Genomics of Megakaryocyte and Platelet Biology

项目总结 我们最近发现ETV6基因突变会导致血小板减少，并伴有出血素质 细胞巨噬细胞增多症和白血病易感性。我们描述了有错义突变的家庭 在ETV6的中心结构域(p.Pro214Leu)和ETS DNA结合域(p.Arg418Gly)中 这导致ETV6的细胞定位异常，转录抑制减少，以及 MK成熟受损。对血小板转录组的深度测序显示有重要意义 ETV6 p.P214L突变患者和正常对照患者的基因表达水平差异 未受影响的家庭成员。此外，外周血单核细胞RNA序列 这些患者的血细胞表达模式发生了显著变化 干扰素反应基因的mRNAs，提示ETV6在维持 骨髓动态平衡。这一提议将检验正常监管的核心假设 而ETV6的功能是控制MK分化和转录事件所必需的 在体内平衡和炎症条件下正常工作的血小板的形成。我们 产生了一只转基因小鼠，在小鼠身上显示了p.P214L突变 同源基因保守位置(Etw6P214L)。带有这种突变(Etw6P214L)的小鼠已经减少 血小板计数，并显示出血小板缺陷。在这项提案中，我们将测试三个目标，它们将 确定ETV6调节巨噬细胞和血小板关键功能的机制。在……里面 具体目标1我们将确定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Etw6P214L、Etw6-/-GATA1-CRE和Etw6-/-PF4-CRE 以确定ETV6基因干扰对MK祖细胞和MKs的影响。这些结果 将与CD34来源的MK中的MK分化和前血小板形成进行比较 来自ETV6突变患者的培养。此外，我们还将研究体外和体内的血小板 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。这一目标将直接检验ETV6病毒的假设 调节MKs中的转录事件。我们将通过以下方式确定依赖ETV6的候选基因 在从Etw6P214L、Etw6-/-GATA1-Cre和Etw6-/-分离的巨噬细胞和血小板中执行RNA-seq PF4-Cre小鼠。这些结果将与Chip-Seq/DNase I序列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。我们将决定 ETV6通过识别调控候选mRNAs转录的机制 ETV6效应器，并对其进行功能测试。最后，在具体目标3中，我们将确定 ETV6干扰对骨髓稳态干扰素反应基因的影响。这一目标 将检验ETV6通过与HDAC3相互作用来调节干扰素反应基因的假设，以及 ETV6功能的中断将产生影响正常的促炎环境 巨核细胞生成和一般的造血。此应用程序的发现将进一步 促进我们对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靶点。

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.