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Identification of an octamer-1 transcription factor binding site in the promoter of the mouse mu-opioid receptor gene
- Source :
- Journal of neurochemistry. 67(4)
- Publication Year :
- 1996
-
Abstract
- 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.
- Subjects :
- Receptors, Opioid, mu
Biology
Transfection
Biochemistry
Polymerase Chain Reaction
Cell Line
Cellular and Molecular Neuroscience
Mice
Neuroblastoma
Genes, Reporter
Gene expression
Consensus Sequence
Tumor Cells, Cultured
Animals
Humans
Electrophoretic mobility shift assay
Histone octamer
Binding site
Nuclear protein
Luciferases
Promoter Regions, Genetic
Transcription factor
DNA Primers
Host cell factor C1
Homeodomain Proteins
Binding Sites
Base Sequence
Molecular biology
Recombinant Proteins
DNA binding site
DNA-Binding Proteins
Mutagenesis, Site-Directed
Host Cell Factor C1
Octamer Transcription Factor-1
Transcription Factors
Subjects
Details
- ISSN :
- 00223042
- Volume :
- 67
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- Journal of neurochemistry
- Accession number :
- edsair.doi.dedup.....d7bed20893b4fc3ea7e38543389a8896