Essential promoter elements are located within the 5' untranslated region of human insulin-like growth factor-I exon I

Multiple mechanisms regulate insulin-like growth factor-I (IGF-I) gene expression in mammals, including transcription from two promoters, alternative RNA splicing, and differential RNA polyadenylation. In previous studies we demonstrated that IGF-I promoter 1, the major human promoter, initiated transcription within a dispersed 158 nt segment of exon 1, and showed that full promoter activity required the entire 322 nt 5' untranslated region (UTR) of exon 1. We now have examined the functional significance of this highly conserved region by testing the activity of hybrid promoter-reporter genes containing various portions of the 5' UTR after transient transfection into the IGF-I-producing SK-N-MC cell line. Recombinant plasmids containing the entire 322 nt 5' UTR of exon 1 and a 1630 nt segment of 5' flanking sequence stimulated luciferase activity nearly 70 times higher than a promoterless control plasmid. Truncation to +198 had little effect on promoter function, while subsequent 3' deletions (to +111, +51, and +5) led to a stepwise decrease in reporter gene expression. Internal deletions of nt +6 to +50, +52 to +110, and +112 to +197 led to 65, 25, and less than 10% decreases in promoter function, respectively. Removal of the entire segment from +6 to +197 caused a complete loss of activity. Analysis for DNA-protein interactions by in vitro DNase-I footprinting identified a broad region of protection extending from nt -12 to +38. Further characterization by gel mobility shift assays indicated that several specific DNA-protein complexes could be formed in this region with nuclear protein extracts from SK-N-MC cells. Substitution mutations within the footprinted segment had deleterious effects on promoter function, and one mutation involving nt +10 to +20 resulted in a greater than 70% decline in reporter gene expression. Our results demonstrate that the initial portion of the 5' UTR of human IGF-I exon 1 is required for high level basal transcription of promoter 1 and provide a starting point for defining the nuclear factors regulating this component of IGF-I gene expression.