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Regulation of DNA Methylation and Hydroxymethylation in Postmitotic Neurons and Other Mammalian Cells

  • Author(s): Le, Thuc Minh
  • Advisor(s): Faull, Kym F
  • Fan, Guoping
  • et al.
Abstract

DNA methylation and hydroxymethylation are two major covalent modifications that occur to the cytosine bases in the mammalian genome. A family of DNA methyltransferase (Dnmt), including maintenance Dnmt1 and de novo Dnmt3a and Dnmt3b, transfers a methyl group onto the 5th carbon of the cytosine base to form 5-methyl-cytosine (5mC). Further modification on the 5mdC is catalyzed by ten-eleven translocation enzymes in which a hydroxyl group is added onto the methyl group of the 5mC to form 5-hydroxymethyl-cytosine (5hmC). During development both Dnmt and Tet act in concert to regulate the pattern and extent of DNA methylation and hydroxymethylation, respectively. By understanding how the two DNA modifications are related in their levels and the kinetics of these processes, factors that have direct impact on development and disease can be ascertained. Methods using combined liquid chromatography and tandem mass spectrometry were established to measure both the global levels and kinetics of 5mC and 5hmC in mouse embryonic stem cells (ESCs) and neurons. Using mouse ESCs lines that encompass all eight possible combinations from the three major Dnmts, the global levels DNA methylation and hydroxymethylation are robustly related. This finding indicates that 5mdC is a rate limiting factor in 5hmdC formation. In addition, the kinetic rate of DNA methylation determined by the genetic combination of the Dnmts was positively correlated to its DNA methylation level. The combination of the de novo and maintenance Dnmts has the fastest kinetic rate than either de novo or maintenance Dnmts combination alone.

For many years, DNA methylation studies were done on cancer and development because it was known that the DNA methylation is very active and that the DNA methylation activity is diminished in terminally differentiated cells. Using the mass spectrometric methods, I measured levels of DNA methylation in control and Dnmt mutant mice. The forebrain excitatory neurons of the conditional Dnmt1 and Dnmt3a knockout (DKO) mutant mice had a loss of about 15% of the DNA methylation level when compared to age-matched control littermate. This loss was accompanied by abnormal synaptic plasticity in the hippocampus and behavioral deficits in learning and memory tasks.

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