Association between OREBP, histone acetylation and histone eviction.

Abstract

<p>(A) Role of OREBP in histone H4 acetylation. WT and OREBP^−/− MEFs were maintained under isotonicity or treated with hypertonic medium for 1 h. ChIP was conducted with antibody against acetylated histone H4 and histone H4, respectively. DNA was analyzed by real-time PCR using AR_ORE primer pair. The ratio of immunoprecipitated DNA to total input DNA for each of the antibodies was determined. The value for acetylation histone H4 level was divided by H4 occupancy value. The value of WT MEFs under isotonic conditions was set to be 1.0. *, p<0.001 by one-way ANOVA. I, isotonic medium; H, hypertonic medium. (B-E) Effect of TSA treatment on histone eviction at ORE and AR expression. WT MEFs were treated with DMSO or TSA (1 µg/ml) for 2 hr. ChIP was conducted with antibody against (B) acetylated histone H4, (C) histone H3, and (D) OREBP. In (B), DNA was analyzed by real-time PCR using AR_ORE and AR_INTER primer pairs. The ratio of immunoprecipitated DNA to total input DNA for each of the antibodies was determined. Values of DMSO treatment were set to be 1.0. *, p<0.05 by one-way ANOVA. In (C) and (D), DNA was analyzed by real-time PCR using AR_ORE primer pair. The ratio of immunoprecipitated DNA to total input DNA for each of the antibodies was determined. The results were normalized to the ratio of immunoprecipitated DNA to total input DNA of DMSO treatment. *, p<0.01 by one-way ANOVA. (E) Quantitative analysis of AR transcriptions by RT-PCR. Cells were treated with DMSO or TSA (1 µg/ml) for 8 h. Total RNA was prepared and relative AR mRNA level was determined. The result was expressed as fold change relative to the expression level of WT MEFs treated with DMSO. Data are the mean ± SEM (n = 3). *, p<0.001 by one-way ANOVA.</p