Promoter of the Na,K-ATPase alpha3 subunit gene is composed of cis elements to which NF-Y and Sp1/Sp3 bind in rat cardiocytes.

Na,K-ATPase alpha subunit has three isoforms whose expression is regulated developmentally and hormonally. Na,K-ATPase alpha3 subunit gene (Atpla3) is expressed only in brain and neonatal heart in a rat. The purpose of this study is to analyze cis-acting elements and trans-acting factors regulating the transcription of Atpla3 in cultured neonatal rat cardiocytes. Transient transfection assays with Atpla3-luciferase chimeric construct and a series of 5' sequential deletion mutations revealed the existence of positive regulatory elements from -74 to -59 and from -59 to -39. A factor was identified to bind across -59 by gel retardation assay. Methylation interference and DNase I footprinting analyses revealed the binding region from -74 to -53 (positive regulatory element (PRE) 1). The binding factor was identified to be NF-Y by gel retardation assay using specific antibody. Gel retardation and methylation interference analyses revealed that factors bind to two other elements from -54 to -43 (PRE2) and from -25 to -13 (PRE3). The binding factors were identified to be Sp1/Sp3 using specific antibodies. The functions of above-mentioned three elements were examined by transient transfection assay with various combinations of mutations. They all regulated the transcription positively and a synergistic enhancement of it was observed. Roles of NF-Y in the transcriptional activation and synergy are discussed.