Fig 2: PDS5A and PDS5B localize to shared sites across the mESC genome. A Genome browser tracks of PDS5A, PDS5B, RAD21, and CTCF ChIP-seq signal near the promoter of Sox2 in WT mESCs (Z-score normalized). B Average signal plots of PDS5A and PDS5B ChIP-seq signal at all PDS5A peaks and all PDS5B peaks (Z-score normalized). C Number of PDS5A and PDS5B peaks overlapping relevant functional elements across the genome: CTCF sites, enhancers, promoters, or other (none of the above). D Clustered heatmaps of PDS5A, PDS5B, RAD21, and CTCF ChIP-seq signal at a union list of PDS5A and PDS5B peaks, ordered by RAD21 signal (k-means = 3) (Z-score normalized). E Western blot analysis following co-immunoprecipitation of IgG (negative control), PDS5A, PDS5B, and RAD21 in WT nuclear lysates, under low and high stringency conditions

Fig 3: STAG2 residues predicted to be putative interacting sites for protein–protein interfaces based on consensus classifier. a Residue colors based on Spider score47 from white to blue, with residues closer to blue with the greater the likelihood to be involved in mediating a protein–protein interaction. SCC1 is shown as cartoon in red. b Non-covalent interactions made by WT residue. Ser327 forms a hydrogen bond with Asn325 that is substituted to a donor-pi interaction with Tyr328 in the mutant model. SCC1 is shown as cartoon in blue. The mutant residue is shown with dark gray carbons

Fig 4: PDS5 proteins localize to the same genomic sites as STAG proteins. A Clustered heatmaps of PDS5A, PDS5B, STAG1, STAG2, and RAD21 ChIP-seq signal at a union list of PDS5A, PDS5B, STAG1, and STAG2 peaks, ordered by RAD21 signal (k-means = 2) (Z-score normalized). B Western blot analysis following co-immunoprecipitation of IgG (negative control), PDS5A, PDS5B, and RAD21 in WT nuclear lysates, under low and high stringency conditions. Control blots for this experiment are in Fig. 1E. C Clustered heatmap of log2 fold change in expression for a combined list of DEGs in Pds5a-/- siGLO, Pds5a-/- siStag1, Pds5a-/- siStag2, and Pds5a-/- siPds5b mESCs relative to WT siGLO mESCs. D Violin plot of log2 fold change in expression for all DEGs, those within Super-enhancer Domains, and those within Polycomb Domains for Pds5a-/- siGLO, Pds5a-/- siStag1, Pds5a-/- siStag2, and Pds5a-/- siPds5b mESCs relative to WT siGLO mESCs. Significance was determined using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Asterisks indicate significant differences between groups (* p < 0.05, **** p < 0.0001). E Bar graphs of log2 fold change in expression of pluripotency genes (Pou5f1, Sox2, Nanog), ectodermal lineage genes (Pax6 and Nestin), and endodermal lineage genes (Gata6 and Sox17) in Pds5a-/- siGLO, Pds5a-/- siStag1, Pds5a-/- siStag2, and Pds5a-/- siPds5b mESCs. Asterisks indicate significant differences from WT siGLO mESCs determined using DESeq2 (padj < 0.01)

Fig 5: Loss of Pds5 does not alter cohesin loading on chromatin. (A) Levels of Rad21 bound to chromatin after depletion of Pds5A (top) or Pds5B (bottom). Chromatin Fractionation of Pds5 depleted U-2 OS cells. Fractions separated included whole cell, chromatin bound and soluble cytoplasmic proteins. Samples were probed with Pds5A, Pds5B, Rad21, Tubulin and H3 antibodies. (B) Relative intensity of Rad21 bound to chromatin after immunofluorescence staining of Pds5A or Pds5B depleted cells. Cells were pre-extracted to remove soluble proteins, leaving chromatin bound proteins intact. Quantification of the Rad21 relative intensity signal. At least 100 cells were scored for each data set. n = 2. Data are represented as mean ± SEM. Statistics: Mann–Whitney; ****P < 0.0001.