Tissue culture
Complemented p65(-/-) NIH 3T3 MEFs stably expressing p65 wild type or the acetylation-deficient mutants were generated by lentiviral complementation as previously described in [18]. Briefly, HEK 293T cells were transfected with the packaging plasmid, the envelope plasmid and pTV-myc-RelA/p65 wild type, RelA/p65 K/R mutants or the control pTV vector. p65(-/-) MEFs were infected with the viral supernatant and split into selective medium (2.5 μg/ml Blasticidin) after thirty-six hours. Expression of recombinant proteins was confirmed by western blot analysis. Pools of cells were used for further analysis. They, as well as the p65(-/-) and (+/+) parental NIH 3T3 MEFs kindly provided by A. Beg [27], were maintained in DMEM supplemented with 10% FCS, 100 units/ml penicillin/streptomycin (Gibco) and non-essential amino acids (Gibco). HEK 293T cells were maintained in DMEM supplemented with 10% FCS and 100 units/ml penicillin/streptomycin.
Plasmids
Plasmids for the mammalian overexpression of human p65 wild type and mutants K310R, K314/315R and KTR in HEK 293T cells were described elsewhere [18]. Briefly, pph-CMV-Km-RelA/p65 wild type, previously described in [28], was used as template vector for the generation of the p65 mutants by site-directed mutagenesis according to the QuickChange protocol (Stratagene). All introduced mutations were confirmed by sequencing.
Reagents and antibodies
Human TNFα, Trichostatin A (TSA), Nicotinamide (Nam) and acetyl-Coenzyme A (acetyl Co-A) were purchased from Sigma. Recombinant mouse TNFα was either purchased from Sigma or generated in our laboratory. Sodium fluoride (NaF) and beta-glycerophosphate were obtained from Fluka. The acetyl-specific antibodies for p65 anti-acetyl K314 (ab18727) and anti-acetyl K315 (ab19869) were generated by Abcam. The following antibodies were purchased from Santa Cruz Biotechnologies: anti-p65 (sc-372) and anti-p300 (sc-585). The anti-myc antibodies were either purchased from Roche (11-667-149-001) or purified from hybridoma cells.
Generation of recombinant proteins
The recombinant proteins were expressed by baculovirus in Sf21 cells using either the FastBac or the BacPAK systems (Clontech). His-tagged proteins were purified over Ni2+-beads (ProBond, Invitrogen).
Micro array
Complemented cell lines p65 wild type or acetylation-deficient mutants were starved overnight before either left untreated or stimulated with 30 ng/ml TNFα for 3 hours. Total RNA from three biological replicates per sample was isolated at different days using the 'Total RNA isolation mini kit' (Agilent Technologies). RNA quality was measured on the 2100 Bioanalyzer (Agilent Technologies). Microarray experiments were performed using 'Whole Mouse Genome (4 × 44 K) Oligo Microarray Kit' (Agilent Technologies) and 'One-color micro array-based gene expression analysis' (Agilent Technologies) following the manufacturer's protocol. Cy3-labeled cRNA was purified with the RNeasy kit (Qiagen). Dye incorporation was assessed with the ND-1000 Spectrophotometer (NanoDrop Technologies). Per sample, 1.65 μg cRNA from each of the three biological replicates was hybridized to independent arrays according to the manufacturer's protocol. Hybridized slides were scanned with the Agilent DNA Microarray scanner and quantified using the Agilent Feature Extraction software. The data analysis was performed using 'Rosetta Resolver® Gene Expression Data Management and Analysis System' (Rosetta Biosoftware). Briefly, data was processed and normalized with default settings. Then, low-signal genes with signal intensities < 0.1 were filtered out. Differential expression between two conditions was assessed based on the average ratio and significance. All genes with expression ratios < 0.556 and >1.8, and a p-value < 0.05 were selected to generate the tables of significantly regulated genes. These sequence data have been submitted to the GEO database http://www.ncbi.nlm.nih.gov/geo under accession number GSE15196.
Gene expression by quantitative RT-PCR
Complemented MEFs were starved overnight before treatment with 30 ng/ml recombinant TNFα for different time points. Total RNA was isolated from at least two biological samples at different days with the 'Total RNA isolation mini kit' (Agilent Technologies). RNA quantity was assessed with the ND-1000 Spectrophotometer (NanoDrop Technologies). 2 μg RNA was subsequently retro-transcribed using the 'High-capacity cDNA reverse transcription kit' (Applied Biosystems) following the manufacturer's protocol. Real-time PCR was performed using the Rotor-Gene 3000 (Corbett Life Science, now Qiagen) and TaqMan assays from Applied Biosystems for the following genes: Cfb, Mpa2l, Mmp10, Mmp13, Rps6 and CanX, according to Applied Biosystems' protocol. The last two genes were used as internal controls to normalize for RNA input. RNA from at least two biological replicates per sample was measured and analyzed with REST [29]. Each experiment was run three independent times, the mean value and ± SD was calculated and blotted into graphs. A 2-tails, paired t-Test was performed with the log of the values to additionally calculate the p-values.
In vitro acetylation assay
1 μg of recombinant p65 wild type or the acetylation-deficient mutants were incubated with 500 ng recombinant p300 in HAT buffer (50 mM Tris HCl pH8, 100 mM NaCl, 10% glycerol, 1 mM PMSF, 1 mM DTT, 1 μg/ml bepstatin, 1 μg/ml leupeptin, 1 μg/ml pepstatin and 1 mM sodium butyrate) with or without addition of 150 μM acetyl-CoA. After 1 hour at 30°C, samples were resolved on SDS-PAGE and analyzed by western blot.
Acetylation assays in cells
HEK 293T cells were transfected with expression plasmids for p300 and either myc-tagged p65 wild type, the acetylation-deficient mutants or an empty vector, using the calcium phosphate precipitation method. After 23 hours, cells were treated with 10 ng/ml human TNFα for 30 minutes. Then, whole cell extracts were prepared and 40 μg protein was analyzed by SDS-PAGE and western blot.
Immunoprecipitation
Whole cell extracts from the complemented cells untreated or stimulated with 10 ng/ml TNFα for 40 minutes were used to immunoprecipitate p65. 750 μg of extract were incubated with 1.5 μg of antibody for 1 hour at 4°C in Co-IP buffer (20 mM HEPES pH 7.9, 100 mM NaCl, 2.5 mM MgCl2, 0.05% NP-40, 1 mM PMSF, 1 μg/ml pepstatin, 1 μg/ml bestatin, 1 μg/ml leupeptin, 1 μM TSA, 5 mM Nam). Protein G sepharose was added and samples were incubated for another 2 h at 4°C. Samples were washed three times 5 minutes in Co-IP buffer containing 100 mM NaCl before being subjected to 10% SDS-PAGE, followed by western blot.
Chromatin Immunoprecipitation
p65(-/-) or (+/+) MEFs were stimulated with 10 ng/ml mouse TNFα for the indicated time points and fixed with 1% formaldehyde (Calbiochem) for 10 minutes. After extensive washing, the plasma membrane was first lysed with lysis buffer 1 (50 mM Tris HCl pH8, 2 mM EDTA pH8, 0.1% NP-40, 10% glycerol, 1 mM PMSF, 0.5 mM DTT, phosphatase and HDAC inhibitors) and then the nuclear membrane with lysis buffer 2 (50 mM Tris HCl pH8, 5 mM EDTA pH8, 1% SDS, 1 mM PMSF, 0.5 mM DTT, phosphatase and HDAC inhibitors). Chromatin fragmentation was achieved with the Bioruptor (Diagenode). Sonified chromatin was diluted with 9 volumes of dilution buffer (50 mM Tris HCl pH8, 5 mM EDTA pH8, 0.5% NP-40, 200 mM NaCl and 1 mM PMSF) and pre-cleared for 1 hour with Protein A Agarose/salmon sperm DNA (Millipore). 1% of input was saved and the remaining chromatin was then incubated overnight with the specific antibodies. After 30 additional minutes of incubation with Protein A Agarose/salmon sperm DNA, the immuno-complexes were extensively washed with washing buffer (20 mM Tris HCl pH8, 2 mM EDTA pH8, 1% NP-40, 0.1% SDS, 500 mM NaCl and 1 mM PMSF) and then with buffer TE (10 mM Tris HCl pH8 and 1 mM EDTA pH8). Chromatin was eluted with 2% SDS in TE buffer and incubated at 65°C for at least 6 hours. DNA was purified with 'QIAquick PCR purification kit' (Qiagen) following the manufacturer's recommendations and measured by real-time PCR using SYBR Green and the Rotor-Gene 3000 (Corbett Life Science, now Qiagen). The following primers were used: Mpa2l_forward (CAGCCCCTTTTATAGTGAGTC), Mpa2l_reverse (TAC AAAATCCGGGAGTATTGC), Cfb_forward (CACCTGTGAAGCAAGTCTCTCTCT), Cfb_reverse (TTTGTGCAGCAAGGACTCTGACCT), IP-10_forward (GCAATGCCCT CGGTTTACAG), IP-10_reverse (GGCTGACTTTGGAGATGACTCA), Glucagon_ forward (GAGTGGGCGAGTGAAATCAT) and Glucagon_reverse (TGAGCTGCGA ACAGGTGTAG) Samples were normalized to input chromatin and expressed as % input. Each experiment was independently repeated at least three times. Mean values ± SD of three independent real-time PCR runs from one independent ChIP are shown.