Mechanisms Regulating DNA Methyltransferases

The expression of specific genes that are regulated by transcription factors and epigenetic modifications is critical for normal cellular functioning and homeostasis. DNA methylation is an epigenetic modification that in mammals is crucial for the regulation of gene expression in a specific cell type. Aberrant loss or gain of DNA methylation is linked to cancer and several epigenetic disorders. DNA methylation is established by the de novo DNA methyltransferases Dnmt3a and Dnmt3b; however, the mechanisms underlying their target specificity remain to be fully understood. Dnmt3a and Dnmt3b can be regulated through interactions with other proteins, including histone modifications, and by their catalytic properties. The N-terminal domain of Dnmt3a interacts with chromatin and can potentially regulate its specific activity. Enzymatic studies have shown that the ADD domain of Dnmt3a autoinhibits its catalytic activity, which is relieved by binding of the ADD domain to an unmethylated H3K4 histone peptide. Using embryonic stem cell differentiation as a model system to study the regulation of Dnmt3a and Dnmt3b activity, we show that Lsd1-mediated H3K4 demethylation acts as an epigenetic switch to activate Dnmt3a specifically at the enhancers of pluripotency genes during differentiation. We show that Dnmt3b had only a minor role at these sites, in agreement with the previously published work showing distinct functions of these enzymes. Dnmt3a and Dnmt3b activity is involved in the development of several cancers, including Acute Myeloid Leukemia (AML). A specific mutation in the catalytic domain of Dnmt3a is highly prevalent in AML. Our studies show that this mutation (R878H) results in the loss of cooperative activity of the enzyme at multiple target sites on the DNA. To support these studies, we also developed an assay to quantify DNA methylation using the methylation dependent restriction enzyme MspJI to cleave DNA that is then used as a template for a quantitative PCR reaction. Use of MspJI increased the accuracy of this method as compared to using a traditional, methylation sensitive enzyme, HpaII. These studies contribute to our understanding of the basis of Dnmt3 target specificity and provide a framework to explain the basis of diseases with aberrant DNA methylation. They also provide both a basis to determine new therapeutic strategies and to evaluate DNA methylation as a biomarker for various disease states