Journal: bioRxiv

Article Title: CBP/EP300 acetylates and stabilizes the stress-responsive Heat Shock Factor 2, a process compromised in Rubinstein-Taybi syndrome

doi: 10.1101/481457

Figure Lengend Snippet: (A) The ectopically expressed YFP-HSF2 protein is acetylated by exogenous HA-CBP or EP300. Representative immunoblots (n=5 independent experiments). HEK 293 cells were transfected with different combinations of YFP-HSF2, HA-CBP, HA-EP300 constructs, and mock-HA or -GFP constructs. YFP-HSF2 was immunoprecipitated using anti-GFP-trap antibody (IP GFP-Trap) and its acetylation status was determined by WB analyses, using an anti-pan-acetyl-lysine antibody (AcK; left panels). Immunoprecipitated HSF2 was detected using an anti-GFP antibody (WB: GFP). The total amounts of the proteins in the input samples were detected with anti-GFP or anti-HA antibodies (inputs; right panels). Actin, loading control. C TA :Trap®-A beads were used as a negative control (see Experimental Procedures). (B) The HSF2-Myc protein is not acetylated by a dominant-negative form of CBP (DNCBP-HA). HEK 293 cells were transfected as in ( A ), except that HSF2-Myc was used instead of HSF2-YFP, and immunoprecipitation was performed using anti-Myc-trap antibody (IP Myc-Trap). Representative immunoblots (n=2 experiments). C TA : Trap®-A beads were used as a negative control (see Experimental Procedures). HSP90: loading control. (C) Schematic representation of the eight main acetylated lysine residues of the HSF2 protein. Purified mouse Flag-HSF2, co-expressed with HA-EP300, immunoprecipitated and subjected to MS analysis for detection of acetylated lysine residues. The three lysine residues K128, K135, K197, located in the oligomerization domain (HR-A/B), are enlightened in red and K82, located in the DBD, in blue; the other four lysine residues (K209/K210, K395/K401) are indicated in regular black. The DNA-binding domain (DBD, orange); the oligomerization domain (HR-A/B; green) and the domain controlling oligomerization (the leucine-zipper-containing HR-C; green); as well as the N-terminal domain (activation domain TAD; red) are illustrated. The boundaries of each domain are indicated in bold and blue. Bold and blue numbers correspond to the number of the amino acids located at boundaries of the domains of the mouse HSF2 protein, numbered from the +1 (ATG); the equivalent in the human HSF2 protein, if different, are indicated in bold and black. These four (K82, K128, K135, K197) or three lysine residues (K128, K135, K197) were mutated into glutamines (4KQ or 3KQ, respectively) or arginines (4KR or 3KR, respectively; see also Figure S2A). (D) The mutations of three or four lysine (K) residues to arginine (3KR or 4KR) or glutamine residues (3KQ or 4KQ) decrease global HSF2 acetylation levels. HEK 293 cells were co-transfected with EP300-HA and wild-type (WT) or mutated human HSF2-Myc on the indicated lysine residues. After immunoprecipitation of HSF2, using anti-Myc antibody, its acetylation was analysed by WB using an anti-AcK antibody. HSC70, loading control. n=3 independent experiments. (See also Figure S2B,C). (E) In vitro acetylation of HSF2 peptides containing the K135 or K197 residues by recombinant CBP Full-HAT . Time course of reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of the acetylation of HSF2K197 (upper panel) and HSF2K135 peptides (lower panel) by CBP Full-HAT. Aliquots of the reaction were collected at 0 (black), 1 (red) or 2 (green) hours and elution of peptides was monitored by fluorescence emission at 530 nm (excitation: 485 nm, uV: arbitrary unit of fluorescence; see Figure S2D for HSF2K82 peptide). (F) Quantification of the in vitro acetylated HSF2 peptides containing K82, K135, and K197 residues. The AUC (area under the curve) of the acetylated K82, K135 and K197 peptides was quantified and converted in product concentration using a calibrated curve of various known concentrations of peptides. Note that it was not possible to investigate the acetylation of the HSF2 K128 peptide by CBP, because this peptide was repeatedly insoluble at the synthesis steps (Manufacturer’s information; see also Figure S2D-F).

Article Snippet: Cells were lysed in Lysis buffer (50 mM Hepes pH 8, 100 mM NaCl, 5 mM EDTA, Triton X-100 0.5 %, Glycerol 10 %, VPA (1 mM), DTT 1 mM, PMSF 1 mM, proteases inhibitors, phosphatase inhibitors (Roche)) and then, HSF2 was immunoprecipitated using anti-GFP- or anti-Myc-trap antibody, or as a control Trap®-A control (ChromoTek).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Negative Control, Dominant Negative Mutation, Purification, Binding Assay, Activation Assay, In Vitro, Recombinant, Liquid Chromatography, Fluorescence, Concentration Assay