Date of Award
Doctor of Philosophy (PhD)
Epigenetic mechanisms are critical for precisely controlling gene expression programs to allow for the development and function of the brain, and disruptions of these processes are highly associated with neurodevelopmental disease. DNA methylation is one mechanism used to regulate gene expression, and neurons employ a unique form of DNA methylation particularly at CA dinucleotides (mCA), that is not widely observed in other somatic cell types. The DNA methyltransferase DNMT3A is required to establish mCA, and mutations in DNMT3A are associated with autism spectrum disorder (ASD) and an overgrowth and intellectual disability disorder called Tatton-Brown Rahman Syndrome (TBRS). However, it remains unclear how these disease-associated mutations alter DNMT3A function to impact the neuronal epigenome and disrupt nervous system function.Using in vitro and in vivo models, we systematically analyzed the functional consequences of DNMT3A mutations and defined effects that can contribute to disease. We modeled diverse missense mutations identified in TBRS and ASD in vitro and revealed that many of these mutations are functionally null and result in depletion of neuronal mCA. Mutations within different canonical protein domains resulted in distinct mechanisms of disruption such as reducing protein expression or ablating catalytic activity. We then examined the effects of loss-of-function mutations in vivo by generating and characterizing a DNMT3A heterozygous KO mouse model. This mutant recapitulated many aspects of TBRS, including increased bone length, progressive obesity, and behavioral disruptions. When we measured changes in neuronal DNA methylation, we observed a striking ~50% loss of neuronal mCA with site-specific changes in mCG. These changes in DNA methylation were enriched at critical gene regulatory elements such as promoters, gene bodies, and enhancers. Mutation of the methyl-binding protein MeCP2 which binds to mCA has been shown to disrupt enhancer activity and alter expression of long neuronal genes, and we observed a similar disruption in enhancer activity in the DNMT3A mutant. Furthermore, the DNMT3A mutant exhibited neuronal gene expression changes that overlap with MeCP2 mutants, as well as other neurodevelopmental disorders including ASD. Together, this work established a foundation for understanding the consequences of DNMT3A mutation in the nervous system. Phenotypic heterogeneity is commonly observed in neurodevelopmental disorders, and DNMT3A-disordres are just one of many examples of disruptions in epigenetic regulators resulting in a wide spectrum of diagnoses ranging from ASD to severe intellectual disability. However, the molecular mechanisms driving this variable disease presentation within a single monogenic disorder remain unclear. Notably, the majority of mutations identified in DNMT3A-associated neurodevelopmental disease are missense mutations, therefore we next assessed how different mutations may contribute to variable disease severity. To achieve this, we characterized and compared two new missense mutant models: the R878H mutation, which hematopoietic studies have indicated may be more severe than other mutations, and the P900L mutation which mimics a common mutation observed in both TBRS and in individuals with a primary diagnosis of ASD. While both mutations cause disease-relevant overgrowth and obesity phenotypes, the R878H mutation resulted in more severe behavioral disruptions than the P900L, thus demonstrating that this mutation can cause increased disease severity. To uncover the mechanisms that may be driving these differences, we performed extensive genomic analysis and revealed that though the P900L mutation resulted in a similar 50% loss of neuronal mCA to the heterozygous null, the R878H mutant had a more dramatic 75% reduction in methylation. Furthermore, the R878H mutation resulted in more extensive disruptions of enhancer activity and gene expression than the P900L. Next, we identified shared transcriptional disruption across multiple DNMT3A mutants that likely contribute to common TBRS pathology and revealed convergent transcriptional disruption between TBRS models and models of other neurodevelopmental diseases such as Rett Syndrome and Sotos Syndrome. We also defined mutation-specific changes in gene expression that suggest that mild ASD-like phenotypes are associated with changes in synaptic and axonal genes, whereas severe phenotypes are observed in mutants exhibiting disruptions in key biological processes such as protein folding and transport. These findings not only shed light on the mechanisms driving DNMT3A disorders, but also suggest biological processes that may be contributing to phenotypic heterogeneity. Finally, we investigated how loss of DNMT3A in postmitotic neurons contributes to disease-relevant phenotypes. Individuals with intellectual disability and autism often have a number of comorbidities, and similarly, TBRS patients also report musculoskeletal, cardiovascular, and endocrine disruptions. To begin to uncover how changes in neuronal DNMT3A may be contributing to these phenotypes, we generated a neuronal-specific heterozygous DNMT3A KO and measured obesity and overgrowth phenotypes. Our analysis revealed a surprising sex-specific effect of neuronal DNMT3A on fat regulation and demonstrated that DNMT3A disruption in the germline and the nervous system result in opposing effects on bone length. These findings indicate that DNMT3A mutations have a nuanced contribution to overgrowth and obesity phenotypes, likely through discrete germline and neuronal mechanisms. Overall, this work reveals key functions of neuronal DNA methylation in regulating gene expression pathways and uncovers mechanisms contributing phenotypic heterogeneity in neurodevelopmental disease. These findings serve as the foundation for future investigations of cellular effects of DNMT3A disruption in diverse tissues and processes and paves the way for development of future therapeutics. We also uncover convergent transcriptional disruption between DNMT3A mutants and other neurodevelopmental disease models, indicating a targetable epigenetic pathway for the treatment of multiple disorders, and highlighting the importance of neuronal DNA methylation for development and function of the nervous system.
Chair and Committee
Harrison W. Gabel
Joseph C. Corbo, Joseph D. Dougherty, Kristen L. Kroll, Jason J. Yi,
Beard, Diana Christian, "Understanding the Spectrum of DNMT3A Mutations in Neurodevelopmental Disease" (2023). Arts & Sciences Electronic Theses and Dissertations. 2831.
Available for download on Monday, April 14, 2025