Analysis of the regulatory promoter region of the human α1 (I) collagen-encoding gene (COL1A1) gene indicated the presence of G + C-rich sequence elements that are potential binding sites for the transcription factor Sp1. As a step toward understanding transcriptional regulation of the human COL1A1, we examined Spl binding in the promoter region using DNase I footprinting, and analyzed the effect of Spl expression on COL1A1 promoter activity in transiently transfected Drosophila melanogaster cells in vivo. The results indicated that recombinant human Spl interacted specifically with two G + C-rich sequences within the COL1A1 promoter. Binding of factors in nuclear extracts prepared from human dermal fibroblasts to a 22-nucleotide deoxyribonucleotide (oligo) spanning the 5′ G + C-rich sequence required Zn2+, and was abolished by excess Spl consensus binding site oligos, or by anti-Spl antibodies. Studies in which a series of progressively 5′-deleted COL1A1 promoter::cat constructs were co-expressed with an Spl expression plasmid in a cellular background devoid of Spl homology demonstrated that Spl markedly enhanced the COL1A1 promoter activity. These results suggest that the transcriptional activity of the human COL1A1 can be positively regulated by Spl.
- DNase footprinting
- Drosophila Schneider cells
- Gene expression
- electrophoretic mobility shift assay
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