Abstract
Opioid receptors are expressed in a cell type-specific manner. Here we show that the mouse delta-opioid receptor (mDOR) gene is regulated by promoter region CpG methylation. The mDOR promoter containing a putative CpG island is highly methylated in Neuro2A cells, correlating with the repression of this gene in these cells. This is in contrast with the unmethylated state of the mDOR promoter in NS20Y cells, which express a high level of mDOR. Repression of mDOR transcription in Neuro2A cells could be partially relieved by chemically induced demethylation with 5-aza-2'-deoxycytidine. In addition, in vitro methylation of the luciferase reporter gene driven by the mDOR promoter resulted in an inhibition of transcription in NS20Y cells. Methyl-CpG-binding protein complex 1 (MeCP1) has been implicated in methylation-mediated transcriptional repression of several genes. Electrophoretic mobility shift assays showed that fully methylated, but not unmethylated, mDOR promoter fragment formed a MeCP1-like protein complex that contained methyl-CpG-binding domain protein 2 (MBD2) and Sp3. Furthermore, the expression level of Sp3 was decreased when Neuro2A cells were demethylated with 5-aza-2'-deoxycytidine, and increasing Sp3 levels in Schneider's Drosophila line 2 cells led to the repression of mDOR promoter activity when the promoter was methylated. These results demonstrate that Sp3 and MBD2 are involved in the transcriptional repression of mDOR in Neuro2A cells through binding to the methylated CpG sites in the promoter region and may play a role in the cell type-specific expression of mDOR.
Highlights
Central nervous system, it is found in the peripheral nervous system and some immune cells
The expression level of Sp3 was decreased when Neuro2A cells were demethylated with 5-aza-2-deoxycytidine, and increasing Sp3 levels in Schneider’s Drosophila line 2 cells led to the repression of mouse ␦-opioid receptor (mDOR) promoter activity when the promoter was methylated. These results demonstrate that Sp3 and methyl-CpG-binding domain protein 2 (MBD2) are involved in the transcriptional repression of mDOR in Neuro2A cells through binding to the methylated CpG sites in the promoter region and may play a role in the cell type-specific expression of mDOR
We report that the suppression of mDOR expression in Neuro2A cells is a result of promoter region CpG methylation and that Sp3 and MBD2 are involved in transcriptional repression of the mDOR promoter through binding of methylated CpGs and may play a role in mDOR cell type-specific expression
Summary
Central nervous system, it is found in the peripheral nervous system and some immune cells. Understanding the molecular mechanisms underlying the cell type-specific expression of DOR will provide insights into the regulation of the pharmacological benefits of ␦-opioids by manipulation of the DOR expression levels in certain cell types. Methylation of the cytosine residue in the 5Ј-CpG-3Ј sequence is an epigenetic modification involved in the establishment and maintenance of cell type-specific gene expression [7]. The mDOR gene core promoter has a G ϩ C content of more than 80%, raising the possibility that the CpG methylation may play a role in the regulation of cell typespecific expression of mDOR. We report that the suppression of mDOR expression in Neuro2A cells is a result of promoter region CpG methylation and that Sp3 and MBD2 are involved in transcriptional repression of the mDOR promoter through binding of methylated CpGs and may play a role in mDOR cell type-specific expression. The expression of the ␦-opioid receptor (DOR) is mainly confined to certain regions of the adult
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