Figure 3

Sumoylation deficient STAT1 mutant shows enhanced DNA-binding on STAT1 dependent promoters and enhanced histone H4 acetylation at Gbp-1 promoter. (A) The binding of phosphorylated STAT1 WT, E705Q and Y701F to Gbp-1- and Irf-1-oligos. Plasmids were transfected to U3A cells as indicated. Cells were either stimulated or left unstimulated with IFN-γ for 1h and osmotic shock (OS) for 15 minutes to induce STAT1 Tyr701 phosphorylation and STAT1 sumoylation, respectively. Equal amounts of total cell lysates were subjected to oligoprecipitation with Gbp-1- and Irf-1-oligos following immunodetection with pSTAT1 antibody (upper and middle panel, respectively). Tyr701 phosphorylated STAT1 WT and E705Q from the whole cell lysates (0.5% input) were blotted using anti-pSTAT1 antibody (lower panel). (B) The Western blot membranes from 3A were stripped and reprobed with anti-HA antibody to detect DNA bound STAT1 and the expression level of STAT1s in the lysates. (C) Sumoylation defective STAT1 K703R shows enhanced acetylation of histone H4 at the IFN-γ-dependent Gbp-1 promoter. Stably transfected STAT1 WT and STAT1 K703R U3A clones were starved overnight and stimulated as indicated. Immunoprecipitation of cross-linked chromatin was performed with anti-acetyl H4 antibody and anti rabbit IgG as a control antibody. DNA was extracted and analysed by qPCR using primers specific for IFN-γ inducible Gbp-1 promoter sequence. The values from immunoprecipitation were normalized to the total input DNA. The mean qPCR values ± SD from three independent experiments are shown. IB: immunoblot, IP: immunoprecipitation, OP: oligoprecipitation, OS: osmotic shock.