Identification of an octamer-1 transcription factor binding site in the promoter of the mouse mu-opioid receptor gene.
In a previous study we showed that a region from -182 to +10 bp in the mouse mu-opioid receptor (MOR) promoter exhibited strong promoter activity. To identify protein-DNA interactions in this fragment, gel shift and DNase I footprint analyses were performed using nuclear extracts from mouse brain and the human neuroblastoma cell line, SK-N-SH. Two regions, nucleotide (nt) -121 to -100 and nt -42 to -22, were identified as being specific protein binding sites. The protein-DNA interaction in the nt -42 to -22 region was characterized in detail in this study. Methylation interference analysis of this region showed that nuclear protein from SK-N-SH cells contracted nucleotides within the sequence ATG-CAAAT, which is a binding motif for octamer trans-acting factors. An octamer-1 (Oct-1)-specific antibody super-shifted the protein-DNA complex in a gel shift assay. A UV cross-linking experiment showed that a nuclear protein, whose molecular weight is similar to that of the Oct-1 factor, bound to the octamer element in the nt -42 to -22 region. Mutagenesis of four base pairs within the octamer cis-acting element eliminated the specific protein binding in vitro. When the MOR-luciferase reporter construct (-182 to +10 bp) with the same four base pairs mutated was transiently transfected into SK-N-SH cells, a 200% increase in transcriptional activity was observed. Collectively, these data suggest that Oct-1 is binding to the octamer motif in the MOR gene and negatively modulating MOR gene expression.
Liang
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Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Carr
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D016384:Consensus Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000378:metabolism*; D051863:Host Cell Factor C1; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009447:Neuroblastoma; D050811:Octamer Transcription Factor-1; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017450:Receptors, Opioid, mu / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; C497693:Hcfc1 protein, mouse; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; C494166:Pou2f1 protein, mouse; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D014157:Transcription Factors; D008156:Luciferases