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 act ivity. To identify protein-DNA interactions in this fragment, gel shif t 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 identifi ed as being specific protein binding sites. The protein-DNA interactio n in the nt -42 to -22 region was characterized in detail in this stud y, Methylation interference analysis of this region showed that nuclea r protein from SK-N-SH cells contacted 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 tha t 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-lucife rase reporter construct (-182 to +10 bp) with the same four base pairs mutated was transiently transfected into SK-N-SH cells, a 200% increa se 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.