title: Transcriptional regulation of mouse delta-opioid receptor gene by CpG methylation: involvement of Sp3 and a methyl-CpG-binding protein, MBD2, in transcriptional repression of mouse delta-opioid receptor gene in Neuro2A cells.
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.
authors.affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA. wangx212@umn.edu
authors.forename: Guilin
authors.initials: G
authors.lastname: Wang
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Wei
authors.forename: Horace H
authors.initials: HH
authors.lastname: Loh
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D018899:CpG Islands; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000077209:Decitabine; D004330:Drosophila; D017930:Genes, Reporter; D006655:Histone Deacetylases; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; C115368:MBD2 protein; C489690:MeCP1 histone deacetylase complex, mouse; D012333:RNA, Messenger; D017465:Receptors, Opioid, delta; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D012313:RNA; D000077209:Decitabine; D004247:DNA; D006655:Histone Deacetylases; D001374:Azacitidine