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(347) Analysis of promoter methylation of Japanese medaka estrogen receptor and aromatase genes.
Contractor, Rooha1, Willett, Kristine*,1, Li, Shuanfang2, Chiang, Tung-chin3, Foran, Christy4, 1 University of Mississippi, University, MS, USA2 National Cancer Institute, Bethesda, MD, USA3 Tulane University, New Orleans, LA, USA4 West Virginia University, Morgantown, WV
ABSTRACT- Changes in methylation patterns of promoter regions play a key role in several chemical-induced alterations in gene expression. In order to explore a link between gene expression and environmental contaminants, we are determining methylation patterns in the promoter region of the estrogen receptor and the aromatase genes from tissues of the Japanese medaka, Oryzias latipes. Primers were designed to span the CpG islands of the promoter regions where there were suspected sites of methylation. Genomic DNA extracted from livers, gonads, and brains were treated with sodium bisulfite (resulting in a change of all non-methylated cytosine residues to uracil), amplified, cloned and sequenced. In the estrogen receptor, the female liver, brain and ovary had 13 and the male liver had 15 CpG islands in the promoter respectively, all of which were methylated. The male gonad and brain only had 12 and 13 CpG islands respectively, 11 of which were methylated. Because the overwhelming majority of the CpG islands showed methylation, we had hypothesized that one possible mechanism for the increase in transcription of the estrogen receptor was through a loss of methylation at these sites. Unexpectedly, the remaining three non-methylated islands (1 from gonad and 2 from brain) were methylated in fish exposed to 500 ng/L ethynylestradiol, but not in control males. The 2 unmethylated sites in the male brain were also methylated in fish exposed to 10 g/L benzo(a)pyrene. In contrast to the estrogen receptor, the aromatase gene had only 6 CpG islands in the amplified portion of the promoter region of the wild type male and female tissues. Further studies will look at the functional significance of the changes in methylation status.
Key words: medaka, methylation, estrogen receptor, aromatase
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