then further increased the AcH3/H3 levels in all regions examined (Fig. S2A, and DEX/DMSO fold induction in Fig. 4A). Conversely, DEX treatment potentiated AcH4/H4 levels in the genomic region from 21815 to 2543, but had no effect in the region from 2160 to +215 (Fig. S2B and Fig. 4B). Next, we wished to determine the histone acetyltransferase (HAT) that acetylates H3 and H4 at the DUSP1 gene. Several coactivators for GR, including GCN5, PCAF, CBP/p300, have been shown to contain histone acetyltransferase (HAT) activity. We reason that if a coactivator is directly involved in glucocor- ticoid-activated DUSP1 gene transcription, it should be present on the DUSP1 promoter upon DEX treatment. To determine which of these factors are directly involved in glucocorticoid-activated DUSP1 gene transcription, we treated A549 cells with DMSO or DEX for 10 minutes, then ChIPed for GCN5, PCAF, CBP and p300 in the areas surrounding the DUSP1 GRE. Of these, p300 was present before treatment with glucocorticoids (relative to ChIP Figure 4. Glucocorticoids increased histone H3 and H4 acetylation of the DUSP1 gene promoter. A A549 cells were treated with DMSO or DEX (0.5 mM) for 10 min, and ChIPs were performed with antibodies against both total histone H3 and multiple acetylated lysines on histone H3 (AcH3). qPCR was performed with the same six primers used for ChIP qPCR in Figure 1B. AcH3 data was normalized to total H3 values and represents the SEM of the fold induction (DEX-treated cells divided by DMSO-treated cells) from at least four experiments. B Identical experiments and calculations as in A, with ChIP antibodies against both total histone H4 and multiple acetylated lysines on histone H4 (AcH4). doi:10.1371/journal.pone.0013754.g004 Glucocorticoid-Induced DUSP1 with IgG control), with levels increasing approximately 16 fold after only 10 minutes of DEX treatment (Fig. 5A). In contrast, CBP was absent without treatment, but was enriched approxi- mately 1.9 fold with 10 minutes of DEX treatment (Fig. 5A). We used RNA interference (RNAi) to reduce the expression of p300 and CBP in A549 cells (Fig. 5B), then tested whether this compromised the ability of DEX to stimulate the expression of the DUSP1 gene (Fig. 5C). Compared to cells transfected with negative control siRNA, p300 knockdown resulted in approxi- mately a two-fold increase of basal expression of DUSP1 gene (Fig. S3). However, the ability of DEX to induce DUSP1 gene expression was decreased approximately 40% (Fig. 5C). These results indicate that under basal conditions, p300 inhibits DUSP1 gene expression, while upon DEX stimulation it contributes to induction of DUSP1 gene expression. CBP RNAi affected neither basal nor DEX-stimulated expression of DUSP1 gene (Fig. S3 and Fig. 5C). p300 is involved in glucocorticoid-induced hyperacetylation of H3 in genomic regions surrounding GRE We used RNAi to decrease the expression of p300 to further investigate its role in DEX-induced histone hyperacetylation. As shown in Fig. 6A, the levels of AcH3/H3 in p300 RNAi cells were similar to those of control RNAi cells before DEX treatment. However, while a 10 minute DEX treatment significantly in- duced AcH3/H3 levels in control RNAi cells, the ability of DEX to increase AcH3/H3 levels was markedly compromised in p300 knockdown cells (Fig. 6A). These results suggest that p300 is involved in DEX-induced histone H3 hyperacetylation surround- ing the DUSP1 GRE, though it does not participate in acetylation of H3 before DEX treatment. Interestingly, AcH4/H4 levels in p300 RNAi cells were similar to those of control RNAi cells before and after 10 min DEX treatment (Fig. 6B). Thus, decreasing p300 expression does not affect DEX-induced histone H4 hyperacetylation. To further confirm the importance of p300-induced histone acetylation in DEX-induced DUSP1 gene transcription, we compared the ability of wild type (WT) p300 and a p300 mutant with reduced histone acetyltransferase (HAT) activity (p300DHAT) to coactivate DEX-induced pDUSP1 reporter gene expression (Fig. 6C). We found that overexpression of p300WT reduced basal reporter activity by 47% (Fig. S4). These data support our p300 RNAi results showing that p300 has a repressive role in basal DUSP1 gene expression. In contrast to p300WT, overexpression of p300DHAT reduced basal reporter activity approximately 24% (Fig. S4). Overexpression of p300WT potentiated the DEX-induced reporter gene expression response from 5.8 fold to 9.5 fold (Fig. 6C), whereas overexpression of p300DHAT increased the DEX response to 7.8 fold (Fig. 6C). These data confirm that in response to glucocorticoids, the mechanism of p300 coactivation of DUSP1 gene transcription involves, but is not limited to, p300 HAT activity. Finally, we performed ChIP-reChIP experiments to determine if p300 and GR are recruited to the same genomic region of DUSP1. GR antibodies were first used to pull down GR- associated DNA fragments. Antibodies against p300 were then used to isolate GR-associated DNA fragments that also associate with p300. Compared to ChIP-reChIP using IgG control antibodies, ChIP-reChIP using GR and then p300 showed significant enrichment of the DUSP1 GRE (23 fold, Fig. 6D). These results suggest that GR and p300 are in the same protein complex recruited to the DUSP1 GRE. PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13754