title: The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription.
abstract: BACKGROUND
  The NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 5'-regulatory area following CIE treatment.
  
  
  METHODS
  Primary cortical cultured neurons were subjected to ethanol treatment in a CIE paradigm. Bisulfite conversion followed by pyrosequencing was used for quantitative measurement and analysis of CpG methylation status within the 5'-regulatory area of the NR2B gene; chromatin immunoprecipitation (ChIP) assay was used to examine DNA levels associated with methylation and transcription factor binding. Electrophoretic mobility shift assay (EMSA) and in vitro DNA methylation assays were performed to determine the direct impact of DNA methylation on the interaction between DNA and transcription factor and promoter activity.
  
  
  RESULTS
  Analysis of individual CpG methylation sites within the NR2B 5'regulatory area revealed three regions with clusters of site-specific CpG demethylation following CIE treatment and withdrawal. This was confirmed by ChIP showing similar decreases of methylated DNA in the same regions. The CIE-induced demethylation is characterized by being located near certain transcription factor binding sequences, AP-1 and CRE, and occurred during treatment as well as after ethanol withdrawal. Furthermore, the increase in vitro of methylated DNA decreased transcription factor binding activity and promoter activity. An additional ChIP assay indicated that the CIE-induced DNA demethylation is accompanied by increased occupation by transcription factors.
  
  
  CONCLUSIONS
  These results suggest an important role of DNA demethylation in mediating CIE-induced NR2B gene up-regulation, thus implicating a novel molecular site of alcohol action.
authors.affiliation: Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America. qiang@uthscsa.edu
authors.forename: Mei
authors.initials: M
authors.lastname: Qiang
authors.forename: Ashley
authors.initials: A
authors.lastname: Denny
authors.forename: Jiguo
authors.initials: J
authors.lastname: Chen
authors.forename: Maharaj K
authors.initials: MK
authors.lastname: Ticku
authors.forename: Bo
authors.initials: B
authors.lastname: Yan
authors.forename: George
authors.initials: G
authors.lastname: Henderson
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D044127:Epigenesis, Genetic; D000431:Ethanol / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects*; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D017931:DNA Primers; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D000431:Ethanol