Reprogramming Gene Regulatory Networks to a Hematopoietic Stem Cell State

项目摘要/摘要 造血是通过有组织的活动产生血细胞的连续过程。 造血干细胞(HSC)。尽管造血干细胞具有巨大的临床实用价值，因为它们能够 通过移植重建造血系统，他们的好处仍然受到缺乏匹配的限制 捐赠者。最近，通过诱导重编程因子将内皮细胞直接重编程为HSCs 成为一种很有前途的替代方案。我们建议的总体目标是通过以下方式揭示分子机制 哪些重编程因子FosB、GFI1、RUNX1和SPI1(FGRS)使内皮细胞恢复功能 重新编程的HSC(重新编程的HSC)。理解遗传网络为此重新布线的基础 深刻的细胞类型转换将提供对不同形式的重新编程、开发和 疾病。我们发现，在重新编程过程的早期，fgrs直接协调两项任务：选择 多能HSC增强子的激活以及内皮增强子和转录因子的破坏 (TFS)。我们假设FGRS对内皮细胞TF结合的影响与重新编程一样关键 多效性增强剂的激活，我们建议剖析其潜在的分子机制 流程。利用单细胞多组体(scRNA和atac-seq)图谱，我们进一步发现在中间 重新编程，相对同质的起始内皮细胞被异种HSC取代 人口。体细胞(内皮细胞)到多潜能(HSC)调节程序的转变是如何发生的 正在进行体外重新编程的单个细胞仍然未知。为了加强体内重新编程，我们 产生了一种新的转基因小鼠模型，允许在所有身体组织中持续表达fgrs，并 便于记录导致HSC建立和维护的所有关键分叉事件。基于 在我们的研究中，我们建议剖析fgrs促进细胞命运的分子和细胞机制。 内皮细胞到肝星状细胞重新编程的背景变化。在我们的第一个目标中，我们将发现分子 FGRS靶向和调控内皮细胞和HSC基因调控网络的机制。在第二个 目的：我们将描述促进内皮细胞到肝星状细胞的内源性和外源性信号通路。 重新编程。我们希望我们的计划将对哺乳动物细胞的控制产生基本的见解 并可能导致新的策略，以高效地产生治疗相关的造血干细胞。

PROJECT SUMMARY/ABSTRACT Hematopoiesis is a continuous process of blood-cell production occurring through the orchestrated activity of hematopoietic stem cells (HSCs). Although HSCs have tremendous clinical utility due to their ability to reconstitute the hematopoietic system by transplantation, their benefit remains limited by the lack of matched donors. Direct reprogramming of endothelial cells into HSCs via induction of reprogramming factors has recently emerged as a promising alternative. The overall goal of our proposal is to reveal the molecular mechanisms by which the reprogramming factors FOSB, GFI1, RUNX1, and SPI1 (FGRS) revert endothelial cells to functional reprogrammed HSCs (reHSCs). Understanding the basis by which the genetic networks become rewired for this profound cell type conversion will provide insights into diverse forms of reprogramming, development, and disease. We discovered that early in the reprogramming process, FGRS directly coordinate two tasks: selection and activation of multipotent HSC enhancers and disruption of endothelial enhancers and transcription factors (TFs). We hypothesize that the effect of FGRS on endothelial TF binding is as crucial for reprogramming as the activation of multipotency enhancers, and we propose to dissect the underlying molecular mechanisms for these processes. Using single-cell multiomic (scRNA & ATAC-seq) profiling, we further discovered that in intermediate reprogramming, the relatively homogenous starting endothelial cells are replaced by heterogeneous HSC populations. How the transition from somatic (endothelial) to multipotent (HSC) regulatory programs occurs in individual cells undergoing in vitro reprogramming remains unknown. To potentiate in vivo reprogramming, we generated a novel transgenic mouse model that allows constant FGRS expression in all somatic tissues and facilitates the recording of all key bifurcating events that lead to HSC establishment and maintenance. Based on our studies, we propose to dissect the molecular and cellular mechanisms by which FGRS promote cell fate changes in the context of endothelial-to-HSC reprogramming. In our first aim, we will uncover the molecular mechanisms by which FGRS target and modulate endothelial and HSC gene regulatory networks. In the second aim, we will delineate the intrinsic and extrinsic signaling pathways that promote endothelial-to-HSC reprogramming. We expect that our program will yield fundamental insights into the control of mammalian cell identity and may lead to novel strategies to generate therapeutically relevant HSCs with high efficiency.