1, 3, and 4, which are more divergent, may play accessory roles that could contribute to hormone response under some conditions. GRE containing GLS2 has active response element chromatin marks To further understand the mechanisms underlying glucocorti- coid-regulated DUSP1 gene transcription, we investigated the effects of glucocorticoids on the chromatin structure of the DUSP1 gene. Chromatin architecture plays a critical role in the control of eukaryotic gene transcription in vivo because it determines the accessibility of genomic regulatory sequences to the transcriptional machinery. Based on their sensitivity to cleavage when intact nuclei are exposed to DNA-modifying agents, such as endonucle- ase DNase I, we can analyze the accessibility of the chromatin structure of specific genomic regions. We first tested whether glucocorticoids affect DNase I accessibility within the DUSP1 promoter. Nuclei from A549 cells treated with DMSO or DEX for 10 minutes were subject to cleavage by DNase I for 5 minutes. DNA fragments were then isolated and analyzed with qPCR using primers from the previously described ChIP experiments. We found that the region from 22000 to +1 (TSS) of DUSP1 was already sensitive to DNase I before DEX treatment (compared to DNA not digested by DNase I, Fig. 3A). In contrast, the promoter region of a- fetoprotein, a liver-specific gene not expressed in A549 cells, was sensitive neither to DNase I digestion nor DEX treatment (Fig. S1). With DUSP1, DEX treatment markedly increased DNase I accessibility in all areas examined except 2160 to 264 (Fig. 3A), which was already very sensitive to DNase I prior to DEX treatment (Fig. 3A). These results indicate that DEX treatment and GR binding can further ‘‘open up’’ the chromatin structure of most genomic regions surrounding the DUSP1 promoter. This opening of chromatin within the GRE suggests that nucleosomes have been repositioned or remodeled. Glucocorti- coids have previously been shown to induce chromatin remodel- ing; thus, they must either disrupt a nucleosome assembly or change the position of nucleosomes in the genome (41). To test this, we used MNase to map the position of nucleosomes surrounding the DUSP1 gene. We treated A549 cells with DMSO or DEX for 10 min. Nuclei were isolated, cross-linked with formaldehyde, and digested with MNase. The MNase sensitivity of the chromatin was then analyzed using qPCR with primers designed to anneal to the DNA approximately every 50 bp from 21550 to 21050 of the DUSP1 TSS. Because the nucleosomal regions are less accessible to MNase, we recovered more DNA fragments from these regions. In contrast, the recovery of DNA fragments from linker regions was lower, as most of these fragments were digested by MNase. In untreated cells, three solid nucleosome positions are evident: from 21500 to 21350 (nucleosome 1), 21350 to 21200 (nucleosome 2), and 21200 to 21050 (nucleosome 3) (Fig. 3B). Consistent with our DNase sensitivity assay, addition of DEX appeared to increase the sensitivity to MNase in nucleosomes 1 and 2 (Fig. 3B). These data indicate that a glucocorticoid-dependent remodeling of chromatin structure within the DUSP1 GRE is associated with gene activation. Glucocorticoids increase histone H3 and H4 acetylation of the DUSP1 gene promoter An increase in the acetylation of histones H3 and H4 has been correlated with both gene transcription and active DNA control elements such as response elements and enhancers [30]. We treated A549 cells for 10 minutes with DMSO or DEX, then Glucocorticoid-Induced DUSP1 Figure 3. DUSP1 DNaseI accessibility and nucleosome mapping in response to glucocorticoids. A DNase I accessibility assay was performed with A549 cells treated for 10 minutes with DMSO or DEX (0.5 mM). DNase I digestion lasted 5 min, and fragments were analyzed by qPCR using the same primers (p1-p6) used for ChIP qPCR in Figure 1B. 50 ng DNA was used per well for qPCR. Results are shown as a percent increase of DNase I accessibility relative to DNA not digested by DNase I, where 100% accessibility represents a complete digestion of DNA. Data represent the SEM of percentage increase (DNaseI-cut cells divided by uncut cells) from at least four experiments. B Nucleosome positioning was analyzed with micrococcal nuclease (MNase) digestion for the 21550 to 21050 region of the DUSP1 promoter. A549 cells were treated for 10 minutes with DMSO or DEX (0.5 mM) before digestion with MNase for 10 min. qPCR was performed with primers annealing approximately every 50 bp. Results are shown as the amount of each genomic fragment in the MNase sample, calculated based on a standard curve from genomic DNA titrations with each primer set. C Three potential nucleosomal regions are located at approximately 21500 to 21350 (nucleosome 1), 21350 to 21200 (nucleosome 2), and 21200 to 21050 (nucleosome 3) of the human DUSP1 gene. GLS2 is located between nuclesomes 1 and 2. doi:10.1371/journal.pone.0013754.g003 performed ChIP experiments using antibodies that recognize multiple acetyl lysines in the tails of either histone H3 (AcH3) or H4 (AcH4) to examine whether glucocorticoids affect the overall histone acetylation status of the DUSP1 gene promoter. Total H3 and H4 levels were also monitored by ChIP, as the levels of AcH3 and AcH4 are associated with the overall density of histone in each genomic region. Thus, the ratio of AcH3/H3 or AcH4/H4 accurately reflects the change of histone acetylation. In compar- ison to ChIP done with control IgG antibodies, we found that AcH3/H3 and AcH4/H4 levels were already enriched before DEX treatment in all genomic regions tested (Fig. S2). These results indicate that the 22000 to +1 region of the DUSP1 gene was already acetylated before DEX treatment. DEX treatment PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13754