Gene expression analysis to identify Runx1 target genes in GMP

Disrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1- deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSC-Meg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and granulocyte/monocyte (G/M) maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML. GMP were isolated either from Runx1+/+-Tg(vav-Cre) and Runx1fl/fl-Tg(vav-Cre) mice or from mice transplanted with Runx1fl/fl-Tg(vav-Cre) progenitors engineered to express GFP with RUNX1-ERt2 or ERt2. RUNX1-ERt2 activity was induced by i.p. injection of tamoxifen on 3 consecutive days prior to GMP isolation.RUNX1基因的破坏性突变在10%的骨髓增生异常综合征（myelodysplasia, MDS）患者与30%的急性髓系白血病（acute myeloid leukemia, AML）患者中被检出。既往研究发现，条件性Runx1敲除（Runx1-knockout, KO）小鼠体内的造血干细胞（hematopoietic stem cells, HSCs）与多能祖细胞（multipotent progenitor, MPP）数量增多，但具体分子机制尚未明确。本研究聚焦于敲除小鼠的髓系祖细胞（myeloid progenitor, MP）群体，推测HSC/MPP层面的异常可能在下游细胞中被进一步放大。研究证实，Runx1敲除小鼠的髓系祖细胞群体扩增超过5倍，且显著向巨核细胞（megakaryocyte, Meg）祖细胞倾斜。Runx1缺陷型粒细胞-巨噬细胞祖细胞具有以下特征：克隆形成能力增强、向成熟细胞分化受阻，且呈现造血干细胞与巨核细胞的转录组特征。在Runx1缺陷小鼠体内，表达巨核细胞标志物的HSC/MPP亚群数量同样有所增加。挽救实验结合转录组分析与Runx1 DNA结合实验表明，细胞谱系定型过程中，Runx1会抑制编码黏附与运动蛋白的基因（Tek、Jam3、Plxnc1、Pcdh7及Selp），这些蛋白可介导造血干细胞与巨核细胞和骨髓（bone marrow, BM）微环境的相互作用。体外实验证实，在造血干细胞/多能祖细胞中强制表达Tek可提升巨核细胞的生成量。值得注意的是，除了上述调控祖细胞谱系决定与细胞数量的关键阻遏功能外，本研究还揭示了Runx1在红细胞分化中发挥的关键激活功能，补充了其已知的在巨核细胞以及粒细胞/单核细胞（granulocyte/monocyte, G/M）成熟过程中的重要作用。综上，Runx1在多个造血阶段与谱系中同时具备阻遏与激活功能，这可能是其在骨髓增生异常综合征与急性髓系白血病中发挥肿瘤抑制活性的分子基础。本研究中，粒细胞-巨噬细胞祖细胞（granulocyte-macrophage progenitors, GMP）分别从两种小鼠中分离：一种为Runx1+/+-Tg(vav-Cre)与Runx1fl/fl-Tg(vav-Cre)小鼠，另一种为经工程改造以表达绿色荧光蛋白（green fluorescent protein, GFP）并携带RUNX1-ERt2或ERt2的Runx1fl/fl-Tg(vav-Cre)祖细胞移植的受体小鼠。在分离GMP前的连续3天，通过腹腔注射（intraperitoneal, i.p.）他莫昔芬以诱导RUNX1-ERt2的活性。