Kusui C, Kimura T, Ogita K, Nakamura H, Matsumura Y, Koyama M, Azuma C, Murata Y
Division of Obstetrics and Gynecology, Department of Specific Organ Regulation, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan.
Biochem Biophys Res Commun. 2001 Dec 7;289(3):681-6. doi: 10.1006/bbrc.2001.6024.
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 HpaII. 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 SssI 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.
在人类催产素受体（OTR）基因中，转录起始位点（TSS）上游140 bp至下游2338 bp存在一个CpG岛。我们研究了该区域的甲基化状态是否会影响OTR基因的转录。源自人肝母细胞瘤的HepG2细胞系中OTR基因转录受到抑制，用去甲基化剂5-氮杂胞苷（Aza-C）处理2天。半定量逆转录聚合酶链反应（RT-PCR）表明，Aza-C处理后OTR信使核糖核酸（mRNA）以剂量依赖方式显著增加。我们评估了外周血白细胞、未孕子宫肌层、足月子宫肌层和肝脏中OTR基因CpG岛内的甲基化水平。翻译起始位点上游5'端的一个1.5 kb区域被分为四个片段。用甲基化敏感限制酶HpaII完全消化后，通过聚合酶链反应（PCR）扩增每个片段。聚合酶链反应产物量在肝脏中最大，表明OTR基因中的这个CpG岛在肝脏中甲基化程度最高，该基因在肝脏中始终处于失活状态。我们比较了CpG岛的体内甲基化对OTR报告质粒转录活性的影响。用SssI甲基转移酶甲基化后，含有CpG岛的表达质粒-2860/+1342-GL3在HepG2细胞中的报告基因活性被抑制至对照水平的30.6%，而不含CpG岛的-2840/+144-GL3的报告基因活性仅被抑制至81.4%。肝脏和子宫之间甲基化水平差异最大的片段（MT2）缺失（-2860/+1342（del）MT2-GL3）后，抑制率恢复至68.0%。这些结果表明，人类OTR基因启动子中CpG岛的甲基化至少在肝脏中抑制了其转录，并可能调节器官间的组织特异性基因表达。
