title: Methylation of a single intronic CpG mediates expression silencing of the PMP24 gene in prostate cancer.
abstract: BACKGROUND
  We previously demonstrated that a putative anti-tumor gene, peroxisomal membrane protein 4, 24 kDa (PMP24 or PXMP4), is silenced via DNA methylation of a CpG island in its 5' flanking region (5'-CGI) in prostate cancer (PCa) cells.
  
  
  METHODS
  To identify demethylation hypersensitive site(s) in PMP24 5'-CGI, PC-3 cells with methylated 5'-CGI were treated with a low-dose of 5-aza-2'-deoxycytidine (5-aza-dC) just sufficient to reactivate gene expression, referred as the limited demethylation approach. Gel shift assays and promoter analyzes were performed to demonstrate the role of the hypersensitive site in PMP24 gene regulation. Transfection of a methylated oligonucleotide corresponding to the hypersensitive site was conducted to determine the effect of site-specific methylation on the gene expression. Bisulfite sequencing analysis was performed to reveal the methylation status of PMP24 promoter in cultured cells and microdissected samples. In situ hybridization was applied to determine expression positivity of PMP24 mRNA.
  
  
  RESULTS
  A 5-aza-dC hypersensitive site encompasses two CpG dinucleotides in intron 1 was identified. Methylation of the first, but not the second, CpG dinucleotide of this site disrupted DNA-protein interactions and suppressed the gene expression. Using archival specimens, we found the first CpG dinucleotide of the hypersensitive site is hypermethylated with a loss of PMP24 mRNA expression in microdissected PCa cells when compared to normal prostatic epithelial cells.
  
  
  CONCLUSIONS
  These findings support a critical role for a single intronic CpG dinucleotide in PMP24 gene regulation through DNA methylation. The data suggest that methylation-mediated silencing of PMP24 is a molecular event associated with prostate carcinogenesis.
authors.affiliation: Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhang
authors.forename: Mengchu
authors.initials: M
authors.lastname: Wu
authors.forename: Hong
authors.initials: H
authors.lastname: Xiao
authors.forename: Ming-Tsung
authors.initials: MT
authors.lastname: Lee
authors.forename: Linda
authors.initials: L
authors.lastname: Levin
authors.forename: Yuet-Kin
authors.initials: YK
authors.lastname: Leung
authors.forename: Shuk-Mei
authors.initials: SM
authors.lastname: Ho
mesh_terms: D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D020868:Gene Silencing / Q000502:physiology*; D006801:Humans; D017403:In Situ Hybridization; D007438:Introns / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D008565:Membrane Proteins; C120047:PXMP4 protein, human