Two putative GATA motifs in the proximal exon 1 promoter of the rat insulin-like growth factor I gene regulate basal promoter activity

The insulin-like growth factor I gene is transcribed from two promoters, which direct synthesis of alternative first exons (exon 1 and exon 2) in insulin-like growth factor I messenger RNAs (mRNAs). An exon 1 promoter construct extending from +75 to +192 (the most upstream exon 1 start site was designated as +1) showed significant promoter activity in C6, OVCAR-3, and SK-N-MC cells. Within the +75 to +192 region, there are two perfect matches to the consensus binding site for GATA transcription factor, at +108 (GATA-A) and at +183 (GATA-B). Mutations of the GATA-A or GATA-B sequences resulted in slight (or no) effect on exon 1 promoter activity in both C6 and OVCAR-3 cells. However, mutation of the GATA-A sequence stimulated exon 1 promoter activity by 68% in SK-N-MC cells. Mutation of the GATA-B sequence inhibited exon 1 promoter activity by 4.4-fold in SK-N-MC cells. Electrophoretic mobility shift assays showed that there were nuclear proteins in SK-N-MC cells capable of specifically binding to the GATA-A and GATA-B elements and that this binding was GATA-sequence specific. GATA-2, GATA-3, and GATA-4 are the only GATA proteins that have been reported to be expressed in neurons. None of the antibodies against these three GATA proteins were capable of inhibiting or supershifting the bands formed by the nuclear proteins and oligonucleotides containing GATA-A or GATA-B elements. A GATA-1 expression vector was used to perform cotransfection experiments. The GATA-A mutation abolished the stimulatory effect of the GATA-1 factor on promoter activity. In contrast, the GATA-B mutation enhanced the stimulatory effect of GATA-1 protein. Anti-GATA-1 antibody was also incapable of inhibiting or supershifting the bands formed by the nuclear proteins and oligonucleotides containing the GATA-A or GATA-B elements. In conclusion, the GATA-A element seems to bind an inhibitory endogenous factor(s) in SK-N-MC cells, whereas the GATA-B element may bind a stimulatory factor(s). These factors seem to be related to GATA transcription factors but are immunologically distinct from GATA-2, GATA-3, or GATA-4. GATA-1 has the potential to transactivate the exon 1 promoter through the GATA-A element but is unlikely to be the endogenous protein binding to the GATA-A or the GATA-B motifs in SK-N-MC cells.