Date of Award
Doctor of Philosophy (PhD)
Heterozygous loss-of-function mutations in the DNMT3A gene are the most common cause of clonal hematopoiesis, and among the most common initiating events for Acute Myeloid Leukemia (AML). Meanwhile, mutant DNMT3A cause an overgrowth syndrome, DNMT3A Overgrowth Syndrome (DOS, also known as Tatten-Brown-Rahmen syndrome, TBRS). Amino acid R882H mutation is the most frequent variance of DNMT3A, while the rest of the mutations spread out on the entire gene of DNMT3A. Over 70% of DNMT3A mutants cause loss of function change. A reduction of DNMT3A activity causes a canonical, focal hypomethylation phenotype in hematopoietic cells, which is associated with immortalization of hematopoietic stem cells, and age-dependent myeloid skewing. Over a period of several months, the methylation defect can be partially reversed with restoration of physiologic levels of DNMT3A expression. In Chapter 2, we characterize the DNA methylation phenotypes of bone marrow cells from mice with hematopoietic cell deficiency of Dnmt3a, Dnmt3b (or both enzymes), or expressing the dominant negative Dnmt3aR878H mutation (R882H in humans; the most common mutation found in AML patients). Using these bone marrow cells as substrates, we define the patterns and completeness of DNA remethylation after "adding back" supraphysiologic levels of wild type DNMT3A1, DNMT3B1, DNMT3B3 (an inactive splice isoform of DNMT3B), or DNMT3L (a catalytically inactive "chaperone" for DNMT3A and DNMT3B in early embryogenesis). High level expression of DNMT3A for two weeks can accurately reverse the hypomethylation phenotype of Dnmt3a deficient cells, or cells expressing the R878H mutation. Remarkably, overexpression of DNMT3L (which is not expressed in AML cells) can likewise correct the hypomethylation phenotype of Dnmt3aR878H/+ bone marrow cells ex vivo and in vivo, probably by augmenting the activity of WT DNMT3A encoded by the residual WT allele. These data suggest that reactivation of DNMT3L may represent a novel approach for restoring DNMT3A activity in AML initiated by DNMT3A mutations.The severity of hypomethylation phenotypes caused DNMT3A mutants varies, but they still cause AML and DOS with variant phenotypes. We hypothesized that DNMT3A mutants may have contribute to diseases with other mechanisms besides hypomethylation, such as altered DNA binding ability, disruption of protein-protein interactions, or decreased binding methyl-donor SAM. In Chapter 3, we chose 21 single nucleotide variants in DNMT3A found in AML and/or DOS. To get a comprehensive view of the interactome of WT and mutant DNMT3A, we used TurboID biotin ligase proximity labeling system followed by mass spectrometry analysis. We were able to identify most of previously reported DNMT3A interacting proteins. Meanwhile, we also found some unstable DNMT3A mutants have increased interactions with ubiquitin ligases and heat shock proteins, and decreased interactions with methylated DNA binding proteins and histone modifiers. Last, some of DNMT3A mutants have disrupted interactions with DNMT3B. The dominant isoform of DNMT3B in somatic cells are an inactive DNMT3B3, which is like DNMT3L can interacted with DNMT3A and stimulate the methyltransferase activity. It is possible in hematopoietic cells, DNMT3B3, taking place of DNMT3L, interact with DNMT3A and facilitate the methylation process. Loss of interaction of DNMT3B3 in some of the mutant DNMT3A could lead to further reduction of enzymatic activity. Overall, our data suggested that besides intrinsic loss of function, DNMT3A mutants have many other mechanisms, including protein instability, disrupted protein-proteins interactions and protein mislocalization.
Chair and Committee
Timothy J. Ley
Stuart Kornfeld, Daniel Link, David Spencer, Matthew Walter,
Li, Yang, "Evaluating the function of DNMT3A mutations associated with Acute Myeloid Leukemia and DNMT3A Overgrowth Syndrome" (2023). Arts & Sciences Electronic Theses and Dissertations. 2877.