MATERIALS AND METHODS

Cell culture and reporter gene constructs
NS20Y and HeLa cells were routinely grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2. PC12 cells were cultured in 10% CO2 in DMEM with 10% donor horse serum and 5% FBS (7).
The pGL4.7 (−4744 to +1, the translation start site was designated as +1) was generated by ligation of the PCR product (−249 to +1) with the BamHI and NcoI digested pL4.7K (−4744 to −249) (19). PCR was performed using genomic DNA from mouse NS20Y cells as a template and an upstream sense oligonucleotide (5′-GCCTCTGGATCCCTCACAGCCCAT-3′), containing a BamHI site, and a downstream antisense oligonucleotide (5′-GGCGCTGCTGTCCATGGTTCTGAA-3′) containing a NcoI site. The pGL4.7NRSP, pGL4.7preNRmSP, pGL4.7NRSPm and pGL4.7preNRmSPm constructs were generated by ligation of the pGL4.7 DNA digested with NcoI and the double-strand oligomers (pGL4.7NRSP; wild type of NRSE and Sp-binding sequence, pGL4.7NRmSP; mutated NRSE and wild type of Sp-binding sequence, pGL4.7NRSPm; wild type of NRSE and mutated Sp-binding sequence, pGL4.7NRmSPm; mutated NRSE and mutated Sp-binding sequence) containing NcoI site at both 5′ and 3′ ends (for pGL4.7NRSP: 5′-TTCAGAACCATGGACAGCAGCGCGCCGGCCCATGGATTCTTC-3′; for pGL4.7preNRmSP: 5′-TTCAGA ACCATGGA ATAGTTGCGCGCCGGCCCATGGATTCTTC-3′; for pGL4.7NRSPm: 5′-TTCAGAACCATGGACAGCAGCGCATATGCCCATGGATTCTTC -3′; for pGL4.7preNRmSPm: 5′-TTCAGAACCATGGAATAGTTGCGCATATGCCCATGGATTCTTC-3′) (The underlines indicate mutated nucleotides for NRSE and Sp3 core binding sites).
The pGL4.7NRmSP and pGL4.7NRmSPm constructs were finally generated by PCR site-directed mutagenesis using high-fidelity Pfu DNA polymerase according to the manufacturer's protocol (Quikchange TM; Stratagene). In vitro mutagenesis was carried out on MOR promoter linked to luciferase gene reporter (pGL4.7preNRmSP and pGL4.7preNRmSPm) using primers as follows: for pGL4.7NRmSP: 5′-TTCAGAACCATAAAATAGTTGCGCGCCGGCCCATGGATTCTTC-3′; 5′-GAAGAATCCATGGGCCGGCGCGCAACTATTTTATGGTTCTGAA-3′; for pGL4.7NRmSPm: 5′-TTCAGAACCATAAAATAGTTGCGCATATGCCCATGGATTCTTC-3′; 5′-GAAGAATCCATGGGCATATGCGCAACTATTTTATGGTTCTGAA-3′). The mutated nucleotides are underlined.

Total RNA preparation and RT–PCR analysis
Total RNA was isolated according to the supplier's protocol (TRI Reagent; Molecular Research Center, Inc.). For RT–PCR, 2 μg of total RNA was reverse transcribed and PCRs were carried out with MOR-specific primers at the same tube using one-step RT–PCR reagent (Qiagen) in a GeneAmp 9600 PCR machine (Perkin-Elmer). The PCR cycle conditions for MOR consisted of 95°C for 45 s, 60°C for 45 s and 72°C for 45 s, followed by a 10 min extension at 72°C (37 cycles for NS20Y, 33 cycles for PC12 cell). PCR products were separated in a 2.0% agarose gel with TBE buffer. The mouse MOR transcript was amplified with primer set P2Ss (5′-CTCCGTGTACTTCTAAGGTGGGAG-3′) and TM2as (5′-GGCTAAGGCATCTGCCAGAGCAAG-3′). Similar reactions were carried out using primers for β-actin as an internal control. Quantitative analyses were carried out using ImageQuant 5.2 (Amersham) software.

Transient transfection and reporter gene assay
For luciferase assays, 1 × 106 cells/well were cultured overnight before transfection. Various reporter constructs at equimolar concentrations were transfected using Effectene transfection reagent (Qiagen, Valencia, CA) as described previously (11). After 48 h of transfection, cells were washed twice with phosphate-buffered saline (PBS) and lysed with lysis buffer (Promega). For all the assays, pCH110 (β-galactosidase; Amersham Bioscience Inc.) was also co-transfected and measured to normalize transfection efficiency. The luciferase and β-galactosidase activities were determined according to the manufacturer's instructions (Promega and Tropics, Madison, WI).

Real-time PCR
Total RNA was extracted using the TRIzol reagent (Molecular Research Center, Inc.) from NS20Y cells. DNase I-treated RNA (2 μg) was subjected to RT (Roche) using oligo(dT) primer. One-fortieth of this reaction was used for real-time PCR analysis using SYBR Green I dye chemistry. PCR product accumulation was monitored using a iCycler iQ Real-Time detection system (Bio-Rad). The mean cycle threshold value (Ct) from triplicate samples was used to calculate gene expression level. PCR products were normalized to levels of β-actin. Relative gene expression levels were determined as described in User's resource guide from Bio-Rad.

Co-immunoprecipitation and western blots
Cells were lysed in a buffer containing 1% Triton X-100, 10 mM Tris, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, supplemented with 1 mg/ml antipain, 1 mg/ml leupeptin, 100 nM PMSF and 100 nM sodium orthovanadate. Approximately 1 mg of the clarified cell lysate was incubated overnight at 4°C with an anti-Sp1, anti-Sp3 or anti-NRSF monoclonal antibody. Immunoprecipitates were recovered on protein G–Sepharose beads, washed extensively and separated on SDS–PAGE. Proteins were transferred on to PVDF membrane (Amersham Biosciences). Membranes were incubated with anti-Sp3 (sc-644; Santa Cruz Biotechnology), anti-Sp1 (sc-59; Santa Cruz Biotechnology) and anti-NRSF antibody (12C11; kindly provided by Dr David Anderson) and signals were detected using a Storm 840 PhosphorImager system (Amersham Biosciences).

Electrophoretic mobility shift assay
In vitro translation of NRSF and Sp3 was performed using the TNT SP6 and T7 Quick Coupled Transcription/Translation System as per the manufacturer's protocol (Promega) and nuclear extracts from NS20Y cells were prepared as described previously (11). Double-stranded oligonucleotides containing a copy of the MOR NRSE/GC box (5′-CAGCAAGCATTCAGAACCATGGACAGCAGCGCCGGCCCAGGGA-3′) were synthesized and end-labeled using the Klenow fragment of DNA polymerase I in the presence of [γ-32P]dATP. Free nucleotides were separated by centrifugation through a G-25 column (Roche). The end-labeled DNA probes were incubated with in vitro translated NRSF or Sp3 or both and with nuclear extract from NS20Y cells. For competition analysis, a 100-fold molar excess of cold competitor oligonucleotide was added to the mixture before adding the probe. For supershift assay, antibodies or pre-immune serum were pre-incubated for 30 min on ice before adding the labeled probe. Radiolabeled probe was then added and the mixture was incubated for further 20 min on ice. Samples were subjected to electrophoresis on a native 4% polyacrylamide gel run in 0.5× TBE buffer for 2 h at 180 V. Monoclonal antibody against NRSF was obtained from Dr D. J. Anderson. Anti-IRF4 antibody was purchased from Santa Cruz Biotechnology.

Chromatin immunoprecipitation (ChIP) and re-precipitation (re-ChIP)
ChIP assay was performed as described by Kim et al. (11). Briefly, cells were incubated for 15 min in a medium containing 1% formaldehyde at room temperature and cross-linking was quenched by adding glycine to 125 mM. Cells were washed three times with ice-cold Tris-buffered saline (150 mM NaCl and 20 mM Tris–HCl, pH 7.6), resuspended in lysis buffer and disrupted on ice with a sonicator followed by centrifugation at 15 000 g for 10 min to remove cell debris. Gel electrophoresis indicated that a substantial fraction of DNA fragments at this stage are from 0.3 to 3.0 kb in length. ChIP was performed by incubating the cell lysate overnight at 4°C with 4 μg of anti-Sp1, anti-Sp3, anti-HDAC1, anti-HDAC2 or anti-IRF4 (as a non-specific antibody) followed by incubation with protein G Plus–agarose (Santa Cruz Biotechnology) for 2 h. The beads were rinsed four times sequentially with lysis buffer 500 (0.1% deoxycholic acid, 1 mM EDTA, 50 mM HEPES, pH 7.5, 500 mM NaCl and 1% Triton X-100), LiCl/detergent solution (0.5% deoxycholic acid, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40 and 10 mM Tris–HCl, pH 8.0) and TE buffer (10 mM Tris–HCl, pH 8.0 and 1 mM EDTA, pH 8.0). The beads were then incubated for 10 min at 65°C with elution buffer (10 mM EDTA, 1% SDS and 50 mM Tris–HCl, pH 8.0).
For re-ChIP, re-ChIP elution buffer (10 mM EDTA, 50 mM Tris–HCl, pH 8.0 and 0.7 M NaCl) was used. To reverse the cross-linking and purify the DNA, the precipitates were incubated at 65°C overnight and then treated with proteinase K solution for 2 h at 37°C. DNA samples were recovered and resuspended in TE buffer. PCRs were performed using 4 μl of immunoprecipitated chromatin sample with the primers spanning mouse MOR promoter region in 50 μl of total volume. Primers used were NSCF forward, 5′-CTGTGAGAGGAAGAGGCTG-3′ and NSCR reverse, 5′-AAGTTGAGCCAGGAGCCAGGT-3′, which produces 188 bp PCR products.