Modulation of plasmid DNA methylation and expression in zebrafish embryos

Gene expression is under the influence of DNA methylation and assembly of chromatin structure. This paper reports the modulation of transgene expression in zebrafish embryos by altering DNA methylation with 5-azacytidine and heterochromatin formation with sodium butyrate, an inhibitor of histone deacetylation, A CMV promoter-luciferase fusion gene construct (pCMVL) microinjected into zebrafish eggs becomes gradually methylated during development, starting at similar to 12 h post-injection. When methylated in vitro by HpalI methylase prior to injection, the construct is rapidly demethylated in vivo before being de novo methylated. Demethylation is independent of DNA replication, indicating that it is an active DNA repair process. Demethylating activity has been characterized in zebrafish embryo nuclear extracts, in which this activity is heat-labile, sensitive to protease and RNase and requires ATP hydrolysis, Demethylating activity in vitro is dependent on the developmental stage of the embryo from which extracts are prepared. In vivo, luciferase transcripts are detected prior to de novo plasmid methylation. Furthermore, incubation of pCMVL-injected embryos with 5-azacytidine or butyrate immediately after injection inhibits plasmid methylation and extends the period of luciferase expression, When applied after de novo methylation has occurred, both inhibitors prevent methylation of newly replicated DNA and promote transgene expression. These data suggest that methylation of the injected construct during early development induces repression of the transgene, perhaps by converting the construct to a repressive chromatin structure.