Regulation of Splicing During Hematopoietic Stem and Progenitor Cell Formation

项目摘要/摘要 造血干细胞和祖细胞(HSPC)通过自我更新和 分化成各种血细胞类型。它们在早期胚胎发育过程中通过调节和保守的 这一过程被称为内皮向造血细胞的转变(EHT)。在EHT期间，内皮细胞的一个亚群 (ECS)调整造血转录程序以形成血源性内皮(HE)，随后 形态变化，以形成HSPC。由于HSPC的从头产生只发生在胚胎早期 在发育过程中，HSPC的形成对所有胚胎和成人的造血都有深远的影响。缺陷 在EHT中，调节因子普遍存在于血液系统疾病中，因此通过研究EHT学到的经验教训可能 告知导致这些疾病的途径。研究EHT调节是理解过程关键的关键 从胚胎到成年的造血健康。虽然一些控制EHT的因素是已知的，但我们的 关于HE/HSPC监管机构的知识仍然知之甚少。在之前的研究中，鲍曼实验室确定 适当的前mRNA剪接是EHT所必需的，因为HES在斑马鱼突变体中大部分缺失 剪接体成分剪接因子3b，亚基1(SF3B1)。这些数据表明，剪接对于 他的形成，但调节剪接变化的机制对EHT至关重要，在很大程度上是未知的。至 探索这一过程，我首先定义了胚胎斑马鱼EC和 他/HSPC。顺式作用调控元件内的前mRNA引导剪接异构体的选择，从而鉴定 在EHT过程中控制选择性剪接的潜在机制，我调查了 EC和HE/HSPC比较剪接调控序列特征的差异。通过这个初步的分析，我 发现EHT替代事件因较弱的剪接位点和较短的内含子长度而丰富 提示这些特征可能在决定EHT特异性剪接异构体的选择方面具有调节功能。在……里面 除了序列驱动的机制外，选择性剪接还可以通过转录检查点进行修改 如启动子-近端停顿。在试点研究中，我证明了药物或遗传抑制 启动子-近端停顿因子可减少HE/HSPC的产生。根据我的数据和文献，我会 检验EHT特异性剪接异构体选择由不同的顺式作用调控元件引导的假设 (目标1)，并受启动子近端暂停因子调节(目标2)。这些细胞类型特异性剪接的研究 在复杂的多细胞背景下的调控将使人们能够理解剪接如何微调EHT的命运 决定。这项研究的完成将揭示HSPC发生的关键规律。

Project Summary/Abstract Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis through self-renewal and differentiation into all blood cell types. They form during early embryogenesis through a regulated and conserved process termed the endothelial-to-hematopoietic transition (EHT). During the EHT, a subset of endothelial cells (ECs) adapt a hematopoietic transcriptional program to form the hemogenic endothelium (HE) followed by morphological changes to make HSPCs. As de novo production of HSPCs occurs solely during early embryonic development, HSPC formation has profound consequences on all embryonic and adult hematopoiesis. Defects in EHT regulators are prevalent in hematologic disorders, therefore lessons learned by studying EHT could inform the pathways driving these diseases. Studying EHT regulation is critical for understanding processes key to hematopoietic health from embryo to adult life. Though some factors controlling EHT are known, our knowledge of HE/HSPC regulators remains poorly understood. In prior studies, the Bowman lab determined that proper pre-mRNA splicing is required for EHT as HEs were largely absent in zebrafish mutants for the spliceosomal component splicing factor 3b, subunit 1 (sf3b1). These data indicate that splicing is important for HE formation, but the mechanisms regulating the splicing changes critical for EHT are largely unknown. To explore this process, I first defined the alternative splicing signature between embryonic zebrafish EC and HE/HSPC. Cis-acting regulatory elements within pre-mRNA guide splice isoform selection thus, to identify potential mechanisms controlling alternative splicing during EHT, I surveyed alternative splicing events between EC and HE/HSPC for differences in splicing regulatory sequence features. Through this preliminary analysis, I uncovered that the EHT alternative events were enriched for weaker splice sites and shorter intron length suggesting these features could have a regulatory function in dictating EHT specific-splice isoform choice. In addition to sequence-driven mechanisms, alternative splicing can be modified by transcriptional checkpoints such as promoter-proximal pausing. In pilot studies, I showed that pharmacological or genetic inhibition of promoter-proximal pausing factors can diminish HE/HSPC production. Based on my data and the literature, I will test the hypothesis that EHT-specific-splice isoform selection is guided by distinct cis-acting-regulatory elements (aim 1) and regulated by promoter proximal pausing factors (aim 2). These studies of cell-type specific splicing regulation in a complex, multicellular context will enable understanding of how splicing fine-tunes the EHT fate decision. Completion of this study will reveal critical regulation for the genesis of HSPC.