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ETV 6突变导致血小板减少伴出血素质，红细胞减少， 细胞巨红细胞症和白血病易感性我们描述了有错义突变的家庭 在ETV 6的中心结构域（p.Pro214Leu）和ETS DNA结合结构域（p.Arg418Gly）中 这导致ETV 6的异常细胞定位，降低转录抑制， MK成熟受损。血小板转录组的深度测序显示， ETV 6 p.P214L突变患者和 未受影响的家庭成员。此外，外周血单核细胞的单细胞RNA序列 来自这些患者的血细胞表现出以下表达模式的显著变化： 干扰素（IFN）应答基因的mRNA，表明ETV 6在维持 骨髓稳态这一提议将检验正常监管的核心假设， ETV 6的功能对于控制MK分化的转录事件是必需的， 在体内平衡和炎症条件下正常发挥功能的血小板的形成。我们 产生了转基因小鼠，其中Etv 6在小鼠中表现出p.P214L突变 直向同源物保守位置（Etv 6P 214 L）。具有该突变的小鼠（Etv 6P 214 L）具有降低的 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ETV 6调节MK和血小板关键功能的机制。在 具体目标1我们将确定ETV 6在调节MK分化、血小板形成和血小板生成中的作用。 和血小板功能。为此，我们将使用Etv 6 P214 L、Etv 6-/-Gata 1-Cre和Etv 6-/-Pf 4-Cre 小鼠以确定Etv 6基因破坏在MK祖细胞和MK中的作用。这些结果 将与CD 34+衍生的MK中的MK分化和前血小板形成进行比较， 从ETV 6突变的患者身上培养出来的。此外，我们将在体外和体内研究血小板， 这些老鼠的反应。在具体目标2中，我们将描述ETV 6在以下方面的贡献： 调节MK中的转录事件。这一目标将检验ETV 6直接 调节MK中的转录事件。我们将通过以下方法鉴定Etv 6依赖性候选基因： 在分离自Etv 6 P214 L、Etv 6-/-Gata 1-Cre和Etv 6-/-Gata 1-Cre的MK和血小板中进行RNA-seq Pf 4-Cre小鼠。将这些结果与Chip-seq/DNase I seq数据和获得的结果进行比较 来自携带ETV 6 p.P214L和R418 G突变的小鼠和人MK。我们将确定 ETV 6通过鉴定候选mRNA的转录来调节候选mRNA的转录的机制 ETV 6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 ETV 6破坏对骨髓稳态中IFN应答基因的影响。这一目标 将检验ETV 6通过与HDAC 3相互作用调节IFN应答基因的假设， ETV 6功能的破坏将产生促炎性环境，其影响正常 巨核细胞生成和造血。该应用程序的发现将进一步 推进我们对MK和血小板生物学的理解，并将提供潜在的治疗方法， 血小板数量和功能紊乱的靶点。

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