9 research outputs found
Inducible Nucleosome Depletion at OREBP-Binding-Sites by Hypertonic Stress
Background: Osmotic Response Element-Binding Protein (OREBP), also known as TonEBP or NFAT5, is a unique transcription factor. It is hitherto the only known mammalian transcription factor that regulates hypertonic stress-induced gene transcription. In addition, unlike other monomeric members of the NFAT family, OREBP exists as a homodimer and it is the only transcription factor known to bind naked DNA targets by complete encirclement in vitro. Nevertheless, how OREBP interacts with target DNA, also known as ORE/TonE, and how it elicits gene transcription in vivo, remains unknown. Methodology: Using hypertonic induction of the aldose reductase (AR) gene activation as a model, we showed that OREs contained dynamic nucleosomes. Hypertonic stress induced a rapid and reversible loss of nucleosome(s) around the OREs. The loss of nucleosome(s) was found to be initiated by an OREBP-independent mechanism, but was significantly potentiated in the presence of OREBP. Furthermore, hypertonic induction of AR gene was associated with an OREBPdependent hyperacetylation of histones that spanned the 59 upstream sequences and at least some exons of the gene. Nevertheless, nucleosome loss was not regulated by the acetylation status of histone. Significance: Our findings offer novel insights into the mechanism of OREBP-dependent transcriptional regulation and provide a basis for understanding how histone eviction and transcription factor recruitment are coupled. Β© 2009 Tong et al.published_or_final_versio
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<sub>INTER</sub> (a), AR<sub>ORE</sub> (b), AR<sub>PP</sub> (c), and AR<sub>EX2</sub> (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<sub>ORE</sub> 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<sub>ORE</sub>. The results were normalized to the ratio of immunoprecipitated DNA to total input DNA at time β=β0. (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 β=β0. *, p<0.05 by Student's t-test. For A-G, data are the mean Β± SEM (nβ=β3).</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<sub>INTER</sub> (a), AR<sub>ORE</sub> (b), AR<sub>PP</sub> (c), and AR<sub>EX2</sub> (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<sup>β/β</sup> 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<sub>ORE</sub> 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<sup>β/β</sup> 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 <sup>32</sup>P-labeled probe spanning the ORE (ORE<sub>MNase</sub>, <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 Ξ²-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 Β± SEM (nβ=β3).</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<sub>INTER</sub>, AR<sub>ORE</sub>, AR<sub>PP</sub> and AR<sub>EX2</sub> 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<sub>MNase</sub>) 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<sub>ORE</sub> primer pair. The results are normalized to the ratio of immunoprecipitated DNA to total input DNA at time 0. Data are the mean Β± SEM (nβ=β3). *, 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 Ξ²-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 Β± SEM (nβ=β3).</p
Association between OREBP, histone acetylation and histone eviction.
<p>(A) Role of OREBP in histone H4 acetylation. WT and OREBP<sup>β/β</sup> 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<sub>ORE</sub> 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 Β΅g/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<sub>ORE</sub> and AR<sub>INTER</sub> 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<sub>ORE</sub> 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 Β΅g/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 Β± SEM (nβ=β3). *, p<0.001 by one-way ANOVA.</p