DNase Hypersensitivity Assay A549 cells were grown to confluence in 6-well plates then treated with DMSO or 0.5 mM 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 4uC. Pellets were resuspended in 100 ml 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 37uC, and reactions were stopped by addition of equal volume (100 ml) 26 Stop Reaction Buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 200 mM NaCl, 12.5 mM EDTA) with 200 mg/ml Proteinase K, and incubated at 65uC 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. RNA interference (RNAi) 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 transfec- tion was done in a 24-well plate. Forty-eight hours post- transfection, cells were treated with either DMSO or 0.5 mM 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 mM DEX, this time for 10 minutes. Western Blot 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), b-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). Micrococcal Nuclease (MNase) A549 cells were grown to confluence and treated with DMSO or 0.5 mM DEX for 10 minutes. Cells were cross-linked with 1% formaldehyde for 3 minutes at 22uC, 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 4uC. 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 Glucocorticoid-Induced DUSP1 Staphylococcus aureus, Sigma-Aldrich, N5386-200UN) for 10 minutes at 25uC. Reactions were stopped by addition of 80 ml MNase Digestion Buffer with CaCl2, 20 ml MNase Stop Buffer (100 mM EDTA, 10 mM EGTA), 75 mg Proteinase K, and 20 ml 10% SDS, and incubated at 65uC for 4 hours or overnight. Samples were then column purified using a PCR purification kit (QIAGEN). RNaseA (Millipore) was diluted to 0.1 mg/ml in TE, and 20 units were added to each sample for 2 hours at 37uC. 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 mg, 0.25 mg, 0.0625 mg, and 0.015625 mg of human genomic DNA. The amount of a particular genomic fragment in the MNase sample was then calculated based on the standard curve. Nuclear Run-on A549 cells were grown to confluence and treated with DMSO or 0.5 mM 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 4uC for 5 minutes. Nuclei were then isolated by centrifugation at 170 x g at 4uC 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 ml 26 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 ml biotin-UTP (Roche), and the other in 100 ml 26 in vitro transcription buffer plus 8 ml UTP (negative control) for 30 minutes at 29uC. 6 ml of 250 mM CaCl2 and 6 ml of RNAse free DNase (Roche) (10 U/ml) 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 ml (40 U) RNasin per 100 ml of beads. 50 ml of beads (in binding/wash buffer) were then added to RNA, incubated at 42uC 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 ml 15% formamide plus 26 saline-sodium citrate (SSC) buffer, twice with 1 ml 26SSC buffer, then resuspended in 30 ml RNase and DNase free water. 10 ml of beads were used for each reverse transcription (RT) reaction prior to qPCR. RNA, RT, 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 mg of total RNA (10 ml), 4 ml of 2.5 mM dNTP, and 2 ml of random primers (New England Biolabs) were mixed and incubated at 70uC for 10 min. A 4-ml cocktail containing 25 U of Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase (New England Biolabs), 10 units of RNasin (Promega), and 2 ml of 106 reaction buffer (New England Biolabs) was then added. The reaction was incubated at 42uC for 1 h, then 95uC for 5 min. The resultant cDNA was diluted to 200 ml with water, and 2.5 ml was used to perform qPCR using EVA QPCR SuperMix Kit (Biochain) per the manufacturer’s protocol. qPCR was PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13754