Fig. 1

WIZ and WAPL differentially regulate cohesin localization. A Overlap of RAD21 ChIP-seq peaks in WT and Wiz^del mESCs. Heatmap shows the log2 ratio of ChIP-seq signal in Wiz^del vs WT. Peaks identified in Wiz^del cells but not WT cells, which displayed a negative log2 ratio of ChIP-seq signal were removed from further analysis. B Bar graph showing overlap of shared (grey) and ectopic (orange) RAD21 peaks in Wiz^del cells with CTCF sites, DNA loop anchors, enhancers, and promoters. C Overlap of RAD21 ChIP-seq peaks in WT and WAPL-AID mESCs. Heatmap shows the log2 ratio of ChIP-seq signal in WAPL-AID / WT. Peaks identified in WAPL-AID cells but not WT cells, which displayed a negative log2 ratio of ChIP-seq signal were removed from further analysis. D Overlap of shared (grey) and ectopic (purple) RAD21 peaks in WAPL-AID cells with CTCF sites, DNA loop anchors, enhancers, and promoters. E Overlap of ectopic RAD21 peaks in Wiz^del cells and ectopic RAD21 peaks WAPL-AID cells. Overlap is defined as within 2 kb. F Histogram showing the distance from an ectopic RAD21 peak in WAPL-AID cells to the nearest ectopic RAD21 peak in Wiz^del cells. Median (red solid line) and mean (red dashed line) of the distribution are indicated. G Heatmaps showing the log2 ratio of RAD21 signal (perturbation / WT) at ectopic RAD21 peaks identified in Wiz^del cells only (left heatmaps, sorted from highest to lowest signal in Wiz^del column), peaks identified as ectopic in both Wiz^del cells and WAPL-AID cells (center heatmaps, sorted from highest to lowest signal in Wiz^del column), and peaks identified only in WAPL-AID cells (right heatmaps, sorted from highest to lowest signal in WAPL-AID column). H Bar graph showing the percent of cells in each indicated cell cycle phase for WT and Wiz^del cells. Data represent n = 5 (WT) and n = 6 (Wiz^del cells) replicates. Asterisks represent p < 0.01(*) and p < 0.001 (**) measured using an unpaired t-test. I Immunoblots for indicators of apoptosis and markers of the DNA damage response in WT and Wiz^del whole cell extracts. Positive controls for DNA damage and apoptosis were generated as described in Methods; GAPDH serves as a loading control for each membrane (top and bottom set of blots). Purple indicates saturated signal. J Immunoblots for indicators of apoptosis in WT and Wiz^del whole cell extracts. GAPDH serves as a loading control. K Quantification of immunoblots in panels I-J and Fig. S1G-I as fold-change relative to WT after normalization to protein loading controls as described in Methods. Data are presented as mean ± SEM, n = 3 biological replicates. Technical replicates, where additional analysis of existing lysates was performed with additional gels and immunoblotting: Gamma H2AX n = 3, Phospho-Chk1 n = 2, Total Chk1 n = 2, Phospho-p53 n = 2, Total p53 n = 1, Cleaved Caspase 3 n = 2, and Caspase 3 n = 2. Paired, two-tailed t-tests were performed to determine the statistical significance of differences in protein levels between WT and Wiz^del cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001