Transcriptional analysis of acetylcholine receptor α3 gene promoter motifs that bind Sp1 and AP2

In this study, we performed an analysis of the neuronal nicotinic acetylcholine receptor α3 subunit gene promoter region, -238/+47, to identify cis and trans elements that are important for basal activity in PC12 cells. Sequence analyses of the α3 promoter and footprint assays revealed an Sp1 binding site between -79 and -57 (termed the α3 GA motif) and an AP2 binding site between -30 and -7. Using mobility shift analysis, we found that PC12 cell extracts contain proteins that specifically bind to the α3 GA motif and are immunologically related to Sp1. Mutation of the α3 GA motif, which prevented binding of Sp1, resulted in a 75% decrease in promoter activity. Mutation of the AP2 site resulted in only a minor loss of promoter activity, which is consistent with the lack of AP2 binding activity in PC12 extracts. In Drosophila Schneider line 2 (S2) cell cotransfection assays, Sp1 activated the α3 promoter in a GA motif-dependent manner. Furthermore, multimerization of the GA motif upstream of the β-globin TATA box conferred Sp1 responsiveness. Our results indicate that Sp1 can activate transcription through direct interaction with the α3 GA motif and that this motif plays a major role in α3 promoter basal activity in PC12 cells.