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.