Demethylation and hydroxymethylation in the mouse zygote
Résumé
In the mouse zygote, DNA methylation patterns undergo important changes and differ in the maternal and paternal pronucleus. Demethylation of the paternal genome has been described as an active and replication-independent process, although the mechanisms responsible for it are not yet fully understood. Recently, 5-hydroxymethylcytosine has been suggested as an intermediate in active demethylation. To investigate this question, we have quantified DNA methylation and hydroxymethylation in both pronuclei of the mouse zygote with a focus on the replication period and examined their patterns on the pericentric heterochromatin using 3D immuno-FISH. Our results demonstrate that 5-methylcytosine and 5-hydroxymethylcytosine localizations on the pericentric sequences are not complementary; indeed we observe no enrichment of either marks on some regions and an enrichment of both on others. In addition, we show that DNA demethylation continues during DNA replication, and is inhibited by aphidicolin. Finally, we observe notable differences in the kinetics of demethylation and hydroxymethylation; in particular, a peak of 5-hydroxymethylcytosine, unrelated to any change in 5-methylcytosine level, is observed after completion of replication. Together our results support the hypothesis that 5-hydroxymethylcytosine is not a simple intermediate in an active demethylation process and could play a role of its own during early development.