Synergism of the ATF/CRE site and GC box in the housekeeping Na,K-ATPase alpha1 subunit gene is essential for constitutive expression.
Na,K-ATPase alpha1 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 alpha1 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 by in vitro transcription 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 alpha1 subunit genes and in other constitutive promoters, we propose that this element is a critical unit for constitutive expression.
Kobayashi
M
M
Department of Biology, Jichi Medical School, Minamikawachi, Tochigi, Kawachi, 329-0498, Japan.
Kawakami
K
K
D051696:Activating Transcription Factors; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D016384:Consensus Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D003596:Cytosine; D006147:Guanine; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics*; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D011401:Promoter Regions, Genetic; D051381:Rats; D016415:Sequence Alignment; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000235:genetics*; D016385:TATA Box; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D051696:Activating Transcription Factors; D001798:Blood Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D007527:Isoenzymes; D046911:Macromolecular Substances; D014157:Transcription Factors; D006147:Guanine; D003596:Cytosine; D000254:Sodium-Potassium-Exchanging ATPase