E1A ACTIVATION OF INSULIN-RECEPTOR GENE-EXPRESSION IS MEDIATED BY SP1-BINDING SITES

E1a adenoviral oncoproteins have been known to modulate genes importan t for the growth and differentiation of cells. Our laboratory is inter ested in understanding how insulin promotes the growth and proliferati on of cells. In this report, we have examined the ability of E1a to mo dulate the insulin receptor gene expression. In HepG2 cells, expressio n of the 243-amino acid E1a protein stimulated expression of the chlor amphenicol acetyltransferase reporter under the control of the insulin receptor promoter. 5'-Deletion analysis of the insulin receptor promo ter indicated that the region between -630 and -607 is important for r egulation by E1a. This region contains two GA and four overlapping GC boxes that are putative Sp1-binding sites. A DNA fragment containing t hese sites was used as a probe in gel retardation assays. Three specif ic protein-DNA complexes were detected with HepGP nuclear extract. The se complexes could be competed partially by the DNA fragments with mut ations in either the GA or GC boxes, but not by the DNA fragment with a mutation in both the GA and GC boxes. In addition, mutation of each of these sites lowered the basal activity of the promoter and partiall y reduced transactivation by E1a. Simultaneous mutation in both GA and GC boxes further reduced the basal activity and abrogated transactiva tion by E1a. Taken together, these results indicate that the loss of b inding ability of Sp1 (or Sp1-like factors) is concomitant with reduct ion of the basal activity and the loss of E1a inducibility of the gene . Therefore, the GA and GC boxes are responsible for transactivation o f the insulin receptor gene promoter by E1a. We also propose that tran sactivation of insulin receptor gene by E1a is mediated by Sp1 transcr iption factor.