Genomics of Megakaryocyte and Platelet Biology

项目概要 我们最近发现 ETV6 突变会导致伴有出血素质的血小板减少症、红色 细胞巨红细胞增多症和白血病倾向。我们描述了具有错义突变的家族 位于 ETV6 的中央结构域 (p.Pro214Leu) 和 ETS DNA 结合结构域 (p.Arg418Gly) 导致 ETV6 细胞定位异常、转录抑制减少，以及 MK 成熟受损。血小板转录组的深度测序揭示了重要的 ETV6 p.P214L 突变患者和 ETV6 p.P214L 突变患者之间 mRNA 表达水平的差异 未受影响的家庭成员。此外，外周单核细胞的单细胞 RNA 序列 这些患者的血细胞显示出表达模式的显着变化 干扰素 (IFN) 反应基因的 mRNA，表明 ETV6 在维持 骨髓稳态。该提案将检验正常监管的中心假设 ETV6 的功能对于控制 MK 分化和 形成在稳态和炎症条件下正常发挥作用的血小板。我们 产生了转基因小鼠，其中 Etv6 在小鼠中表现出 p.P214L 突变 直系同源物保守位置(Etv6P214L)。具有这种突变（Etv6P214L）的小鼠减少了 血小板计数并表现出血小板缺陷。在本提案中，我们将测试三个目标 确定 ETV6 调节 MK 和血小板关键功能的机制。在 具体目标 1 我们将定义 ETV6 在调节 MK 分化、血小板形成中的作用 和血小板功能。为此，我们将使用 Etv6P214L、Etv6-/- Gata1-Cre 和 Etv6-/- Pf4-Cre 小鼠以确定 Etv6 基因破坏对 MK 祖细胞和 MK 的影响。这些结果 将与 CD34 衍生的 MK 中的 MK 分化和前血小板形成进行比较， 培养自具有 ETV6 突变的患者。此外，我们将研究体外和体内血小板 这些小鼠的反应。在具体目标 2 中，我们将描述 ETV6 在 调节 MK 中的转录事件。该目标将检验 ETV6 直接 调节 MK 中的转录事件。我们将通过以下方式识别 Etv6 依赖的候选基因 对从 Etv6P214L、Etv6-/-Gata1-Cre 和 Etv6-/- 分离的 MK 和血小板进行 RNA 测序 Pf4-Cre 小鼠。这些结果将与 Chip-seq/DNase I seq 数据和获得的结果进行比较 来自携带 ETV6 p.P214L 和 R418G 突变的小鼠和人类 MK。我们将确定 ETV6 通过识别来调节候选 mRNA 转录的机制 ETV6 效应器并测试它们的功能。最后，在具体目标 3 中，我们将确定 ETV6 破坏对骨髓稳态中 IFN 反应基因的影响。这个目标 将检验 ETV6 通过与 HDAC3 相互作用调节 IFN 应答基因的假设，以及 ETV6 功能的破坏会产生促炎环境，影响正常 一般巨核细胞生成和造血作用。该应用程序的发现将进一步 增进我们对 MK 和血小板生物学的理解，并将提供潜在的治疗 血小板数量和功能紊乱的目标。

PROJECT SUMMARY We recently found that mutations in ETV6 lead to thrombocytopenia with bleeding diathesis, red cell macrocytosis, and predisposition to leukemia. We described families with missense mutations in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6 that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant differences in mRNA expression levels between patients with the ETV6 p.P214L mutation and non-affected family members. Additionally, single cell RNA-sequence of peripheral mononuclear blood cells from these patients demonstrated significant changes in the expression patterns of mRNAs of Interferon (IFN) Response Genes, suggesting a critical role for ETV6 in maintaining bone marrow homeostasis. This proposal will test the central hypothesis that normal regulation and function of ETV6 is essential for transcriptional events that control MK differentiation and formation of platelets that function properly under homeostatic and inflammatory conditions. We generated a transgenic mouse in which Etv6 exhibits the p.P214L mutation at the mouse orthologue conserved position (Etv6P214L). Mice with this mutation (Etv6P214L) have reduced platelet counts and exhibit a platelet defect. In this proposal, we will test three aims that will determine the mechanisms by which ETV6 regulates critical functions of MKs and platelets. In Specific Aim 1 we will define roles for ETV6 in regulating MK differentiation, platelet formation and platelet function. For this purpose, we will use Etv6P214L, Etv6-/- Gata1-Cre and Etv6-/- Pf4-Cre mice to determine the effects of Etv6 gene disruption in MK progenitors and MKs. These results will be compared to MK differentiation and proplatelet formation in CD34+- derived MK that are cultured from patients with ETV6 mutations. Additionally, we will study in vitro and in vivo platelet responses from these mice. In Specific Aim 2 we will delineate the contributions of ETV6 in modulating transcriptional events in MK. This aim will test the hypothesis that ETV6 directly regulates transcriptional events in MKs. We will identify Etv6-dependent gene candidates by performing RNA-seq in MKs and platelets isolated from Etv6P214L, Etv6-/-Gata1-Cre, and Etv6-/- Pf4-Cre mice. These results will be compared to Chip-seq/DNase I seq data and results obtained from mice and human MKs that carry the ETV6 p.P214L and R418G mutations. We will determine the mechanisms by which ETV6 regulates the transcription of candidate mRNAs by identifying ETV6 effectors and test them functionally. Finally, in Specific Aim 3 we will determine the consequences of ETV6 disruption on IFN response genes in bone marrow homeostasis. This aim will test the hypothesis that ETV6 regulates IFN response genes by interacting with HDAC3, and disruption of ETV6 function will generate a proinflammatory milieu that affects normal megakaryopoiesis and hematopoiesis in general. Discoveries from this application will further advance our understanding of MK and platelet biology, and will provide potential therapeutic targets for disorders of platelet number and function.