REGULATION OF HEMATOPOIETIC PROGENITORS BY DE NOVO DNA METHYLATION

项目总结/摘要 DNA甲基转移酶3A（DNMT 3A）已成为造血的关键调节因子。我们展示 DNMT 3A的缺失抑制造血干细胞（HSC）分化同时有利于自我更新， 建立了一种范式，通过这种范式，人体中体细胞DNMT 3A突变的无数影响， 目前查看。DNMT 3A是克隆性造血（CH）中最常见的突变基因，也是一种重要的肿瘤基因 抑制器。虽然DNMT 3A的广泛重要性是明确的，但它参与的机制 HSC分化仍然知之甚少。我的实验室的一个主要的长期兴趣是解开 DNMT 3A功能的分子机制。在过去4年的资助中，我的实验室为 在多个方面推进我们对DNMT 3A的理解，包括发现表型 DNMT 3A缺失的HSC，揭示了DNMT 3A与TET 1和TET 2之间的复杂相互作用， 描述了人类嵌合现象的表观遗传学改变，以及DNA甲基化峡谷作为非常长- 范围的相互作用。在下一阶段，我们将在这项工作的基础上，探索机制， DNMT 3A在HSC中起作用。我们将剖析DNMT 3A亚型在调节HSC中的不同作用， 分化和基因表达。我们亦会透过以下途径研究有关机制： DNMT 3A蛋白的稳定性如何以及对造血的影响。我们的初步数据显示 约1/3的DNMT 3A突变导致CH，导致蛋白质稳定性丧失，我们已经确定 一种参与其周转的E3-泛素连接酶复合物。这些研究提供了调节 突变体和WT DNMT 3A的降解具有潜在的治疗影响。最后，我们将研究 DNMT 3A突变体在骨髓和发育过程中的细胞竞争。使用细胞 使用具有不同预测适应性的不同DNMT 3A突变，我们将产生骨髓嵌合体， 研究DNA甲基化活性丧失与竞争优势程度之间的关系。 我们还将产生胚胎嵌合体，并检查突变与WT细胞对多种组织的贡献。 和血统。这些研究将揭示细胞竞争的基本原理，从而深入了解 克隆造血和干细胞动力学。总的来说，拟议的工作将促进我们对以下问题的理解： 造血中的克隆动力学和DNMT 3A在维持适当造血中的作用。研究 可能最终导致在病理条件下调节克隆动力学的策略。

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.