Binding of upstream stimulatory factor to an E-box in the 3'-flanking region stimulates alpha 1(I) collagen gene transcription

Since several lines of evidence implicate the 3'-flanking region in regulating alpha 1(I) collagen gene transcription, we analyzed 12.4-kilobase pairs of 3'-flanking sequence of the murine alpha 1(I) collagen gene for transcriptional elements. A region of the 3'-flanking region stimulated expression of the heterologous beta-globin gene promoter in an enhancer trap plasmid and of the alpha 1(I) collagen gene promoter in a collagen-luciferase reporter gene construct when located 3' to the luciferase reporter gene. DNase I footprinting analysis demonstrated the presence of three regions where DNA binding proteins specifically interact within this 3'-stimulatory region. Inspection of the DNA sequence revealed a consensus E-box, a binding site for basic helix-loop helix proteins, in one of the protein binding sites. Mobility shift assays demonstrated that upstream stimulatory factors (USF) USF-1 and USF-2 bind to this E-box. Mutating the E-box in the context of the 3'-flanking region confirmed that it contributes to the enhancement of transcriptional activity of the alpha 1(I) collagen gene promoter. Mutations in all three protein binding sites abolished transcriptional activation by the 3'-flanking region, suggesting a complex interaction among the trans-acting factors in enhancing transcriptional activity. Thus, a region of the 3'-flanking region of the alpha 1(I) collagen gene stimulates transcription of the alpha 1(I) collagen gene promoter, and USF-1 and USF-2 con tribute to this transcriptional stimulation.