Regulation of Hematopoietic Progenitors by de novo DNA Methylation

描述（由申请人提供）：DNA甲基化是一种重要的表观遗传修饰，用于保护基因组免受繁殖突变的影响，并调节基因表达。异常DNA甲基化越来越被认为是在许多病理状态下发现的一种主要的表观遗传扰动，并且在骨髓增生异常综合征（MDS）的背景下变得特别有趣。MDS是一种造血功能失调的状态，其特征是贫血增加并伴有高骨髓母细胞计数。MDS的发病率随着年龄的增长而增加，具有实质性的病理影响，有时是急性髓性白血病（AML）的前兆。MDS患者造血细胞DNA甲基化谱的数据支持异常DNA甲基化与MDS的相关性，以及阿扎胞苷和地西滨的临床价值，这些抑制DNA甲基化的治疗至少显示出一定的临床疗效。然而，DNA甲基化失调的机制，以及DNA甲基化扰动导致血液学紊乱的方式，是不透明的。在这里，我们将使用小鼠作为模型来研究当DNA甲基转移酶特异性改变时发生的血液学功能扰动。我们推测，适当的Dnmt3a或Dnmt3b表达的缺失会导致DNA甲基化异常，改变基因的表达，破坏自我更新和分化之间的平衡，最终导致无能的干细胞群体无法促进正在进行的血液形成。我们有两个广泛的目标在功能和分子水平上探索这个问题。我们将确定Dnmt3s在调节小鼠造血干细胞功能中的作用。我们将通过干细胞纯化和骨髓移植诱导Dnmt3基因的缺失来检测HSC的分化和自我更新特性。我们将确定在小鼠中Dnmt3异常表达重现mds样疾病的程度。用DNA甲基化抑制剂治疗小鼠将揭示对造血祖细胞的功能影响以及由此导致的甲基化模式的变化。我们还将使用DNA甲基化的全局分析来确定从头DNA甲基转移酶丢失的分子后果。这些变化将与Dnmt3a或Dnmt3b基因表达的改变相关。我们还将研究关于结合其他全球表观遗传调控因子的后果。这些研究将使我们能够确定dnmt3在HSC中的作用模式，以及确定在dnmt3缺失的HSC中赋予改变表型的特定候选基因。通过分析造血干细胞中Dnmt3缺失的表型和功能后果，我们将首次详细描述Dnmt3在任何成年细胞类型中的作用。这些数据将有助于深入了解DNA甲基化改变在病理状态中的作用，特别是MDS。更深入地了解DNA甲基化如何调节干细胞自我更新和分化，以及鉴定介导这种影响的关键基因，可能为未来的治疗发展提供新的靶点。

DESCRIPTION (provided by applicant): DNA methylation is an important epigenetic modification that serves to protect the genome from propagating mutations, and to regulate gene expression. Aberrant DNA methylation is increasingly being recognized as a major epigenetic perturbation found in many pathologic states, and has become of particular interest in the context of myelodysplastic syndrome (MDS). MDS is a state of dysregulated hematopoiesis that is characterized by increasing anemia accompanied by high marrow blast counts. MDS incidence increases with age, has substantial pathologic impact, and is sometimes a precursor to acute myeloid leukemia (AML). The relevance of aberrant DNA methylation to MDS has been supported by data on the DNA methylation profiles on hematopoietic cells from MDS patients, as well the clinical value of azacytidine and decitibine, treatments that inhibit DNA methylation and have shown at least some clinical efficacy. However, the mechanisms of DNA methylation dysregulation, as well as the manner in which perturbations of DNA methylation lead to hematologic disturbance, are opaque. Here, we will use mice as a model to study the perturbations of hematologic function that occur when specific alterations in DNA methyltransferases are induced. We hypothesize that loss of appropriate Dnmt3a or Dnmt3b expression leads to aberrant DNA methylation, altering the expression of genes that disrupt the balance between self-renewal and differentiation, ultimately leading to an impotent stem cell population unable to contribute to ongoing blood formation. We have two broad aims to explore this at functional and at molecular levels. We will determine the roles of Dnmt3s in regulation of murine hematopoietic stem cell function. We will induce deletion of the Dnmt3 genes to examine the differentiation and self-renewal properties of HSC, using stem cell purification and bone marrow transplantation. We will determine the extent to which aberrant Dnmt3 expression recapitulates an MDS-like disease in mice. Treatment of mice with DNA methylation inhibitors will reveal the functional consequences on hematopoietic progenitors and the resulting changes in methylation patterns. We will also determine the molecular consequences of loss of de novo DNA methyltransferases using global analysis of DNA methylation. These changes will be correlated with alterations in gene expression that accompany loss of Dnmt3a or Dnmt3b. We will also examine the consequences with regard to binding of additional global epigenetic regulators. These studies will allow us to determine the mode of action of the Dnmt3s in HSC, as well as to identify specific genes that are candidates for conferring the altered phenotype in Dnmt3-deleted HSCs. By analyzing the phenotypic and functional consequences of Dnmt3 loss in HSCs, we will provide the first detailed portrait of the role of Dnmt3s in any adult cell type. These data will offer insights into the role of DNA methylation alterations in pathologic states, particularly MDS. A deeper understanding of how DNA methylation regulates stem cell self-renewal and differentiation, and identification of the key genes that mediate the effects, may offer new targets for future therapeutic development.