Structural and functional analyses of the promoter of the murine multidrug resistance gene mdr3/mdr1a reveal a negative element containing the AP-1 binding site.
We have previously shown that the multidrug-resistance/P-glycoprotein gene, mdr3/mdr1a, is activated in mouse hepatocellular carcinomas (HCC). In this study, we show that in a number of HCC-derived cell lines (Hepa1c1c, Hepa1c1c-BprC1, and Hepa1-6) mdr3 is expressed at high levels. To investigate transcriptional regulation of mdr3 in these cells, we have isolated a DNA fragment containing the 5' portion of the mouse mdr3 gene and performed a functional analysis of its promoter. Transient transfection assays using various lengths of the promoter sequence to direct expression of the chloramphenicol acetyltransferase (CAT) reporter gene revealed that the sequence located -94 nucleotides upstream from mouse mdr3 transcription start site functions as a negative element in mouse hepatoma cells. A canonical AP-1 binding sequence TGA-GTCA located at -117 is at least in part responsible for the negative effect from the following observations: (i) Alteration of this AP-1 sequence by site-directed mutagenesis enhanced CAT expression. (ii) Expression of CAT reporter gene was elevated when double-stranded DNA containing the AP-1 sequence, but not mutated sequences, was used as a competitor in cotransfection experiment. (iii) Enhancement of the CAT expression was also seen in cotransfection experiments using recombinant plasmid DNA expressing the c-jun/c-fos proteins, which interact with AP-1 sequences. Interestingly, the proximal region of the hamster pgp1 promoter shares striking sequence similarity with that of the mouse mdr3 gene, including the AP-1 site, but the AP-1 site in the hamster promoter serves as a positive regulator. Although previous studies have demonstrated that positive and negative transcription factors can modulate gene expression through interactions with c-jun/c-fos, this is the first study to show that an AP-1 site functions as a negative cis-element in the regulation of gene expression.
Department of Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
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Ikeguchi
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Teeter
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Eckersberg
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Ganapathi
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Kuo
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D002678:Chimera; D002874:Chromosome Mapping; D006224:Cricetinae; D004351:Drug Resistance / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D008113:Liver Neoplasms / Q000235:genetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000502:physiology; D016755:Proto-Oncogene Proteins c-jun / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun