EPIGENTIC REGULATION OF HEMATOPOIETIC STEM CELL FUNCTION AND TRANSFORMATION

 描述(申请人提供)：所有的造血血统都来自于骨髓中的造血干细胞库。造血干细胞的特点是能够自我更新以维持种群，并分化以再生造血系统。在自我更新和世系承诺之间的选择受到外在和内在因素的调节，包括表观遗传决定因素，如DNA甲基化。为了直接研究DNA甲基化在HSC中的作用，我们建立了一个条件基因敲除小鼠模型，表明DNA甲基转移酶DNMT3A的缺失会损害HSC的分化，并强制实施自我更新计划。然而，潜在的分子机制仍然不清楚，我们的团队和其他人还没有观察到DNA甲基化模式改变和基因表达变化之间的相关性。在这项提案中，我们正在研究DNA甲基化以外的表观遗传修饰作为驱动表型的主要效应因素。我们已经确定了一组关键的调控基因，这些基因在正常的HSCs中受到抑制染色质标记H3K27me3的保护，但在DNMT3A缺失的HSCs中失去了这一标记。我们假设，抑制染色质的减少会诱导DNMT3A缺失的HSC中HSC自我更新基因的上调，从而阻碍分化。此外，我们的初步研究表明，抑制H3K27me3去甲基酶KDM6B可以挽救DNMT3A缺失的HSCs的许多功能缺陷。这项建议的目的是了解DNMT3A和KDM6B如何调节HSCs的基因表达，以维持自我更新和谱系承诺之间的动态平衡。我们将使用遗传小鼠工具和临床可翻译试剂的组合来验证我们的假设：特定目标1：确定KDM6B在HSC自我更新中的功能。特定目的2：研究DNMT3A和KDM6B对二价基因的调控。具体目的3：KDM6B在DNMT3A功能失调性造血功能障碍中的作用。表观遗传失调是许多造血疾病的基础，包括骨髓增生异常综合征(MDS)，一种以血细胞异常增生和无效造血为特征的异质性造血疾病集合。MDS源于HSCs中的基因突变，这种突变会损害分化并导致骨髓中的克隆性扩张，而MDS中最常见的突变基因之一是DNMT3A。拟议研究的长期目标之一是通过了解潜在的HSC功能障碍来确定DNMT3A突变患者(与DNMT3A野生型患者相比，他们的预后较差)的定向表观遗传治疗的靶点。由于MDS患者的前景不佳(根据疾病亚型的不同，中位生存期从5个月到6年不等)，迫切需要替代治疗策略。鉴于MDS在人群中的发病率不断上升，这些研究对公共卫生和NIDDK的使命具有很高的意义。

 DESCRIPTION (provided by applicant): All hematopoietic lineages are derived from a pool of hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs are characterized by their ability to self-renew to sustain the population, and differentiate to regenerate the hematopoietic system. The choice between self-renewal and lineage commitment is regulated by extrinsic and intrinsic factors, including epigenetic determinants such as DNA methylation. To directly address the role of de novo DNA methylation in HSCs, we generated a conditional knockout mouse model to show that loss of the DNA methyltransferase enzyme Dnmt3a impairs HSC differentiation and imposes a self-renewal program. However, the underlying molecular mechanisms remain obscure, and our group and others have not observed a correlation between altered DNA methylation patterns and gene expression changes. In this proposal, we are investigating epigenetic modifications outside of DNA methylation as the major effectors driving the phenotype. We have identified a critical set of regulatory genes that are safeguarded by the repressive chromatin mark H3K27me3 in normal HSCs, but lose this mark in Dnmt3a-null HSCs. We hypothesize that reduced repressive chromatin induces upregulation of HSC self-renewal genes in Dnmt3a-null HSCs, thereby impeding differentiation. Furthermore, our preliminary studies show that inhibition of the H3K27me3 demethylase Kdm6b can rescue many of the functional defects of Dnmt3a-null HSCs. The goal of this proposal is to understand how Dnmt3a and Kdm6b regulate gene expression in HSCs to maintain homeostatic balance between self-renewal and lineage commitment. We will test our hypothesis with the following specific aims using a combination of genetic mouse tools and clinically translatable agents; Specific Aim 1: Define the function of Kdm6b in HSC self-renewal. Specific Aim 2: Characterize the regulation of bivalent genes by Dnmt3a and Kdm6b. Specific Aim 3: The role of Kdm6b in Dnmt3a loss-of-function dysplastic hematopoiesis. Epigenetic dysregulation underlies many hematopoietic disorders, including myelodysplastic syndromes (MDS), a heterogeneous collection of hematopoietic diseases characterized by blood cell dysplasia and ineffective hematopoiesis. MDS arises from genetic mutations in HSCs that impair differentiation and lead to a clonal expansion in the bone marrow, and one of the most commonly mutated genes in MDS is DNMT3A. One of the long-term goals of the proposed studies is to identify targets for directed epigenetic therapies in DNMT3A-mutation patients (who have poor prognosis compared to DNMT3A wild-type patients) by understanding the underlying HSC dysfunction. As the outlook for MDS patients is poor (median survival varying from 5-months to 6-years depending on disease subtype), alternative treatment strategies are desperately needed. Given the increasing incidence of MDS in the population, these studies have a high significance for public health and the mission of the NIDDK.