Figure 5. p300 is a transcriptional coactivator for GR-regulated DUSP1 gene transcription. A Glucocorticoid treatment increased the level of p300 and CBP at the DUSP1 GLS2 region. A549 cells were treated with DMSO or DEX (0.5 mM) for 10 min, and ChIP was performed with antibodies against p300, CBP and IgG (control). Data is shown from qPCR analyses with primers specific to the DUSP1 GLS2. B RNAi knockdown causes substantial reduction of p300 protein and CBP protein. A549 cells reverse transfected with p300, CBP or scramble siRNA were harvested 48 h post-transfection, then probed by Western blot using an antibody against p300 or CBP, with b-actin as a loading control. C RNAi against p300 but not CBP decreased DEX-induced DUSP1 gene expression. p300, CBP or scramble (control) siRNA was transfected into A549 cells. 48 h post-transfection, cells were treated with DMSO or DEX (0.5 mM) for 5 h. Total RNA was isolated and converted to cDNA. qPCR was used to monitor the expression of DUSP1 gene. The expression of Rpl19 gene was used as an internal control. Data represents the SEM of DEX-induced DUSP1 gene expression (DEX- treated cells divided by DMSO-treated cells), as a percent of the control siRNA response from at least three experiments. doi:10.1371/journal.pone.0013754.g005 Glucocorticoid-Induced DUSP1 Discussion Multiple mechanisms contribute to the anti-inflammatory effects of glucocorticoids. One of them is the ability of glucocorticoids to induce the transcription of DUSP1. Thus, understanding the regulatory mechanism of glucocorticoid-acti- vated DUSP1 gene transcription is vital for future therapeutic interventions against inflammatory diseases. In this study, we systematically investigated the mechanisms of GR-regulated DUSP1 gene transcription. First, we determined that GR regulates the expression of DUSP1 through transcriptional initiation (Fig. 1A); then, we identified the GRE at the human DUSP1 promoter that is responsible for this regulation (Fig. 1B, 1C). The GRE appeared to be in organized nucleosomal structures, within which GR is bound at a consensus GR binding site (GLS2) at the GRE (Fig. 2 and Fig. 3). We also showed that the induction of DUSP1 gene transcription by glucocorticoids involves the opening up of chromatin structure in the DUSP1 promoter region corresponding with changes in the nucleosomal status (Fig. 3). While histone H3 and H4 of the DUSP1 promoter are already acetylated in the basal state, glucocorticoids further increased acetylated histone H3 and H4 levels (Fig. S2 and Fig. 4). Finally, we showed that p300 acts as a transcriptional coactivator for GR in the induction of DUSP1 gene transcription (Fig. 5 and Fig. 6). GR and p300 appear to be in the same protein complex recruited to the DUSP1 GRE (Fig. 6D), and reducing the expression of p300 in cells specifically decreased glucocorticoid-stimulated histone H3 acetylation (Fig. 6A). Overall, these observations provide a solid foundation that will allow us to further dissect this important transcriptional regulatory mechanism. A recent study using genomic conservation analyses identified multiple GREs upstream of the human DUSP1 gene [31]. These GREs include the one reported here, located ,1.3 kb upstream of the TSS. In a reporter gene that contains this GRE and another located 4.6 kb upstream of the TSS, Tchen et al. showed that the mutation of either GRE reduces hormonal response by approx- imately 50%. Thus, both GREs play a role in glucocorticoid- regulated DUSP1 gene transcription. This confirms the impor- tance of our studies on the role of the presently characterized GRE in overall transcriptional activation of the DUSP1 gene. The locations of other GREs identified by Tchen et al. are far away from the TSS (29, 28 and 24 kb), and genome-wide analysis of GR binding regions indeed reveals that many potential GREs are located far away from the TSS [32,33]. However, the importance of these long-range GREs in DUSP1 gene regulation is unclear at the moment, as these GREs have not been studied in their natural configuration. Future studies should address these questions. In addition to GLS2, we identified three other GLS elements that may play accessory roles in the regulation of DUSP1 gene transcription. Interestingly, only one of these three is required for a complete glucocorticoid response, and any of these three can play the accessory role. It is unclear whether the mechanisms governing the functional interactions between GLS2 and GLS1, 3, or 4 are the same. This will require detailed studies of the role of these accessory elements in glucocorticoid response. At present, we cannot be sure if they directly associate with GR. It is possible that GLS2, which fits the consensus sequence of a canonical GR binding site, serves as an essential site for the binding of GR while the other GLS elements, which are more divergent, can only bind to the receptor when GR:GLS2 interacts. Alternatively, these GLS elements may bind to other transcriptional regulators. The requirement of functional interactions between GR and other DNA-binding transcriptional regulators to confer a complete glucocorticoid response has been previously reported. These PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13754