ChIP and re-ChIP assay revealed that Sp3 and NRSF were presented together on 188 bp MOR DNA segment for synergic downregulation of MOR expression
To validate the functional Sp3 binding to MOR promoter in vivo, we carried out the in vivo ChIP assay using several antibodies. After cross-linking the proteins and DNAs with formaldehyde, cell lysates from NS20Y cells were subjected to immunoprecipitation with NRSF, HDAC1, HDAC2, Sp1, Sp3 and IRF-4 (as a negative control). The precipitated DNA fragments were PCR-amplified with specific primers design to generate a 188 bp fragment covering the NRSE and Sp3-binding GC box in the MOR gene. As shown in Figure 7A and B, ChIP PCR products were detected with HDAC2, NRSF and Sp3 antibody, but were not detected when PI, no antibody or IRF-4 antibody was tested. In addition, we performed the Re-ChIP assay to verify the Sp3 and NRSF are specifically co-localized on MOR DNA (Figure 7C). First, ChIP was performed using anti-NRSF antibody. Then, prior to reversal of protein–DNA cross-linking, the chromatin fragments were subjected to re-precipitation using anti-Sp1 or anti-Sp3 antibody. During subsequent PCR only those MOR DNA sequences that are simultaneously bound to both NRSF and Sp3 proteins should be amplified. As shown in Figure 7C, Sp3, but not Sp1 and NRSF are specifically co-localized on the 188 bp MOR promoter sequence containing NRSE and a GC box. The results clearly demonstrate that NRSF and Sp3 factor bind to the short MOR promoter segment with close proximity in vivo.