agreement with the DNase I accessibility findings discussed above and are not surprising, as DUSP1 gene expression is not silent in the absence of glucocorticoid treatment. The fact that glucocor- ticoid treatment further increases the acetylation status of histone H3 and H4 indicates that HAT(s) are correlated with the glucocorticoid response on DUSP1 gene expression. We found two HATs, p300 and CBP, whose recruitment to the DUSP1 GRE is increased upon DEX treatment. RNAi knockdown of CBP fails to affect basal or glucocorticoid-stimulated DUSP1 gene expression, both of which are affected by p300 RNAi (Fig. 5C and Fig. S3). Although p300 and CBP frequently exert redundant functions in vitro, their roles in DEX-activated DUSP1 gene transcription may be distinct. Alternatively, the CBP protein remaining after RNAi knockdown may be sufficient to maintain normal effects of CBP on DUSP1 gene expression. While further investigation is necessary to examine these possibilities, our current study better illuminates the role of p300. We found that p300 is present in the DUSP1 gene promoter in the basal state, and glucocorticoid treatment further augments its occupancy. It is possible that p300 is recruited to the promoter region near the DUSP1 GRE through existing DNA-binding transcriptional regulators. Upon glucocorticoid treatment, GR binds to the GRE and further recruits p300 to the GRE. Interestingly, the results from our RNAi experiments indicate that p300 has dual roles on DUSP1 gene expression. RNAi knockdown of p300 increases basal gene expression of DUSP1, but also limits the ability of glucocorticoids to potentiate DUSP1 gene transcrip- tion. Thus, p300 likely acts as a corepressor for basal DUSP1 expression, but as a coactivator for GR-stimulated DUSP1 gene transcription. Other results in this report support this model. Supporting the corepressor role, p300 overexpression reduced the activity of a reporter gene containing the 21672 to 2959 region of DUSP1 gene. This suggests that the element(s) responsible for repressive effects of p300 is located in this genomic region. Notably, it has been reported that p300 can reduce the transcriptional activity of certain DNA-binding transcription factors [48,49], and it will be interesting to identify these element(s) and their associated proteins in future studies. Supporting the coactivator role of p300, overexpression of p300 increased DEX- induced reporter activity from 5.8 to 9.5 fold, whereas a p300 mutant lacking HAT activity only stimulated the reporter activity to 7.8 fold. Together, these results indicate that the HAT activity of p300 plays a role in DEX-induced DUSP1 gene expression. RNAi knockdown of p300 in A549 cells abolished DEX-induced H3 acetylation, which further supports the role of HAT activity in p300 but does not exclude other potential mechanisms of p300 activity. Finally, ChIP-reChIP experiments showed GR and p300 are in the same protein complex that associates with the DUSP1 GRE, which also supports the role of p300 as a GR coactivator. Glucocorticoids are an essential tool in combating inflammatory disorders, however their use is limited by multiple serious side effects. There is a conception that glucocorticoids’ anti-inflamma- tory effects are attributed to GR-regulated transcriptional repression, while the side effects reflect transcriptional activation by GR. However, recent studies clearly demonstrate the importance of glucocorticoid-activated DUSP1 gene expression in mediating the anti-inflammatory effects of glucocorticoids. Thus, an important topic in glucocorticoid pharmacology will be how to dissociate transcriptional activation of GR-induced anti- References Glucocorticoid-Induced DUSP1 inflammatory actions from the undesired side effects. To do this, it is critical to identify the specific components of the mechanisms governing GR-activated transcription of DUSP1 and other genes involved in the induction of unwanted side effects. Supporting Information Figure S1 DNaseI accessibility of a-fetoprotein gene in response to glucocorticoids. An example of a gene whose chromatin structure is not opened up in response to glucocorticoids. a- fetoprotein (AFP) gene schematic shows the location of three primers used (black boxes labeled A1–A3) for qPCR analyses. The regions of the AFP gene (relative to the TSS) amplified by these primers are: A1 (2729 to 2652), A2 (2405 to 2324), and A3 (2183 to 299). DNase I accessibility assay and calculations were performed as described in Figure 3. Data represent the SEM of percentage increase (DNaseI-cut cells divided by uncut cells) from at least three experiments. Found at: doi:10.1371/journal.pone.0013754.s001 (0.29 MB TIF) Figure S2 Basal and glucocorticoid-induced histone H3 and H4 acetylation of the DUSP1 gene promoter. A AcH3/H3 ChIP experiments presented in Figure 4A, with results shown relative to IgG ChIP. B AcH4/H4 ChIP experiments presented in Figure 4B, with results shown relative to IgG ChIP. The data represent the SEM of the fold induction (DEX-treated cells divided by DMSO- treated cells) from at least four experiments. Found at: doi:10.1371/journal.pone.0013754.s002 (0.45 MB TIF) Figure S3 Effects of p300 and CBP siRNA on basal DUSP1 gene expression. RNAi knockdown experiments presented in Figure 5C, showing RNAi against p300 and CBP increased basal (DMSO-treated) DUSP1 gene expression relative to scramble (control). Data represent the SEM of fold induction (p300 or CBP siRNA divided by scramble control) from at least three experiments. Found at: doi:10.1371/journal.pone.0013754.s003 (0.16 MB TIF) Figure S4 Effects of p300WT and p300DHAT overexpression on basal activity of pDUSP1 reporter gene. pDUSP1 reporter assay presented in Figure 6C, showing both pCI-p300WT and pCI-p300DHAT decreased basal (DMSO-treated) reporter activ- ity. Data represent the SEM of fold induction (pCI-p300WT or pCI-p300DHAT divided by pCI control) from five experiments. Found at: doi:10.1371/journal.pone.0013754.s004 (0.15 MB TIF) Table S1 Primers. Found at: doi:10.1371/journal.pone.0013754.s005 (0.04 DOC) MB Acknowledgments We thank Dr. Alex So for reviewing the manuscript and Dr. Mike Stallcup for generously providing pCI-p300 wild type and pCI-p300DHAT constructs. Author Contributions Conceived and designed the experiments: LS CYY MP PZ DS JCW. Performed the experiments: LS JL CYY PZ DS. Analyzed the data: LS JL CYY PZ DS JCW. Contributed reagents/materials/analysis tools: MP. Wrote the paper: LS MP JCW. 1. Barnes PJ (2006) Corticosteroids: the drugs to beat. Eur J Pharmacol 533: 2– 14. 2. Rhen T, Cidlowski JA (2005) Antiinflammatory action of glucocorticoids–new mechanisms for old drugs. N Engl J Med 353: 1711–1723. PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13754