Identification and characterization of a hepatoma cell-specific enhancer in the mouse multidrug resistance mdr1b promoter.
The expression of multidrug resistance/P-glycoprotein genes mdr1b(mdr1) and mdr1a(mdr3) is elevated during hepatocarcinogenesis. To investigate the regulation of mdr1b gene expression, we used transient transfection expression assays of reporter constructs containing various 5'-mdr1b flanking sequences in hepatoma and non-hepatoma cells. We found that nucleotides -233 to -116 preferentially enhanced the expression of reporter gene in mouse hepatoma cell lines in an orientation- and promoter context-independent manner. DNase I footprinting using nuclear extracts prepared from hepatoma and non-hepatoma cells identified four protein binding sites at nucleotides -205 to -186 (site A), -181 to -164 (site B), -153 to -135 (site C), and -128 to -120 (site D). Further analyses revealed that, while site B alone played a major part for the enhancer function, sites A and B combined conferred full enhancer activity. Site-directed mutagenesis results also supported these results. Gel retardation experiments using oligonucleotide competitors revealed that the site B contains a dominant binding protein. This is the first report demonstrating a cell type-specific enhancer in the mdr locus. The role of this enhancer in the activation of mdr1b gene during hepatocarcinogenesis is discussed.
Song
R
R
Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Ikeguchi
M
M
Zhou
G
G
Kuo
M T
MT
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D008113:Liver Neoplasms / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D014018:Tissue Distribution; D014162:Transfection; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004268:DNA-Binding Proteins