PROJECT SUMMARY DNA methylation is an epigenetic modification that plays a key role in regulating stem cells, development and many diseases. Abnormal DNA methylation has been observed in cancer for more than two decades, with many investigations focusing on promoter hypermethylation, which silences tumor suppressor genes. Additionally, DNMT3A, one of de novo DNA methyltransferases, is frequently mutated in a spectrum of hematological malignancies. Our lab has demonstrated that Dnmt3a loss impairs hematopoietic stem cell (HSC) differentiation, while expanding HSC numbers in bone marrow, suggesting DNMT3A may have a role in tumorigenesis and stem cell regulation. However, DNA methylation profiling of leukemia patient samples shows DNA methylation correlates poorly with gene expression across the genome, highlighting our limited understanding of the specific functions of DNA methylation. Recent studies using a murine model of the most frequent DNMT3A mutation in hematological malignancies, DNMT3AR882, demonstrated that DNMT3AR882 cooperates with FLT3-­ITD and NPM1c mutations to contribute to leukemic transformation. Nevertheless, our knowledge of which remaining DNMT3A mutations lead to leukemogenesis and the mechanisms by which they contribute to cancer formation remains lacking. Therefore, the long-­term goal of the proposed research is to understand how DNMT3A affects gene regulation in cancer, and how DNMT3A mutants predispose stem cell expansion. In Aim 1, I established a novel DNA epigenome editing tool (dCas9-­SunTag-­DNMT3A system) to investigate the causal relationship between DNA methylation and gene expression. Using pan-­cancer analysis of genome-­wide profiles, we have identified DNA hypermethylation occurring in the gene-­body regions of canyons (broad and undermethylated regions) with activation of corresponding gene expression. In Aim 2, using a Dnmt3a mutant murine model I developed, I will elucidate the role of one Dnmt3a mutant in priming stem cell expansion. The findings from this proposed research will shed the light on abnormal DNA methylation in cancer and molecular mechanisms of DNMT3A-­associated malignancies. Little is known about how mutations in epigenetic modifiers affect the 3D genomic structure in cancer. Therefore, in Aim 3, I plan to use my postdoctoral studies to understand how epigenetic modifiers shape the genomic landscape in cancer and their underlying mechanism. This training program is tailored to give me a comprehensive education in basic science research that will be extremely useful in achieving my long-­term career goal of becoming an independent cancer researcher.

PROJECT NARRATIVE DNA methylation, an epigenetic modification with widespread effects on gene expression, controls tissue specification during development. Aberrant DNA methylation has been observed in cancer for more than two decades, however, the role of abnormal DNA methylation in tumor formation is still largely unclear. Previously, our lab has demonstrated that Dnmt3a loss impairs hematopoietic stem cell (HSC) differentiation, while expanding HSC numbers in bone marrow, indicating potential roles of Dnmt3a in HSC regulation. However, whether a hypomorphic mutation in Dnmt3a leads to hematopoietic stem/progenitor cell expansion is unknown. Here I propose to use a novel DNA epigenome editing tool developed during my graduate school studies to investigate the relationship between DNA methylation and gene expression. I will also elucidate the role of a specific Dnmt3a in stem cell expansion. The overall goal of this research plan is to uncover novel therapeutic targets for epigenetic modifier-­associated diseases.