Identification And Functional Analysis Of Methyltransferases In Mammals

Epigenetic modifications are comprised of stable and heritable changes in gene function that occur without a change in DNA sequence. DNA methylation is an important epigenetic modification of the mammalian genome that regulates gene expression and chromatin organization. Aberrant DNA methylation has been found to be associated with a number of developmental disorders and cancers. DNA methylation is catalyzed by various methyltransferases, which are responsible for the establishment and maintenance of DNA methylation patterns during development. Studies of the enzymes will provide further insights into the regulation of this process.Part I Cbx4 promotes SUMO modification of DNA methyltransferase 3aDnmt3a is one of de novo DNA methyltransferases in mammals, which are responsible for the generation of somatic methylation patterns during embryogenesis and gametogenesis. It catalyzes transfer of a methyl group (-CH3) from S-adenosyl-L-methionine to the C-5 position of a cytosine residue. Inactivation of Dnmt3a by gene targeting blocks de novo methylation in early embryos and disrupts imprinting in the germ cells. Dnmt3a knockout mice died shortly after birth, indicating the critical role of the enzyme during development.Posttranslational protein modifications have been shown to regulate the functions of target proteins. A number of proteins are modified by covalent attachment of small ubiquitin-related modifier, SUMO. Sumoylation regulates various cellular processes, such as transcription, protein localization, protein-protein interaction and DNA repair. In our studies, we found that Dnmt3a can be modified by SUMO both in vivo and in vitro. Sumoylation occurs in the N-terminal regulatory region of Dnmt3a, suggesting it regulates the interaction of Dnmt3a with other proteins. We identified a Polycomb group protein, Cbx4, as a specific interaction partner of Dnmt3a. Cbx4 enhances sumoylation of Dnmt3a in vivo and in vitro, indicating it functions as a SUMO E3 ligase toward Dnmt3a.Part II Targeted disruption of a putative adenine methyltransferase gene N6amt1 results in embryonic lethalityC-5 methylcytocine, N-4 methylcytocine and N-6 methyladenine are three types of modified bases found in the genome of prokaryotes. Adenine methylation plays important roles in restriction-modificaton system, DNA replication, DNA mismatch repair and gene expression regulation in prokaryotes. Although over 1% of adenine is methylated in bacteria, methyladenine has not been found in mammalian genomes so far. However, some indirect evidences suggest the existence of methyladenine in higher eukaryotes. Our research focused on a putative mammalian DNA methyltransferase N6amt1, which is a homolog to N-6 adenine methyltransferases in bacteria. It is conserved in various eukaryotes, suggesting an important role of the protein. To determine whether N6amt1 methylates adenine residues in DNA, we examined its activity by in vitro assay. However, N6amt1 lacks DNA methyltransferase activity. To investigate the role of N6amt1 during development, we inactivated the gene by gene targeting in mice. We found that N6amt1-deficient embryos die at the early postimplantation stage, indicating its essential role during development.