PROTEIN-DNA INTERACTIONS AT A DRUG-RESPONSIVE ELEMENT OF THE HUMAN APOLIPOPROTEIN-A-I GENE

Previously, we demonstrated that when two human hepatoma cell lines, H ep3B and HepG2, were exposed to gemfibrozil, a hypolipidemic drug, a 2 -fold induction in apolipoprotein A-I (apoA-I) mRNA levels resulted, T o determine if mRNA stabilization Nas responsible for the changes in a poA-I mRNA levels, the half-lives for apoA-I mRNA were measured in the presence of actinomycin D with and without gemfibrozil. These experim ents revealed no differences in stability, However, nuclear runoff ass ays indicated that the transcription rate of the apoA-I gene was incre ased 2-fold in gemfibrozil-treated cells, Transient transfection exper iments also indicated that the induction of apoA-I mRNA level in respo nse to gemfibrozil is mediated at the transcriptional level, We have i dentified two copies of the ''drug-responsive element'' (DRE) in the a poA-I promoter region that may be responsible for the increase in apoA -I transcriptional activity by gemfibrozil, Using gel mobility shift a ssays with a synthetic DRE oligonucleotide, we have demonstrated that exposure of Hep3B and HepG2 cells to gem fibrozil resulted in strong i nduction of a protein-DNA complex, The formation of this complex is hi ghly sequence-specific as indicated by the DNA competition experiments , The drug-inducible nuclear proteins bind to the DRE of the human apo A-I gene with an apparent K-d of 4.1 nM. Methylation interference expe riments have localized the contact sites of nuclear factors to the DRE region, Southwestern blot analyses have identified two groups of drug -inducible nuclear proteins with molecular masses of approximately 30 and 15 kDa. When a copy of synthetic DRE oligonucleotide was inserted upstream of the thymidine kinase promoter and luciferase reporter cons truct, a significant 2-fold induction in luciferase activity was obser ved in the presence of gemfibrozil following transient transfection of two human hepatoma cell lines, HepG2 and Hep3B, However, a plasmid co ntaining one copy of mutated apoA-I-DRE oligomer did not confer respon siveness to gemfibrozil treatment, Furthermore, pGL2 (apoA-I -250 muta nt DRE), which carried an internal mutation of the DRE in the human ap oA-I proximal promoter region, showed no increase in luciferase activi ty in response to gemfibrozil, These results implicate protein-DNA int eractions at the DRE region in the transcriptional induction of human apoA-I gene expression by gemfibrozil.