Nucleic Acids Research, Vol 26, Issue 19 4454-4461, Copyright © 1998 by Oxford University Press
P Collas
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 approximately 12 h
post-injection. When methylated in vitro by Hpa II 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.
ARTICLES
Modulation of plasmid DNA methylation and expression in zebrafish embryos
Norwegian College of Veterinary Medicine, Department of Biochemistry, PO Box 8146 Dep., 0033 Oslo, Norway. philippe.collas@veths.no
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