FIG. 3.
Effects of dexamethasone (Dex) on IL-1β-induced histone acetylation. (A) PCAF acetylates specific histone residues. Cells were treated with IL-1β (1 ng/ml) for 1 h before total cellular proteins were extracted. PCAF was immunoprecipitated under stringent IP conditions and incubated with histones and acetyl-CoA (see Materials and Methods). Using antibodies against specific acetylated lysine residues the level of histone acetylation in the IP sample was measured by immunoassay. Histone acetylation at each lysine residue is expressed in units (1 U is equivalent to the absorbance produced by 50 ng of TSA-treated hyperacetylated histone). Results are expressed as mean ± the SEM (n = at least three independent experiments). (B) CBP acetylation of histone H4 lysine residues. Cells were treated with IL-1β (1 ng/ml) for 1 h before total cellular proteins were extracted. CBP was immunoprecipitated under stringent IP conditions and incubated with histones and acetyl-CoA (see Materials and Methods). Using antibodies against specific acetylated lysine residues the level of histone acetylation in the IP sample was measured by immunoassay. (C) CBP-associated proteins acetylate specific lysine residues. Cells were treated with IL-1β (1 ng/ml) for 1 h before total cellular proteins were extracted. CBP was immunoprecipitated under mild IP conditions and incubated with histones and acetyl-CoA (see Materials and Methods). Using antibodies against specific acetylated lysine residues the level of histone acetylation in the IP sample was measured by immunoassay. (D) Dexamethasone inhibits IL-1β-induced histone acetylation in total cell extracts. Cells were pretreated with dexamethasone for 30 min before incubation with IL-1β (1 ng/ml) for 1 h in the presence of 0.05 mCi of [3H]acetate. Histones were isolated and separated by SDS-PAGE, and [3H]acetate incorporated histones were counted and normalized to the protein level. The data represent the means ± the SEM of three independent experiments. ∗∗, P < 0.01. (E) Western blot analysis of dexamethasone actions on IL-1β-stimulated histone acetylation. Cells were incubated with IL-1β (1 ng/ml) for 6 h in the presence of increasing concentrations of dexamethasone. Protein extracts were obtained and examined for pan-acetylated histone H4 lysine residues and for specific K5, K8, K12, and K16 acetylation by Western blotting. Lanes: control (lane 1); IL-1β stimulation (lane 2); IL-1β stimulation in the presence of dexamethasone at 10−12 M (lane 3), 10−10 M (lane 4), 10−8 M (lane 5), and 10−6 M (lane 6); and dexamethasone, 10−6 M alone (lane 7). The results are representative of three independent experiments.