Fig. 3: Bromodomain 4 of PBRM1 is required for recognition of acetylated K382 on p53.

a Schematic depiction of the functional domains of PBRM1 and truncated constructs. WT: wild-type, BAH: bromo-adjacent homology domain, HMG: high-mobility group domain. b, c HEK293T cells were transfected with vectors, Flag-WT PBRM1 and Flag-PBRM1 truncated constructs (b), and each individual or all the PBRM1 bromodomains (BD1–6, c). After immunoprecipitation with Flag-M2 beads, the inputs and eluates were analyzed by immunoblots. d Schematic of key amino acid residues of binding pockets in PBRM1 bromodomains 4 and 5. The critical YN residues are underlined and mutations are red. e–g H1299 PBRM1 KO cells were transfected with Flag-PBRM1 bromodomains 4 and 5 (Flag-BD45) containing mutations (BD4* or BD5*) that abolish acetyl-lysine recognition in each domain (e), Flag-BD45 containing tumor-derived mutations (f) or full-length PBRM1 containing the BD4* mutation (g). Lysates were incubated with biotinylated p53 peptides with lysine acetylation at the indicated sites. The peptides were pulled down with streptavidin beads and the associated proteins were immunoblotted. h Vectors, Myc-p53, HA-p300, and Flag-PBRM1 with or without the BD4* mutation were transfected into HEK293T PBRM1 knockout cells. The lysates were subjected to anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were analyzed in immunoblots. Source data are provided as a Source Data file.