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 th...