ANOMALOUS INTERACTION OF SP1 AND SPECIFIC BINDING OF AN E-BOX-BINDINGPROTEIN 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-ATPas e alpha2 subunit gene (Atp1 a2) using transient transfection assays in L6 rat skeletal muscle myoblast cells. By 5'-deletion mutation analys is, the region between nucleotide positions -175 and -108 was identifi ed as a positive regulatory region. In the region, the distal E box (n ucleotides -144 to -139) acts as a negative regulatory,element, and th e Spl consensus sequence (nucleotides -123 to -118) and the GGGAGG seq uence (nucleotides -114 to -109) act as positive regulatory elements. Gel-retardation analysis revealed that binding factors are an E-box-bi nding protein and Sp1. DNase I footprinting and methylation-interferen ce analyses revealed that Sp1 binds to the region from nucleotides -12 2 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 Sp 1 activates the Atp1 a2 promoter is discussed.