Mice and cells
Mice deficient for Ifnar1 , Tyk2 , Stat1 , Irf1  and IFNβ  were crossed for at least ten generations onto C57BL/6 background. Wild type (WT) mice (C57BL/6) were purchased from Charles River Laboratories. Mice were housed under specific pathogen-free conditions according to FELASA guidelines, except for Oct-6+/- mice which were housed conventionally. Oct-6+/- mice were of mixed background , Oct-6+/+ littermates were used as WT controls. All animal experiments were discussed and approved by the institutional ethics committee and the Austrian laws (GZ 68.205/0204-C/GT/2007 and GZ 68.205/0233-II/10b/2009). Bone marrow-derived macrophages (BMMs) were grown in the presence of L929 conditioned medium as described  and used for experiments on day 7 after isolation. Foetal liver-derived macrophages (FLMs) were isolated/grown by culturing foetal liver cell suspensions (day 13.5-14.5 post conception) under the same conditions as BMMs and were used for experiments on day 6 after isolation. Primary murine embryonic fibroblasts (pMEFs) were isolated and grown from individual embryos at day 13.5-14.5 post conceptionem according to standard procedures . MEF cell lines were grown as described . SW10 cells were from ATCC (Cat. No. CRL-2766; ) and propagated as recommended.
Reagents, treatments and infections
Cells were treated for the indicated time points with IFNβ, IFNγ or IL-6 (all purchased from Calbiochem), or with polyinosinic-polycytidylic acid (poly(I:C), GE Healthcare). IL-6 was used at a concentration of 125 ng/ml, poly(I:C) at 50 μg/ml, IFNβ and IFNγ at 1000 U/ml (if not stated otherwise). Infections with Murine Cytomegalovirus (MCMV) and plaque assays were done as described previously .
Whole cell extracts and immunoprecipitations (IP)
Cells were lysed in 50 mM Tris.HCl pH8, 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 2 mM DTT, 0.1 mM EDTA, 0.2 mM Na3VO4, 25 mM NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 mM PMSF. Cell debris was removed by centrifugation. 1 mg whole cell extract/ml was incubated overnight with 2 μg anti-Oct-6 antibody (C-20, Santa Cruz) and purified with protein-G PLUS agarose (Santa Cruz). Precipitates were separated on 8% SDS polyacrylamide gels. Proteins were blotted onto nitrocellulose membranes (GE Healthcare), detection was performed with a previously described rabbit polyclonal anti-Oct-6 antibody . As an input control for IPs, whole cell extracts were analysed for panERK (pan-extracellular signal regulated kinases) expression. panERK antibody was from BD Transduction Laboratories. Anti-rabbit- and anti-mouse-IgG horse-raddish peroxidase-conjugated secondary antibodies and the ECL™-detection system were from GE Healthcare.
Electrophoretic mobility shift assays (EMSAs)
EMSAs were done as described previously  using 15 μg whole cell extract and an octamer consensus motif containing oligonucleotide . For supershifts, cell extracts were incubated with 1 μl of the respective antibody prior to the binding reaction: anti-Oct-6 (C-20), anti-Oct-1 (12F11), and anti-Oct-2 (C-20) (all Santa Cruz).
Cells were grown and stimulated on glass slides, and fixed with 4% formaldehyde for 15 min (Histofix, Roth). Formaldehyde was quenched by glycine (100 mM, 15 min), cells were permeabilised by methanol treatment (-20°C, 5 min). Nonspecific binding was blocked with 1% BSA in PBS for 1 hour. Oct-6 was detected by incubating the slides with anti-Oct-6 antibody (C-20, Santa Cruz; 4 μg/ml in blocking solution; 4°C overnight) and a fluorescently labelled a-goat IgG secondary antibody (Alexa-Fluor™ 488; 1:200 in PBS; 1 hour at room temperature). Goat IgG (Invitrogen) was used as isotype control. Nuclei were counterstained with DAPI (100 ng/ml).
Alignment of Oct-6 upstream sequences and transcription factor binding site (TFBS) prediction
For the alignments, mouse, rat and human sequences from +1 to -5 kb were used (Blast 2 sequences: http://blast.ncbi.nlm.nih.gov/bl2seq/wblast2.cgi; March 2008). Mouse sequence accession number NC_000070.5 (M.m. C57BL/6, chromosome 4, reference assembly; base 124334896 = +1 on the +strand). Human sequence accession number NC_000001_10 (H.s. chromosome 1, GRCh37 primary reference assembly; base 38512450 = +1 on the -strand). Rat sequence accession number NC_005104.2 (R.n., chromosome 5, reference assembly; base 143981547 = +1 on the +strand). Homologous sequence parts, i.e. -4367 to -4500, -1598 to -3233, and -161 to -637 (base counts correlate to the murine sequence), were submitted to TFBS analysis analysed using "Patch" (http://www.gene-regulation.com/pub/programs.html; August 2008), which scans input sequences for potential TFBSs based on the Transfac database. A number of Stat1- and ISGF3-binding sites were predicted for each sequence part. We decided to concentrate on the homologous sequence part nearest to the transcription start site (-161 to -637) of Oct-6, based on a report showing that the regions -500 bp upstream of the transcription start site of IFN-inducible genes are enriched in predicted binding sites for Stat1 and ISGF3 .
Chromatin immunoprecipitation (ChIP)
ChIP for Stat1 was performed as described  with minor modifications. Sonication (Sonopuls HD70, MS72 sonotrode; Bandelin) was performed at 50% power and 90% duty cycle for 10 times 15 sec with 1 min break between the pulses. Equal amounts of lysate were used for Stat1 IP (4 μl Stat1-C antibody/500 μl lysate; a kind gift from Pavel Kovarik, MFPL, University of Vienna; ) and a control reaction using nonspecific rabbit serum (Sigma). DNA was isolated following a phenol:chloroform extraction protocol and subjected to PCR analysis. PCRs were run in a final volume of 25 μl containing 300 nM primer, 2 mM MgCl2, 200 μM dNTPs (Fermentas), 1× Biotaq buffer, 2 U Biotaq DNA polymerase (Agrobiogen) under following cycling conditions: 5 min at 95°C for initial denaturation, followed by 35 cycles of 95°C for 30 sec and 61°C for 1 min. PCRs were done from all samples of the anti-Stat1 IP, the control IP (nonspecific rabbit serum) and from an aliquot of the initial sample input prior to the IP (input DNA). Following primers were used: ChIP_Oct6-F: GTCTCTGCTCGGAACCCGA, ChIP_Oct6_S-R: CCCACGTTCCACACAAGCT, ChIP_Oct6_L-R: GCCCGCGTACACATTCAC; ChIP_Irf1-F: GCACAGCTGCCTTGTACTTCC, ChIP_Irf1-R: TCGGCCTCATCATTTCGG.
RNA isolation and reverse transcription (RT)
Total RNA was isolated following the TRIZOL (Invitrogen) protocol. Prior to cDNA synthesis, RNA was treated with 1 U/μg RNA RQ1 DNase I (Promega) in order to digest contaminating genomic DNA. cDNA was prepared from 1 μg total RNA per 20 μl reaction using the iScript First Strand cDNA synthesis kit (BioRad), including controls for DNA contamination (reactions without addition of reverse transcriptase).
Real-time quantitative PCR (qPCR) analysis of gene expression
Target gene expression was assessed by qPCR with ubiquitin-conjugating enzyme E2D2 (Ube2d2) as endogenous control gene. Assays for IFNβ and Ube2d2 were described previously . Taqman® probes labelled with 6-carboxyfluorescein (FAM) at the 5'end, and a black-hole-quencher (BHQ1) at the 3'end were used. EvaGreen (Biotium) assays were used for the quantification of Oct-6, Egr2, Pmp22, Lsm10, and Stk40. Following primers were used (5' to 3'): panIFNa:fwd-CCACAGGATCACTGTGT(A/T)CCTGAGA, rev-CTGATCACCTCCCAGGCACAG, probe-AG+AA+GAA+A+C+AC+AG+CC (locked nuclear acids (LNAs) are indicated by a "+" in front of the respective base; );
Oct-6: fwd-AGGTCCTGTTGGAGATGATATGTT, rev-TTGGGAAATGAATTGTCAAGAAA;
Egr2: fwd-GGTGACCATCTTCCCCAATG, rev-TTGATCATGCCATCTCCCG;
Pmp22: fwd-CCGGTTTTACATCACTGGATTCT, rev-TGTAGATGGCCGCTGCACT;
Lsm10: fwd-CCTCCAAAAGGCCATGAGACT, rev-CGGGAGTTGGCTCAGAACAC;
Stk40: fwd-CTCTCAGTGCCATCATTGCATC, rev-CACCTTTGCCTCCTGGGA.
Taqman qPCR assays were run in a final volume of 25 μl containing 300 nM primer (Invitrogen), 100 nM probe (Sigma or Metabion), 200 μM dNTPs (Fermentas), 4 mM MgCl2, 1× HotFire buffer B, and 1U HotFire DNA polymerase (all Solis BioDyne). The conditions were the same for the EvaGreen assays, except for the use of 0.2× EvaGreen dye instead of the probe in the presence of only 2.5 mM MgCl2. All qPCRs were run on a Mastercycler® ep Realplex (Eppendorf) applying following cycling conditions: 15 min at 95°C for initial denaturation, then 40 to 45 cycles of 95°C for 20 sec and 60°C for 1 min. For EvaGreen assays, the PCR was followed by a melting curve analysis in order to confirm assay specificity. Data were analysed using the Realplex software (Eppendorf) and relative target gene expression levels (i.e. n-fold expression levels) were calculated following the standard curve method [66, 67].
Statistical analysis of RT-qPCR data. RT-qPCR gene expression data were investigated for differences among genotypes and time after challenge. Univariate regression was calculated with the log of the transformed target to endogenous control gene expression ratio as dependent variable. Linear contrasts were encoded such that for each time point Oct6-/- were compared to WT cells. Differences among experiments were controlled for. Data were analysed with SPSS 17.0 for Mac OS-X.
WT and Oct-6-/- FLMs were treated with 50 μg/ml poly(I:C) for 8 hours in three independent experiments. RNA integrity was assessed by capillary electrophoresis using a Bioanalyser2100 (Agilent Technologies), and photometric analysis (OD260 nm/280 nm ration of ~2.1 for all samples). RNA integrity numbers (RIN) ranged between 9.2 and 9.4 indicating high quality of RNA samples. ABI1700 Mouse Genome Survey Microarrays (Applied Biosystems) in combination with the RT direct labelling kit (Applied Biosystems) were used according to the manufacturer's recommendations to generate gene expression profiles. 20 μg of input total RNA was used for direct labelling and microarray hybridisation. Data transformation and normalisation: expression values less than 10 were set to 10. Data were normalised to the 50th percentile (intra-array normalisation) and each gene was normalised to the median expression (inter-array normalisation). Data were pre-filtered based on the signal to noise ratio (cut-off level: signal above noise > two-fold in all samples of the respective biological replicate group) and non-changing genes (normalised expression levels from 0,667 to 1,334 in at least 4 of 4 conditions) were subtracted. The remaining data set was tested for differentially expressed genes using ANOVA (GeneSpring Expression Analysis 7.3.1 tool, Agilent Technologies). A p-value of p < 0.05 (Welch t-test) was considered significant. Expression differences of at least two-fold were considered relevant. Not fully annotated probes, i.e. probes that did not correspond to a NCBI RefSeq, EST or RIKEN cDNA, were excluded. Functional annotation and clustering of the differentially regulated genes was performed using the Gene Functional Classification tool of the database for annotation, visualisation and integrated discovery (DAVID; http://david.abcc.ncifcrf.gov/[68, 69]). The array data have been deposited in the Gene Expression Omnibus database, http://www.ncbi.nlm.nih.gov/geo (GEO accession no. GSE22691).