REGULATION OF HEMATOPOIETIC PROGENITORS BY DE NOVO DNA METHYLATION

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Project Summary/Abstract DNA METHYLTRANSFERASE 3A (DNMT3A) has emerged as a key regulator of hematopoiesis. We showed that loss of DNMT3A inhibited hematopoietic stem cell (HSC) differentiation while favoring self-renewal, establishing the paradigm through which the myriad effects of somatic DNMT3A mutations in humans are currently viewed. DNMT3A is the most frequently mutated gene in clonal hematopoiesis (CH) and a critical tumor suppressor. While the broad importance of DNMT3A is clear, the mechanisms through which it participates in HSC differentiation remain poorly understood. A major long-term interest of my lab is to unravel the molecular mechanisms of DNMT3A function. In the previous 4-years of funding, my lab has contributed to advancing our understanding of DNMT3A in multiple facets, including discovery of immortalization of phenotypic HSCs with DNMT3A loss, uncovering the complex interactions between DNMT3A and TET1 and TET2, description of epigenetic alterations with human mosaicism, and DNA methylation canyons as sites of very long- range interactions. In the next phase, we will build on this work, exploring the mechanisms through which DNMT3A functions in HSCs. We will dissect the distinct roles of DNMT3A isoforms in regulating HSC differentiation and gene expression using unique knock-out mice. We will also examine the mechanisms through which stability of DNMT3A protein is maintained and the impact on hematopoiesis. Our preliminary data indicate that about 1/3 of DNMT3A mutations that contribute to CH lead to loss of protein stability and we have identified an E3-ubiquitin ligase complex putatively involved in its turnover. These studies offer the opportunity to modulate degradation of mutant and WT DNMT3A with potential therapeutic impact. Finally, we will examine the basis of cell competition of DNMT3A mutants in the context of both bone marrow and during development. Using cells with different DNMT3A mutations with distinct predicted fitness, we will generate bone marrow chimeras and investigate the relationship between loss of DNA methylation activity and the degree of competitive advantage. We will also generate embryo chimeras and examine the contribution of mutant vs WT cells to multiple tissues and lineages. These studies will reveal fundamental principles governing cell competition and thus insights into clonal hematopoiesis and stem cell dynamics. Overall, the proposed work will advance our understanding of clonal dynamics in hematopoiesis and the role of DNMT3A in maintaining proper hematopoiesis. The studies could ultimately lead to strategies to modulate clonal dynamics in pathologic conditions.