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) Identification of HDAC1 as a HSF2 protein partner in TAP-TAG/MS analysis in HeLa-S3 cells . After sequential immunoprecipitation of nuclear extracts of HeLa-S3 expressing CTAP-HSF2α and CTAP-HSF2β, using two tags (G-protein and Streptavidin-binding peptide; see Figure S6A), eluates were analyzed by MS. The number of unique peptides from each identified protein and their UniProt Knowledgebase (UniProtKB) codes are indicated. (B) Interaction between ectopically expressed HDAC1-GFP and HSF2-Flag in F3H assays . As in , except that BHK cells were triple transfected with LacI fused to the GFP-binder, HDAC1-GFP, and HSF2-Flag constructs. Chromatin was counterstained using DAPI. All the negative controls are shown in Figure S6E. n=3 independent experiments. Scale Bar, 10 µM. (C) The ectopically expressed exogenous HDAC1 interacts with HSF2. (Upper panel) GFP-Trap co-immunoprecipitation of HDAC1-GFP and HSF2-Myc in transfected HEK 293 cell extracts. (Middle and lower panels) Immunoblot showing total HDAC1 (WB GFP) or HSF2 levels (WB Myc) in inputs, respectively. n=2 independent experiments. Actin was used as a loading control. ns: non-specific band. (D) Overexpression of HDAC1 markedly reduces the acetylation of HSF2 by CBP . HEK 293 cells were transfected by the following constructs: CBP-HA and HSF2-Flag, and HDAC1-Flag, HDAC2-Myc, or HDAC3-Myc and the acetylation status of the HSF2-Flag protein was checked by using an anti-AcK antibody. n=5 independent experiments. Actin was used as a loading control. (E) The stability of HSF2 3KQ is increased upon HS, compared with HSF2 WT or HSF2 3KR . (Upper panel) Representative gel analysis of HSF2 protein decay in a SNAP-TAG pulse-chase experiment. As in , except that cells were submitted to HS at 42°C for the indicated times. (Lower panel) Quantification of the fluorescent signal intensity, relative to the control samples (t0) and normalized to the loading control H3.3. n=7 independent experiments. Standard deviation. * p<0.05; *** p<0.001. SNAP-H3.3 was used as a loading control. (F) Class I HDAC inhibitor VPA prevents the decrease in endogenous HSF2 protein levels induced by HS . N2A cells were pretreated or not with 1 mM valproic acic (VPA) for 3 h and subjected to HS 42°C for 2h30. (Upper panel) Representative immunoblot. (Lower panel) Quantification on the signal intensity normalized to actin levels (n=4 independent experiments). * p<0.05.

Article Snippet: Cell culture, transfections and treatments: murine Neuro2A (N2A, neuroblastoma, DSMZ # ACC 148), Hamster BHK (kindly provided by Dr.Leonhardt H and cultured as described ( Herce et al., 2013 ), human HEK 293T (ATCC®, CRL-11268TM), U2OS (osteosarcoma, ATCC®, HTB-96TM), U2OS-Crispr HSF2 KO (2KO), SH-SY5Y (neuroblastoma, ATCC® CRL-2266TM) and HeLa-S3 cells (kindly provided by Dr. Slimane Ait-Si-Ali and cultured as described (Yahi et al ., 2008)) were grown in DMEM (Lonza Group Ltd.) supplemented with 4,5 g/L glucose and 10% fetal bovine serum (FBS, Life technology) in humidified atmosphere with 5 % CO2 at 37°C.

Techniques: Immunoprecipitation, Expressing, Binding Assay, Transfection, Construct, Western Blot, Control, Over Expression, Pulse Chase, Standard Deviation