Establishing the role and mechanisms of LSD1 during megakaryocytic and erythroid fate commitment

项目摘要/摘要 在血液中发现的各种细胞类型都是由造血作用产生的。在成年人中，这一过程主要是 发生在骨髓中，以连续几轮分化为特征，以 造血干细胞通过向世系承诺。这一过程涉及到血统的逐步缩小。 作为祖细胞的潜能最终致力于生产单细胞谱系。造血功能紊乱 会导致良性和恶性的病理。我们关注的是双能巨核红系祖细胞 (MEP)，它有可能分化为血统承诺的红系祖细胞(ERP)或血统 承诺的巨核系祖细胞(MKP)。MEP命运规范已经被研究到了有限的程度， 只发现了对这一过程有贡献的一小部分影响。人类一个未被充分研究的方面 MEP命运规范是表观遗传学的作用。已知KDM1A(LSD1)在红系中起重要作用 成熟，但它在MEP命运规范中的作用尚不清楚。使用原代人体细胞的初步数据显示 抑制原代人类事件相关电位中的LSD1导致红系祖细胞能够经历 粒细胞-单核细胞和巨核细胞承诺。相反，抑制LSD1并不影响MKP 对巨核细胞命运的承诺。这表明LSD1只拮抗另一种谱系潜力 在红细胞生成过程中，而不是巨核细胞生成过程中，即使LSD1 mRNA在 MEP、ERP和MKP。这项提案的目标是建立LSD1促进红系的机制 承诺，同时沉默替代髓系和巨核细胞系的潜力。我将实现这些目标 利用功能(细胞培养)、遗传和表观遗传学的混合方法来确定LSD1在哪里以及如何 调节MEP、ERP和MKP中的基因表达和命运规范。计算分析将确定 候选基因靶点和潜在的基因组位置，以使用不同的融合来强制表观遗传修改 基于催化失活的Cas9核酸内切酶(DCas9)的蛋白质。这些结果将确立 控制LSD1介导的红系谱系承诺的表观遗传学机制。它们还将揭示 关于不同的谱系特有的表观遗传机制，调节和维持命运的决定 造血术。

PROJECT SUMMARY/ABSTRACT Hematopoiesis is responsible for producing the varied cell types found in blood. In adults, this process primarily takes place in the bone marrow and is characterized by successive rounds of differentiation beginning with the hematopoietic stem cell through to lineage commitment. This process involves progressive narrowing of lineage potency as progenitors eventually commit to the production of a single cell lineage. Disruption of hematopoiesis can lead to benign and malignant pathologies. We focus on the bipotent megakaryocytic-erythroid progenitor (MEP), which has the potential to differentiate into a lineage committed erythroid progenitor (ErP) or a lineage committed megakaryocytic progenitor (MkP). MEP fate specification has been studied to a limited extent, uncovering only a small number of influences that contribute to this process. One understudied aspect of human MEP fate specification is the role of epigenetics. KDM1A (LSD1) is known to be important for erythroid maturation, but its role in MEP fate specification is unknown. Preliminary data using primary human cells show that inhibition of LSD1 in primary human ErP results in the ability of erythroid committed progenitors to undergo granulocytic-monocytic and megakaryocytic commitment. In contrast, LSD1 inhibition does not affect MkP commitment to the megakaryocytic fate. This suggests that LSD1 only antagonizes alternative lineage potential during erythropoiesis, and not megakaryopoiesis, even though LSD1 mRNA is expressed at similar levels in MEP, ErP, and MkP. The goal of this proposal is to establish the mechanism by which LSD1 promotes erythroid commitment while silencing alternative myeloid and megakaryocytic lineage potential. I will achieve these goals utilizing a mix of functional (cell culture), genetic and epigenetic approaches to determine where and how LSD1 regulates gene expression and fate specification in MEP, ErP, and MkP. Computational analyses will determine candidate gene targets, and potential genomic sites to force epigenetic modifications using different fusion proteins based on the catalytically inactive Cas9 endonuclease (dCas9). These results will establish the epigenetic mechanisms that govern erythroid lineage commitment mediated by LSD1. They will also shed light on the distinct lineage-specific epigenetic mechanisms that mediate and maintain fate specification in hematopoiesis.