBL or WAPL separate the role of cohesin in chromatin folding from chromosome segregation.

(A) Cartoon depicting the roles of the two opposing cohesin regulators; NIPBL loads cohesin onto chromatin and is required for loop extrusion whereas WAPL opens the ring and removes it. (B) Fluorescent western blot to NIPBL (top band, see arrow) and WAPL in nuclear (nuc) and chromatin-bound (chr) subcellular protein fractionations of RNAi control, NIPBL, or WAPL depleted HCT116 cells. All bands are from the same blot. (C) Mean fold change (%) of NIPBL and WAPL bound to chromatin in each respective knockdown. Each symbol represents a biological replicate, error bars represent standard deviation. (D) Fluorescent western blot to RAD21 in nuclear (nuc) and chromatin-bound (chr) subcellular protein fractionations of RNAi control, NIPBL, or WAPL depleted HCT116 cells. All bands are from the same blot. (E) Mean fold change (%) of RAD21 bound to chromatin in each respective knockdown. Each symbol represents a biological replicate, error bars represent standard deviation. (F) Cell growth measured in 24-hour increments following RNAi or auxin treatment. Each bar represents the mean of 3 biological replicates and error bars represent the standard deviation. (G) Representative immunofluorescence images of mitotic cells stained for α-tubulin (cyan) and phospho-Histone H3 (PH3; red). Top row are HCT116 cells following 72 hour treatment with RNAi against control, NIPBL, or WAPL. Bottom row are HCT116-RAD21-AID cells -/+ auxin for 6 or 24 hours. Scale bar, 5μm. (H) Average percentage of abnormal mitotic cells in RNAi control, NIPBL, or WAPL depleted HCT116 cells and HCT116-RAD21-AID cells -/+ auxin for 6 or 24 hours. Each symbol represents a biological replicate, error bars represent standard deviation. (I) Oligopaint design for three neighboring domains at chr2:217-222Mb. (J) Representative FISH images for three domains at chr2:217-222Mb in RNAi control, NIPBL, and WAPL depleted HCT116 cells. Dashed line represents nuclear edge, scale bar, 5μm (above) or 1μm (below). (K) Cumulative frequency distribution of overlap between the neighboring domains D1 and D2 on chr2 in RNAi control (n = 1,170 chromosomes), NIPBL (n = 1,177 chromosomes), or WAPL (n = 1,136 chromosomes) depleted HCT116 cells. Two-tailed Mann-Whitney test, **** p < 0.0001. (L) Cumulative frequency distribution of overlap between the neighboring domains D2 and D3 on chr2 in RNAi control (n = 1,202 chromosomes), NIPBL (n = 1,284 chromosomes), or WAPL (n = 1,149 chromosomes) depleted HCT116 cells. Two-tailed Mann-Whitney test, **** p < 0.0001. (M) Change in contact frequency across 18 domain pairs in NIPBL, or WAPL depleted HCT116 cells and HCT116-RAD21-AID cells treated with auxin for 6 hours. Each dot represents the median of ≥ 4 biological replicates at each locus.