Mechanisms of Flow-driven Transcriptional Control of Hematopoietic Stem Cell Development by YAP

YAP 流驱动转录控制造血干细胞发育的机制

• 批准号: 10283499
• 负责人: Wade William Sugden
• 金额: $ 15.53万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10283499
• 关键词: Achievement; Address; Adult; Advisory Committees; Algorithms; Animals; Aorta; Arteries; Award; Behavior; Blood; Blood Cells; Blood Vessels; Blood flow; Boston; Candidate Disease Gene; Cell Nucleus; Cells; Cellular biology; Chemicals; Clinical; Complex; Coupled; Coupling; Cues; DNA; DNA Binding; DNA-Protein Interaction; Data; Development; Distant; Dorsal; Embryo; Embryonic Development; Endothelial Cells; Endothelium; Enhancers; Environment; Erythroid; Event; Exhibits; Fetal Liver; Fishes; Future; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Goals; Growth; Growth and Development function; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Cell Production; Hematopoietic Neoplasms; Hematopoietic Stem Cell Specification; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Home; Human; Image; Immune; In Vitro; Individual; Integrin beta Chains; Integrins; Investigation; Laboratories; Learning; Link; Logic; Lymphoid; Mammals; Mechanics; Mediating; Mediator of activation protein; Membrane; Membrane Proteins; Mentors; Mentorship; Methods; Molecular; Morphogenesis; Morphology; Myelogenous; Nuclear; Organism; Patients; Pediatric Hospitals; Periodicity; Phase; Phenotype; Piezo 1 ion channel; Piezo ion channels; Postdoctoral Fellow; Production; Protein Activation Pathway; Proteins; Protocols documentation; RUNX1 gene; Regulation; Regulator Genes; Research; Research Training; Scientist; Signal Transduction; Signaling Protein; Solid; Stem Cell Development; Stretching; System; Therapeutic; Therapeutic Uses; Tissues; Training; Transcriptional Regulation; Translations; Transplantation; Viscosity; Work; Zebrafish; career development; cell type; chemical genetics; cofactor; developmental genetics; endothelial stem cell; genetic manipulation; genome-wide; hematopoietic differentiation; hematopoietic gene; hematopoietic stem cell differentiation; hematopoietic stem cell expansion; hematopoietic stem cell fate; hematopoietic stem cell formation; hematopoietic tissue; hemodynamics; hemogenic endothelium; improved; in vivo; in vivo evaluation; induced pluripotent stem cell; interest; knock-down; leukemia; leukemia/lymphoma; link protein; mechanical force; medical schools; new technology; non-genetic; novel; programs; protein activation; response; self-renewal; shear stress; stem cell biology; stem cell therapy; success; transcription factor; transcriptomics; vertebrate embryos

Project Summary/Abstract Hematopoietic stem cells (HSCs) are capable of producing all erythroid, myeloid and lymphoid blood cells of an organism. Coupled with their unique capacity for self-renewal, successful transplantation of healthy HSCs is the only therapy currently available that can completely replace and restore the blood system in patients with leukemia and lymphoma. Despite this need, HSCs presently cannot be efficiently created or cultured in vitro, suggesting that extrinsic factors supporting their growth and development in vivo are lacking from existing protocols. Previous work from our lab demonstrated that blood flow is an essential non-genetic environmental cue required for HSC production in vertebrate embryos, mediated in part by stimulating mechanical activation of the Yes-associated protein (YAP) transcription factor (TF). This proposal intends to resolve the physical, genetic and molecular mechanisms underlying mechanically-activated, YAP-driven HSC production. YAP, while a potent co-activator of gene expression, lacks DNA-binding ability of its own. To understand the molecular logic behind flow/YAP-driven hematopoiesis, the goal of the first aim is to employ chemical, physical and genetic perturbation of shear stress and cyclic stretch in live zebrafish embryos to assess the impact of these individual components of hemodynamic force on HSC production from hemogenic endothelium (HE). To this will be added tissue- specific transcriptomic and genome-wide YAP/DNA interaction profiling from sorted HE from wildtype zebrafish, flow-deficient and yap-/- animals (with normal blood flow) in order to discriminate flow-dependent gene regulatory modules and transcriptional targets that rely on YAP. Hypothesis-driven candidate TFs will be tested in vivo and in vitro to evaluate YAP-interaction ability and uncover key partners required for normal YAP-dependent hematopoiesis. In the second aim, the zebrafish system will be used to investigate candidate membrane- localized proteins, Piezos and Integrins, as components linking hemodynamic forces with YAP activation. These studies stand to provide a comprehensive “membrane-to-nucleus” paradigm for how blood flow activates YAP to guide developmental hematopoiesis, which may improve current efforts to generate or expand HSCs. As a postdoctoral fellow, Dr. Sugden will conduct his research in the laboratory of Dr. Trista North at Boston Children's Hospital. Her expertise in extrinsic regulation of developmental hematopoiesis, together with dedicated co-mentorship by Dr. George Daley (an expert in stem cell biology and hematology) and a strong advisory team provide an exceptionally well-supported environment for career development and research training. Dr. Sugden will build on a solid background in developmental genetics and live-imaging, by adding new technologies in transcription factor/DNA interaction profiling, transcriptomics and in vitro methods to study protein interactions. A rigorous research and training plan lay the groundwork for success, both in the mentored and independent phases of the award. The environment at Boston Children's Hospital and Harvard Medical School will provide the ideal surroundings to support Dr. Sugden to become a successful independent scientist.

项目概要/摘要 造血干细胞 (HSC) 能够产生所有红系、髓系和淋巴系血液 有机体的细胞。加上它们独特的自我更新能力，成功移植健康的 HSCs是目前唯一可以完全替代和恢复患者血液系统的疗法 患有白血病和淋巴瘤。尽管有这样的需求，HSC 目前还不能在体外有效地产生或培养， 这表明现有的技术缺乏支持其体内生长和发育的外在因素。 协议。我们实验室之前的工作表明，血流是一种重要的非遗传环境 脊椎动物胚胎中 HSC 产生所需的线索，部分通过刺激机械激活来介导 Yes 相关蛋白 (YAP) 转录因子 (TF)。该提案旨在解决身体、遗传问题 以及机械激活、YAP 驱动的 HSC 生产的分子机制。 YAP，虽然是一个强大的 基因表达的共激活剂，缺乏其自身的 DNA 结合能力。了解背后的分子逻辑 流/YAP驱动的造血，第一个目标是利用化学、物理和遗传扰动 活体斑马鱼胚胎中的剪切应力和循环拉伸，以评估这些单独成分的影响 血流动力学对造血内皮 (HE) 生成 HSC 的影响。为此将添加组织- 来自野生型斑马鱼的分选 HE 的特定转录组和全基因组 YAP/DNA 相互作用分析， 血流缺陷和 yap-/- 动物（血流正常），以区分血流依赖性基因调控 依赖 YAP 的模块和转录目标。假设驱动的候选转录因子将在体内进行测试 体外评估 YAP 相互作用能力并发现正常 YAP 依赖所需的关键伙伴 造血作用。在第二个目标中，斑马鱼系统将用于研究候选膜- 局部蛋白、压电蛋白和整合素，作为将血流动力学与 YAP 激活联系起来的成分。这些 研究将为血流如何激活 YAP 提供全面的“膜到核”范例 指导发育性造血，这可能会改善目前生成或扩大 HSC 的努力。 作为博士后研究员，Sugden 博士将在 Trista North 博士的实验室进行研究 波士顿儿童医院。她在发育造血的外在调节方面的专业知识，以及 George Daley 博士（干细胞生物学和血液学专家）的专门共同指导和强大的 顾问团队为职业发展和研究提供非常良好的支持环境 训练。 Sugden 博士将在发育遗传学和实时成像方面建立坚实的背景，通过添加新的 转录因子/DNA 相互作用分析、转录组学和体外蛋白质研究方法等技术 互动。严格的研究和培训计划为成功奠定了基础，无论是在指导还是在 奖项的独立阶段。波士顿儿童医院和哈佛医学院的环境 将为萨格登博士成为一名成功的独立科学家提供理想的环境。