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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

项目概要/摘要造血干细胞和祖细胞（HSPC）通过自我更新和维持终生造血功能分化为所有血细胞类型。它们在早期胚胎发生过程中通过受调控和保守的这个过程称为内皮细胞向造血细胞的转变（EHT）。在 EHT 期间，内皮细胞的一个子集（EC）调整造血转录程序以形成造血内皮（HE），然后形态变化以制造 HSPC。由于 HSPC 的从头生产仅发生在胚胎早期发育过程中，HSPC 的形成对所有胚胎和成人造血都有深远的影响。缺陷 EHT 调节剂在血液系统疾病中很常见，因此通过研究 EHT 获得的经验教训可以告知驱动这些疾病的途径。研究 EHT 监管对于理解流程至关重要从胚胎到成年的造血健康。尽管控制 EHT 的一些因素是已知的，但我们的人们对 HE/HSPC 监管机构的了解仍然知之甚少。在之前的研究中，鲍曼实验室确定 EHT 需要正确的前 mRNA 剪接，因为斑马鱼突变体中基本上不存在 HE 剪接体成分剪接因子 3b，亚基 1 (sf3b1)。这些数据表明拼接对于 HE 的形成，但调节对 EHT 至关重要的剪接变化的机制在很大程度上尚不清楚。到为了探索这个过程，我首先定义了胚胎斑马鱼 EC 和他/HSPC。前 mRNA 内的顺式作用调节元件指导剪接亚型选择，从而识别在EHT期间控制选择性剪接的潜在机制，我调查了之间的选择性剪接事件 EC 和 HE/HSPC 剪接调控序列特征的差异。通过这个初步分析，我发现 EHT 替代事件因剪接位点较弱和内含子长度较短而丰富表明这些特征可能在决定 EHT 特异性剪接亚型选择方面具有调节功能。在除了序列驱动机制之外，选择性剪接还可以通过转录检查点进行修改例如启动子近端暂停。在初步研究中，我表明药理学或遗传抑制启动子近端暂停因子可以减少 HE/HSPC 的产生。根据我的数据和文献，我将检验以下假设：EHT 特异性剪接亚型选择是由不同的顺式作用调节元件引导的（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环境中进行调控将有助于理解剪接如何微调 EHT 的命运决定。这项研究的完成将揭示 HSPC 发生的关键调控机制。