Glucocorticoid-Induced DUSP1 lysates (Fig. 1C). These results indicate that the 21672 to 2959 region of the DUSP1 gene promoter is a functional GRE. To further resolve binding in this region, we looked for direct GR binding sites by searching for a consensus GR binding sequence. Based on a compilation of known GR binding sequences (S. Cooper and K. Yamamoto, unpublished data), we searched for motifs matching the loose consensus sequence XGXACXxxxXGTXCX. We identified a 15 bp sequence that closely matched this consensus GRE in the 21672 to 2959 region of the human DUSP1 promoter (GLS2). The search yielded four more degenerate potential binding sites (GLS 1, 3, 4, 5, Fig. 2A). To gauge the importance of each GLS to GRE activity, we compared the wild type sequence to ones mutated at each of the GLSs at position 5, where GR makes direct contact and is critical for specific binding [29]. We transfected these mutant constructs into A549 cells and found that mutating GLS2 (pDUSP1m2) severely reduced the glucocorticoid response, whereas a mutation at GLS5 (pDUSP1m5) had no effect (Fig. 2B). Mutations at GLS1, GLS3, and GLS4 (pDUSP1m1, pDUSP1m3, pDUSP1m4) showed a modest reduction of glucocorticoid response (Fig. 2B). To verify the contribution of these sites, we performed double mutations of GLS1 and GLS3 (pDUSP1m1m3), GLS1 and GLS4 (pDUSP1m1m4), and GLS3 and GLS4 (pDUSP1m3m4). These double mutations did not further compromise DEX response, nor did the triple mutant (GLS1, GLS3 and GLS4, pDUSP1m1m3m4). In all cases, the response of multiple mutations to DEX was similar to single mutations within GLS1, GLS3, and GLS4 (Fig. 2B). Overall, this mutational analysis indicates that GLS2, which best fits the canonical GRE consensus, is essential for conferring glucocorticoid response, whereas GLSs Figure 2. Mutational analyses of GRE-like sequences (GLS) in the DUSP1 gene. A Five GLSs identified in the 21672 to 2959 region of the human DUSP1 promoter. The locations of five GRE like sequences are: GLS1 (21382 to 21368), GLS2 (21337 to 21323), GLS3 (21254 to 21240), GLS4 (21154 to 21140), GLS5 (21030 to 21018). The position of each nucleotide in each GLS is indicated. The specific point mutations made in each GLS are shown in red. In GLS1-GLS4, position 5 was mutated from C to A. In GLS5, position 5 was mutated from C to G. The consensus GR binding sequence is indicated in blue. B Wild type and mutant reporter plasmids were transfected into A549 cells along with a human GR expression vector. 24 h post-transfection, cells were treated with DMSO or DEX (0.5 mM) for 18–20 h. Luciferase assay data represent the SEM of the fold induction of luciferase activity (DEX-treated cells divided by DMSO-treated cells) from at least three experiments. Results are shown as a percent of wild type DUSP1 response. For mutant plasmids, the mutation at distinct GLSs is indicated (e.g. m1 indicates a mutation at GLS1). doi:10.1371/journal.pone.0013754.g002 PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13754