CHARACTERIZATION OF AN INTRONIC HORMONE RESPONSE ELEMENT OF THE RAT-LIVER SKELETAL-MUSCLE 6-PHOSPHOFRUCTO-2-KINASE FRUCTOSE-2,6-BISPHOSPHATASE GENE/

The glucocorticoid response element of the rat liver/skeletal muscle 6 - phosphofructo-2-kinase/fructose-2,6-bisphostatase gene was character ized. The element is composed of two tandem hormone receptor binding s ites separated by 12 base pairs. Addition of dexamethasone to HeLa cel ls transiently transfected with a chloramphenicol acetyl transferase ( CAT) reporter plasmid containing the hormone response element and cotr ansfected with glucocorticoid receptor stimulated transcription 24-fol d in an orientation- and position-independent manner. Deletion or muta tion of essential G/C pairs of the distal binding site abolished hormo ne-stimulated CAT activity, whereas deletion or mutation of the proxim al binding site decreased the hormone-stimulated response only slightl y. Mutation of both distal and proximal binding sites resulted in comp lete loss of hormone-stimulated CAT activity. Experiments carried out using testosterone and progesterone with their respective receptors re vealed qualitatively similar results to those seen with glucocorticoid . Binding of glucocorticoid receptor or androgen receptor DNA binding domains to the hormone response element, visualized by gel mobility sh ift, was unaffected in the proximal binding site mutant, markedly decr eased in the distal binding site mutant, and abolished in the double m utant. In gel mobility shift analysis of separate distal and proximal binding sites, only the native distal site demonstrated high affinity binding to glucocorticoid and androgen receptor DNA binding domains. T he results demonstrate that this element is responsible for glucocorti coid, androgen, and progesterone stimulation of transcription of the p hosphofructo-2-kinase/fructose-2,6-bisphosphatase gene and that the di stal receptor binding site is dominant. (C) 1997 Elsevier Science Irel and Ltd.