Role of the ETV6 transcription factor in hematopoietic stem cell function

项目摘要 造血干细胞和祖细胞（HSPC）是造血系统的根源，必须产生 所有的血细胞，同时也准备立即响应侮辱。因此，彻底 当开发新的和更有效的治疗疾病时，了解HSPC生物学是必不可少的。 血液和免疫系统。E26转化特异性变体6（ETV 6）是一种转录因子， 在HSPC中表达并对其调节至关重要，在小鼠中条件性敲除该转录因子 导致这一种群的完全丧失。然而，对基因网络和直接靶点知之甚少， HSPC内受ETV 6调控的基因及其支配的细胞过程。2015年，Nichols博士 其他人在常染色体显性血小板减少症家族中鉴定出致病性生殖系ETV 6变异体 和易患恶性血液病（一种现在称为血小板减少症5的综合征）。更好地 为了了解这些变异如何影响造血，尼科尔斯实验室开发了一种新的小鼠， 携带杂合Etv 6 R355 X变体的菌株，该变体等同于 血小板减少症患者5. Etv 6 R355 X/+小鼠的研究表明，它们是活的，可以建立 HSPC人群。然而，当与Etv 6 +/+同窝小鼠相比时，Etv 6 R355 X/+小鼠具有显著更少的 HSPCs是一种随着年龄增长而逐渐衰退的表型。此外，当来自Etv 6 R355 X/+小鼠的HSPC被诱导时， 与竞争性移植相比，它们显示出显著降低的植入潜力。 Etv6+/+ HSPC。基于这些发现，我假设血小板减少症5相关的变异ETV 6 R355 X蛋白通过改变造血所需的关键下游靶基因的表达来损害造血 HSPC维护。为了解决这个假设，我将完成以下具体目标。在目标1中，我将 定量和功能表征胎肝和骨髓中的Etv 6 +/+和Etv 6 R355 X/+ HSPC 在动物的整个生命周期中以及在诱导造血应激之后。接下来，在目标2中，我将执行 在来自Etv 6 +/+和Etv 6 R355 X/+小鼠的HSPC上进行单细胞RNA测序（RNA-seq），以鉴定 在特定HSPC亚群内差异表达。此外，我将使用 Etv 6 +/+和Etv 6 R355 X/+ HSPC。这项新技术允许使用蛋白质-DNA相互作用的映射， 抗体靶向微球菌核酸酶控制的DNA切割，以鉴定推定的直接靶基因 (in这是ETV 6的情况下）。总之，这些研究将确定HSPC中的基因网络和靶基因， Etv 6 R355 X变异体失调，我将通过qRT-PCR验证。然后我会在体外和体内 由鉴定的感兴趣的靶基因的已知功能和在细胞中观察到的表型指导的功能测定法。 我们的小鼠模型来定义受ETV 6 R355 X变体影响的细胞过程。成功完成 本项目的一部分将描述HSPCs中必需转录因子ETV 6的新作用，并阐明 生殖系变异如何导致造血系统疾病

PROJECT SUMMARY Hematopoietic stem and progenitor cells (HSPCs) are at the root of the hematopoietic system and must give rise to all blood cells while also being prepared to respond immediately to insults. Accordingly, a thorough understanding of HSPC biology is essential when developing new and more effective treatments for diseases of the blood and immune system. E26 transforming-specific variant 6 (ETV6) is a transcription factor that is highly expressed in HSPCs and critical for their regulation, with conditional knock-out of this transcription factor in mice leading to a complete loss of this population. However, little is known about the gene networks and direct target genes that are regulated by ETV6 within HSPCs and the cellular processes that they govern. In 2015, Dr. Nichols and others identified pathogenic germline ETV6 variants in families with autosomal dominant thrombocytopenia and predisposition to hematologic malignancies (a syndrome now known as Thrombocytopenia 5). To better understand how these variants impact hematopoiesis, the Nichols laboratory has developed a novel mouse strain harboring a heterozygous Etv6R355X variant that is equivalent to a recurrent ETV6R359X variant identified in individuals with Thrombocytopenia 5. Studies of Etv6R355X/+ mice reveal that they are viable and can establish HSPC populations. However, when compared to Etv6+/+ littermates, Etv6R355X/+ mice have significantly fewer HSPCs, a phenotype that progressively worsens with age. Furthermore, when HSPCs from Etv6R355X/+ mice are challenged with competitive transplantation, they show significantly reduced engraftment potential compared to Etv6+/+ HSPCs. Based on these findings, I hypothesize that the Thrombocytopenia 5-associated variant ETV6 R355X protein impairs hematopoiesis by altering the expression of key downstream target genes needed for HSPC maintenance. To address this hypothesis, I will complete the following Specific Aims. In Aim 1, I will quantify and functionally characterize Etv6+/+ and Etv6R355X/+ HSPCs in the fetal liver and in the bone marrow throughout the lifetime of the animal and after induction of a hematopoietic stress. Next, in Aim 2, I will perform single-cell RNA-sequencing (RNA-seq) on HSPCs from Etv6+/+ and Etv6R355X/+ mice to identify genes that are differentially expressed within specific HSPC sub-populations. Additionally, I will perform CUTandRUN using Etv6+/+ and Etv6R355X/+ HSPCs. This new technique allows for mapping of protein-DNA interactions using antibody-targeted controlled cleavage of DNA by micrococcal nucleases to identify putative direct target genes (in this case of ETV6). Together, these studies will identify gene networks and target genes in HSPCs that are dysregulated by the Etv6R355X variant which I will validate by qRT-PCR. I will then perform in vitro and in vivo functional assays guided by known functions of identified target genes of interest and phenotypes observed in our mouse model to define the cellular processes impacted by the ETV6 R355X variant. Successful completion of this project will describe novel roles for the essential transcription factor ETV6 within HSPCs and elucidate how germline variants contribute to hematopoietic disease.