Genomic imprinting is a molecular process that distinguishes, with different epigenetic marks, the maternal and paternal alleles of genes. Central to this process is the creation of differentially methylated domains (DMDs) in which one parental allele is methylated and the opposite allele is unmethylated. DMDs develop through a two- step mechanism in which a DNA cytosine methylation pattern (imprint) is first placed on one of the parental alleles (in oocyte or sperm depending on the gene) and this epigenetic modification is then maintained (inherited). DMDs govern the imprinting of most, if not all, of the imprinted genes in the developing fetus and associated extraembryonic tissues. Our results indicate that the maintenance of methylation marks on DMD sequences during preimplantation development is crucial to the imprinting process and thus to the developing embryo. The main objective of this proposal is to study the roles of the DNMT1 DNA cytosine methyltransferases, DNMT1o and DNMT1s, in this maintenance process. DNMT1s is expressed in somatic cells and DNMT1o is an oocyte-derived DNMT1 variant that is expressed in oocytes and also during preimplantation development. The studies are divided into two Aims, each addressing a specific hypothesis: (1) We hypothesize here that the inheritance of genomic imprints during preimplantation development depends on maintenance methylation activities of both oocyte- and zygote-derived DNMT1 proteins. Hypotheses regarding the preimplantation roles of DNMT1o and DNMT1s will be tested in a variety of experiments examining the effect of Dnmt1 gene mutations on the maintenance of genomic imprints in mouse ES cells. (2) We hypothesize here that specific regions of the DNMT1o and DNMT1s proteins are involved in the inheritance of genomic imprints, and that the specific functions of these regions can be deduced by studying appropriate DNMT1 mutants in vivo.