Genomic methylation patterns are established during gametogenesis and early development and propogated in somatic cells by clonal inheritance; disruption of methylation patterns causes fulminating expression of endogenous retroviruses, ectopic X inactivation, biallelic expression of imprinted genes, and death of differentiating cells. Dnmt1, the predominant DNA (cytosine-5)-methyltransferase of mammals, is involved in both the establishment and maintenance of methylation patterns. The Dnmt1 gene has sex-specific 5'exons and promoters that are expressed only in germ cells; these alternative exons may be involved in the establishment of sex-specific methylation patterns. Exon 1o (oocyte) is present only in postnatal growing oocytes and causes the accumulation of enourmous amounts of truncated but active Dnmt1 protein in the cytoplasm, whereas exon 1p (pachytene spermatocyte) blocks translation and eliminates all Dnmt1 protein at the pachytene stage of male meiosis even though large amounts of mRNA are present. Exon 1s (somatic) is used in all somatic cells. The sex-specific exons in Dnmt1 are proposed to play a role in the re-setting of the epigenetic program in the germline and in sex-specific DNA methylation that underlies the phenomenon of genomic imprinting. We will determine the stage of oogenesis and spermatogenesis at which imprinted loci acquire sex- specific methylation patterns and will examine primordial germ cells and cell lines derived from them (EG cells) for alternative splicing of 5' exons and cytoplasmic localization of Dnmt1 protein that could explain the dominant demethylating properties of EG cells in somatic cell hybrids. We will determine the roles of the sex-specific exons by targeted Cre-loxP deletion, and by forcing expression of the somatic form of Dnmt1 at stages where alternative forms are normally expressed. Remarkably little is known of the mechanisms that control the formation of methylation patterns in germ cells and the early embryo; the experiments described here will give important insights into an important regulator of genome organization that is unique to large- genome organisms: vertebrates and flowering plants.
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