title: Location-specific epigenetic regulation of the metallothionein 3 gene in esophageal adenocarcinomas.
abstract: BACKGROUND
  Metallothionein 3 (MT3) maintains intracellular metal homeostasis and protects against reactive oxygen species (ROS)-induced DNA damage. In this study, we investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs).
  
  
  RESULTS
  Using quantitative bisulfite pyrosequencing, we detected unique DNA methylation profiles in the MT3 promoter region. The CpG nucleotides from -372 to -306 from the transcription start site (TSS) were highly methylated in tumor (n = 64) and normal samples (n = 51), whereas CpG nucleotides closest to the TSS (-4 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from -139 to -49 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, -log10(FDR)>3.0]. The DNA methylation levels from -127 to -8 CpG sites showed the strongest correlation with MT3 gene expression (r = -0.4, P<0.0001). Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively). The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells. Treatment of OE33 cells with 5-Aza-deoxycitidine restored MT3 expression with demethylation of its promoter region and reversal of the histone modifications towards active histone marks.
  
  
  CONCLUSIONS
  In summary, EACs are characterized by frequent epigenetic silencing of MT3. The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.
authors.affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.
authors.forename: Dunfa
authors.initials: D
authors.lastname: Peng
authors.forename: Tian-Ling
authors.initials: TL
authors.lastname: Hu
authors.forename: Aixiang
authors.initials: A
authors.lastname: Jiang
authors.forename: Mary Kay
authors.initials: MK
authors.lastname: Washington
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Moskaluk
authors.forename: Regine
authors.initials: R
authors.lastname: Schneider-Stock
authors.forename: Wael
authors.initials: W
authors.lastname: El-Rifai
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine / Q000494:pharmacology; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D044127:Epigenesis, Genetic / Q000187:drug effects; D004938:Esophageal Neoplasms / Q000235:genetics* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D052138:Genes, Neoplasm / Q000235:genetics*; D006657:Histones / Q000378:metabolism; D006801:Humans; D008207:Lymphatic Metastasis / Q000235:genetics; D000090023:Metallothionein 3; D008875:Middle Aged; D009367:Neoplasm Staging; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012097:Repressor Proteins / Q000378:metabolism
chemical_list: D006657:Histones; D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D012097:Repressor Proteins; D001374:Azacitidine