Splicing and Epigenetics Regulation of Hematopoietic Stem Cells

造血干细胞的剪接和表观遗传学调控

• 批准号: 8769909
• 负责人: Teresa V Bowman
• 金额: $ 8.35万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-04 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8769909
• 关键词: Acute; Address; Adult; Alternative Splicing; Anemia; Biochemical; Blood Cell Count; Blood Cells; Cell Lineage; Cells; Chronic Lymphocytic Leukemia; Complex; Data; Defect; Development; Disease; Dysmyelopoietic Syndromes; Elements; Environment; Epigenetic Process; Exons; Functional disorder; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Screening; Genomics; Goals; Health; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Hemorrhage; Heterozygote; Homeostasis; Human; In Vitro; Infection; Injury; Laboratories; Lead; Life; Maintenance; Malignant - descriptor; Malignant Neoplasms; Messenger RNA; Modification; Multipotent Stem Cells; Mus; Mutate; Mutation; Myeloproliferative disease; Natural regeneration; Organism; Outcome; Pancytopenia; Pathway interactions; Patients; Pattern; Phenotype; Phosphotransferases; Population; Process; Production; RNA Sequences; RNA Splicing; Recovery; Regulation; Rest; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Specificity; Spliceosomes; Stem cells; Syndrome; System; Techniques; Testing; Transcript; Transcriptional Regulation; Work; Zebrafish; beta catenin; cell type; chemotherapy; chromatin modification; genome-wide; hematopoietic stem cell fate; improved; in vivo; leukemia; mutant; research study; response; response to injury; stem; stem cell biology; stem cell population; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) produce all mature blood cells throughout the life of an organism. Following hematologic injuries that greatly reduce blood cell numbers, such as chemotherapy, a slow HSC response can result in life threatening complications from infection, bleeding, and anemia. Furthermore, malfunctioning HSCs can also result in bone marrow failure disorders or malignancies. Mounting evidence shows that proper gene expression control in HSCs is essential for maintenance of hematopoietic homeostasis. Most prior work centered on studying transcriptional control in HSCs. Recent human genomics studies of patients with hematologic diseases uncovered splicing as a putative regulator of HSCs, but how or why mutated spliceosomal components lead to aberrant hematopoiesis is unclear. In previous work studying gene expression in murine HSCs, we observed a change in splicing dynamics between resting and injury-activated HSCs. This process of HSC activation can be modulated by stimulation of the Wnt signal transduction pathway, which has been implicated in splicing regulation. Moreover, in developing zebrafish, we and others have data demonstrating that mutations in several spliceosomal components result in diminished production of hematopoietic stem and progenitor cells (HSPCs). Together these data suggest splicing regulation is important for HSCs during development, regeneration, and disease. As spliceosomal components are ubiquitously expressed, in this proposal we plan to test the hypothesis that external signals and intrinsic elements drive cell- specific outcomes of splicing i HSCs. Aim 1 will examine how the Wnt signaling pathway alters splicing in HSCs. We will perform RNA-sequencing experiments in purified murine HSCs in the presence and absence of Wnt pathway activation and identify which transcripts and exons are differentially spliced. Specific inhibition of various steps of the Wnt pathway will then be performed to define which step is important for the effects on splicing. Aim 2 will address which epigenetic factors are central for the splicing defects observed in HSPC formation in zebrafish. We will execute a synthetic lethal screen where we knockdown the levels of select epigenetic factors in zebrafish carrying mutations in spliceosomal components and then look for alterations in HSPC levels. We will then employ biochemical and genomics techniques to determine the mechanism through which the epigenetic and splicing components interact. This work will uncover elements of cell-type specific splicing regulation in HSCs.

描述（由申请人提供）：造血干细胞（HSC）在生物体的整个生命过程中产生所有成熟的血细胞。在大大减少血细胞数量的血液损伤（如化疗）后，缓慢的HSC反应可导致感染、出血和贫血等危及生命的并发症。此外，造血干细胞功能障碍也可能导致骨髓衰竭或恶性肿瘤。越来越多的证据表明，适当的基因表达控制造血干细胞是维持造血稳态必不可少的。大多数先前的工作集中在研究HSC中的转录控制。最近对血液病患者的人类基因组学研究揭示了剪接作为HSC的假定调节因子，但突变的剪接体组分如何或为什么导致异常造血尚不清楚。在先前研究小鼠HSC基因表达的工作中，我们观察到静息和损伤激活的HSC之间剪接动力学的变化。HSC激活的这一过程可以通过刺激Wnt信号转导途径来调节，该途径与剪接调节有关。此外，在开发斑马鱼的过程中，我们和其他人的数据表明，几种剪接体成分的突变导致造血干细胞和祖细胞（HSPC）的产生减少。总之，这些数据表明剪接调控在发育，再生和疾病期间对HSC很重要。由于剪接体组分普遍表达，在本提案中，我们计划测试外部信号和内在元件驱动HSC剪接的细胞特异性结果的假设。目的1将研究Wnt信号通路如何改变HSC中的剪接。我们将在存在和不存在Wnt通路激活的情况下在纯化的鼠HSC中进行RNA测序实验，并鉴定哪些转录本和外显子被差异剪接。然后将对Wnt途径的各个步骤进行特异性抑制，以确定哪个步骤对剪接的影响是重要的。目的2将解决哪些表观遗传因素是斑马鱼HSPC形成中观察到的剪接缺陷的核心。我们将进行一项合成致死筛选，在该筛选中，我们敲低了携带剪接体组分突变的斑马鱼中选择的表观遗传因子的水平，然后寻找HSPC水平的改变。然后，我们将采用生物化学和基因组学技术，以确定通过表观遗传和剪接组件相互作用的机制。这项工作将揭示HSC中细胞类型特异性剪接调控的要素。