Establishing CUX1 as a determinant of Hematopoietic Stem cell heterogeneity

项目总结/摘要 造血干细胞（HSC）在再生能力、谱系偏好、 和细胞周期参与。从目的论上讲，异质性是干细胞使基本的 关于组织的群体的决定。干细胞行为的差异与 细胞表面和细胞质标记物的表达，而没有机制解释。HSC异质性 以前没有被归因于转录因子。我们认为观察到的HSC异质性可以 可以通过剂量依赖性、含有同源结构域的转录因子CUX 1的作用来解释。三 一系列证据表明CUX 1是HSC异质性的假定协调者：它在HSC异质性中反复起作用， 在发育系统中呈剂量依赖性，在高危造血系统疾病中反复丢失， 及其在染色质调节中的作用。 我报告了CUX 1 mCherry报告小鼠的产生，以研究CUX 1在造血中的作用。的 Cux 1 mCherry小鼠是内源性CUX 1基因座处的符合读框的C-末端mCherry标签的结果。的 标签的添加没有产生异常的造血表型，这表明这是一个合适的模型， 建议的研究。免疫表型长期HSC（LT-HSC）区室具有最大的 CUX 1表达的差异。在LT-HSC区室的CUX 1蛋白水平上，我们报告了几个 相关性强有力地表明，观察到的CUX 1蛋白的变化导致了有意义的差异， 干细胞行为例如，CUX 1Bright HSC比CUX 1 Int和CUX 1Dim更可能循环 HSC处于稳态。因此，我们的研究表明，CUX 1在小鼠中发挥剂量依赖性作用， 造血干细胞和祖细胞（HSPC）。该建议旨在（i）确立CUX 1在以下方面的作用： 谱系偏倚和再增殖能力，以及（ii）确定CUX 1发挥剂量- 依赖角色。完成所提出的研究将阐明一个重要的范式，在发展 生物学：一小群干细胞如何平衡自我更新和分化，以产生所有成熟的干细胞。 组织中的细胞。对干细胞行为的病因学理解将为干细胞的生物学行为提供新的见解。 为HSC中出现的许多疾病开发新疗法。 我在这里提出的项目是伴随着我和我的导师开发的培训计划， 描述了四个目标，我需要完成，以推动我成为一个成功的独立 物理学家兼科学家这四个目标包括获得造血方面的专业知识， 生物信息学，发展科学交流能力，整合科学和临床 我的训练方面。实现这些目标将给我的技能，我需要成为一个医生，科学家以及- 有能力解决与儿童灾难性疾病有关的有意义的生物学问题。

Project Summary/Abstract Hematopoietic stem cells (HSCs) exhibit heterogeneity with respect to repopulating capacity, lineage bias, and cell cycle participation. Teleologically, heterogeneity is the result of stem cells making fundamental decisions regarding the population of the tissue. Differences in stem cell behavior have been linked to the expression of cell surface and cytoplasmic markers without mechanistic explanation. HSC heterogeneity has not previously been attributed to a transcription factor. We propose the observed heterogeneity in HSCs can be explained by the actions of a dose-dependent, homeodomain-containing transcription factor, CUX1. Three lines of evidence suggest CUX1 to be a putative orchestrator of HSC heterogeneity: it recurrently acts in a dose-dependent manner across developmental systems, its recurrent loss in high-risk hematopoietic disease, and its role in chromatin regulation. I report the generation of a CUX1mCherry reporter mouse to study the role of CUX1 in hematopoiesis. The Cux1mCherry mouse is the result of an in-frame, C-terminal mCherry tag at the endogenous CUX1 locus. The addition of the tag creates no aberrant hematopoietic phenotype, suggesting this is a suitable model for the proposed studies. The immunophenotypic long-term HSC (LT-HSC) compartment has among the greatest variances in CUX1 expression. Across CUX1 protein levels in the LT-HSC compartment, we report several correlations to strongly suggest that the observed variation in CUX1 protein results in meaningful differences in stem cell behavior. For example, CUX1Bright HSCs are more likely to be cycling than CUX1Int and CUX1Dim HSCs at steady state. Thus, our studies suggest that CUX1 is playing a dose-dependent role in murine hematopoietic stem and progenitor cells (HSPCs). This proposal aims to (i) establish the role of CUX1 in lineage bias and repopulating capacity and (ii) determine the mechanism by which CUX1 exerts a dose- dependent role. Accomplishing the proposed studies will illuminate an important paradigm in developmental biology: how a small pool of stem cells balance self-renewal and differentiation to give rise to all the mature cells in a tissue. An etiological understanding of stem cell behavior will provide new insights into the development of new therapies for the many diseases that arise in HSCs. The project I propose here is accompanied by a training plan developed by me and my mentors that delineates four goals I will need to accomplish to propel me towards becoming a successful independent physician-scientist. These four goals include gaining expertise in hematopoiesis, gaining expertise in bioinformatics, developing proficiency in scientific communication, and integrating the scientific and clinical aspects of my training. Realizing these goals will give me the skills that I need to be a physician-scientist well- equipped to address meaningful biological questions related to the catastrophic illnesses of childhood.