Splicing and Epigenetics Regulation of Hematopoietic Stem Cells

描述(由申请人提供)：造血干细胞(HSCs)在生物体的整个生命过程中产生所有成熟的血细胞。在血液系统损伤后，血细胞数量会大大减少，如化疗，HSC反应缓慢可能会导致感染、出血和贫血等危及生命的并发症。此外，功能失常的造血干细胞也可能导致骨髓衰竭或恶性肿瘤。越来越多的证据表明，适当的基因表达调控对维持造血系统内环境的稳定至关重要。大多数先前的工作集中在研究HSCs的转录调控。最近对血液病患者的人类基因组学研究发现，剪接可能是HSCs的调节因子，但剪接体成分突变如何或为什么会导致异常造血尚不清楚。在之前研究小鼠HSCs基因表达的工作中，我们观察到静息和损伤激活的HSCs之间剪接动力学的变化。这一激活HSC的过程可以通过刺激Wnt信号转导通路来调节，Wnt信号转导通路与剪接调控有关。此外，在斑马鱼的发育过程中，我们和其他人有数据表明，几个剪接体成分的突变会导致造血干细胞和祖细胞(HSPC)的产生减少。综上所述，这些数据表明，剪接调控在造血干细胞的发育、再生和疾病过程中非常重要。由于剪接体组分普遍表达，在这项提议中，我们计划测试外部信号和内在元件驱动剪接I HSCs的细胞特异性结果的假设。目标1将研究Wnt信号通路如何改变HSCs的剪接。我们将在有无Wnt通路激活的情况下对纯化的小鼠HSCs进行RNA测序实验，并确定哪些转录本和外显子是差异剪接的。然后将对Wnt途径的各个步骤进行特定的抑制，以确定哪个步骤对剪接的影响是重要的。目标2将阐述哪些表观遗传因素是在斑马鱼HSPC形成中观察到的剪接缺陷的中心。我们将进行人工致死筛选，在斑马鱼中敲除携带剪接体成分突变的选定表观遗传因子的水平，然后寻找HSPC水平的变化。然后，我们将使用生化和基因组学技术来确定表观遗传和剪接成分相互作用的机制。这项工作将揭示HSCs中细胞类型特异性剪接调控的元件。

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.