Regulation of Splicing During Hematopoietic Stem and Progenitor Cell Formation

造血干细胞和祖细胞形成过程中剪接的调节

• 批准号: 10678816
• 负责人: Ilana N Karp
• 金额: $ 4.77万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-26 至 2025-04-25
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678816
• 关键词: 3' Splice Site; Adult; Alternative Splicing; Automobile Driving; Binding Proteins; Blood Cells; Cells; Cis-Acting Sequence; Complex; Computer Analysis; Data; Defect; Disease; Embryo; Embryonic Development; Endothelial Cells; Endothelium; Event; Genetic; Genetic Transcription; Health; Hematologic Neoplasms; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Introns; Knowledge; Learning; Length; Life; Literature; Measures; Morphology; Pathway interactions; Pilot Projects; Play; Process; Production; Protein Isoforms; RNA Binding; RNA Splicing; RNA-Binding Proteins; Regulation; Regulatory Element; Reporter; Role; Sampling; Site; Spliceosomes; Surveys; Testing; Therapeutic; Trans-Activators; Validation; Zebrafish; cell type; cis acting element; differential expression; experience; experimental study; genetic manipulation; hemogenic endothelium; in vivo; mRNA Precursor; mutant; pharmacologic; programs; promoter; recruit; self-renewal; tool; transcriptome sequencing

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.

项目摘要/摘要 造血茎和祖细胞（HSPC）通过自我更新和 分化为所有血细胞类型。它们在早期的胚胎发生过程中形成 过程称为内皮到山层过渡（EHT）。在EHT期间，内皮细胞的子集 （EC）适应造血转录程序形成血液内皮（HE），然后是 形态学变化以制造HSPC。由于HSPC的从头产生仅发生在早期胚胎中 开发，HSPC的形成对所有胚胎和成人造血作用都有深远的影响。缺陷 在EHT调节剂中，血液学疾病很普遍，因此通过研究EHT学到的经验教训可以 通知驱动这些疾病的途径。研究EHT调节对于理解过程关键至关重要 从胚胎到成人生活的造血健康。尽管已知某些控制EHT的因素，但我们的 对HE/HSPC监管机构的了解仍然很少了解。在先前的研究中，鲍曼实验室确定 EHT需要适当的前MRNA剪接，因为HES在斑马鱼突变体中基本不存在 剪接小组分量剪接因子3B，亚基1（SF3B1）。这些数据表明剪接对于 他的形成，但是调节剪接变化的机制在很大程度上是未知的。到 探索这个过程，我首先定义了胚胎斑马鱼EC和 他/HSPC。在前MRNA指导剪接同工型选择中的顺式作用调节元件因此，以识别 控制EHT期间替代剪接的潜在机制，我调查了替代剪接事件 EC和HE/HSPC的剪接调节序列特征差异。通过此初步分析，我 发现EHT的替代事件富含较弱的剪接位点和较短的内含子长度。 提出这些特征可以在决定EHT特异性同工型选择方面具有调节功能。在 在序列驱动的机制中，可以通过转录检查点来修改替代剪接 例如启动子暂停。在试点研究中，我表明药理或遗传抑制 启动子抗性暂停因子可以减少HE/HSPC的产生。根据我的数据和文献，我将 测试以下假设，即EHT特异性分类同工型选择以不同的顺式作用元件为指导 （AIM 1），并由启动子近端暂停因素（AIM 2）监管。这些细胞类型特异性剪接的研究 在复杂的多细胞上下文中的调节将使您了解如何微调EHT命运 决定。这项研究的完成将揭示对HSPC起源的关键调节。