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

项目摘要 造血干细胞（hematopoietic stem cells，HSCs）具有复杂的维持体内稳态的机制 以平衡自我更新、增殖和分化。HSC在发育过程中和感染后 调整自我更新与差异化，以满足但不超过 有机体了解HSC如何调节这种平衡对于体外操纵HSC至关重要 用于改进移植中的用途。为了提高我们对HSC自我更新与 其他需要，我们已经研究了与原癌基因MLL 1相关的分子通路，由于其 独特的能力，以调节HSC丰富的转录程序和潜力，成为 急性白血病中染色体易位的致白血病性。我们用老鼠的损失- 功能模型表明，Mll 1在胚胎发生期间对HSC的发育以及 在成人骨髓中维持。这一作用是通过维持一个 转录调控子网络，包括Hoxa 9、Mecom/Evi 1、Prdm 16和Meis 1。因此我们 假设MLL 1蛋白水平或活性的增加可以协调地增强这一点， HSC的自我更新和防止分化。测试 根据这一假设，我们创造了一种新的动物模型，首次克服了耐药性。 过表达野生型MLL 1。使用单拷贝整合到强力霉素中- 诱导位点，我们显示增加造血输出从胚状体和胚胎 hMLL 1诱导后的祖细胞。在这个单一目标闪耀II提案中，我们试图评估这一点， 动物模型，以确定a）发育和分化阶段， 允许hMLL 1诱导的造血扩增，和B）鉴定 通过相关造血群体中MLL 1水平的增加而改变。我们期望 产生将增加的MLL 1与增强的造血联系起来的原理证明数据， 确定潜在的机制将指导未来的努力，实验操纵MLL 1， 人类干细胞和祖细胞在移植中扩增其数量并改善其功能 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.