expression of DUSP1 attenuates p38 MAPK signaling, which disrupts the signal leading to the induction of pro-inflammatory gene expression. Mouse knockout studies support this role. Macrophages isolated from mice lacking DUSP1 gene (Dusp12/2) have prolonged activation of JNK and p38 MAPK, resulting in overexpression of inflammatory genes [13,14]. Similarly, in LPS-induced endotox- emia and collagen-induced arthritis (experimental models of acute or chronic inflammation, respectively), Dusp12/2 mice show exaggerated inflammatory responses [15]. Lastly, in Dusp12/2 macrophages, the ability of glucocorticoids to inhibit the expression of specific pro-inflammatory genes is significantly compromised [15]. Overall, studies of various inflammatory disease models with Dusp12/2 animals support the critical role of DUSP1 in regulating anti-inflammatory responses mediated by glucocorticoids [16,17,18]. Despite the importance of DUSP1 in glucocorticoid-regulated inflammatory responses, how glucocorticoids regulate its expres- sion is poorly understood [6,19]. Glucocorticoids act through the intracellular glucocorticoid receptor (GR) to exert their biological functions. GR is a transcriptional regulator, which, upon binding to cognate ligands, occupies specific genomic regions called glucocorticoid response elements (GREs) [20]. These GREs are typically composite elements, composed of multiple cis-acting elements that work in a combinatorial fashion to direct a complete glucocorticoid response [21,22]. Among these cis-acting elements are often multiple GR binding sites as well as binding sites for other transcriptional regulators. As each of these GREs appear to be different, it is likely that compositionally and/or conforma- tionally distinct transcriptional regulatory complexes assemble at each locus, resulting in distinct mechanisms of transcriptional regulation at each GR target gene. As DUSP1 plays an important role in mediating the anti-inflammatory response of glucocorti- coids, dissecting the mechanisms by which GR activates DUSP1 gene transcription is critical for our fundamental understanding of anti-inflammatory action exhibited by glucocorticoids. In this report, we identified a GRE for the human DUSP1 gene. We also analyzed the effect of glucocorticoids on the chromatin structure and acetylation status of histones surrounding the GRE. Finally, using RNA interference and chromatin immunoprecipitation approaches, we identified a transcriptional cofactor participating in GR-activated DUSP1 gene transcription. Methods Cell Culture A549 cells (type II alveolar lung epithelium cells; ATCC CCL85) [23] were cultured in DMEM (Cellgro) with 5% fetal bovine serum (FBS; J R Scientific). When cells were treated with dexamethasone (DEX, a synthetic glucocorticoid, Sigma) or DMSO (vehicle control), DMEM with 5% charcoal stripped FBS (J R Scientific) was used. ChIP A549 cells were grown to confluence and treated with 0.5 mM DEX or DMSO, then cross-linked by using formaldehyde at a final concentration of 1% at room temperature for 5 minutes. The ChIP protocols were otherwise as previously described [24,25]. ChIP-reChIP A549 cells were grown to confluence (46150 mm plates per treatment) and treated with 0.5 mM DEX or DMSO, then cross- linked by using formaldehyde at a final concentration of 1% at room temperature for 5 minutes. Reactions were quenched with Glucocorticoid-Induced DUSP1 0.125 M glycine for 5 min. Cells were washed with PBS, scraped in cell lysis buffer (50 mM HEPES-KOH at pH 7.4, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100) supplemented with protease inhibitor cocktails (Calbiochem), and incubated for 10 min at 4uC. Crude nuclei were collected by centrifugation at 600 x g for 5 min at 4uC then resuspended in 1.5 ml of ice-cold RIPA buffer (10 mM Tris-HCL at pH 8.0, 1 mM EDTA, 150 mM NaCl, 5% glycerol, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, supplemented with protease inhibitor). Chromatin was fragmented with 18 pulses of 20 sec at 30% power with a Branson Sonifier 250 sonicator at 4uC. To remove insoluble components, samples were centrifuged at 13,000 rpm for 15 min at 4uC, and the supernatant was recovered. For the first round of IP, two aliquots from each treatment were taken. 8 mg of rabbit polyclonal GR (N499) antibody was used to immunoprecipitate GR-bound chromatin or 8 mg of rabbit polyclonal IgG was used as a control for each aliquot, per treatment. The samples rotated overnight at 4uC. 120 ml of 50% protein A/G plus-agarose bead slurry (Santa Cruz Biotechnology) was then added to each immunoprecipitation and rotated for 2 h at 4uC. After incubation, beads were washed with 1 ml of solution per wash as follows: twice with RIPA, twice with RIPA containing 510 mM NaCl, twice with LiCl buffer (20 mM Tris at pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodiumdeox- ycholate), and twice with TE. Between each wash, samples were rotated at room temperature for 5 min, followed by 1 min centrifugation at 5,000 rpm. All washes were supplemented with protease inhibitors. Immmunocomplexes were eluted by adding 75 ml TE with 10 mM DTT and incubated at 37uC for 30 min. The samples were centrifuged at 800 x g for 2 minutes at room temperature, and the supernatant was transferred to a clean 1.5 ml tube. An additional 14 ml of RIPA was added to dilute the sample. For the second round of IP, 8 mg of p300 (rabbit polyclonal, Santa Cruz Biotechnology) was used in all samples and rotated overnight at 4uC. Again, we added 120 ml of 50% protein A/G plus-agarose bead slurry and washed as described, modified only by replacing the final two TE washes with one wash in RIPA buffer. After removing the last wash buffer, 75 ml of proteinase K solution (TE pH 8.0, 0.7% SDS, 200 mg/ml proteinase K) was added to each IP. Reactions were incubated for 3 h at 55uC, then overnight at 65uC to reverse formaldehyde cross-links. DNA was purified using a QIAquick PCR purification kit (Qiagen) and eluted in 60 ml of Elution Buffer (Qiagen). Transfection To transfect A549 cells, we used Lipofectamine 2000 (Invitro- gen) according to the technical manual in the 24-well format. Twenty-four hours post-transfection, cells were treated with either DMSO or 0.5 mM DEX for 18–24 h. Cells were then harvested, and luciferase activity was measured by a Dual-Luciferase Reporter Assay kit (Promega) according to the technical manual. pCI-p300 wild type and pCI-p300DHAT were provided by Mike Stallcup (USC) [26]. Cloning and Site Directed Mutagenesis The human DUSP1 genomic region containing the predicted GR binding site was amplified by PCR using specific primers. The PCR fragment was restriction-digested and subcloned into the pGL4-TATA reporter. A mutagenesis kit (QuikChange) was used to make site-directed mutations per the manufacturer’s instruc- tions (Stratagene). PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13754