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.

项目总结/摘要 造血干细胞和祖细胞（HSPC）通过自我更新和再生维持终身造血。 分化成各种类型的血细胞。它们在早期胚胎发育过程中通过一个受调节的和保守的 这一过程称为内皮细胞向造血细胞转化（EHT）。在EHT期间，内皮细胞亚群 (ECs)调整造血转录程序以形成生血内皮（HE），然后 形态学改变以制造HSPC。由于HSPCs的从头产生仅发生在早期胚胎发育期间， 在胚胎发育中，HSPC形成对所有胚胎和成体造血具有深远的影响。缺陷 EHT调节剂在血液系统疾病中普遍存在，因此通过研究EHT获得的经验教训可以 告知这些疾病的传播途径。研究EHT调节对于理解过程关键至关重要 从胚胎到成人的造血健康。虽然控制EHT的一些因素是已知的，但我们的 对HE/HSPC调节剂的了解仍然很少。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，因为在斑马鱼突变体中， 剪接体成分剪接因子3b，亚单位1（sf 3b 1）。这些数据表明，剪接是重要的 HE的形成，但机制调节剪接的变化，EHT的关键是在很大程度上是未知的。到 为了探索这一过程，我首先定义了胚胎斑马鱼EC和 HE/HSPC。因此，前mRNA内的顺式作用调控元件引导剪接异构体选择，以鉴定 在EHT过程中控制选择性剪接的潜在机制，我调查了EHT和EHT之间的选择性剪接事件。 EC和HE/HSPC的剪接调控序列特征差异。通过初步分析，我 发现EHT选择性事件富含较弱的剪接位点和较短的内含子长度 提示这些特征在决定EHT特异性剪接异构体选择中具有调节功能。在 除了序列驱动的机制外，可变剪接还可以通过转录检查点进行修饰 例如启动子近端暂停。在初步研究中，我表明，药物或遗传抑制， 启动子近端暂停因子可减少HE/HSPC的产生。根据我的数据和文献，我将 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指导的假设 (aim 1）并受启动子近端暂停因子（aim 2）调控。这些关于细胞类型特异性剪接的研究 在一个复杂的多细胞环境中的调控将使我们能够理解剪接是如何微调EHT的命运的 决定这项研究的完成将揭示HSPC发生的关键调控。