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Abstract:
The mechanisms responsible for active DNA demethylation remain elusive
in Metazoa. A previous study that utilized zebrafish embryos provided a
potent mechanism for active demethylation in which three proteins, AID,
MBD4, and GADD45 are involved. We recently found age-dependent DNA
hypomethylation in zebrafish, and it prompted us to examine if AID and
MBD4 could be involved in the phenomenon. Unexpectedly, however, we
found that most of the findings in the previous study were not
reproducible. First, the injection of a methylated DNA fragment into
zebrafish eggs did not affect either the methylation of genomic DNA,
injected methylated DNA itself, or several loci tested or the expression
level of aid, which has been shown to play a role in demethylation.
Second, aberrant methylation was not observed at certain CpG islands
following the injection of antisense morpholino oligonucleotides against
aid and mbd4. Furthermore, we demonstrated that zebrafish MBD4 cDNA
lacked a coding region for the methyl-CpG binding domain, which was
assumed to be necessary for guidance to target regions. Taken together,
we concluded that there is currently no evidence to support the proposed
roles of AID and MBD4 in active demethylation in zebrafish embryos.
