DNA methylation levels in the 5' flanking region of the vitellogenin I gene in liver and brain of adult zebrafish (Danio rerio)--sex and tissue differences and effects of 17alpha-ethinylestradiol exposure.

Vitellogenin is produced in the liver of sexually mature female fish in response to endogenous estrogens. Exogenous estrogens also induce synthesis of vitellogenin in the liver of male and juvenile fish and vitellogenin is a frequently used biomarker for estrogen exposure. The epigenetic state, e.g. histone acetylation and DNA methylation, in the region of a gene or in its 5' flanking region influences the gene expression. DNA methylation positions in multicellular eukaryotes are mostly found on cytosine bases located 5' to guanine, i.e. in CpG sites. Here, we have for the first time analyzed the DNA methylation levels of three CpG sites located in the 5' flanking region of the vitellogenin I gene in liver and brain from adult zebrafish (Danio rerio) utilizing Pyrosequencing technology. This sequencing technique allows determination of methylation levels of multiple individual CpG sites. Our purpose was to assess any differences in methylation levels related to sex, tissue and exposure to estrogen. Out of the seven vitellogenin genes identified in the zebrafish, vitellogenin I is the most highly expressed during vitellogenesis. We found that the methylation levels of all three CpG sites were higher in male liver than in female liver. In brain, which does not express vitellogenin, females and males showed similar, high methylation levels in the analyzed CpG positions. Exposure of adult zebrafish to 17alpha-ethinylestradiol (100 ng/L) for 14 days decreased the methylation levels in the 5' flanking region of vitellogenin I in the liver in both females and males. These results suggest that induced expression of vitellogenin in fish following exposure to estrogens might involve alterations in DNA methylation.