Histone acetylations and histone occupancy at the AR gene in response to hypertonicity.

<p>(A) Histone H4 hyperacetylation at the AR gene in response to hypertonic stress. Cells were induced with hypertonic stress, and ChIP was conducted with antibodies against acetylated histone H4 and histone H4. DNA was analyzed by real-time PCR using AR_INTER (a), AR_ORE (b), AR_PP (c), and AR_EX2 (d) primer pair respectively. The ratio of immunoprecipitated DNA to total input DNA for each of the antibodies was determined. The value for the level of acetylation of histone H4 was divided by H4 occupancy value. Values at time 0 were set to be 1.0. *, p<0.01 by one-way ANOVA. (B) Histone occupancy at the ORE region of WT and OREBP^−/− MEFs. Cells were maintained under isotonicity or treated with hypertonicity for 1 hr. ChIP was conducted with antibody against histone H3. DNA was analyzed by real-time PCR using AR_ORE primer pair. The results are presented as the ratio of immunoprecipitated DNA to total input DNA and normalized to the value of WT MEFs maintained under isotonicity. *, p<0.01 and **, p<0.001 by one-way ANOVA. I, isotonic medium; H, hypertonic medium. Insert, Western blotting analysis of MEFs using anti-OREPB antibodies. (C) MNase assay. (Upper) Nuclei were prepared from WT and OREBP^−/− cells that were induced with hypertonic stress for 0 and 60 min, respectively, and chromatin was subjected to limited digestion with MNase for increasing times. The nucleosome ladder was resolved in agarose gel and visualized by ethidium bromide staining; (Lower). The DNA was subjected to southern blot analysis using a ³²P-labeled probe spanning the ORE (ORE_MNase, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008435#pone-0008435-g001" target="_blank">Fig. 1A</a>). The triangles denote increasing times of MNase digestion. a, mononucleosome; b, dinucleosomes; c, trinucleosomes. (D) Quantitative analysis of AR transcriptions by RT-PCR. WT and OREBP-/- fibroblasts were incubated in isotonic or hypertonic medium for 16 h. Total RNA was prepared. Relative expression level of AR was determined. The expression level of β-actin was used to normalize the AR expression. Results were expressed as fold change relative to the expression level at time 0. Data are the mean ± SEM (n = 3).