Competition analysis of the human alpha2(I) collagen promoter using synthetic oligonucleotides

Previous studies have identified four cis-response elements which mediate the basal transcriptional activity of the human alpha2(I) collagen gene. One of these elements, a pyrimidine-rich region (TCCCCC motif), was shown to be a repressor site, and the other three elements were shown to be activator sites. Furthermore, the repressor site and two of the activator sites were found to constitute binding sites for the transcription factors Sp1 and Sp3. In this study, we further determined the affinity and specificity of the binding of Sp1 and Sp3 to the human alpha2(I) collagen promoter and investigated the function of the pyrimidine-rich region which contains the TCCCCC motif. Functional analyses of Sp1 and Sp3 in Drosophila cells confirmed that Sp1 and Sp3 activate the human alpha2(I) collagen promoter via the GC boxes and the TCCTCC motif, but that binding of Sp1 or Sp3 to the repressor site does not activate or repress the collagen promoter activity. Com- petitive analyses using DNA mobility shift assays showed that the TCCCCC motif which constitutes the repressor site abolished the binding of Sp1 or Sp3 to the GC boxes or the TCCTCC motif, but not the binding of CCAAT-binding factor to the fourth cis-response element (CCAAT-binding factor site). Furthermore, the affinity of Sp1 or Sp3 for the TCCTCC motif was shown to be greater than that of the Sp1 consensus oligonucleotide. In vitro transcription analysis revealed that the addition of each activator site oligonucleotide or repressor site oligonucleotide had an inhibitory effect on the transcription of the collagen gene. These results suggest that the repressor site regulates the transcription of the collagen gene by taking away Sp1 or Sp3 from the activator sites.