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, al ternative RNA splicing, and differential RNA polyadenylation. In previ ous studies we demonstrated that IGF-I promoter 1, the major human pro moter, initiated transcription within a dispersed 158 nt segment of ex on 1, and showed that full promoter activity required the entire 322 n t 5' untranslated region (UTR) of exon 1. We now have examined the fun ctional significance of this highly conserved region by testing the ac tivity of hybrid promoter-reporter genes containing various portions o f the 5' UTR after transient transfection into the IGF-I-producing SK- N-MC cell line. Recombinant plasmids containing the entire 322 nt 5' U TR 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, w hile 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% de creases in promoter function, respectively. Removal of the entire segm ent from +6 to +197 caused a complete loss of activity. Analysis for D NA-protein interactions by in vitro DNase-I footprinting identified a broad region of protection extending from nt -12 to +38. Further chara cterization by gel mobility shift assays indicated that several specif ic DNA-protein complexes could be formed in this region with nuclear p rotein 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% de cline in reporter gene expression. Our results demonstrate that the in itial 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 ge ne expression. (C) 1997 Elsevier Science Ireland Ltd.