CHARACTERIZATION OF PROTEIN-DNA INTERACTIONS WITHIN THE PEROXISOME PROLIFERATOR-RESPONSIVE ELEMENT OF THE RAT HYDRATASE-DEHYDROGENASE GENE

A peroxisome proliferator-responsive element is located in the 5'-flan king region of the gene encoding rat hydratase-dehydrogenase, the seco nd enzyme of the peroxisomal beta-oxidation pathway. DNase I foot prin t analysis with nuclear extracts from proliferator responsive rat H4II EC3 cells revealed two protected regions within the 196-base pair pero xisome proliferator-responsive element. Both regions contained multipl e copies of a motif related to the consensus steroid hormone receptor binding half-site TGACCT, suggesting that peroxisome proliferator-depe ndent activation of this gene is mediated via peroxisome proliferator- activated receptors. Region II contains three TGACCT-like motifs in a direct repeat array. An oligonucleotide corresponding to this region w as sufficient to confer responsiveness to the peroxisome proliferator ciprofibrate onto a heterologous promoter, as determined by transient transfection assays. Gel retardation assays demonstrated that nuclear factors bound to the hydratase-dehydrogenase oligonucleotide. Mutation of a single G residue within the second repeat motif abolished factor binding and consequently the ability of the element to respond to cip rofibrate, directly demonstrating that factor binding is necessary for peroxisome proliferator responsiveness. These results are discussed i n the context of our current understanding of the mechanism of the coo rdinated transcriptional induction of the genes encoding peroxisomal b eta-oxidation enzymes by peroxisome proliferators.