Extended Data Fig. 9: The bromodomain of BRD8 sequesters H2AZ to restrain chromatin accessibility of intrinsic p53 to its targets.

a, Design of Proximity Ligation Assay (PLA) for detecting in situ proximal interactions between BRD8 and H2AZ. b, PLA showing interactions between BRD8 and H2AZ or H2AZac (acetylated H2AZ), using mouse Flag-M2 antibody against Flag-tagged BRD8 and rabbit antibody against H2AZ or H2AZac. Data shown represents three independent results. c, Immunoprecipitation followed by western blotting to detect interactions between H2AZ or H2AZac, and Flag-tagged wild type BRD8 (BRD8-C3F) or bromodomain deleted BRD8 (BDdel-C3F) in A382^WT cells, respectively. Data shown represents two independent results. d, Construction (left) and expression (right) of recombinant GST fusion BRD8 proteins. e and f, Western blot and Coomassie blue staining of purified recombinant GST fusion BRD8 proteins. g, In vitro pulldowns and western blots showing interaction of the BD of BRD8 (Upper) and full length of BRD8 (Lower) with H2AZ and H2AZac. Data shown represents three independent results. h, Gene tracks depicting occupancy of BRD8, p53, H2AZ, H3K27ac, H3K4me3 and chromatin accessibility by ATAC-seq in control (sgNeg), BRD8- or H2AZ-depleted A383^WT GBM cells. i–k, RT-qPCR assays validating enhanced chromatin accessibility at the CDKN1A promoter region after depletion of BRD8 (sgBRD8) and H2AZ (sgH2AZ) compared with control (sgNeg) in A382^WT GBM (i) and two patient-derived primary GBM cells MN03^WT (j) and MN06^WT(k). Plotted is the mean ± s.d. (n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. l and m, Western blot showing expression of indicated proteins at different timepoints following BRD8 knockdown (l) and multiple modifications of p53 at day 5 (m) in A382^WT GBM cells. Data shown represents three independent results.