Chromatin changes at the AR gene in response to hypertonic stress.

<p>(A-C) NIH-3T3 cells were subjected to hypertonic stress, and ChIP was conducted with antibodies against (A) histone H3, (B) histone H2B, and (C) histone H3. DNA was analyzed by real-time PCR using AR_INTER (a), AR_ORE (b), AR_PP (c), and AR_EX2 (d) primer pair respectively as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008435#pone-0008435-g001" target="_blank">Figure 1</a>. *, p<0.01 and **, p<0.001 by one-way ANOVA. (C) Cells were either left untreated (isotonic), or treated with hypertonic medium for 30 min followed by incubation in isotonic medium for 30 min, 6 and 16 h respectively (isotonic recovery). 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 cells maintained under isotonicity. *, p<0.001 by one-way ANOVA. (D) Cells were subjected to hypertonic stress, and ChIP was conducted with antibodies against OREBP. DNA was analyzed by real-time PCR using AR_ORE. The results were normalized to the ratio of immunoprecipitated DNA to total input DNA at time  = 0. (E and F) Cells were treated with hypertonicity for 0 and 8 hr, respectively. ChIP was conducted with antibodies against OREBP (E) or histone H3 (F). DNA was analyzed by real-time PCR using a primer pair specific for the TonEA of the SMIT gene. The results were normalized to the ratio of immunoprecipitated DNA to total input DNA at time  = 0. *, p<0.05 by Student's t-test. For A-G, data are the mean ± SEM (n = 3).</p

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).</p

Evaluation of histone acetylation status at the OREs of AR in response to hypertonicity.

<p>(A) Schematic illustration of the mouse AR genomic DNA showing exon 1 and 2, promoter and upstream region. The region amplified by primer sets AR_INTER, AR_ORE, AR_PP and AR_EX2 are indicated by arrows. Transcriptional start site is indicated by a bold arrow. The DNA probe for southern blotting analysis in the MNase digestion assay (ORE_MNase) is indicated by a grey bar. (B) ChIP assays of AR promoter. ChIP assays were performed with antibodies against acetylated histone H3 or acetylated histone H4 using NIH-3T3 cells that were induced with hypertonicity for 0, 2, 6 and 10 h, respectively. DNA was analyzed by real-time PCR using AR_ORE primer pair. The results are normalized to the ratio of immunoprecipitated DNA to total input DNA at time 0. Data are the mean ± SEM (n = 3). *, p<0.05 by one-way ANOVA test when compared to the value at time 0. (C) Quantitative analysis of AR transcriptions by RT-PCR. NIH-3T3 cells were incubated in hypertonic medium for 0, 3, 6, 8 and 15 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).</p

Association between OREBP, histone acetylation and histone eviction.

<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