Biochemical Investigation of the de novo DNA Methyltransferases DNMT3A and DNMT3B
DNA methylation is an epigenetic modification that is nearly ubiquitous. Eukaryotic DNA methylation contributes to the regulation of gene expression and maintaining genome integrity. In mammals, DNA methylation occurs primarily on the C5 carbon of cytosine in a CpG dinucleotide context and is catalyzed by the DNA methyltransferases, DNMT1, DNMT3A and DNMT3B. While dnmt3a and dnmt3b genes are highly homologous, the enzymes have distinct functions. Some previous reports suggested differences in the enzymatic behavior of DNMT3A and 3B, which could affect their biological roles. The goal of my thesis work was to characterize kinetics mechanisms of DNMT3A and 3B, and to identify the similarities and differences in their catalytic properties that contribute to their distinct biological functions. Given the sequence similarity between the enzymes, we asked whether DNMT3B was kinetically similar to DNMT3A. In a series of experiments designed to distinguish between various kinetics mechanisms, we reported that unlike DNMT3A, DNMT3B methylated tandem CpG on DNA in a processive manner. We also reported that the disruption of the R-D interface, critical for the cooperativity of DNMT3A, had no effect on DNMT3B activity, supporting the non-cooperative mechanism of this enzyme.
DNMT3A is frequently mutated in numerous cancers. Acute Myeloid Leukemia (AML) is a malignancy of hematopoietic stem cells in which numerous patients exhibit a high frequency of the heterozygous somatic mutation Arg882His in DNMT3A. Through thorough consensus motif building, we discovered a strong similarity in CpG flanking sequence preference between DNMT3A Arg882His variant and DNMT3B enzyme. Moreover, we found that the variant enzyme has the same kinetics mechanism as DNMT3B, indicating a gain-of-function effect caused by the mutation. This change is significant because the variant enzyme can aberrantly methylate DNMT3B targets in AML cells and effect global gene expression. In particular, given that DNMT3B has been shown to have oncogenic properties, this suggests that the Arg882His variant can acquire similar oncogenic properties and drive AML development.
Taken together, my thesis work provides novel insights into the relationship between the biochemical properties and the biological functions of DNMT3A and 3B.
Regulation of Dnmt3 Activity at Enhancers of Cell Identity Genes During Differentiation
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