Enhancing hematopoiesis through modulation of a histone methyltransferase: evaluating a new MLL1 gain-of-function animal model

项目总结 为了维持持续的体内平衡，造血干细胞(HSCs)具有复杂的机制 平衡自我更新、增殖和分化。发育过程中和感染后的HSCs 调整自我更新与差异化，以扩展以满足但不超过 有机体。了解造血干细胞如何调节这一平衡对于体外操作造血干细胞至关重要。 用于改善移植中的使用。为了提高我们对HSC自我更新与平衡的理解 为了满足其他需求，我们已经研究了与原癌基因MLL1相关的分子通路，因为它 独特的能力来调节HSC丰富的转录程序，并有可能成为 急性白血病患者染色体易位导致白血病。我们已经展示了使用鼠标丢失- M111在胚胎发育过程中对HSCs的发育是必不可少的功能模型 它们在成人骨髓中的维持。这一角色是通过维护 转录调控因子网络包括Hoxa9、Mecom/Evi1、Prdm16和Meis1。因此我们 假设增加MLL1蛋白水平或活性可以协调地增强这一点 转录网络，从而增强HSC的自我更新，防止分化。为了测试 在这个假设下，我们创造了一种新的动物模型，首次克服了抵抗力 在多种细胞类型中过表达野生型MLL1。使用单拷贝整合到多西环素- 可诱导基因座，我们显示类胚体和胚胎的造血量增加。 祖细胞对hMLL1的诱导。在这个单一的Aim Shine II提案中，我们试图评估这一点 详细的动物模型，以确定a)发育和分化阶段， 允许hMLL1诱导的造血扩张，以及b)确定 在相关的造血群体中，MLL1的水平增加而改变。我们的期望是 生成将增加的MLL1与增强的造血联系起来的原则证明数据 确定潜在的机制将指导未来在实验中操纵MLL1的努力 人类干细胞和祖细胞在移植中扩大数量和改善功能 设置。

PROJECT SUMMARY To maintain ongoing homeostasis, hematopoietic stem cells (HSCs) possess elaborate mechanisms to balance self-renewal, proliferation and differentiation. During development and after infection HSCs adjust self-renewal versus differentiation to expand to meet but not overshoot the needs of the organism. Understanding how HSCs modulate this balance is critical for manipulating HSCs ex vivo for improved use in transplantation. To improve our understanding balancing HSC self-renewal with other needs, we have studied molecular pathways connected to the proto-oncogene, MLL1, due to its unique ability to regulate an HSC-enriched transcriptional program and potential to become leukemogenic upon chromosomal translocation in acute leukemia. We have shown using mouse loss- of-function models that Mll1 is essential for the development of HSCs during embryogenesis and for their maintenance in adult bone marrow. This role is carried out by maintaining expression of a network of transcriptional regulators including Hoxa9, Mecom/Evi1, Prdm16 and Meis1. Therefore we hypothesize that increasing MLL1 protein levels or activity could coordinately enhance this transcriptional network thereby enhancing HSC self-renewal and preventing differentiation. To test this hypothesis, we have created a new animal model that for the first time overcomes the resistance of many cell types to overexpress wild-type MLL1. Using single-copy integration into a doxycycline- inducible locus, we show increased hematopoietic output from embryoid bodies and embryo progenitors upon hMLL1 induction. In this Single Aim Shine II proposal, we seek to evaluate this animal model in detail to determine a) the developmental and differentiation stages that are permissive for hMLL1-induced hematopoietic expansion and b) identify regulatory networks that are altered by increased levels of MLL1 in relevant hematopoietic populations. It is our expectation that generating this proof-of-principle data linking increased MLL1 to enhanced hematopoiesis and determining the underlying mechanisms will guide future efforts to experimentally manipulate MLL1 in human stem and progenitor cells to expand their numbers and improve function in transplantation settings.