Figure 6. p300 contributes to hyperacetylation of H3 but not H4 in DUSP1 GRE-surrounding region. A A549 cells reverse transfected with p300 or scramble (control) RNAi were treated 48 h post-transfection with DMSO or DEX (0.5 mM) for 10 min. ChIPs were performed for AcH3 and H3 as described in Figure 4A, and B for AcH4 and H4 as described in Figure 4B. RNAi knockdown of p300 decreases DEX-induced hyperacetylation of H3 but not H4. Data represent the Glucocorticoid-Induced DUSP1 SEM of fold change relative to IgG ChIP from at least four experiments. C pCI, pCI-p300 wild type (WT), or pCI-p300DHAT mutant was co- transfected with pDUSP1 along with an expression vector for human GR into A549 cells. Cells were treated with DMSO or DEX as described in Figure 1C. Luciferase assay data represents the SEM of the fold induction of luciferase activity (DEX-treated cells divided by DMSO- treated cells) from at least five experiments. *p300WT caused significant induction from pCI control (student t-test, p,0.0001). ** p300DHAT mutation caused significant reduction from p300WT response (student t-test, p = 0.01). D ChIP-reChIP was performe with GR antibody followed by p300 antibody to isolate fragments bound to both GR and p300. Fragments were analyzed by qPCR with primers corresponding to GLS2. Data represent the SEM of the fold enrichment (DMSO- or DEX-treated cells divided by DMSO-treated IgG-ChIP cells) from at least three experiments. doi:10.1371/journal.pone.0013754.g006 functional interactions are required in GREs of rat PEPCK [34,35,36,37], rat tyrosine aminotransferase (TAT) [38,39] and rat glucose-6-phosphatase [40] genes. Thus, these GLS elements could add another layer of control to the regulatory mechanisms of GR-activated DUSP1 gene expression. Notably, within the GRE identified here, there is a CCAAT enhancer binding protein (C/ EBP) site (21312 and 21305) [41], located adjacent to the GR binding sequence (21337 and 21323). This configuration of the DUSP1 GRE is reminiscent of glucocorticoid-activated rat a1- acid glycoprotein (AGP) in hepatocytes. The rat AGP gene GRE is a composite response element that contains both GR and C/EBPb binding sites – requiring both for synergistic activation [42,43]. Intriguingly, transcriptional synergism is still observed even when one of the two factors lacked either its DNA-binding or transcriptional-activation function. Thus, a direct protein-protein interaction between these two distinct transcription factors is probably required to regulate the rat AGP gene [42,43]. Supporting this mechanism, overexpression of a GR mutant defective in DNA binding still potentiates the glucocorticoid response in DUSP1 gene expression [41]. The functional interaction between GR and C/EBPb has also been reported in the regulation of other glucocorticoid target genes, such as phosphoenolpyruvate carboxykinase (PEPCK) and b-casein [44,45]. The role of C/EBP binding site in glucocorticoid- activated DUSP1 gene transcription merits future studies. Chromatin structure plays a critical role in the regulation of gene transcription. Based on our findings from DNase I accessibility experiments, the DUSP1 genomic region up to ,2 kb upstream of the TSS is already opened up prior to treatment with glucocorticoids (Fig. 3A). Glucocorticoid treatment further increases this region’s DNase I accessibility except in the area from 2160 to 264. The results from MNase experiments indicate that nucleosomes 1 and 2 are likely remodeled, increasing the accessibility of DNase I to chromatin in this region. Previous studies have shown that transcriptional activation of several glucocorticoid target genes requires the SWI/SNF chromatin remodeling complex [46,47]. When we knocked down the expression of Brm, an ATPase subunit of SWI/SNF, in A549 cells, the ability of glucocorticoids to stimulate several of their target genes was reduced – intriguingly, DUSP1 escapes this requirement for Brm (data not shown). This suggests that Brg1, another ATPase component of the SWI/SNF complex, may compensate for the loss of Brm in A549 cells. Alternatively, it is possible that chromatin remodeling complexes other than SWI/ SNF are involved in glucocorticoid-regulated DUSP1 gene transcription. We are currently investigating these possibilities. The analyses of histone acetylation status in the DUSP1 gene promoter showed that the genomic region up to 2 kb upstream of the TSS is already acetylated in its basal state. These results are in PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13754