Protein-DNA Interactions at a Drug-responsive Element of the Human Apolipoprotein A-I Gene

Shui-Pang Tam,William Mohan,Zhiwei Wang,Zhang-Qun Chen,Xia Zhang

doi:10.1074/jbc.271.43.27152

Abstract

Previously, we demonstrated that when two human hepatoma cell lines, Hep3B and HepG2, were exposed to gemfibrozil, a hypolipidemic drug, a 2-fold induction in apolipoprotein A-I (apoA-I) mRNA levels resulted. To determine if mRNA stabilization was responsible for the changes in apoA-I mRNA levels, the half-lives for apoA-I mRNA were measured in the presence of actinomycin D with and without gemfibrozil. These experiments revealed no differences in stability. However, nuclear run-off assays indicated that the transcription rate of the apoA-I gene was increased 2-fold in gemfibrozil-treated cells. Transient transfection experiments also indicated that the induction of apoA-I mRNA level in response to gemfibrozil is mediated at the transcriptional level. We have identified two copies of the "drug-responsive element" (DRE) in the apoA-I promoter region that may be responsible for the increase in apoA-I transcriptional activity by gemfibrozil. Using gel mobility shift assays with a synthetic DRE oligonucleotide, we have demonstrated that exposure of Hep3B and HepG2 cells to gemfibrozil resulted in strong induction of a protein-DNA complex. The formation of this complex is highly sequence-specific as indicated by the DNA competition experiments. The drug-inducible nuclear proteins bind to the DRE of the human apoA-I gene with an apparent Kd of 4.1 nM. Methylation interference experiments 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 construct, a significant 2-fold induction in luciferase activity was observed in the presence of gemfibrozil following transient transfection of two human hepatoma cell lines, HepG2 and Hep3B. However, a plasmid containing one copy of mutated apoA-I-DRE oligomer did not confer responsiveness to gemfibrozil treatment. Furthermore, pGL2 (apoA-I -250 mutant DRE), which carried an internal mutation of the DRE in the human apoA-I proximal promoter region, showed no increase in luciferase activity in response to gemfibrozil. These results implicate protein-DNA interactions at the DRE region in the transcriptional induction of human apoA-I gene expression by gemfibrozil.

Highlights

ApoA-I1 is the major constituent of plasma high density lipoprotein (HDL) and participates in cholesterol ester formation as a cofactor for lecithin-cholesterol acyltransferase [1]
Effect of Actinomycin D on the Induction of Apolipoprotein AI mRNA Levels by Gemfibrozil—Previously, we demonstrated that exposure of HepG2 and Hep3B cells to gemfibrozil resulted in a 2-fold induction in apolipoprotein A-I (apoA-I) mRNA levels [11]
To determine whether inhibition of RNA synthesis affects the induction of apoA-I mRNA levels, Hep3B cells were treated both in the absence and presence of increasing amounts of actinomycin D for 30 min prior to the addition of gemfibrozil

Summary

EXPERIMENTAL PROCEDURES

Southwestern Blot Analyses—Equal volumes of nuclear extract and sample buffer (5% SDS, 5 mM Tris/HCl, pH 6.8, 200 mM DTT, 20% glycerol, and 0.5% pyronin Y) were mixed and subjected to electrophoresis on a 10% SDS-polyacrylamide gel at room temperature. The fragment was inserted into the XhoI site of the pGL2-Basic vector (Promega), upstream of the luciferase gene This plasmid is hereafter called pGL2 (apoA-I Ϫ491)luc. Plasmid pGL2 (apoA-I Ϫ250)luc was constructed by releasing a DNA fragment (Ϫ491 to Ϫ251 of the apoA-I promoter) from pGL2 (apoA-I Ϫ491) using SmaI, and the vector was gel-purified and religated. Plasmid pGL2 (apoA-I Ϫ250 mutant DRE)luc was prepared by a PCR-based protocol for extensive mutagenesis of a target DNA sequence as described by Morrison and Desrosiers [32]. The labeled oligonucleotides were subjected to Maxam and Gilbert sequencing [36], and the G ϩ A reactions serve as markers

RESULTS

Competition efficiencya

DISCUSSION