Synergism of the ATF/CRE Site and GC Box in the Housekeeping Na,K-ATPase α1 Subunit Gene Is Essential for Constitutive Expression

Abstract

Na,K-ATPase α1 subunit gene is constitutively expressed in a wide variety of tissues. Our previous studies revealed that the promoter region between −77 and +17 of the transcription initiation site of the rat Na,K-ATPase α1 subunit gene (Atp1a1) is sufficient for the promoter activity. In this region, an ATF/CRE site with an adjacent GC box exists. To elucidate how these sites are involved in the promoter activity, we analyzed effects of point mutations at these sites on transcription byin vitrotranscription assays using nuclear extracts prepared from various rat tissues. Mutation at either site resulted in dramatic reduction of the promoter activity in all nuclear extracts, while mutation at both sites did not lead to further reduction. These results indicate that the ATF/CRE site and GC box are both essential for promoter activity and show synergistic activation. Electrophoretic mobility shift assay indicated that Sp1 and/or Sp3 bind to the GC box, and ATF1-CREB heterodimer binds to the ATF/CRE site. Since an element, ATF/CRE site-GC box, is conserved in mammalian Na,K-ATPase α1 subunit genes and in other constitutive promoters, we propose that this element is a critical unit for constitutive expression.