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Epigenetics. 4th Australian Scientific Conference, Adelaide, Australia, 7 - 09 May 2012
Normal development of the mammalian embryo requires epigenetic reprogramming of the genome. The level of cytosine methylation of CpG-rich (5meC) regions of the genome is a major epigenetic regulator and active global demethylation of 5meC throughout the genome is reported to occur within the first cell- cycle. This is followed by progressive demethylation of the maternal genome. This model of re- programming forms the central dogma of developmental epigenetics and has influenced all thinking in the field. We have reassessed the evidence for this phenomenon of genome-wide demethylation following fertilisation in the mouse. Using conventional methods of immunolocalization 5meC showed a progressive acid-resistant antigenic masking during zygotic maturation which gave the appearance of demethylation. Changing the unmasking strategy to include tryptic digestion revealed a persistence of the methylated state. Analysis of methyl-binding domain 1 protein (MBD1) binding confirmed that the genome remained methylated following fertilisation. The maintenance of this methylated state over the first several cell-cycles required the actions of DNA methyltransferase activity. Co-staining for 5- hydroxymethylC did not show a dynamic pattern of staining and does not provide support for a role for this metabolite in global demethylation. Analysis of somatic cells showed that trypsin-sensitive masking of 5mC also occurs to some degree and should be taken into account when using immune-based methodology. Reports of global demethylation in the zygote primarily results from antigenic masking of 5meC that occurs as a consequence of chromatin remodelling of the genome following fertilisation. The study shows that any 5meC remodelling that occurs in the zygote is not explained by a global active loss of 5meC staining and that global loss of methylation following fertilization is not a major component of epigenetic reprogramming in the mouse zygote. Supported by Grants from the Australian National Health and Medical Research Council 1