Molecular analysis of DNA methyltransferase deficient embryonic stem cells

The mammalian DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, are the enzymes responsible for the establishment and maintenance of DNA methylation patterns in mammalian cells. Previous studies of ES cells deficient in one of more of these enzymes have helped to elucidate the roles of these enzymes in various biological processes such as development, imprinting, and silencing of parasitic sequences. The work presented in this thesis describes a molecular analysis of a panel of DNA methyltransferase knockout cells. Methylation-sensitive AP-PCR analysis was performed on wild-type, Dnmt1 -/-, Dnmt3a-/-, Dnmt3b-/-, and [Dnmt3a -/-, Dnmt3b-/-] ES cells to globally assess their methylation patterns and to assess whether particular sequences are the preferred substrates for any of the three DNA methyltransferases. Our results showed hypomethylation in the Dnmt1 -/- and [Dnmt3a -/-, Dnmt3b-/-] cell lines with the methylation being globally decreased in the Dnmt1-deficient cell lines but more specifically decreased at repetitive sequences in the Dnmt3a/3b-deficient cell lines. The activities of the Dnmts during the cell cycle were analyzed by BrdU pulse-chase experiments. Wild-type and [ Dnmt3a-/-, 3b -/-] ES cells showed similar kinetics of methylation with methylation occurring continuously during the 3 hr after DNA replication while no increase in methylation was observed in the Dnmt1 -/- ES cells during this time. To quantitatively assess the role of DNA methylation on gene inactivation pathways, a cell-culture based assay system using TKNeo concatamers was used to detect and quantitate deletion, methylation, and mutation events in Dnmt1 -/- and Dnmt1 +/- ES cells. Gene inactivation rates due to gene loss and missense mutation events were lower in the Dnmt1-/- cells compared to the Dnmt1+/- cells. A high proportion of the missense mutations identified in the Dnmt1-/- cells were transition mutations in the context of CpG dinucleotides. This suggests the possibility of enzyme-mediated deamination by Dnmt3a, Dnmt3b, or Dnmt2. Observations that mutation rates were enhanced in Dnmt1-/- cells under methionine-deficient conditions lends further support for enzyme-mediated deamination by Dnmt3a, Dnmt3b, or Dnmt2.