REGULATION OF HEMATOPOIETIC PROGENITORS BY DE NOVO DNA METHYLATION

项目摘要/摘要 DNA甲基转移酶3A(DNMT3A)是一种重要的造血调控因子。我们展示了 DNMT3A的缺失抑制了造血干细胞(HSC)的分化，而有利于自我更新， 建立人类体细胞DNMT3A突变的无数影响的范例 当前查看。DNMT3A是克隆性造血中最常见的突变基因，也是一种重要的肿瘤 抑制者。虽然DNMT3A的广泛重要性是显而易见的，但它参与的机制 HSC的分化仍然知之甚少。我的实验室的一个主要长期兴趣是解开 DNMT3A作用的分子机制。在过去4年的资助中，我的实验室致力于 从多方面加深对DNMT3A的理解，包括表型永生化的发现 DNMT3A缺失的HSC，揭示了DNMT3A与TET1和TET2之间的复杂相互作用， 人类嵌合体的表观遗传改变的描述，以及DNA甲基化峡谷作为非常长的- 范围交互作用。在下一阶段，我们将在这项工作的基础上，探索通过以下机制 DNMT3A在HSC中发挥作用。我们将剖析DNMT3A亚型在调节HSC中的不同作用 利用独特的基因敲除小鼠进行分化和基因表达。我们还将通过以下方式研究这些机制 其中DNMT3A蛋白的稳定性维持及对造血的影响。我们的初步数据显示 导致CH的DNMT3A突变中约有三分之一会导致蛋白质稳定性丧失，我们已经确定 一个E3-泛素连接酶复合体，可能与其周转有关。这些研究提供了调整的机会 突变体和WT DNMT3A的降解具有潜在的治疗作用。最后，我们将研究以下基础 DNMT3A突变体在骨髓和发育过程中的细胞竞争。使用单元格 不同的DNMT3A突变具有不同的预测适合度，我们将产生骨髓嵌合体和 研究DNA甲基化活性丧失与竞争优势程度的关系。 我们还将产生胚胎嵌合体，并检查突变的VS WT细胞对多种组织的贡献 和血统。这些研究将揭示管理细胞竞争的基本原则，从而深入了解 克隆性造血和干细胞动力学。总体而言，拟议的工作将促进我们对 造血中的克隆动力学和DNMT3A在维持正常造血中的作用。这些研究 最终可能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.