A CACCC BOX IN THE PROXIMAL EXON-2 PROMOTER OF THE RAT INSULIN-LIKE-GROWTH-FACTOR-I GENE IS REQUIRED FOR BASAL PROMOTER ACTIVITY

The insulin-like growth factor I gene is transcribed from two promoter regions, resulting in alternative first exons in insulin-like growth factor I messenger RNAs. A previous study showed that the sequence fro m -73 to +44 (where +1 is the first nucleotide in the exon 2 transcrip tion initiation cluster) contained an active exon 2 promoter, and that sequences between -73 and -36 were required for promoter activity. In the current study, the roles of two putative cis-acting elements with in the -73 to +44 region in basal exon 2 promoter activity were evalua ted using mutagenesis and nuclear protein-DNA binding assays. Mutation of the CCCCACCC sequence at position -53 to GAAATCCC resulted in a co mplete loss of promoter activity in transient transfection assays in G H(3), OVCAR-3, C6, and Chinese hamster ovary (CHO) cells. A -73/+24 ex on 2 promoter-luciferase construct had partial promoter activity. Muta tion of a putative initiator motif surrounding the major exon 2 start site did not alter the activity of this construct. In electrophoretic mobility shift assays, a P-32-labeled oligomer extending from -73 to 44 in the exon 2 promoter was specifically bound by GH(3) cell nuclear extracts. A P-32-labeled oligomer which extended from -63 to -37 in t he exon 2 promoter was specifically bound by GH(3) and OVCAR-3 cell nu clear extracts. These unlabeled oligomers inhibited the binding of a l abeled -236/+44 exon 2 promoter fragment to OVCAR-3 nuclear extracts. Mutation of the CCCCACCC sequence prevented the unlabeled -73/+44 olig omer from inhibiting the binding of the -236/+44 fragment. An unlabele d oligomer containing a consensus activating protein-2 (AP-2)-binding site inhibited labeled -236/+44, -73/+44, and -63/-37 exon 2 promoter binding with a much lower affinity than did the respective unlabeled o ligomers. Purified AP-2 protein did not bind to the exon 2 promoter fr agment, nor did anti-AP-a antibody alter the binding. Cotransfection o f AP-2 expression vector did not significantly increase exon 2 promote r activity. On the other hand, an oligomer containing a consensus Spl- binding site inhibited labeled -63/-37 exon 2 promoter binding by GH(3 ) cell nuclear extracts with an affinity similar to that of the unlabe led -63/-37 oligomer. A mutation in the Spl-binding site in this same oligomer resulted in a complete loss of binding affinity. Purified Spl bound to the -63/-37 exon 2 promoter oligomer. Addition of polyclonal antibody to Spl resulted in a partial supershift of the complex forme d between GH(3) cell and OVCAR-3 cell nuclear extracts and the labeled -63/-37 oligomer. However, in Drosophila Schneider cells, which are a n experimental model for studying the ability of Spl to activate trans cription, the -73/+44 exon 2 promoter construct was not stimulated by cotransfection with an Spl expression plasmid. UV cross-linking studie s indicated that proteins of approximate molecular mass 125, 76, 47, a nd 38 kDa are bound to the proximal(-236/+44) exon 2 promoter region. It is concluded that the CCCCACCC sequence at -53 is required for exon 2 promoter activity. Moreover, specific binding of nuclear proteins t o the proximal exon 2 promoter region requires the CCCCACCC sequence. Sequences downstream of the exon 2 initiation site from +24 to +44 are required for full promoter activity. However, the putative initiator surrounding the major transcription start site at +1 does not appear t o be important for the strength of the proximal promoter. The CCCCACCC sequence at -53 appears to be a CACCC box, which binds zinc finger tr anscription factors of the Kruppel family such as Spl, although protei n factors in addition to Spl are required to activate exon 2 transcrip tion through the -73/+44 proximal promoter region.