MECHANISTIC ASPECTS OF GENOME-WIDE DEMETHYLATION IN THE PREIMPLANTATION MOUSE EMBRYO

Gene-specific methylation patterns in mammals play a role in a variety of biological processes in the embryo and adult tissues. These patter ns are established during embryo development by a process that involve s genome-wide demethylation in the morula and de novo methylation in t he pregastrula. To elucidate the mechanism of demethylation in the ear ly mouse embryo, we have injected mouse zygotes with gene sequences th at were methylated in vitro by Hpa II methylase and analyzed the methy lation status of specific sites in blastocyst DNA. Because it had been propagated in Escherichia coli, the DNA used for these injections was also methylated at adenine residues in GATC sites. This allowed us to eliminate fully methylated, unintegrated DNA by Dpn I digestion and f ully unmethylated, integrated DNA that underwent several rounds of rep lication by Mbo I digestion. The integrated, originally injected DNA s trands were in a hemimethylated state and survived this treatment. The methylation status of Hpa II sites in these molecules was analyzed by Hpa II digestion of the genomic DNA isolated from blastocysts, follow ed by PCR amplification using appropriate primers. The results demonst rate that demethylation is achieved by an active mechanism and that sp ecific sites in imprinted genes escape demethylation, maintaining a me thylated state throughout preimplantation development.