Regulation of de novo DNA Methylation by Inactive DNMT3B Splice Variants

DNA methylation is a key epigenetic mechanism involved in development, gene regulation, genomic stability, and silencing parasitic DNA elements. In humans and other mammals, three DNA methyltransferases (DNMTs) actively catalyze the transfer of methyl groups to DNA, including DNMT3A and DNMT3B, which primarily act as de novo DNMTs, and DNMT1, which primarily acts as the maintenance DNMT. Together, DNMT3A, DNMT3B, and DNMT1 establish and maintain the cell-type specific DNA methylation patterns in mammalian genomes. DNMT3L, an inactive DNMT, also plays a role in de novo DNA methylation by directly binding to DNMT3A and DNMT3B and stimulating their activity. DNMT3B, unlike DNMT1 and DNMT3A, has a high number of known alternatively spliced isoforms, many of which are catalytically inactive since they are missing key regions in the C-terminal catalytic domain. DNMT3B3, the major splice variant in somatic cells and tumor cell lines, is associated with active DNA methylation despite being inactive. By contrast, overexpression of DNMT3B4 in hepatocellular carcinoma and chronic myeloid leukemia has been linked to DNA hypomethylation. Inactive DNMT3B isoforms are conserved between human and mouse, display developmental and tissue-specific expression, are associated with changes in DNA methylation, and are often overexpressed in cancer, yet their functions in normal and disease states remain unclear.;In order to explain changes in DNA methylation that result from the expression of inactive DNMT3B variants I used DNMT3L as a paradigm for the regulation of de novo DNMTs by inactive variants, and thus tested the hypothesis that inactive DNMT3B variants physically associate with and modulate the activity of catalytically competent DNMT3 molecules. In Chapter 2 of this dissertation I provide evidence that DNMT3B3 binds to catalytically competent DNMT3 molecules, and in doing so, stimulates overall catalytic activity by weakening DNA binding of the complexes. In Chapter 3 of this dissertation I provide evidence that DNMT3B4 also binds to catalytically competent DNMT3 molecules, but in doing so, acts as a dominant-negative inhibitor of DNA methylation by strongly inhibiting DNA binding of the complexes. My results suggest that binding and regulation of active DNMT3 members by in inactive DNMT3 variants might be a general mechanism of how DNMT3 proteins function. This offers a mechanism that could account, at least in part, for some of the changes in DNA methylation profiles observed in development, different tissue types, and disease states like cancer.