performed in either a 7900 HT, 7500 HT, or StepOne PCR System (Applied Biosystems) and analyzed by using the nn-Ct method as supplied by the manufacturer (Applied Biosystems). Rpl19 expression was used for internal normalization. All primer sequences are presented in Table S1. Results Induction of DUSP1 gene transcription by glucocorticoids in human A549 cells Previous studies have shown that DUSP1 gene expression is induced by glucocorticoids in A549 cells [28]. To confirm that the DUSP1 gene is a direct target of GR and to determine the kinetics of glucocorticoid-induced transcription, we performed nuclear run-on experiments. We isolated nuclei from A549 cells that were treated with the synthetic glucocorticoid dexamethasone (DEX) for 10, 30, and 90 minutes. Nascent transcripts from these nuclei were labeled with biotin-UTP, and then isolated using magnetic beads. After random-primed cDNA was synthesized, qPCR was performed to measure the change of transcript levels using DUSP1 specific primers. As shown in Fig. 1A, transcription of DUSP1 is induced rapidly, increasing approximately two fold after just 10 minutes of DEX treatment (compared to 90 minutes DMSO- treated samples and no treatment), with levels continuing to increase after 30 and 90 minutes (Fig. 1A). These results demonstrate that glucocorticoids rapidly initiate transcription of DUSP1, and that it is likely directly regulated by GR. Identification of GREs in the human DUSP1 gene As our data revealed that glucocorticoids stimulate DUSP1 through transcriptional initiation, we wished to further probe the mechanism by which GR induces DUSP1 transcription. To this end, we used a chromatin immunoprecipitation (ChIP) scanning approach [23] to systematically locate the GR binding regions in the vicinity of the human DUSP1 transcription start site (TSS). A549 cells were treated with DMSO or DEX for 10 minutes, when we first observed transcriptional initiation, then subjected to a standard ChIP procedure [25] of formaldehyde cross-linking of A549 cells, shearing of chromatin to approximately 200–500 bp fragments, and precipitating cross-linked GR:DNA complexes with GR-specific antibody. IgG antibody was used as a negative control for all ChIP experiments. We performed qPCR on the precipitated DNA fragments using six pairs of oligonucleotide primers that amplify approximately 100 bp segments (Fig. 1B), spaced at 300–500 bp intervals. As the average size of the sheared DNA fragments is 300–500 bp, this arrangement of amplified sequences can scan effectively for GR occupancy in the first 2 kb upstream of the DUSP1 TSS. We found that two primer pairs, flanking 21421 to 21333 and 21191 to 21118 respectively, exhibited significant enrichment in GR ChIP (Fig. 1B), but not with IgG alone (data not shown). These results reveal that GR binding localizes between 21421 and 21118 of the TSS. We next tested whether this GR binding region was a mediator of glucocorticoid response. We subcloned the genomic region extending approximately 200–300 bp upstream and downstream of our ChIP fragments (21672 to 2959) into an E4 TATA box containing pGL4 luciferase reporter to test whether the identified GR-binding regions are glucocorticoid-responsive (Fig. 1C). We transfected this reporter plasmid (pDUSP1) along with an expression vector encoding human GR cDNA into A549 cells. After 24 hours, cells were treated with DMSO or DEX for 18 to 20 hours. Cells were then lysed, and firefly luciferase assay was performed. We found that firefly luciferase activity in DEX-treated cell lysates was significantly higher than that of DMSO-treated cell Glucocorticoid-Induced DUSP1 Figure 1. Identification of DNA elements mediating glucocor- ticoid response in the human DUSP1 gene. A A549 cells were untreated, treated with DMSO for 90 min, or treated with DEX (0.5 mM) for 10 min, 30 min, and 90 min as shown. Nuclei were used for nuclear run-on experiments with biotin-UTP to monitor in vitro transcription. Newly synthesized RNA was isolated and used to make cDNA. qPCR was performed to monitor changes in transcription rates using primers specific to Dusp1 and Rpl19 (control). Data represent the standard error mean (SEM) of the fold induction (DMSO- or DEX-treated cells divided by untreated cells) from at least three experiments. B Identification of GR binding regions in human DUSP1 using ChIP scanning across the first 2 kb of the human DUSP1 promoter. A549 cells were treated with DMSO or DEX (0.5 mM) for 10 min, and ChIP was performed with GR- specific antibody as described. DUSP1 gene schematic shows the location of six primers used (black boxes labeled p1-p6) for qPCR analyses. The regions of the DUSP1 gene (relative to the TSS) amplified by these primers are: p1 (21815 to 21717), p2 (21421 to 21333), p3 (21191 to 21118), p4 (2662 to 2543), p5 (2160 to 254), and p6 (+98 to +215). Data represent the SEM of the fold enrichment (DEX-treated cells divided by DMSO-treated cells) from at least three experiments. C DUSP1 GR binding region mediates glucocorticoid response. 21672 to 2959 (relative to the TSS) of the human DUSP1 genomic region was subcloned into pGL4-TATA reporter plasmid to create pDUSP1. pDUSP1 was transfected into A549 cells along with an expression vector for human GR. 24 h post-transfection, cells were treated with DMSO or DEX (0.5 mM) for 18–20 h. Luciferase assay data represents the SEM of the fold induction of luciferase activity (DEX-treated cells divided by DMSO- treated cells) from at least three experiments. doi:10.1371/journal.pone.0013754.g001 PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13754