Fig 2: Investigation of the role of DNA damage, WAPL, and CAPD3 in RB-mediated dispersion. RB activation increases chromatin resistance to MNase digestion. (A) PLA images for WT and ?CDK-RB after 24 and 48 h of DOX induction. Just 24 h of ?CDK-RB expression leads to higher RB-TOPIIß PLA interaction foci, when compared with WT cells. A similar increase was observed after 48 h of ?CDK-RB induction. Images show G1 cells. (B) Average numbers of RB-TOPIIß PLA foci per G1 cell were higher for ?CDK-RB after 24 and 48 h of DOX induction, when compared with WT. Negative controls (using only RB or TOPIIß antibody for PLA assay) showed low average numbers of PLA foci in both WT and ?CDK-RB cells, implying that there was very little background signal. Numbers of foci quantified for each sample (n) are as follows: n = 95 (WT, RB-TOPIIß 24 h), 214 (?CDK-RB, RB-TOPIIß 24 h), 29 (WT, RB 24 h), 83 (?CDK-RB, RB 24 h), 70 (WT, TOPIIß 24 h), 74 (?CDK-RB, TOPIIß 24 h), 80 (WT, RB-TOPIIß 48 h), 162 (?CDK-RB, RB-TOPIIß 48 h), 69 (WT, RB 48 h), 110 (?CDK-RB, RB 48 h), 70 (WT, TOPIIß 48 h), 130 (?CDK-RB, TOPIIß 48 h). Error bars show SEM. Nonparametric two-tailed Mann–Whitney U test was performed for pairs of samples indicated on the graph, and asterisks denote P values. (C) Mean skeleton dot lengths (chromosome 7 a-satellite probe) after WT and ?CDK-RB RPE cells were treated with IR or camptothecin (CPT) to induce DNA damage. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): n = 21, 28, 25, 26, 21, 24. Error bars are SEM. One-way ANOVA and Holm–Sidak multiple comparison tests were performed, and asterisks denote P values. (D) Western blot analysis of cells in C show that IR and CPT induced H2A.X Ser139 phosphorylation in both WT and ?CDK-RB–expressing cells. Note that IR- or CPT-induced DNA damage does not affect the ?CDK-RB–induced chromatin dispersion. MW, molecular weight. (E, G, and I) Mean skeleton dot lengths (chromosome 7 a-satellite probe) after WT and ?CDK-RB RPE cells were treated with control and CAPD3 siRNAs (E), control and WAPL siRNAs (G), or DMSO and DRB (I). Note that none of these treatments significantly modified dispersion levels in either WT or ?CDK-RB–expressing RPE cells. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): E, n = 40, 41, 36, 39; G, n = 40, 47, 36, 34; I, n = 29, 51, 58, 53. Error bars are SEM. For E, G, and I, nonparametric two-tailed Mann–Whitney U test was performed for pairs of samples indicated on the graphs, and asterisks denote P values. ns, P > 0.05; *, P = 0.05; ****, P = 0.0001. (F and H) Western blots for WT and ?CDK-RB cells treated with control and WAPL siRNAs (F) and control and CAPD3 siRNAs (H). (J) MNase digestion profile of WT and ?CDK-RB samples. Left: MNase digestion profile for WT and ?CDK-RB nuclei treated with 40 gel units of MNase per reaction. Right: MNase digestion profile for WT and ?CDK-RB cells treated with 1,500 units of MNase per reaction. All other conditions were the same. Note: WT samples resolve into a ladder-like typical MNase digestion pattern as early as 30 s at higher MNase concentrations and 1 min at lower concentrations. The ?CDK-RB sample does not digest into a ladder-like pattern at 30 s at higher MNase concentrations and at even 1 min at lower concentrations (red arrows). A clear MNase digestion ladder is seen at 3 min (left gel) and 2 min (right gel) in the ?CDK-RB samples, much later than the laddering in WT cells. 1 µg DNA was loaded per well. DNA samples were run on 0.8% agarose gel. Dashed lines indicate the cutoffs for defining the categories compact, diffused, and dispersed for chromosome 7 a-satellite. Source data are available for this figure: SourceData FS4.

Fig 3: Expression correlation relationship of menin/MAPs genes and OXPHOS/glycolytic genes. (A) Workflow of the in silico correlation analysis of gene expression in The Cancer Genome Atlas (TCGA) breast cancer cohort. (B) Heatmaps of the expression correlation between MEN1/selected 4 MAP genes and OXPHOS genes (upper) or glycolytic genes (lower) in normal (N) and tumor (T) samples. The genes are arranged from the highest to the lowest according to gene expression correlation coefficients of MEN1–OXPHOS genes or MEN1–glycolytic genes in breast tumors. (C) and (D) Scatter plots and linear regression analyses of MEN1/selected MAPs expression and mean expression of OXPHOS genes (C) or glycolytic genes (D) in normal and tumor samples. (E) Violin plots (lower panel) shows the average expressions of the genes of OXPHOS complexes I-V and glycolysis in the samples of each of the corresponding 4 groups are shown as violin plots. Based on the median values (where ≥median is “high” and KMT2A, MEN12, WAPL, and GATA3) and MEN1, the TCGA breast tumor samples were divided into 4 groups—1: high-low, 2: high-high, 3: low-low, and 4: low-high (upper panel). Letters on top of the violin plot denote statistical significance, where two groups with different letters are significantly different (p < 0.05) and those with the same letter are not.

Fig 4: Bioenergetic dynamics are regulated by menin and MAPs in T47D and MCF-7 cells. (A) and (B) Glycolytic and OXPHOS ATP productions in T47D (A) or MCF-7 (B) cells infected with vehicle, shMEN1, shKMT2A, shMED12, shWAPL, or shGATA3 lentivirus. Statistics represented the difference of glycolytic or OXPHOS ATP production between shRNA knockdown and vehicle controls. (C) and (D) Bar charts representing mitochondrial functions in the single knockdown of MEN1, KMT2A, MED12, WAPL, or GATA3 and their vehicle control in T47D (C) or MCF-7 (D) cells. (E) Schematic summary of mitochondrial dynamics affected by the knockdown of MEN1 or MAPs. (F,G) Bar charts represented the glycolytic functions in T47D (F) or MCF-7 (G) cells subject to gene knockdown by shMEN1, shKMT2A, shMED12, shWAPL, or shGATA3 lentivirus. (H) Schematic summary of glycolytic functions affected by the knockdown of MEN1 or MAPs. Data are presented as mean ± S.D. (n = 15–20 technical-replicate wells). Statistical significance was performed by an unpaired two-tailed Student’s t-test between treated groups and corresponding controls. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Fig 5: Working model.Sororin suppresses WAPL and maintains sister chromatid cohesion in Sororin cohesin cells. In contrast, in PD-L1 overexpressed cells, PD-L1 compensates for the loss of Sororin and competes with WAPL binding to PDS5B, thereby regulating sister chromatid cohesion.