The major promoter of the rat insulin-like growth factor-I gene binds a protein complex that is required for basal expression.
IGF-I gene transcription is regulated by two promoters--the major promoter which is active in all tissues and regulates transcription of IGF-I mRNAs that contain exon 1 and a second promoter which regulates transcription of IGF-I mRNAs that contain exon 2 and from which significant transcription is restricted to the liver. The major promoter is a TATAA-less promoter that lacks both a CAAT box and SP1 binding sites and that utilizes multiple transcription initiation sites. The current studies were designed to delineate the functional elements of the major promoter. Transient transfection assays using rat C6 glioma cells and rat dermal fibroblasts in primary culture demonstrated that basal activity of the major promoter was located between -18 (with +1 defined as the most 5' transcription initiation site in exon 1) and +78 of exon 1. DNase I footprinting, which was performed using nuclear extracts from rat C6 glioma cells, demonstrated protein binding to a sequence that extended from -10 to +9 (termed IGFI-FP1). In gel shift assays, binding of C6 cell nuclear proteins to a 34-basepair (bp) oligonucleotide that contained IGFI-FP1 resulted in the formation of three specific protein-DNA complexes. The functional role of protein binding to IGFI-FP1 was examined by mutating the sequences between either -4 and -2 or -9 and -7 in IGF-I-luciferase fusion genes that contained either 412 or 18 bp of 5'-flanking region and 302 bp of exon 1. Both of these mutations altered protein binding to IGFI-FP1 as demonstrated by gel shift analysis. Transfection of the wild-type and mutant fusion genes into C6 glioma cells demonstrated that mutation of the nucleotides between -4 and -2 decreased luciferase activity to approximately 50% of wild-type activity, whereas mutation of the nucleotides between -9 and -7 decreased luciferase activity to 11-35% of wild-type activity. These data demonstrate that: (i) basal activity of the major promoter of the rat IGF-I gene is localized to the region between -18 and +78 of exon 1; (ii) protein binding sites are present within this region of the major promoter; and (iii) protein binding to this region contributes to basal expression of the IGF-I gene.
An
M R
MR
Department of Medicine, VA Lakeside Medical Center, Chicago, IL 60611, USA.
Lowe
W L
WL
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I; D004247:DNA; D008156:Luciferases