Anomalous interaction of Sp1 and specific binding of an E-box-binding protein with the regulatory elements of the Na,K-ATPase alpha 2 subunit gene promoter.

We identified cis elements in the 5'-flanking region of rat Na,K-ATPase alpha 2 subunit gene (Atp1a2) using transient transfection assays in L6 rat skeletal muscle myoblast cells. By 5'-deletion mutation analysis, the region between nucleotide positions -175 and -108 was identified as a positive regulatory region. In the region, the distal E box (nucleotides -144 to -139) acts as a negative regulatory element, and the Sp1 consensus sequence (nucleotides -123 to -118) and the GGGAGG sequence (nucleotides -114 to -109) act as positive regulatory elements. Gel-retardation analysis revealed that binding factors are an E-box-binding protein and Sp1. DNase I footprinting and methylation-interference analyses revealed that Sp1 binds to the region from nucleotides -122 to -101 and the E-box-binding protein to the region from nucleotides -144 to -136. T4 DNA polymerase footprinting revealed that there are three Sp1-binding sites in the region and that Sp1 binds to one of the three sites in a mutually exclusive manner. The mechanism by which Sp1 activates the Atp1a2 promoter is discussed.