Localization of promoter elements in the human mu-opioid receptor gene and regulation by DNA methylation.
The regulation of mu-opioid receptor gene expression was investigated using several molecular techniques. Genomic clones containing portions of the human mu-opioid receptor gene were sequenced. 5'-RACE analysis of human brain cDNA confirmed the presence of mRNAs up to -313 from the start codon. As was found for the mouse and rat genes, transcription apparently initiates in the absence of a discernable TATA box. To characterize promoter function, portions of the 5'-flanking region were linked to a reporter gene in transient transfection experiments. Two approximately 50 bp adjacent segments had potent, orientation specific promoter activity. More down-stream segments also had promoter activity. None of the 5'-flanking region constructs showed tissue specificity. The potential role of DNA methylation in preventing ectopic expression was investigated by surveying the methylation state of a CpG rich region straddling the start codon. A neural derived cell line (SH-SY5Y) that expresses the mu-opioid receptor lacked virtually any CpG methylation. In contrast, two neural derived cell lines that do not express the mu-opioid receptor were nearly totally methylated while non-neural cell lines had intermediate levels of CpG methylation. Additional transient transfection experiments revealed that CpG methylation of the 5'-flanking region suppressed reporter gene expression. These results indicate that CpG methylation plays an important role in regulating mu-opioid receptor expression in neural cells; however, no association was found with regulation of expression in non-neural cells.
Andria
M L
ML
Department of Psychiatry, New York University Medical Center, Millhauser Labs HN605, New York, NY 10016, USA. andrim01@mcrcr.med.nyu.edu
Simon
E J
EJ
D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D018899:CpG Islands; D019175:DNA Methylation; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007668:Kidney; D007739:L Cells; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D009447:Neuroblastoma / Q000473:pathology; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats; D017450:Receptors, Opioid, mu / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D008156:Luciferases