Competition analysis of the human alpha 2(I) collagen promoter using synthetic oligonucleotides

Previous studies have identified four cis-response elements which mediate t he basal transcriptional activity of the human alpha 2(I) collagen gene. On e 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 activato r sites. Furthermore, the repressor site and two of the activator sites wer e found to constitute binding sites for the transcription factors Sp1 and S p3. In this study, we further determined the affinity and specificity of th e binding of Sp1 and Sp3 to the human alpha 2(I) collagen promoter and inve stigated the function of the pyrimidine-rich region which contains the TCCC CC motif. Functional analyses of Sp1 and Sp3 in Drosophila cells confirmed that Sp1 and Sp3 activate the human alpha 2(I) collagen promoter via the GC boxes and the TCCTCC motif, but that binding of Sp1 or Sp3 to the represso r site does not activate or repress the collagen promoter activity. Competi tive 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 fac tor to the fourth cis-response element (CCAAT-binding factor site). Further more, the affinity of Sp1 or Sp3 for the TCCTCC motif was shown to be great er than that of the Sp1 consensus oligonucleotide. In vitro transcription a nalysis revealed that the addition of each activator site oligonucleotide o r repressor site oligonucleotide had an inhibitory effect on the transcript ion of the collagen gene. These results suggest that the repressor site reg ulates the transcription of the collagen gene by taking away Sp1 or Sp3 fro m the activator sites.