Studies of the dynamic patterns of DNA methylation during early mammalian development

Epigenetic mechanisms of gene regulation utilize the dynamic nature of complex DNA-protein interactions that form the chromatin structure of eukaryotic DNA. The studies described in this thesis were performed under the general hypothesis that DNA methylation plays an important role in the developmental process and is required for normal mammalian development. I have demonstrated dynamic patterns of cytosine methylation that include both allele- and sequence-specific variations during early preimplantation development. It was shown that allele-specific patterns of methylation exist at the Nf1 locus in the 2-cell murine embryo. The bisulfite sequencing of the Nf1 coding region also led to the discovery of allele-specific non-CpG methylation in the oocyte and early preimplantation embryo.; My analysis of genomic patterns of methylation at multiple sites during the 2-cell stage of development has revealed a generalized, possibly genome-wide phenomenon of non-CpG methylation. In addition, it was found that the de novo methyltransferases Dnmt3a and Dnmt3b are expressed in the oocyte and early preimplantation stages of development and hypothesize that these enzymes are responsible for the establishment of non-CpG methylation.; This thesis also tests the general hypothesis that demethylation of gene regulatory regions are associated with transcriptional activation and that methylation is associated with transcriptional silencing. A correlation between transcriptional activation and demethylation of the myogenin promoter has been demonstrated. I have also shown a similar correlation between hypomethylation and active transcription for a myogenin-LacZ transgene, indicating that the relationship between transcriptional activation and demethylation of the myogenin promoter is not restricted to the location of the endogenous gene. I propose that methylation of the myogenin promoter is critical during early development.; In this thesis presents the novel discovery of non-CpG methylation occurring in gametes and the early stages of preimplantation development and suggest that this novel form of developmental methylation is established by the de novo methyltransferases Dnmt3a and Dnmt3b during gametogenesis. It also present an argument in support of the host defense hypothesis for cytosine methylation in mammals and proposes that non-CpG methylation may play a protective role during gametogenesis by acting to decrease the frequency of aberrant recombinational events during meiosis.