CHARACTERIZATION OF THE RAT INSULIN-LIKE GROWTH-FACTOR-I GENE PROMOTERS AND IDENTIFICATION OF A MINIMAL EXON-2 PROMOTER

Insulin-like growth factor I(IGF-I) promoter activity was characterize d in CG, GM(3), OVCAn-3, and Chinese hamster ovary (CHO) cells. Maxima l exon 1 promoter activity was present in the region extending from -1 33 to +362 (where tl is the first transcription start site). Promoter activity was higher in the +75/+362 fragment, which contains exon 1 tr anscription start sites 3 and 4, than in the -133/+74 fragment, which contains exon 1 transcription start sites 1 and 2. Promoter activity w as also observed in constructs containing sequences from -133 to +192, which includes start sites 1, 2, and 3. Inclusion of sequences upstre am of -133 inhibited exon 1 proximal promoter activity in a cell type- specific manner. Exon 2 promoter activity was observed in all cell lin es with a construct containing 73 bp of 5'-flanking sequence and 44 bp of exon 2. Exon 2 promoter activity was abolished when only 36 bp of 5'-flanking sequence and 44 bp of exon 2 were present, suggesting that an essential minimal promoter element(s) is contained within the -73 to -36 region.,4 putative CACCC box was observed within this region at -53. Upstream sequence regulated exon 2 promoter activity in a cell t ype-specific manner. Electrophoretic mobility shift assays revealed a single specifically bound band when the +75/+362 fragment of the exon I promoter was used with nuclear extracts from C6 and GH, cells. Multi ple specifically bound bands with slower mobility were observed when t he -236/+44 exon 2 promoter fragment was incubated with C6, GH(3); CHO , and OVCAR-3 cell nuclear extracts. The exon 1 and exon 2 promoter re gions were able to inhibit each other's binding in electrophoretic mob ility shift assay using GH, cell and OVCAR-3 cell nuclear extracts, re spectively. Oligonucleotides containing consensus activating protein-1 (AP-1) and AP-3 sequences inhibited exon 1 promoter binding by GH, ce ll nuclear extracts. AP-2 and AP-3 sites inhibited exon 2 promoter bin ding. Our data suggest that the sequence surrounding and including sta rt site 3 in exon I functions as a minimal independent promoter. The m inimal exon 2 promoter is contained within the 73 bp upstream and 41 b p downstream of the transcription start site cluster. These minimal pr omoters contain similar and distinct elements that are important for b asal transcription. Upstream sequences may contain cell type-specific silencer elements.