Transcriptional analysis of acetylcholine receptor alpha 3 gene promoter motifs that bind Sp1 and AP2.
In this study, we performed an analysis of the neuronal nicotinic acetylcholine receptor alpha 3 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 alpha 3 promoter and footprint assays revealed an Sp1 binding site between -79 and -57 (termed the alpha 3 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 alpha 3 GA motif and are immunologically related to Sp1. Mutation of the alpha 3 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 alpha 3 promoter in a GA motif-dependent manner. Furthermore, multimerization of the GA motif upstream of the beta-globin TATA box conferred Sp1 responsiveness. Our results indicate that Sp1 can activate transcription through direct interaction with the alpha 3 GA motif and that this motif plays a major role in alpha 3 promoter basal activity in PC12 cells.
Yang
X
X
Department of Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4975, USA.
Fyodorov
D
D
Deneris
E S
ES
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D011950:Receptors, Cholinergic / Q000235:genetics* / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011950:Receptors, Cholinergic; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors