A549 cells (type II alveolar lung epithelium cells; ATCC CCL85) [23] were cultured in DMEM (Cellgro) with 5% fetal bovine serum (FBS; J R Scientific). When cells were treated with dexamethasone (DEX, a synthetic glucocorticoid, Sigma) or DMSO (vehicle control), DMEM with 5% charcoal stripped FBS (J R Scientific) was used.

A549 cells were grown to confluence and treated with 0.5 µM DEX or DMSO, then cross-linked by using formaldehyde at a final concentration of 1% at room temperature for 5 minutes. The ChIP protocols were otherwise as previously described [24], [25].

A549 cells were grown to confluence (4×150 mm plates per treatment) and treated with 0.5 µM DEX or DMSO, then cross-linked by using formaldehyde at a final concentration of 1% at room temperature for 5 minutes. Reactions were quenched with 0.125 M glycine for 5 min. Cells were washed with PBS, scraped in cell lysis buffer (50 mM HEPES-KOH at pH 7.4, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100) supplemented with protease inhibitor cocktails (Calbiochem), and incubated for 10 min at 4°C. Crude nuclei were collected by centrifugation at 600 x g for 5 min at 4°C then resuspended in 1.5 ml of ice-cold RIPA buffer (10 mM Tris-HCL at pH 8.0, 1 mM EDTA, 150 mM NaCl, 5% glycerol, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, supplemented with protease inhibitor). Chromatin was fragmented with 18 pulses of 20 sec at 30% power with a Branson Sonifier 250 sonicator at 4°C. To remove insoluble components, samples were centrifuged at 13,000 rpm for 15 min at 4°C, and the supernatant was recovered.

For the first round of IP, two aliquots from each treatment were taken. 8 µg of rabbit polyclonal GR (N499) antibody was used to immunoprecipitate GR-bound chromatin or 8 µg of rabbit polyclonal IgG was used as a control for each aliquot, per treatment. The samples rotated overnight at 4°C. 120 µl of 50% protein A/G plus-agarose bead slurry (Santa Cruz Biotechnology) was then added to each immunoprecipitation and rotated for 2 h at 4°C. After incubation, beads were washed with 1 ml of solution per wash as follows: twice with RIPA, twice with RIPA containing 510 mM NaCl, twice with LiCl buffer (20 mM Tris at pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodiumdeoxycholate), and twice with TE. Between each wash, samples were rotated at room temperature for 5 min, followed by 1 min centrifugation at 5,000 rpm. All washes were supplemented with protease inhibitors. Immmunocomplexes were eluted by adding 75 µl TE with 10 mM DTT and incubated at 37°C for 30 min. The samples were centrifuged at 800 x g for 2 minutes at room temperature, and the supernatant was transferred to a clean 1.5 ml tube. An additional 14 µl of RIPA was added to dilute the sample.

For the second round of IP, 8 µg of p300 (rabbit polyclonal, Santa Cruz Biotechnology) was used in all samples and rotated overnight at 4°C. Again, we added 120 µl of 50% protein A/G plus-agarose bead slurry and washed as described, modified only by replacing the final two TE washes with one wash in RIPA buffer. After removing the last wash buffer, 75 µl of proteinase K solution (TE pH 8.0, 0.7% SDS, 200 µg/ml proteinase K) was added to each IP. Reactions were incubated for 3 h at 55°C, then overnight at 65°C to reverse formaldehyde cross-links. DNA was purified using a QIAquick PCR purification kit (Qiagen) and eluted in 60 µl of Elution Buffer (Qiagen).

To transfect A549 cells, we used Lipofectamine 2000 (Invitrogen) according to the technical manual in the 24-well format. Twenty-four hours post-transfection, cells were treated with either DMSO or 0.5 µM DEX for 18–24 h. Cells were then harvested, and luciferase activity was measured by a Dual-Luciferase Reporter Assay kit (Promega) according to the technical manual. pCI-p300 wild type and pCI-p300ΔHAT were provided by Mike Stallcup (USC) [26].

The human DUSP1 genomic region containing the predicted GR binding site was amplified by PCR using specific primers. The PCR fragment was restriction-digested and subcloned into the pGL4-TATA reporter. A mutagenesis kit (QuikChange) was used to make site-directed mutations per the manufacturer's instructions (Stratagene).

A549 cells were grown to confluence in 6-well plates then treated with DMSO or 0.5 µM DEX for 10 min. Cells were washed then scraped in 1 ml ice cold DNase I Buffer + NP40 (20 mM HEPES pH 7.4, 0.5 mM CaCl₂, 5% glycerol, 3 mM MgCl₂, 0.2 mM spermine, 0.2 mM spermidine, 0.2% NP40). Cells were vortexed for 10 seconds to homogenize, then centrifuged at 500 x g in a microfuge for 5 minutes at 4°C. Pellets were resuspended in 100 µl DNase I Buffer without NP40, then equilibrated to room temperature for 10 min. Samples were digested with 5 units of DNase I (QIAGEN) for 5 minutes at 37°C, and reactions were stopped by addition of equal volume (100 µl) 2× Stop Reaction Buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 200 mM NaCl, 12.5 mM EDTA) with 200 µg/ml Proteinase K, and incubated at 65°C for 4 hours or overnight. DNA was purified using a PCR purification kit (QIAGEN), and samples were measured and diluted to use 50 ng/well for qPCR analysis.

p300 (QIAGEN), CBP (Santa Cruz Biotechnology), and a scramble sequence (control, QIAGEN) were reverse transfected with HiPerFect transfection reagent (QIAGEN) according to the technical manual. For gene expression analyses, reverse transfection was done in a 24-well plate. Forty-eight hours post-transfection, cells were treated with either DMSO or 0.5 µM DEX for 5 hours, and total RNA was then isolated. For western blot analyses, cells were collected from a 24-well plate 48 hours post-transfection. For ChIP experiments, reverse transfection was done in 10 cm plates. Again, 48 hours post-transfection, cells were treated with either DMSO or 0.5 µM DEX, this time for 10 minutes.

A549 cells transfected with p300, CBP, or scramble (control) siRNA were collected 48 hours post-transfection in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 150 mM NaCl, 5% glycerol, 0.1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, supplemented with protease inhibitors). Proteins were resolved and imaged as described [27. The following antibodies were used: p300 rabbit polyclonal IgG (sc-584; Santa Cruz Biotechnology), CBP mouse monoclonal IgG₁ (sc-7300, Santa Cruz Biotechnology), β-Actin (C4) mouse monoclonal IgG₁ (sc-47778; Santa Cruz Biotechnology), anti-rabbit IgG₁-HRP (Cell Signaling), and anti-mouse IgG₁-HRP (sc-2060; Santa Cruz Biotechnology).

A549 cells were grown to confluence and treated with DMSO or 0.5 µM DEX for 10 minutes. Cells were cross-linked with 1% formaldehyde for 3 minutes at 22°C, and reactions were quenched by adding glycine to a final concentration of 0.125 M. Cells were then rinsed in PBS and scraped in ice-cold MNase NP-40 Lysis Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.5% NP-40, 0.15 mM spermine, 0.5 mM spermidine). Cells were vortexed for 10 seconds to ensure thorough lysis before nuclei were collected by centrifugation at maximal speed in a microfuge for 5 minutes at 4°C. Nuclei were washed in ice-cold MNase Digestion Buffer without CaCl₂ (10 mM Tris pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine) and re-centrifuged. Pelleted nuclei were suspended in ice-cold MNase Digestion Buffer with CaCl₂ (10 mM Tris pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM CaCl₂). Samples were treated with 1 unit of MNase (Nuclease micrococcal from Staphylococcus aureus, Sigma-Aldrich, N5386-200UN) for 10 minutes at 25°C. Reactions were stopped by addition of 80 µl MNase Digestion Buffer with CaCl₂, 20 µl MNase Stop Buffer (100 mM EDTA, 10 mM EGTA), 75 µg Proteinase K, and 20 µl 10% SDS, and incubated at 65°C for 4 hours or overnight. Samples were then column purified using a PCR purification kit (QIAGEN). RNaseA (Millipore) was diluted to 0.1 µg/µl in TE, and 20 units were added to each sample for 2 hours at 37°C. Samples were then purified using a PCR purification kit (QIAGEN) and run on a 1.5% agarose gel. DNA fragments of 150 bp were purified using a gel extraction kit (QIAGEN).

Quantitative real-time PCR was used to measure the amount of distinct genomic fragments surrounding the DUSP1 GRE. For each primer set, a standard curve was generated using 1 µg, 0.25 µg, 0.0625 µg, and 0.015625 µg of human genomic DNA. The amount of a particular genomic fragment in the MNase sample was then calculated based on the standard curve.

A549 cells were grown to confluence and treated with DMSO or 0.5 µM DEX for different times. Cells were then harvested and incubated in lysis buffer (10 mM Tris-HCl pH 7.4, 3 mM MgCl₂, 10 mM NaCl, 150 mM sucrose and 0.5% NP40) at 4°C for 5 minutes. Nuclei were then isolated by centrifugation at 170 x g at 4°C for 5 min. The total nuclei from each of the DMSO or DEX treated samples were counted, and equal numbers of nuclei were used for in vitro transcription. The samples were split into two aliquots. One was incubated in 100 µl 2× in vitro transcription buffer (200 mM KCl, 20 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 4 mM dithiothreitol (DTT), 4 mM each of ATP, GTP and CTP, 200 mM sucrose and 20% glycerol) plus 8 µl biotin-UTP (Roche), and the other in 100 µl 2× in vitro transcription buffer plus 8 µl UTP (negative control) for 30 minutes at 29°C. 6 µl of 250 mM CaCl₂ and 6 µl of RNAse free DNase (Roche) (10 U/µl) were then added to stop the reactions. Total RNA was then isolated using Nucleospin RNA II (Macherey-Nagel).

Dyna beads M-280 (Invitrogen) were washed twice in solution A (0.1 mM NaOH, 0.5 M NaCl) for 5 min, once in solution B (0.1 M NaCl) for 5 min, and then resuspended in binding/wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA and 2 M NaCl) plus 1 µl (40 U) RNasin per 100 µl of beads. 50 µl of beads (in binding/wash buffer) were then added to RNA, incubated at 42°C for 20 min, and then shaken for 2 h at room temperature. Afterward, the beads were isolated and the supernatant discarded. The beads were then washed once (5 min) with 500 µl 15% formamide plus 2× saline-sodium citrate (SSC) buffer, twice with 1 ml 2× SSC buffer, then resuspended in 30 µl RNase and DNase free water. 10 µl of beads were used for each reverse transcription (RT) reaction prior to qPCR.

Total RNA was isolated from cells by using QIAshredder and RNeasy kits (Qiagen) or a NucleoSpin RNAII kit (Macherey-Nagel). To synthesize random-primed cDNA, 0.5 µg of total RNA (10 µl), 4 µl of 2.5 mM dNTP, and 2 µl of random primers (New England Biolabs) were mixed and incubated at 70°C for 10 min. A 4-µl cocktail containing 25 U of Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase (New England Biolabs), 10 units of RNasin (Promega), and 2 µl of 10× reaction buffer (New England Biolabs) was then added. The reaction was incubated at 42°C for 1 h, then 95°C for 5 min.

The resultant cDNA was diluted to 200 µl with water, and 2.5 µl was used to perform qPCR using EVA QPCR SuperMix Kit (Biochain) per the manufacturer's protocol. qPCR was performed in either a 7900 HT, 7500 HT, or StepOne PCR System (Applied Biosystems) and analyzed by using the ▵▵-Ct method as supplied by the manufacturer (Applied Biosystems). Rpl19 expression was used for internal normalization. All primer sequences are presented in Table S1.

Previous studies have shown that DUSP1 gene expression is induced by glucocorticoids in A549 cells [28]. To confirm that the DUSP1 gene is a direct target of GR and to determine the kinetics of glucocorticoid-induced transcription, we performed nuclear run-on experiments. We isolated nuclei from A549 cells that were treated with the synthetic glucocorticoid dexamethasone (DEX) for 10, 30, and 90 minutes. Nascent transcripts from these nuclei were labeled with biotin-UTP, and then isolated using magnetic beads. After random-primed cDNA was synthesized, qPCR was performed to measure the change of transcript levels using DUSP1 specific primers. As shown in Fig. 1A, transcription of DUSP1 is induced rapidly, increasing approximately two fold after just 10 minutes of DEX treatment (compared to 90 minutes DMSO-treated samples and no treatment), with levels continuing to increase after 30 and 90 minutes (Fig. 1A). These results demonstrate that glucocorticoids rapidly initiate transcription of DUSP1, and that it is likely directly regulated by GR.

As our data revealed that glucocorticoids stimulate DUSP1 through transcriptional initiation, we wished to further probe the mechanism by which GR induces DUSP1 transcription. To this end, we used a chromatin immunoprecipitation (ChIP) scanning approach [23] to systematically locate the GR binding regions in the vicinity of the human DUSP1 transcription start site (TSS). A549 cells were treated with DMSO or DEX for 10 minutes, when we first observed transcriptional initiation, then subjected to a standard ChIP procedure [25] of formaldehyde cross-linking of A549 cells, shearing of chromatin to approximately 200–500 bp fragments, and precipitating cross-linked GR:DNA complexes with GR-specific antibody. IgG antibody was used as a negative control for all ChIP experiments. We performed qPCR on the precipitated DNA fragments using six pairs of oligonucleotide primers that amplify approximately 100 bp segments (Fig. 1B), spaced at 300–500 bp intervals. As the average size of the sheared DNA fragments is 300–500 bp, this arrangement of amplified sequences can scan effectively for GR occupancy in the first 2 kb upstream of the DUSP1 TSS. We found that two primer pairs, flanking −1421 to −1333 and −1191 to −1118 respectively, exhibited significant enrichment in GR ChIP (Fig. 1B), but not with IgG alone (data not shown). These results reveal that GR binding localizes between −1421 and −1118 of the TSS.

We next tested whether this GR binding region was a mediator of glucocorticoid response. We subcloned the genomic region extending approximately 200–300 bp upstream and downstream of our ChIP fragments (−1672 to −959) into an E4 TATA box containing pGL4 luciferase reporter to test whether the identified GR-binding regions are glucocorticoid-responsive (Fig. 1C). We transfected this reporter plasmid (pDUSP1) along with an expression vector encoding human GR cDNA into A549 cells. After 24 hours, cells were treated with DMSO or DEX for 18 to 20 hours. Cells were then lysed, and firefly luciferase assay was performed. We found that firefly luciferase activity in DEX-treated cell lysates was significantly higher than that of DMSO-treated cell lysates (Fig. 1C). These results indicate that the −1672 to −959 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 −1672 to −959 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 1, 3, and 4, which are more divergent, may play accessory roles that could contribute to hormone response under some conditions.

To further understand the mechanisms underlying glucocorticoid-regulated DUSP1 gene transcription, we investigated the effects of glucocorticoids on the chromatin structure of the DUSP1 gene. Chromatin architecture plays a critical role in the control of eukaryotic gene transcription in vivo because it determines the accessibility of genomic regulatory sequences to the transcriptional machinery. Based on their sensitivity to cleavage when intact nuclei are exposed to DNA-modifying agents, such as endonuclease DNase I, we can analyze the accessibility of the chromatin structure of specific genomic regions.

We first tested whether glucocorticoids affect DNase I accessibility within the DUSP1 promoter. Nuclei from A549 cells treated with DMSO or DEX for 10 minutes were subject to cleavage by DNase I for 5 minutes. DNA fragments were then isolated and analyzed with qPCR using primers from the previously described ChIP experiments. We found that the region from −2000 to +1 (TSS) of DUSP1 was already sensitive to DNase I before DEX treatment (compared to DNA not digested by DNase I, Fig. 3A). In contrast, the promoter region of α-fetoprotein, a liver-specific gene not expressed in A549 cells, was sensitive neither to DNase I digestion nor DEX treatment (Fig. S1). With DUSP1, DEX treatment markedly increased DNase I accessibility in all areas examined except −160 to −64 (Fig. 3A), which was already very sensitive to DNase I prior to DEX treatment (Fig. 3A). These results indicate that DEX treatment and GR binding can further “open up” the chromatin structure of most genomic regions surrounding the DUSP1 promoter.

This opening of chromatin within the GRE suggests that nucleosomes have been repositioned or remodeled. Glucocorticoids have previously been shown to induce chromatin remodeling; thus, they must either disrupt a nucleosome assembly or change the position of nucleosomes in the genome (41). To test this, we used MNase to map the position of nucleosomes surrounding the DUSP1 gene. We treated A549 cells with DMSO or DEX for 10 min. Nuclei were isolated, cross-linked with formaldehyde, and digested with MNase. The MNase sensitivity of the chromatin was then analyzed using qPCR with primers designed to anneal to the DNA approximately every 50 bp from −1550 to −1050 of the DUSP1 TSS. Because the nucleosomal regions are less accessible to MNase, we recovered more DNA fragments from these regions. In contrast, the recovery of DNA fragments from linker regions was lower, as most of these fragments were digested by MNase. In untreated cells, three solid nucleosome positions are evident: from −1500 to −1350 (nucleosome 1), −1350 to −1200 (nucleosome 2), and −1200 to −1050 (nucleosome 3) (Fig. 3B). Consistent with our DNase sensitivity assay, addition of DEX appeared to increase the sensitivity to MNase in nucleosomes 1 and 2 (Fig. 3B). These data indicate that a glucocorticoid-dependent remodeling of chromatin structure within the DUSP1 GRE is associated with gene activation.

An increase in the acetylation of histones H3 and H4 has been correlated with both gene transcription and active DNA control elements such as response elements and enhancers [30]. We treated A549 cells for 10 minutes with DMSO or DEX, then performed ChIP experiments using antibodies that recognize multiple acetyl lysines in the tails of either histone H3 (AcH3) or H4 (AcH4) to examine whether glucocorticoids affect the overall histone acetylation status of the DUSP1 gene promoter. Total H3 and H4 levels were also monitored by ChIP, as the levels of AcH3 and AcH4 are associated with the overall density of histone in each genomic region. Thus, the ratio of AcH3/H3 or AcH4/H4 accurately reflects the change of histone acetylation. In comparison to ChIP done with control IgG antibodies, we found that AcH3/H3 and AcH4/H4 levels were already enriched before DEX treatment in all genomic regions tested (Fig. S2). These results indicate that the −2000 to +1 region of the DUSP1 gene was already acetylated before DEX treatment. DEX treatment 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 −1815 to −543, but had no effect in the region from −160 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 glucocorticoid-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 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 approximately 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 approximately 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).

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 induced 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 surrounding 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 (p300ΔHAT) 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 p300ΔHAT 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 p300ΔHAT 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.