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.