TRANSCRIPTION FACTOR AP2 IS REQUIRED FOR EXPRESSION OF THE RAT TRANSFORMING GROWTH-FACTOR-ALPHA GENE

DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interac tion (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of sp ecific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alpha LUC) from 50-90% in transi ently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consen sus sequence (5'-GCCNNNGGC-3') as its center, we investigated the poss ibility that AP2 binding is important for TGF alpha promoter activity, A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited b y an excess of known functional AP2-binding sequence. Moreover, a simi lar mobility shift occurred in the presence of purified AP2 protein, a nd the further addition of AP2 antibody produced a supershifted comple x. More refined DNase I footprinting of a smaller, oligonucleotide pro be in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site, Additionally, co-transfe ction of an AP2 expression vector increased TGF alpha LUC expression 2 5-foId in Drosophila Schneider cells. These various findings corrobora te a role for AP2 in TGF alpha promoter activity. The Fp-B region cont ains a T-5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mo bility shift in the presence of a TATA binding protein (TBP)-TFIIA com plex, and the further addition of TBP antibody produced a supershift. These resdts confirm that protein binding within Fp-B is functionally important, and they also indicate that the T-5 motif functions as a TB P binding site.