Role of the ETV6 transcription factor in hematopoietic stem cell function

项目总结 造血干细胞和祖细胞(HSPC)是造血系统的根部，必须产生 所有的血细胞，同时也准备好立即对侮辱做出反应。因此，一个彻底的 了解HSPC生物学对于开发新的更有效的疾病治疗方法是必不可少的 血液和免疫系统。E26转化特异性变异体6(ETV6)是一种高度转录因子 在HSPC中表达并对其调节至关重要，在小鼠中有条件地敲除该转录因子 导致了这一种群的完全丧失。然而，人们对基因网络和直接靶点知之甚少。 HSPC中受ETV6调控的基因及其所支配的细胞过程。2015年，尼科尔斯博士 另一些人发现了常染色体显性遗传性血小板减少症家系中的致病胚系ETV6变异 以及易患恶性血液病(这种综合征现在被称为血小板减少症5)。为了更好地 为了了解这些变异是如何影响造血的，尼科尔斯实验室开发了一种新的小鼠 一株携带杂合子Etw6R355X变异体的菌株，该变异体相当于在 5.对Etw6R355X/+小鼠的研究表明，它们是活的，可以建立 HSPC种群。然而，与Etw6+/+小鼠相比，Etw6R355X/+小鼠的数量明显减少 HSPC，一种随着年龄增长而逐渐恶化的表型。此外，当来自Etw6R355X/+小鼠的HSPC 与竞争移植相比，它们的植入潜力显著降低 ETV6+/+HSPC。基于这些发现，我推测与血小板减少症5相关的变异ETV6 R355X蛋白通过改变关键下游靶基因的表达而损害造血 HSPC维护。为了解决这一假设，我将完成以下具体目标。在《目标1》中，我将 胎肝和骨髓中ECTV6+/+和ETV6R355X/+HSPC的定量和功能特征 在动物的整个生命过程中以及在诱导造血性应激之后。接下来，在《目标2》中，我将表演 对ECTV6+/+和ETV6R355X/+小鼠的HSPC进行单细胞RNA测序(RNA-seq)，以确定 在特定的HSPC亚群中差异表达。此外，我将使用以下命令执行CUT和RUN ETV6+/+和ETV6R355X/+HSPC。这项新技术允许绘制蛋白质-DNA相互作用的图谱 抗体靶向控制微球菌核酸酶切割DNA以确定可能的直接靶基因 (在本例中为ETV6)。总之，这些研究将确定HSPC中的基因网络和靶基因 受Etw6R355X变异体的调控失调，我将通过qRT-PCR进行验证。然后我将在体外和体内进行表演 以已确定的目标基因的功能和观察到的表型为指导的功能分析 我们的小鼠模型来定义受ETV6 R355X变异影响的细胞过程。成功完成 将描述必要的转录因子ETV6在HSPC中的新角色并阐明 生殖系变异是如何导致血液病的。

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.