CHARACTERIZATION OF AN ENHANCER UPSTREAM FROM THE MUSCLE-TYPE PROMOTER OF A GENE ENCODING 6-PHOSPHOFRUCTO-2-KINASE FRUCTOSE-2,6-BISPHOSPHATASE

The muscle-type isozyme of rat 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase is encoded by a mRNA transcribed from the M promoter of a 55-kb gene, which also produces the liver-type isozyme from an alternative promoter. By transient transfection and in vitro protein-DNA binding assays we have delineated, within 4.7 kb of 5' flanking sequence, the M promoter proper and an enhancer located between - 1615 and - 1809. This enhancer stimulated up to 12-fold the activity of the promoter in the context of an intact 5' flanking sequence and close to 900-fold the activity of the minimal (+ 41 to - 40) M promoter cloned directly downstream from it. A functional dissection of the enhancer by site-directed mutagenesis and use of oligonucleotides suggested that its activity involves the cooperative effect of six binding sites for trans-acting factors clustered within 150 bp. These sites contain either an EF-1A/E4TF1 motif (also known to bind the ets oncogene product) or a Spl motif, or both. The activity of the enhancer could be demonstrated in L6 myoblasts and myocytes and in FTO2B hepatoma cells. When left within the intact 5' flanking sequence, however, enhancer activity was inhibited upon differentiation of myoblasts into myocytes.