Fig. 2: HSF2 is acetylated by CBP and EP300 in normal conditions.
From: CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder
a Representative immunoblots of immunoprecipitated HSF2-YFP (IP GFP) from HEK 293 cells transfected with combinations of tagged constructs, YFP-HSF2, HA-CBP, HA-EP300, mock-HA or mock-GFP, showing that ectopically expressed YFP-HSF2 is acetylated by exogenous HA-CBP or HA-EP300 (n = 5). CTA, Trap®-A beads used as a negative control. Inputs, total proteins in input samples. Short and long, different exposure times. b Representative immunoblots of immunoprecipitated HSF2-Myc (IP Myc) from HEK 293 cells transfected with combinations of tagged constructs, HSF2-Myc, HA-CBP, or DNCBP (dominant-negative form of CBP) showing HSF2-Myc protein is acetylated by CBP but not by a dominant-negative form of CBP (n = 2). CTA, Trap®-A beads used as a negative control. c Schematic representation of the eight main acetylated lysine residues of the HSF2 protein. DBD DNA-binding domain, HR-A/B hydrophobic heptad repeat, HR-C leucine-zipper-containing domain controlling oligomerization (TAD, activation domain). The numbers of the amino acids located at domain boundaries are indicated in gray (mouse HSF2) or black (human HSF2, if different). The four (blue box, K82, K128, K135, K197) or three (green box, K128, K135, K197) lysine residues in DBD, and/or HR-A/B were mutated into glutamines (4KQ, 3KQ) or arginines (4KR, 3KR). d Representative immunoblots of immunoprecipitated HSF2-Myc (IP Myc) from HEK 293 cells, co-transfected with EP300-HA and HSF2-Myc wild-type (WT) or HSF2-Myc carrying mutations on the indicated lysine residues showing that concommittant mutations of three or four lysine to arginine (3KR or 4KR) or glutamine (3KQ or 4KQ) residues decrease global HSF2 acetylation levels (n = 3). e Time course elution of HSF2K197 and HSF2K135 peptides detected by reverse phase-ultra-fast liquid chromatography (RP-UFLC) after 0 (black), 1 (red), or 2 (green) hours of acetylation by CBP-Full-HAT, monitored by fluorescence emission at 530 nm. uV arbitrary unit of fluorescence. See Methods for HSF2K197 and HSF2K135 peptide sequences and Supplementary Fig. 2. f Quantification of the in vitro acetylated HSF2 peptides containing K82, K135, and K197 residues detected by RP-UFLC. Source data are provided as a Source Data file.
