PMC:1702488 / 18260-22981 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1702488","sourcedb":"PMC","sourceid":"1702488","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1702488","text":"Sp3 interacts with NRSF and binds G/C box adjacent to NRSE to repress transcription of MOR gene\nCo-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).\nTo 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.\nSince 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.\nAs 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.\nInterestingly, 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.\nTaken 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 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