Extended Data Fig. 4: BRD8 reciprocally regulates transcription of p53 targets at a distinct level from MDM2.

a and b, GFP dropout assays of depleting BRD8 (sgBRD8-1 and sgBRD8-2) in Ctrl or MDM2 overexpression (MDM2OE) A382^WT cells (a), and of depleting MDM2 (sgMDM2-1 and sgMDM2-2) in Ctrl or BRD8 overexpression (BRD8OE) A382^WT cells (b). Plotted is the mean ± s.d. (n = 3 biologically independent samples). c and d, MTT assay of BRD8 depletion using sgBRD8-2 and targeting MDM2 using sgMDM2-2 (c) or the MDM2 inhibitor Nutlin-3a (d) separately or in combination in A382^WT GBM cells. Plotted is the mean ± s.d. (n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. e, Schematic of doxycycline-induced Flag-tagged p53 expression under the control of the tight TRE element (top), with validation of p53 induction by western blot assays (bottom). HSC70 serves as loading control. f, Volcano plot depicting genes that were significantly upregulated and downregulated greater than 1.5-fold after pP53 induction in A382^WT cells. g, GSEA plots of p53 targets and GBM plasticity signatures after p53 induction (P53OE) in A382^WT cells. h, Confocal microscopy in A382^WT cells. Data shown represents three independent results. Scale bar, 25 µm. i, Western blot of BRD8 in empty vector control (Ctrl) and CRISPR-resistant N or C terminal Flag-tagged BRD8 (CR-2-N3F and CR-2-C3F)-expressing A382^WT cells. j, GFP dropout assays of indicated sgRNAs in empty vector control (Ctrl), CR-2-N3F, and CR-2-C3F-expressing A382^WT cells. Plotted is the mean ± s.d. (n = 3 biologically independent samples). k and l, Pie charts showing distribution of high-confidence peaks of BRD8 (k) and p53 (l) in A382^WT cells. m, De novo binding motif of p53 derived from p53 ChIP-seq. E-value (expectancy value) represents the enrichment of the motif around the center of the peak binding regions using the binomial test. n, Venn diagram depicting overlapped genes located near high-confidence peaks of BRD8 and p53. o, Validation of BRD8 and p53 occupancy in a panel of common target loci using ChIP-qPCR. Plotted is data from two biological replicates. p, GSEA plot of p53-ChIPseq-targets signature derived from p53 ChIPseq after p53 induction (P53OE) in A382^WT cells. q, Western blot showing depletion efficiency of p21 in A382^WT cells. r, SA-β-gal assays of Ctrl or p21-deficient A382^WT cells transduced with sgNeg or sgBRD8-2. Data shown represents three independent results. Scale bar, 50 µm. s and t, Pie charts depicting p53 mutation rate in low grade glioma (LGG) from TCGA Firehose Legacy (s), and CDKN1A expression in three subgroups divided based on TP53 status and BRD8 expression level in LGG (t). P values were calculated using two-tailed unpaired Student’s t-tests.