Transcription factor AP2 is required for expression of the rat transforming growth factor-α gene

DNase I footprinting of the rat TGFα promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from −222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGFα promoter-reporter gene (TGFαLUC) from 50 – 90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5′-GCCNNNGGC-3′) as its center, we investigated the possibility that AP2 binding is important for TGFα promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGFαLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGFα promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.