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Role of the ETV6 transcription factor in hematopoietic stem cell function

ETV6转录因子在造血干细胞功能中的作用

基本信息

批准号：
10373938
负责人：
Mackenzie Bloom
金额：
$ 3.32万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL GRADUATE SCHOOL OF BIOMEDICAL SCIENCES, LLC
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2022-10-20
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10373938
关键词：
Ablation Address Adult Affect Age Animals Antibodies Biological Assay Blood Cells Bone Marrow Bone Marrow Transplantation Cell Cycle Cell Death Cell Maintenance Cell physiology Cells Cellular biology DNA DNA Binding DNA-Protein Interaction Defect Disease Embryo Engraftment Family Fetal Liver Flow Cytometry Fluorouracil Frequencies Gene Targeting Genes Genetic Genetic Transcription Genotype Hematologic Neoplasms Hematological Disease Hematology Hematopoiesis Hematopoietic Hematopoietic System Hematopoietic stem cells Human Immune Immune System Diseases Impairment In Vitro Individual Inherited Knock-in Mouse Knowledge Laboratories Life Maintenance Micrococcal Nuclease Mouse Strains Mus Mutate Natural regeneration Nutrient Oxygen Partner in relationship Pathogenicity Phenotype Plant Roots Play Population Positioning Attribute Predisposition Process Proteins Quantitative Reverse Transcriptase PCR Recovery Recurrence Regulation Resources Role Stress Syndrome System Techniques Testing Thrombocytopenia Time Transplantation Variant Yolk Sac angiogenesis base cell regeneration cohort conditional knockout differential expression disease phenotype effective therapy experimental study follow-up gene network gene repression human disease in vivo interest knock-down mouse model novel nuclease overexpression programs single-cell RNA sequencing stem cell function stressor tool transcription factor

项目摘要

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.

项目总结