Splicing and Epigenetics Regulation of Hematopoietic Stem Cells

描述（由申请人提供）：造血干细胞（hsc）在生物体的整个生命周期中产生所有成熟的血细胞。在血液学损伤后，血细胞数量大大减少，如化疗，缓慢的HSC反应可导致威胁生命的并发症，如感染、出血和贫血。此外，造血干细胞功能异常也会导致骨髓衰竭、疾病或恶性肿瘤。越来越多的证据表明，造血干细胞中适当的基因表达控制对于维持造血稳态至关重要。大多数先前的工作集中在研究造血干细胞的转录控制。最近对血液病患者的人类基因组学研究发现，剪接可能是造血干细胞的调节因子，但剪接体成分突变如何或为何导致造血异常尚不清楚。在先前研究小鼠造血干细胞基因表达的工作中，我们观察到静息和损伤激活的造血干细胞之间剪接动力学的变化。这个HSC的激活过程可以通过刺激Wnt信号转导通路来调节，Wnt信号转导通路与剪接调节有关。此外，在发育中的斑马鱼中，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（HSPCs）的产生减少。综上所述，这些数据表明剪接调节在造血干细胞的发育、再生和发病过程中是重要的。由于剪接体成分无处不在地表达，在本提案中，我们计划验证外部信号和内在因素驱动hsc剪接细胞特异性结果的假设。目的1将研究Wnt信号通路如何改变造血干细胞的剪接。我们将在存在和不存在Wnt通路激活的纯化小鼠造血干细胞中进行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通路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在斑马鱼HSPC形成中观察到的剪接缺陷的中心。我们将执行一个合成致死筛选，我们将在剪接体成分中携带突变的斑马鱼中敲低选择的表观遗传因子的水平，然后寻找HSPC水平的变化。然后，我们将采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。这项工作将揭示造血干细胞中细胞类型特异性剪接调节的要素。

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.