DNA Methylation of the Human Oxytocin Receptor Gene Promoter Regulates Tissue-Specific Gene Suppression

Abstract In the human oxytocin receptor (OTR) gene, there is a CpG island from 140 bp upstream to 2338 bp downstream of the transcription start site (TSS). We investigated whether the methylation state of this region affects the transcription of the OTR gene. HepG2 derived from human hepatoblastoma, in which OTR gene transcription was suppressed, was treated with a demethylating agent, 5-azacytidine (Aza-C) for 2 days. Semiquantitative RT-PCR indicated that OTR mRNA was significantly increased by Aza-C treatment in a dose-dependent manner. We estimated the level of methylation within the CpG islands of the OTR gene in peripheral blood leukocytes, nonpregnant uterine myometrium, term uterine myometrium and liver. A 1.5-kb region located 5′ upstream of the translation start site was divided into four fragments. Each was amplified by PCR after complete digestion with methylation-sensitive restriction enzyme Hpa II. The amount of PCR products was largest in the liver, suggesting that this CpG island in the OTR gene is most highly methylated in liver, where the gene is always inactivated. We compared the effect of in vivo methylation of the CpG island on transcriptional activity of an OTR-reporter plasmid. The reporter gene activity of expression plasmid −2860/+1342-GL3, containing the CpG island, in HepG2 cells was suppressed to 30.6% of the control level after methylation with Sss I methylase, while that of −2840/+144-GL3, without the CpG island was suppressed only to 81.4%. The deletion of the segment (MT2) where the level of methylation was most different between liver and uterus (−2860/+1342 del MT2-GL3) rescued the suppression rate to 68.0%. These results indicate that the methylation of the CpG island in the human OTR gene promoter suppressed its transcription at least in liver and may regulate tissue specific gene expression among organs.