Fig 2: Overexpression of H2A.Z isoforms affects the oncogenic phenotype of PDAC cells.a Expression of the three H2A.Z isoforms analyzed by RT-qPCR in the KD cell lines PZT-1 and PZT-2 transfected with plasmids coding for H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2 alone or together (Z3s). The values are normalized to the non-transfected PZT-2 and PZT-1 KD cell lines. GAPDH was used as house-keeping gene. The means ± SEM of three independent experiments is shown. Expression of CCNB1, E2F1 (b) and SCD1 (c) analyzed by RT-qPCR in the KD clones PZT-1 and PZT-2 transfected with H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2 alone or together as analyzed in (a). n = 3. d SA-ß-galactosidase assay performed in the knockdown clones PZT-1 and PZT-2 overexpressing either H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2 alone or all isoforms together (Z3s). Representative images at 20× are shown. Scale bars = 100 µm. e Quantification of the SA-ß-galactosidase assays (means ± SEM of three independent experiments). f Cytotoxicity assay using gemcitabine in the KD clones PZT-1 and PZT-2 transfected with H2A.Z.1 H2A.Z.2.1, and H2A.Z.2.2 alone or together. Cells were incubated in logarithmic concentrations of gemcitabine for 72 h. The IC50 of three biological replicates in two independent experiments are shown. ANOVA analysis gave p values of: *p = 0.05; **p = 0.01; ***p = 0.001 ****p = 0.0001.

Fig 3: The three H2A.Z isoforms are overexpressed in PDAC cell lines and in PDAC patients.a Histone-enriched protein extracts from PDAC cell lines and the non-cancerous hTERT-HPNE cell line were analyzed by western blot using an anti-H2A.Z antibody. H3 was used as loading control. The graph shows the WB quantification depicting means ± SEM of four biological replicates. Statistical differences between two groups were evaluated using two-way ANOVA. *p value = 0.05. b Normal and PDAC tissues were analyzed by IHC using an anti-H2A.Z antibody (color brown) and an anti-CK7 antibody (pink). Two representative images of PDAC and control samples at 50× are shown. The magnifications at 200× on the right correspond to the squares on the left. Scale bars = 50 µm. c IHC scores of H2A.Z staining in normal and PDAC tissues. Statistical differences between the two groups were evaluated by unpaired two-tailed Student’s t test. *p value = 0.05. d Percentage of H2A.Z-positive cells. e Corrected total cell fluorescence of H2A.Z in different stages of PDAC progression. Statistical differences between two groups were evaluated using two-way ANOVA. **p value = 0.01; ***p value = 0.001. f Kaplan–Meier curves generated using the H2A.Z IHC score showing the average survival of Mexican PDAC patients. Log-Rank analysis gave a p value = 0.01777. g Kaplan–Meier curves generated using expression data from TCGA database. The graph shows the average survival of 90 PDAC patients with low levels of both H2A.Z isoforms (gray) and 53 PDAC patients with high levels of both H2A.Z isoforms (red). Log-Rank analysis gave a p value = 0.027. h Expression of the isoforms H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2 were analyzed by RT-qPCR in the cell lines CAPAN-1, PANC-1 and Mia-PaCa-2. Relative expression values normalized to the non-cancerous cell line hTERT-HPNE (dotted line). The means ± SEM of three biological replicates is shown. Two-way ANOVA analysis gave p values of: **p = 0.0085; ***p = < 0.001 ****p = 0.0001. i Expression analysis of the three H2A.Z isoforms from the 179 PDAC samples available in the TCGA database, and 171 normal tissues available in the GTEx database. Statistical differences were evaluated by unpaired two-tailed Student’s t test.

Fig 4: Quantitative analyses of the associated detection of H2A.Z chromosomal asymmetry with respective H3K27me3 and H3K4me3 chromosomal asymmetry during nonrandom co-segregation. Strain 3C5 (a, c, d) and strain 5B8 (b) murine hair follicle DSCs were evaluated under conditions that either promoted (Xn-free; open circles) or suppressed (Xn-supplemented; closed circles) nonrandom co-segregation. After CD-arrest, binucleated cells were evaluated by quantitative ISIF with anti-H2A.Z antibodies in combination with respective anti-H3K27me3 antibodies (a, b) or anti-H3K4me3 antibodies (d). The percent difference in mean fluorescence intensity (%difference) for H2A.Z detection between sister nuclei in each examined binucleated cell is plotted against the corresponding %difference for either H3K27me3 detection (a, b) or H34Kme3 detection (d). In c, the corresponding distribution of the %difference in DAPI mean fluorescence intensity for the same binucleated cells evaluated in a was use to set thresholds for chromosomal detection patterns. Based on this analysis, a %difference ?30% was used to define a symmetric chromosomal detection pattern; and a %difference >30% defined an asymmetric chromosomal detection pattern. Quadrant 2 data correspond to binucleated cells exhibiting dual asymmetry. P-values indicate the level of statistical confidence, from the two-tailed Fisher's exact test, that the observed reduction in binucleated cells with dual asymmetry under conditions that suppress nonrandom co-segregation is not a chance occurrence

Fig 5: Distinct properties of HA-H2A.Z.1- and MYC-H2A.Z.2-containing nucleosomes. A, Representative image of immunocytochemical analysis of HA-H2A.Z.1 and MYC-H2A.Z.2 expression in unstimulated neurons. Approximately 70% neurons expressed both hypervariants. Scale bar, 20 µm. B, Co-IP assay from nuclear lysate preparation of neurons exogenously expressing a control vector or HA-H2A.Z.1 and MYC-H2A.Z.2. N = 3. C, Representative image of genomic DNA obtained from untreated or MNase-treated nuclei of neurons treated as described above. The band size of ~150 bp indicates that MNase digestion predominantly produced mononucleosomes. N = 3. D, Co-IP assay from single nucleosome preparation of neurons exogenously expressing HA-H2A.Z.1 and MYC-H2A.Z.2. Antigen-antibody complex was resolved by electrophoresis and blotted for indicated histones. The 15-kDa protein ladder band is indicated in all panels. Arrow indicates unique band found only in anti-MYC precipitate. N = 3.