Transcriptional Regulation of Mouse δ-Opioid Receptor Gene by CpG Methylation

Abstract

Opioid receptors are expressed in a cell type-specific manner. Here we show that the mouse ␦-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.Opioids display strong analgesic effects as well as addictive properties through the activation of three major types of Gprotein-coupled receptors referred to as -, ␦-, and -opioid receptors (1). Whereas all three opioid receptors mediate opioid-induced analgesia, each receptor type exerts a distinct pharmacological profile as well as a unique cell type-specific expression pattern (2, 3). The expression of the ␦-opioid receptor (DOR) 1 is mainly confined to certain regions of the adult central nervous system, although it is found in the peripheral nervous system and some immune cells. It has been reported that the expression levels of DOR determine the activities of the DOR agonist, and the localization of DOR generally matches the pharmacological action sites of ␦-opioids. Thus 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. We reported previously (4) the isolation of mouse genomic clones of DOR (mDOR), and we identified transcription upstream stimulatory factors Sp1/Sp3 and Ets-1 that functionally interact with the mDOR promoter and activate it in a mouse neuronal cell line NS20Y (5, 6). As yet, no data are available on the mechanisms involved in the cell type-specific expression of mDOR.Methylation of the cytosine residue in the 5Ј-CpG-3Ј seq...

CpG methylation regulates allelic expression of GDF5 by modulating binding of SP1 and SP3 repressor proteins to the osteoarthritis susceptibility SNP rs143383

“…Thus, the increased chondrocyte GDF5 expression associated with AZA- and siRNA-mediated promoter demethylation presumably results from increased binding of transcription factors that are inhibited by methylation and through prevention of binding by methyl-binding proteins such as MeCP1 and MeCP2. It is also possible that the levels of proteins that regulate GDF5 expression are altered in these cells, and AZA treatment has previously been reported to decrease levels of SP3, a transcriptional repressor of GDF5 , in human and mice cells (Lee et al 2004; Wang et al 2003). …”

“…Methylation also has variable effects on SP3 binding affinity, with methylation inhibiting binding of SP3 to the chondrocyte-specific ChM - 1 promoter (Aoyama et al 2004), leading to gene repression. In contrast, methylation increases binding of SP3 and the methyl-binding protein MBD2 to the mouse δ-Opioid receptor ( mDOR ) promoter (Wang et al 2003), and SP3 can recruit chromatin remodelling proteins leading to gene repression (Won et al 2002). As well as altering binding affinity, methylation can also alter the function of SP3, with SP3 increasing the SP1-mediated activity of the unmethylated mDOR promoter but inhibiting SP1-mediated promoter activity of the methylated mDOR promoter (Wang et al 2003).…”

“…In contrast, methylation increases binding of SP3 and the methyl-binding protein MBD2 to the mouse δ-Opioid receptor ( mDOR ) promoter (Wang et al 2003), and SP3 can recruit chromatin remodelling proteins leading to gene repression (Won et al 2002). As well as altering binding affinity, methylation can also alter the function of SP3, with SP3 increasing the SP1-mediated activity of the unmethylated mDOR promoter but inhibiting SP1-mediated promoter activity of the methylated mDOR promoter (Wang et al 2003). A similar mechanism may occur at rs143383, with methylation increasing the repressive activity of SP3 despite reducing SP3 binding affinity, and this could explain why SP3 represses the methylated 5′UTR luciferase plasmid more strongly than the unmethylated plasmid.…”

Transcriptional and Epigenetic Regulation of Opioid Receptor Genes: Present and Future

“…All these studies have been conducted using reporter constructs in a heterologous cellular background; therefore, the physiological relevance of these regulatory signals again remains to be validated. In this regard, an examination of DNA methylation of the endogenous Oprd locus has established that it is epigenetically regulated (93, 94) (see Section 4).…”

Epigenetic control of the expression of opioid receptor genes

“…For the DOR gene, a MeCP1-like molecule rendered DNA methylation on multiple CG sites of its promoter. 19,20 For the MOR gene, siRNA experiment provided the evidence for a role of MeCP2 in methylating CG sites of this promoter. 21 The chromatin of MOR regulatory region in P19 appeared to maintain a stable, and regularly spaced nucleosome array even in later stages of differentiation when MOR transcription was highly active.…”