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Localisation of DNA methyltransferase isoforms during preimplantation embryogenesis (#110)

Michelle Kay Yi Seah ¹ , Yan Li ¹ , Chris O'Neill ¹

Centre for Developmental and Regenerative Medicine, Kolling Institute of Medical Research, Sydney Medical School, University of Sydney, St Leonards, NSW, Australia

During early embryogenesis, temporal and spatial regulation of gene expression controlling cell fate and differentiation is mediated by epigenetic modification such as DNA methylation. DNA methyltransferase (DNMT) enzymes mediate methylation of CpG dinucleotides through two processes: (i) maintenance methylation (DNMT1); and (ii) de novo methylation (DNMT3A and 3B). The accepted paradigm of epigenetic reprogramming in embryos holds that there is preferential demethylation of the paternal pronucleus while the maternally-derived DNA undergoes passive demethylation. Recent analyses show that the presumed loss of methylation following fertilization is accounted for by a progressive onset of antigenic masking during zygotic maturation (). Full antigenic retrieval by tryptic digestion showed that high levels of DNA methylation persisted throughout zygotic maturation and subsequent mitoses to the 8-cell stage. Thereafter, cytosine methylation was lost from the inner cells of the embryo. In the blastocyst, the trophectoderm remained hypermethylated while the nuclei of the inner cell mass were substantially hypomethylated. In order to understand the mechanisms mediating the differential methylation patterns of the inner cell mass and trophectoderm, protein localization of the various DNMT isoforms were examined by immunolocalization. Each DNMT enzyme showed characteristic patterns at each stage of development. DNMT1 was restricted to the cytoplasm of blastomeres at each stage of preimplantation development. DNMT3a was expressed in the nucleus of cleavage stage embryos but was progressively lost from the 8-cell stage, so that blastocysts were largely devoid of DNMT3A. DNMT3B was absent from the early stage embryos but increased in the nuclei from the 8-cell stage through to blastocysts. Interestingly, DNMT3B was only found to be localized to the nuclei of the outer cells of the morula and the trophectoderm of the blastocyst, little was detected in the inner cells at each stage. The patterns of expression of the DNMT isoforms suggest a role for DNMT3A as the dominant methylating enzyme in the cleavage stage embryo and DNMT3B as the dominant enzyme after the 8-cell stage. The differential localisation of DNMT3B in the inner and outer cells of the embryo may account for the lineage-specific pattern of differential methylation in the early embryo.