Characterization of the integrin alpha(v) gene promoter in mice

To characterize a promoter for integrin alpha(v) in mice, we cloned a 5'-flanking region of the mouse integrin alpha(v) gene. The nucleotide sequence of the promoter region lacks both TATA or CCAAT boxes, and has the consensus sequence for Sp1, Ets, AP-2 and GATA-1. The transcription initiation site was mapped as 287 base pairs (bp) upstream from the translation initiation site by primer extension analysis. To analyze the promoter activity of the integrin alpha(v) gene, the successive 5'-deletions which were cloned upstream of the luciferase reporter gene were transfected into mouse melanoma B16F10 cells with high metastatic properties. The luciferase assay reveals that a region (positions -108 to +22) has important cis-acting elements for the promoter activity. Further, three distinct protein-DNA complexes in the promoter region were detected in a gel shift assay using nuclear extracts from B16F10 cells. To identify positions where the complexes are formed, the promoter region (positions -108 to +97) was subjected to DNaseI footprinting analysis. The analysis showed that a 17 bp-element (positions -31 to -15) interacts with putative transcription factors. Deletion of the 17 bp-element from the promoter resulted in a reduction of about 80% in the promoter activity when compared with the wild type promoter, and a gel shift assay using the deleted mutant of the promoter showed only one protein-DNA complex. These results reveal that the 17 bp-element contains cis-acting elements, and plays an important role in the transcription initiation of the integrin alpha(v) gene.