THE SOMATIC FORM OF THE MOUSE DNMT1 MAINTENANCE METHYLTRANSFERASE IS EXPRESSED THROUGHOUT PREIMPLANTATION DEVELOPMENT

CHAILLET, JR; CIRIO, MC; NAVARA, C

Congreso; Society for the Study of Reproduction (SSR); 2007

The inheritance of gametic epigenetic modifications of DNA isessential to the process of genomic imprinting, which in turnis essential for normal mammalian development. Patterns of CpGdinucleotide methylation on imprinted genes appear during gametogenesisdue to the action of de novo cytosine methyltransferase activity.Although the inheritance of these methylation patterns duringpost-implantation development is due to the potent maintenancemethyltransferase activity of the somatic form of the Dnmt1cytosine methyltransferase (Dnmt1s), the molecular mechanismfor maintaining imprinted methylation patterns during preimplantationdevelopment remains largely unknown. The goal of this studywas to determine if Dnmt1s is a possible candidate enzyme forthis mechanism by determining if Dnmt1s is expressed in wild-typepreimplantation embryos. For these studies we used the UPT82rabbit polyclonal antibody, which recognizes epitopes specificto Dnmt1s protein, but does not detect the shorter, oocyte-specificDnmt1o protein. Improved, sensitive techniques of Dnmt1s immunodetectionwith UPT82 were used. On immunoblots of extracts from 100 stagedoocytes or staged embryos, UPT82 detects Dnmt1s in fully-grownoocyte (GV and MII stages), and in embryos at the 1-cell, 2-cell,4-cell, 8-cell, morula and blastocyst stages. Using immunostainingand confocal microscopy, we showed that Dnmt1s is present inthe cytoplasm at all stages, and in the nuclei of all stagesexcept the 1-cell, pronuclear-stage embryos. Dnmt1s also failedto appear in the maternal and paternal pronuclei even in pronuclear-stageembryos derived from mouse strains that overexpress Dnmt1s inoocytes, and show greatly increased cytoplasmic Dnmt1s staining.Because DNA replication is very likely to have been completedat the time of pronuclear membrane breakdown, these observationsindicate that DNA replicated at the first embryonic cell cycleis probably not exposed to Dnmt1s until well after the replicationevent. Using crosses between wild-type mice and mice carryingthe Dnmt1V allele, which express only the Dnmt1o form of Dnmt1,we showed that Dnmt1s protein expressed in 1-cell and 2-cellembryos is derived from the oocyte, whereas Dnmt1s expressedafterwards is synthesized de novo during embryogenesis. Thesefindings indicate that Dnmt1s is expressed in all diploidnuclearstages of mouse preimplantation embryos, and suggest thereforethat Dnmt1s is a strong candidate for the maintenance methyltransferaseactivity required for the inheritance of methylation imprintsin the early mouse embryo.