Cells and materials
HEK293 cells stably expressing the IL-1 receptor (HEK293IL-1R) (35
), human epithelial carcinoma KB cells (36
), A549 lung epithelial carcinoma cells (catalogue number ACC 107, German Collection of Microorganisms and Cell Cultures), mouse embryonic fibroblasts (Mefs) with a tamoxifen-inducible deletion of HDAC3 (33
), p65 NF-κB-deficient and reconstituted Mefs (15
), and all other Mefs lines were cultured in Dulbecco’s modified Eagle’s medium, complemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. For the experiments shown in and Supplementary Figures S3
, Hdac3 fl/+
or Hdac3 fl/−
Mefs were immortalized by the 3T3 protocol. For the experiments shown in Supplementary Figure S2
, wild-type Mef lines obtained from Matthias Gaestel (37
) and from Mitchell A. Lazar (38
) were used.
Figure 2. Deletion of the HDAC3 genes inhibits IL-1-triggered Cxcl2 transcription. (A) Immortalized murine embryonic fibroblast (Mef) lines were generated from mice harboring floxed Hdac3 alleles and the tamoxifen-inducible Cre recombinase gene (33). These cells (more ...)
Antibodies against the following proteins or peptides were used: β-actin (JLA20; EMD), p65 (C-20, sc-372), HDAC1 (C-19, sc-6298), HDAC3 (H-99, sc-11417), p300 (N15, sc-584) and mouse IgG (sc-2025) from Santa Cruz; GFP (clone 7.1 and 13.1; 11814460001) and MYC (9E10) from Roche; acetyl-NF-κB p65 (K310) (3045) and acetylated lysine antibody (9441) from Cell Signaling; acetyl-histone H3 (K9/14) (06-599; Upstate), acetyl-NF-κB p65 (K310) (ab52175) from Abcam; FLAG (clone M2; F1804) and β-tubulin (clone TUB2.1; T4026) from Sigma and HDAC3 (mouse monoclonal: MCA4831Z; AbD Serotec). Antibodies against acetyl-NF-κB p65 (K314) and acetyl-NF-κB p65 (K315) were generated by Michael O. Hottiger and have been described in (12
TSA, apicidin, tamoxifen and puromycin were from Sigma. Human recombinant IL-1α was used at 10 ng/ml in all experiments and was a kind gift from Jeremy Saklatvala, London, UK. Recombinant human TNFα was from R&D Systems or Hoelzel. Ni2+-NTA agarose was from Qiagen (1018244), and True Blot anti-mouse Ig IP beads were from eBioscience (00-8811-25).
Plasmids, transfections, reporter gene assays and selection of stable cell lines
For reporter gene assays the following expression vectors were used: pUHC13-3-IL-8 promoter and pSV40-ß-galactosidase (SV40-ß-gal) (39
). His-p65, YFP (yellow fluorescent protein)-CBP, HA-p65 and its various point mutated derivatives (HA-p65 K310Q / K310R, HA-p65 K4Q / K4R, HA-p65 K5Q / K5R) were described (15
). Additional K-Q mutants of p65, His-p65 E39I and His-p65 K5R were generated by site-directed mutagenesis as described (40
). HDAC3-HA was a kind gift of Dr. Edward Seto, Tampa, Florida. Expression vectors for p300, HDAC1, HDAC2, HDAC5, HDAC8 have been described.
Transient transfections by the calcium phosphate method and determination of luciferase reporter gene activity were performed as described previously (40
). Equal amounts of plasmid DNA within each experiment were obtained by adding empty vector.
For establishing a stable knockdown of HDAC3, HEK293IL-1R cells were transfected with Sure Silencing™ shRNA plasmids against human HDAC3 (clone ID3) (Superarray Biosciences; KH05911P) or an empty control vector (pSuper-Puro) as negative control, using the calcium phosphate method. Twenty-four hours post transfection, selection was started using 0.75 µg/ml puromycin, and stable cell lines were isolated. KB cells were stably transfected with the same plasmids using Lipofectamine (Invitrogen™) and also selected in 0.75 µg/ml puromycin.
Cell lysis and coimmunoprecipitation
HEK293IL-1R cell lines with a partial shRNA-mediated knockdown of HDAC3 (A) were harvested after washing in cold phosphate-buffered saline (PBS) and collected by centrifugation. The pellet was directly lysed in a modified lysis buffer (50 mM HEPES pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 2 mM Na3VO4, 50 mM NaF, 20 mM β-glycerophosphate, 4 µM TSA, 1 mM PMSF, 1× protease inhibitor cocktail from Roche (11697498001) containing TSA for inhibiting HDAC. After rotating for 30 min at 4°C, the lysates were centrifuged (15 min, 13 000 r.p.m., 4°C) and used for Western blotting or were stored frozen.
Figure 1. Interference with HDAC3 inhibits IL-8 transcription. (A) HEK293IL-1R cells were stably transfected with an empty shRNA vector (pS-Puro, black bars) or with a plasmid encoding a HDAC3-specific shRNA (pS-HDAC3, white bars) to generate shRNA-mediated HDAC3 (more ...)
Transiently transfected HEK293IL-1 R cells (D and E, B, B and C) were harvested after washing in cold PBS and collected by centrifugation. The pellet was directly lysed in β-galactosidase lysis buffer as described (36
Figure 3. Overexpression of HDAC3 enhances mRNA expression of inflammatory genes. (A) HEK293IL-1R cells were transiently transfected to express wild-type HA-tagged p65 along with p300 and HA-tagged HDAC3. Twenty-four hours later, cells were lysed, and mRNA expression (more ...)
Figure 5. Acetylation mimicking mutants of p65 suppress IL-8 gene expression. (A) HEK293T cells were transfected with expression plasmids encoding His-tagged p65 wild-type or a mutated version thereof where K122, K123, K310, K314 and K315 (K5R) were mutated to (more ...)
Mefs with a tamoxifen-inducible deletion of HDAC3 (A) were harvested after washing in cold PBS and collected by centrifugation. The pellet was directly lysed in cell lysis buffer (10 mM Tris pH 7.05, 30 mM Na4O7P2 × 10H2O, 50 mM NaCl, 1% Triton X-100, 2 mM Na3VO4, 50 mM NaF, 20 mM β-glycerophosphate, 1 µg/ml pepstatin, 10 µg/ml leupeptin, 1 mM PMSF, 1 µM microcystin), incubated for 15 min on ice, and, after centrifugation, the lysate was further used for Western blotting.
For coimmunoprecipitation experiments (B), cells were harvested after washing in PBS and collected by centrifugation. The pellet was directly lysed in cell lysis buffer (50 mM HEPES pH 7.4, 50 mM NaCl, 1% Tween20, 2.5 mM EGTA, 1 mM EDTA, 1 mM NaF, 10 mM β-glycerophosphate, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM DTT, 1× protease inhibitor cocktail from Roche) and incubated for 20 min on ice. The DNA was sheared by three sonication steps of 20 s, and lysates were cleared by ultracentrifugation for 20 min at 100.000g and 4°C. HDAC3 was immunoprecipitated from 0.5 mg precleared cell extracts using 2 µg mouse monoclonal anti-HDAC3 antibody (AbD Serotec) or 2 µg normal mouse IgG (Santa Cruz) coupled for 2 h to 25 µl True Blot anti-mouse Ig IP beads (eBioscience). After rotating for 2 h at 4°C, the supernatant was discarded, and the beads were washed three times with high salt wash buffer (50 mM HEPES pH 7.4, 450 mM NaCl, 1% Tween20, 2.5 mM EGTA, 1 mM EDTA, 1 mM NaF, 10 mM β-glycerophosphate, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM DTT, 1× protease inhibitor cocktail from Roche). The precipitated proteins were eluted by boiling in 2× Roti-Load for 10 min before analyzing by western blotting.
Figure 4. HDAC3 binds to and deacetylates p65 at lysines K310, K314 and K315. (A) HEK293IL-1R cells were transiently transfected to express wild-type His-tagged p65 along with YFP-tagged CBP and HA-tagged HDAC3. Cells were lysed under denaturing conditions, proteins (more ...)
NF-κB-p65-deficient and reconstituted Mefs (A) were harvested after washing in cold PBS and collected by centrifugation. The pellet was directly lysed in cell lysis buffer (50 mM HEPES pH 7.4, 50 mM NaCl, 1% Tween20, 2.5 mM EGTA, 1 mM EDTA, 1 mM NaF, 10 mM β-glycerophosphate, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM DTT, 1× protease inhibitor cocktail from Roche) and incubated for 20 min on ice. The DNA was sheared by three sonication steps of 20 s, and, after ultracentrifugation for 20 min at 100.000g at 4°C, the lysates were further used for western blotting.
Figure 9. Specific inhibitory effects of p65 NF-κB acetylation-mimicking mutants on IL-1-induced gene expression. (A) NF-κB p65-deficient Mefs (p65−/−) were reconstituted to express p65 NF-κB wild-type (HA-p65) and its mutant (more ...)
For preparation of nuclear protein extracts (Supplementary Figure S4
), cells were harvested after washing in cold PBS and collected by centrifugation. The pellet was suspended in extraction buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2
, 300 µM Na3
, 20 mM β-glycerophosphate, 10 µM E64, 5 mM DTT, 2.5 µg/ml leupeptin, 1 µM pepstatin, 300 µM PMSF, 1 µM microcystin), centrifuged (5 min, 2000 r.p.m., 4°C) and resuspended again in extraction buffer A containing NP-40 with a final concentration of 0.1%. After incubation on ice for 10 min, samples were centrifuged (5 min, 13 000 r.p.m., 4°C), and the supernatants (corresponding to cytoplasmic extracts) were collected. Nuclear extracts were prepared by resuspending pellets in extraction buffer B (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl2
, 200 µM EDTA, 300 µM Na3
, 20 mM β-glycerophosphate, 10 µM E64, 5 mM DTT, 2.5 µg/ml leupeptin, 1 µM pepstatin, 300 µM PMSF, 1 µM microcystin, 25% glycerol) followed by mixing, incubation on ice for 30 min and centrifugation (5 min, 13 000 r.p.m., 4°C). The supernatants correspond to nuclear extracts.
Cytosolic and soluble nuclear fractions were prepared as described (43
) with the following modifications. Cells were washed twice with ice-cold PBS and harvested by centrifugation (500g
, 5 min, 4°C). For separation of soluble and insoluble nuclear fractions (B), cells were lysed in lysis buffer 1 (20 mM HEPES pH 8.0, 10 mM KCl, 1 mM MgCl2
, 0.1% Triton X-100, 20% glycerol, 50 mM NaF, 1xRoche protease inhibitor mix, 1 µM microcystin, 1 mM Na3
) for 10 min on ice and centrifuged for 1 min at 2300g
at 4°C. The supernatant (C, cytosol) was removed, and the pellet was lysed in lysis buffer 2 (20 mM HEPES pH 8.0, 2 mM EDTA, 400 mM NaCl, 0.1% Triton X-100, 20% Glycerol, 50 mM NaF, 1xRoche protease inhibitor mix, 1 µM microcystin, 1 mM Na3
) for 20 min on ice, vortexed twice and centrifuged at 20 400g
at 4°C for 5 min. The supernatant (N1, soluble nuclear fraction) was removed, and the pellet was lysed in buffer 3 (20 mM Tris (pH 7.5), 2 mM EDTA, 150 mM NaCl, 1% SDS, 1% NP-40, 50 mM NaF, 1xRoche protease inhibitor mix, 1 µM microcystin, 1 mM Na3
). The DNA was sheared by sonication for 20 s, and lysates were incubated for 50 min on ice, repeatedly vortexed and centrifuged at 20 400g
at 4°C for 5 min. The supernatant (N2, insoluble nuclear fraction) was collected.
Figure 6. Suppression of HDAC3 affects p65 recruitment and phosphorylation of RNA polymerase II at the IL-8 promoter. (A) Human KB cells were stably transfected with an empty shRNA vector (pS-Puro, gray bars) or with a plasmid encoding a HDAC3-specific shRNA (pS-HDAC3, (more ...)
Identification of TSA- and IL-1-regulated genes in HDAC3 knockdown cells
In all, 29 421 probes with EntrezGeneID showed hybridization signals more than the 20% percentile. Of these, 24 755 probes corresponding to 16 412 genes were measurable in at least 4 of the 16 microarray hybridizations. This data set was used for further analyses ( and ). The pS-Puro and pS-HDAC3 data sets were filtered separately for genes showing differential regulation by IL-1 by at least 1.5-fold, revealing a total of 75 probes corresponding to 70 genes with consistent regulation. Expression values for these genes above background had to be measurable in at least the stimulated condition (for upregulated genes) or in the unstimulated condition (for downregulated genes). Differential expression of genes in this final set was significant (P ≤ 0.01) in at least one out of four comparisons according to feature extraction software algorithms.
In all, 5045 probes showed differential hybridization signals of at least 2-fold in TSA stimulated pS-Puro or pS-HDAC3 cells. This data set was filtered for consistent regulation resulting in 4241 genes: The probes were separated into three subsets: (i) TSA-regulated, HDAC3 knockdown dependent, not IL-1-regulated; (ii) TSA-regulated, HDAC3 knockdown independent, IL-1-regulated; and (iii) TSA-regulated, HDAC3 knockdown independent, not IL-1-regulated. In case of multiple probes per gene in the same subset, only one probe was selected for further analysis. Probe values were removed from other subsets, if probe values for the same gene occurred in intersections of data sets (i–iii). Probes measuring identical genes but having been assigned to different subsets were removed from the data sets.