RESULTS

Mithramycin A up-regulates the endogenous MOR transcription
Previously, we reported that the MOR transcription is suppressed by NRSE in the mouse MOR promoter through binding of NRSF (11). In the current study, we have studied the role of G/C box sequences (GGGGGCGGGGC), identified in mouse, rat and human in adjacent to NRSE, which is a consensus nucleotide-binding sites for the Sp transcription factors (Figure 1A). To assess the functionality of the putative Sp factor binding site on the expression of the MOR genes, NS20Y cells were treated with mithramycin A, a DNA-binding blocking drug that inhibits the binding of transcription factor to G/C-specific regions of DNA. Mithramycin A is a cell-permeable agent that binds GC-rich DNA sequences and is frequently used to explore the sequence dependency of DNA-binding factors (20). As shown in Figure 1B and D, RT–PCR and real-time RT–PCR revealed that the mRNA levels of the MOR were increased by mithramycin A treatment in a dose-dependent manner in the NRSF positive NS20Y cells. However, in the NRSF negative PC12 cells, MOR transcript level was not affected by mithramycin A treatment (Figure 1C). In accordance with other studies, mithramycin A did not change mRNA levels of β-actin (21). These results indicated that the putative Sp-binding site may mainly act as a negative element for the expression of MOR gene in NS20Y cells.

The Sp-binding site and NRSE synergistically repress the expression of MOR gene
Independent studies have shown that Sp1 has functional implication in expression of some of NRSF target genes including N-methyl-d-aspartate receptors, dynamin I, synaptophysin and glutamate receptor ionotropic kainate-5 genes (22–24). For MOR gene regulation, Sp1 factor acts as a major positive regulator by binding two cis-elements (distal and proximal promoters) located at −340 through −300 residues from the translation start site while Sp3 has a minor effect by binding to one of these elements (25). As mentioned above, a computer search identified a G/C box sequence at the downstream of NRSE region. To verify the functionality of this motif in MOR transcription, we generated various MOR/luciferase reporter constructs containing pGL4.7NRSP (wild type), pGL4.7NRmSP (mutated NRSE and wild-type Sp-binding site), pGL4.7NRSPm (wild-type NRSE and mutated Sp-binding site), pGL4.7NRmSPm (mutated both in NRSE and Sp-binding site) (Figure 2A). All the constructs had been generated in more than 4.7 kb MOR promoter region containing distal and proximal elements and possibly other unidentified elements that might have roles in the promoter activity. Thus, the promoter assay in the current study may accurately reveal the role of newly identified GC box in the MOR gene regulation.
The constructs were transiently transfected in NS20Y and PC12 cells separately and the promoter activity was examined by measuring the signal of the reporter gene. As expected, the mutation in either NRSE or GC box sequence increased the expression of reporter gene in NS20Y cells (Figure 2B). Interestingly, when both elements were mutated at the same time, the promoter activity (14-fold in NS20Y cell) was repeatedly greater than the sum of activities from the constructs where each element was mutated singly (4-fold in Sp site mutation and 6-fold in NRSE mutation). Therefore, these data indicate that NRSE and Sp-binding site may cooperate to repress the MOR promoter activity. Meanwhile, the activities of the same reporter constructs in the NRSF negative PC12 cells (judged from the western blot in Figure 2C) had little effect (Figure 2B) on the promoter activity. These data suggest that a putative Sp-binding site acts as a synergic repressor with NRSE in NS20Y cells and NRSF and NRSE plays an important role to decide the function of the newly identified Sp-binding site as a repressive cis-element.
In addition, we also investigated whether the transcriptional repression activity of the putative Sp-binding site in the MOR promoter can be relieved in the NS20Y cells after mithramycin A treatment. NS20Y cells were transiently transfected either with pGL4.7NRSP or pNRSPm constructs (Figure 3A). Twenty-four hours after transfection, the cells were treated with either 100 or 400 nM mithramycin A. When the wild-type promoter sequence (pGL4.7NRSP) was transfected, a dose-dependent increase in the promoter activity was observed (Figure 3B). In contrast, when the putative Sp-binding site was mutated (pGL4.7NRSPm), the high promoter activity was detected even without treatment of mithramycin A strongly supporting the notion that this putative Sp-binding site downregulates expression of the MOR gene. Interestingly, at the highest concentration, mithramycin A started to decrease the promoter activity. At this condition, mithramycin A could interfere the binding of positive transcriptional regulators such as Sp1 within the promoter region.

Sp3 interacts with NRSF and binds G/C box adjacent to NRSE to repress transcription of MOR gene
Co-immunoprecipitation was performed to identify a subclass of Sp factors responsible for the observed repressor activity in the transcription of MOR gene. The lysates from HeLa cells were immunoprecipitated with Sp1, Sp3, NRSF antibody and pre-immune serum (PI) (as a negative control) and subjected to SDS–PAGE followed by western blot analysis with NRSF and Sp3 antibody. The endogenous Sp3 factor and NRSF were co-immunoprecipitated with NRSF and Sp3 antibody, respectively. In contrast, Sp1 did not co-precipitate with NRSF antibody (Figure 4A). The same result was obtained using c-Myc-tagged NRSF expressed in NS20Y cells implying that Sp3 binds the GC box and a direct interaction between Sp3 and NRSF is required for a synergistic repression of MOR gene expression (Figure 4B). In addition, immunoprecipitation experiment revealed that only the full-length Sp3 factor, other than two short Sp3 isoforms (M1 and M2), interact with NRSF (Figure 4C).
To confirm the binding of Sp3 to the putative GC box, we performed a supershift assay with in vitro translated proteins (myc-NRSF, Sp3) and nuclear extract from NS20Y and PC12 cells. In the electrophoretic mobility shift assay (EMSA) with in vitro translated proteins, both NRSF (Figure 5A) and Sp3 (Figure 5B) formed a binary complex with the labeled probe (from −18 to +24 of MOR). In both cases, cold competitor completely eliminated the binary complex indicating the sequence-specific binding of NRSF and Sp3 to NRSE/GC box. Moreover, the super shift of the binary complex were observed when incubated with anti-c-myc or anti-Sp3 antibody demonstrating the specific binding of two transcription factors to the probed DNA sequences. The incubation with PI or IRF-4 (non-specific antibody as a negative control) had no effect in either NRSF or Sp3 bound complex.
Since the binding site in DNA for both NRSF and Sp3 are slightly overlapped and co-immunoprecipitation experiment suggests that NRSF and Sp3 interact with each other, we tested whether both NRSF and Sp3 can bind the labeled probe at the same time without any interference. As shown in Figure 5C, incubation of increased amount of Sp3 to the fixed amount of NRSF generated a new ternary complex indicating that NRSF and Sp3 bind this short MOR promoter sequence. The fact that the NRSF binary complex was remained at the highest amount of Sp3 tested may indicate that either an additional protein(s) is needed for the efficient binding in vivo or the two short isoforms of Sp3 produced during in vitro translation (data not shown) decreased the actual concentration of the full-length Sp3.
As an additional test of specific binding of NRSF and Sp3 to NRSE/GC box of MOR gene, we performed EMSA with NS20Y nuclear extracts. As shown in Figure 6, three major DNA bound complexes were observed (lane 2). The upper complex was eliminated in the presence of monoclonal NRSF or Sp3 antibody (lanes 5 and 6, respectively), whereas incubation with PI had no effect on the complex (lane 4). The two lower complexes were not affected (lanes 5 and 6) by both antibodies, suggesting that these are not related to NRSF or Sp3. Competitive binding experiments were also conducted in this EMSA using a 100-fold molar excess of a self NRSP cold competitor, NRmSP and NRSPm competitor (lanes 7 and 8, respectively). The self NRSP competitor competed for protein–DNA interaction efficiently (lane 3) and also mutated competitor NRmSP and NRSPm significantly competed for upper complex indicating the specific binding of NRSF and Sp3 (lanes 7 and 8). The signal left after the treatment of the mutated competitor represents the complex with either NRSF (lane 7) or Sp3 (lane 8). In PC12 cells (NRSF negative but Sp3 positive), the NRSF-associated complex (the upper complex in lane 7) was not detected (data not shown). The subtle shift in mobility (lane 7) or no apparent mobility shift (land 8) may be due to the presence of various nuclear proteins that were recruited by both NRSF and Sp3 and/or due to the binding of other nuclear proteins which have affinity for the probe DNA sequences that could now be available after the displacement of NRSF or Sp3 by the mutated competitors.
Interestingly, two lower complexes were dramatically decreased only when NRSPm competitor was used, suggesting that the bound proteins have specificity to NRSE region. Studies investigating the identity of this complex are underway.
Taken altogether, the co-immunoprecipitation and EMSA studies demonstrate that NRSF and Sp3 factor interact with each other and physically bind to the repressive element of the MOR promoter, NRSE/GC box.

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.