Figure 2: PTEN represses oncogene expression by controlling H3.3 deposition on chromatin and H3.3-DAXX interaction.

(a) Co-immunoprecipitation analysis of anti-PTEN followed by anti-H3.3 or anti-DAXX immunoblotting were performed using nuclear protein (NCL) from patient-derived glioma spheres. (b) Sequential immunoprecipitation from nuclear extracts of HK281-PTEN GBM-spheres. First IP anti-DAXX and second IP anti-PTEN, and immunoblots anti-DAXX, anti-PTEN and anti-H3.3. (c) Confocal immunofluorescence analysis of endogenous PTEN, H3.3 and DAXX proteins in TS576 GBM-spheres (Pearson’s coefficient in colocalized region equal to 0.4168). Scale bar, 5 μm. (d) Nuclear proteins from MEF pten+/+ and MEF pten−/− (left), and U87 glioma cells (right) transfected with empty vector pEV or PTEN-WT were immunoprecipitated with anti-DAXX followed by immunoblotting with anti-H3.3. (e,f) mRNA expression analysis of CCND1, MYC, FOS and BCL2 by RT–qPCR in PTEN-WT or PTEN-deficient cells (n=3 biological samples with three replicates each, **P<0.001, ***P<0.0001, one-way ANOVA). (g,h) ChIP-qPCR of H3.3 in PTEN-WT or PTEN-deficient cell, using different sets of primers that anneal within 1–2 Kb of the transcription start site. For ChIP assays, bar graphs indicate fold enrichment of H3.3 over input (n=3 biological samples with three replicates each, ***P<0.0001, one-way ANOVA). (i) Cellular fractionation was carried out in MEF cells followed by anti-DAXX and anti-PTEN immunoblotting. DAXX expression was quantified by densitometry analysis. (j,k) Representative immunofluorescence images of endogenous DAXX expression in MEFs (j) or GBM-spheres (k). For j top scale bar is 20 μm and bottom scale bar is 50 μm. For k scale bar is 5 μm. (l) Percentage of DAXX-positive cells in different cellular compartments in Pten-wt and Pten-null MEFs. NCL: nuclear; CYT/NCL: cytosolic and nuclear; CYT: cytosolic; NS: no significant differences. Error bars represent s.e.m. from three different experiments, ***P<0.0001, one-way ANOVA.