Regulation of Hematopoietic Progenitors by de novo DNA methylation

 描述（由申请人提供）：造血干细胞（HSC）通过其自我更新和分化的能力在整个生命过程中再生外周血。DNA甲基转移酶3a（DNMT 3A）在小鼠和人的HSC中有高表达，其条件性缺失促进了HSC的自我更新，但同时也损害了HSC的分化。在人类中，发现DNMT 3A在约10%的骨髓增生异常综合征（MDS）患者和约20%的骨髓性或淋巴性白血病患者中发生突变。DNMT 3A突变似乎出现在HSC中，赋予突变细胞优势，因为这些突变存在于非恶性和恶性克隆以及所有谱系的HSC后代中。最后，携带DNMT 3A突变的细胞在50岁以上的个体的外周血中是常见的，这些个体在其外周血中表现出克隆优势。总之，来自小鼠和人类的这些研究无可争议地指出了DNMT 3A在维持正常造血中的核心作用。然而，这些突变如何赋予HSC优势并促进克隆扩增以及MDS和白血病的发展仍不清楚。深入了解这些机制是本项目的首要目标。在这里，我们将确定DNMT 3a突变是否会导致分化受阻，DNA甲基化功能本身是否对造血影响至关重要，以及不同的DNMT 3A突变是否具有相似的作用。具体而言，我们将通过绘制整个HSC分化过程中DNA甲基化变化，确定DNMT 3A蛋白在整个基因组中的定位，调节DNMT 3a-KO HSC中参与自我更新或分化的基因，以及通过调节DNA甲基化来研究分化中的作用。此外，我们将确定DNMT 3A在HSC中发挥作用是否需要DNA甲基化。我们将向KO HSC中添加保留或缺乏催化功能的特异性DNMT，并检查后代的DNA甲基化和HSC活性。最后，我们将检查常见的DNMT 3A突变体，如R882形式，以确定它们对DNA甲基化和造血的影响。总之，这些研究将明确地将分化抑制与DNMT 3A功能丧失联系起来，并揭示了可用于治疗策略的关键靶点。这些研究还将证明催化性DNA甲基转移酶活性是否是完整HSC功能所必需的，并深入了解一些常见DNMT 3A突变的影响。我们的实验室处于独特的位置，可以为这个不断增长的领域做出贡献，因为我们对DNMT 3A在HSC中的作用进行了开创性的工作，并且我们开发了几种新的工具和试剂来解决所提出的问题。

 DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) regenerate the peripheral blood throughout life via their capacity to both self-renew and differentiate. A critica role for DNA methyltransferases in maintaining effective HSC function has recently come to light through a series of studies in mouse and man. Our laboratory recently found that that DNA methyltransferase 3a (DNMT3A) was highly expressed in murine HSCs, and its conditional loss promoted dramatic self-renewal at the expense of differentiation. In humans, DNMT3A was found to be mutated in around 10% of myelodysplastic syndrome (MDS) patients, and around 20% of patients with either myeloid or lymphoid leukemia. DNMT3A mutations appear to arise in the HSCs, conferring an advantage to mutant cells, as these mutations are found in non-malignant as well as malignant clones, and HSC progeny of all lineages. Finally, cells bearing DNMT3A mutations are common in the peripheral blood of individuals over the age of 50 who exhibit a clonal dominance in their peripheral blood. Together, these studies from both mouse and humans incontrovertibly point to a central role for DNMT3A in maintaining normal hematopoiesis. Nevertheless, how these mutations confer an advantage to HSCs and contribute to clonal expansion as well as to the development of MDS and leukemia remains unclear. Gaining insight into these mechanisms is the overarching goal of this project. Here, we will determine whether DNMT3a mutations lead to blocked differentiation, whether DNA methylation function per se is critical to the hematopoietic impact, and whether different DNMT3A mutations have similar roles. Specifically, we will examine the role in differentiation by mapping DNA methylation changes throughout HSC differentiation, determining the localization of DNMT3A protein throughout the genome, modulating genes involved in self-renewal or differentiation in DNMT3a-KO HSCs, and by modulating DNA methylation. In addition, we will determine whether DNA methylation is required for DNMT3A to exert its role in HSCs. We will add back to KO HSCs specific DNMTs that retain or lack catalysis function, and examining the progeny for DNA methylation and HSC activity. Finally, we will examine commonly occurring DNMT3A mutants such as the R882 form to determine their impact on DNA methylation and hematopoiesis. Together, these studies will definitively link differentiation inhibition with DNMT3A loss-of-function and reveal key targets that could be modulated for therapeutic strategies. These studies will also demonstrate whether catalytic DNA methyltransferase activity is required for full HSC function, and lend insights into the impact of some of common DNMT3A mutations. Our lab is uniquely placed to contribute to this growing area given our pioneering work on the role of DNMT3A in HSCs and our generation of several novel tools and reagents to pursue the proposed questions.