Genomics of Megakaryocyte and Platelet Biology

巨核细胞和血小板生物学基因组学

• 批准号: 10554387
• 负责人: Jorge A Di Paola
• 金额: $ 56.19万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10554387
• 关键词: Affect; Biology; Blood Coagulation Disorders; Blood Platelet Disorders; Blood Platelets; Bone Marrow; CD34 gene; Candidate Disease Gene; Cell Line; Cells; Cessation of life; ChIP-seq; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coagulation Process; Coupled; DNA Binding Domain; Data; Defect; Deoxyribonuclease I; Disease susceptibility; Dysmyelopoietic Syndromes; ETV6 gene; Emergency Situation; Environment; Erythrocytes; Erythroid; Event; Exhibits; Family; Family member; Generations; Genes; Genetic; Genetic Transcription; Genomics; HDAC3 gene; Hematopoiesis; Hemorrhage; Homeostasis; Human; Impairment; In Vitro; Inflammatory; Interferons; Lead; Lymphoblastic Leukemia; Maintenance; Malignant Neoplasms; Manipulative Therapies; Measures; Megakaryocytes; Megakaryocytopoieses; Messenger RNA; Missense Mutation; Molecular; Mus; Mutation; Names; Oncogene Deregulation; Patients; Pattern; Peripheral Blood Mononuclear Cell; Platelet Activation; Platelet Count measurement; Positioning Attribute; Predisposition; Process; Production; RNA Sequences; Regulation; Role; Testing; Thrombocytopenia; Transcriptional Regulation; Transgenic Mice; deep sequencing; gene interaction; gene repression; genetic manipulation; in vivo; induced pluripotent stem cell; inflammatory marker; inflammatory milieu; leukemia; mRNA Expression; molecular phenotype; novel; platelet function; progenitor; response; therapeutic target; tool; transcriptome; transcriptome sequencing

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.

项目摘要 我们最近发现，ETV6中的突变导致血小板减少症，并具有出血性临床，红色 细胞大细胞增多症和白血病的倾向。我们描述了带错义突变的家庭 在中央域（P.Pro214leu）和ETS DNA结合域（P.Arg418gly）中 这导致ETV6的异常细胞定位，转录抑制降低，并且 MK成熟受损。血小板转录组的深度测序显示出显着的 ETV6 P.P214L突变患者之间的mRNA表达水平差异和 不受影响的家庭成员。另外，周围单核的单细胞RNA序列 这些患者的血细胞表现出明显的变化 干扰素（IFN）反应基因的mRNA，这表明ETV6在维持 骨髓稳态。该提案将检验正常调节的中心假设 ETV6的功能对于控制MK分化和的转录事件至关重要 在稳态和炎症条件下正常发挥作用的血小板的形成。我们 产生了一种转基因小鼠，其中ETV6在小鼠处表现出P.P214L突变 直系同源的位置（ETV6P214L）。这种突变的小鼠（ETV6P214L）降低了 血小板计数并表现出血小板缺陷。在此提案中，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 特定目的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 小鼠确定MK祖细胞和MK中ETV6基因破坏的影响。这些结果 将与CD34+衍生的MK中的MK分化和预言形成进行比较 从ETV6突变患者中培养。此外，我们将在体外和体内研究 这些小鼠的反应。在特定目标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直接的假设 调节MKS中的转录事件。我们将通过 在从ETV6P214L，ETV6 - / - GATA1-CRE和ETV6 - / - 中分离的MK和血小板中进行RNA-SEQ PF4-CRE小鼠。这些结果将与chip-seq/dnase I Seq数据进行比较，并获得了结果 来自带有ETV6 P.P214L和R418G突变的小鼠和人类MK。我们将确定 ETV6通过鉴定来调节候选mRNA的转录的机制 ETV6效应子并在功能上测试它们。最后，在特定目标3中，我们将确定 ETV6破坏骨髓稳态中IFN反应基因的后果。这个目标 将检验以下假设：ETV6通过与HDAC3相互作用，调节IFN响应基因 ETV6功能的破坏将产生影响正常的促炎环境 一般而言，巨型摩卡去和造血。该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 血小板数和功能疾病的目标。

项目成果

CRISPR-edited megakaryocytes for rapid screening of platelet gene functions.

CRISPR 编辑的巨核细胞用于快速筛选血小板基因功能。

• DOI: 10.1182/bloodadvances.2020004112
• 发表时间: 2021
• 期刊: Blood advances
• 影响因子: 7.5
• 作者: Montenont,Emilie;Bhatlekar,Seema;Jacob,Shancy;Kosaka,Yasuhiro;Manne,BhanuK;Lee,Olivia;Parra-Izquierdo,Ivan;Tugolukova,Emilia;Tolley,NealD;Rondina,MatthewT;Bray,PaulF;Rowley,JesseW
• 通讯作者: Rowley,JesseW