RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis.
RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis.
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DOI：
10.1371/journal.pgen.1005814
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发表时间：
2016-01
期刊：
影响因子：
4.5
通讯作者：
Lacaud G
中科院分区：
文献类型：
--
作者：
Draper JE;Sroczynska P;Tsoulaki O;Leong HS;Fadlullah MZ;Miller C;Kouskoff V;Lacaud G
核心结合因子(CBF)蛋白RUNX1是最终造血的主要调节因子，在个体发育过程中对造血干细胞(HSC)的出现至关重要。RUNX1在成年小鼠中也扮演着重要的角色，在调节许多血统的正确规格方面发挥着重要作用。与其他哺乳动物的RUNX基因类似，RUNX1有两个启动子P1(远端)和P2(近端)，它们产生不同的蛋白质亚型。这两个启动子在成人造血中的活性和具体相关性仍有待充分阐明。利用双报告小鼠模型，我们证明远端P1启动子在成人造血干细胞和祖细胞(HSPC)群体中广泛活跃。相比之下，近端P2启动子的活性受到更多的限制，在未成熟的谱系-Sca1高cKithigh(LSK)和双潜能前巨核/红系祖细胞(PreMegE)群体中，其上调与红系丢失(Ery)规范一致。因此，根据RUNX1 P2的活性，PreMegE群体可以被前瞻性地分为“前红系”和“前巨核细胞”群体。RUNX1、P2+和P2-细胞群体的基因表达比较分析表明，CD34表达水平可以替代P2活性来区分野生型(WT)骨髓中的这两个细胞群体。这两个群体的前瞻性分离将使进一步研究巨核/红系(Mk/Ery)细胞命运决定的分子机制成为可能。在表征了P1的广泛活性之后，我们利用P1-GFP纯合子小鼠模型来分析成年小鼠完全缺乏RUNX1 P1表达的影响，并观察到T细胞谱系的严重缺陷。最后，我们研究了白血病融合蛋白AML1-ETO9a如何影响RUNX1启动子的使用。AML1-ETO9a在骨髓中的短期诱导导致P2的优先上调，表明其表达对于建立白血病前环境可能是重要的。转录因子RUNX1被认为是成人和胚胎血细胞产生的主要调节因子。RUNX1的突变会导致人类患者和小鼠模型中不同血统的缺陷，包括白血病和由于缺乏产生血小板的巨核细胞而导致的凝血缺陷。与哺乳动物中存在的其他RUNX基因一起，RUNX1由两个启动子表达，这两个启动子产生几个不同的RNA转录本和蛋白质亚型。为了研究这两个启动子(称为远端和近端)的表达时间和定位，我们建立了一个报告基因在RUNX1启动子控制下表达的小鼠模型。我们之前描述了RUNX1启动子在发育中的胚胎中启动血液生产的活动。我们现在研究这两个启动子在成人器官中的输出，包括骨髓、脾和胸腺。我们在这里表明，RUNX1的远端启动子高度表达，但近端的启动子受到更多的限制，特别是标志着成人血液生产中红细胞和巨核细胞途径分离的点。因此，这两个RUNX1启动子产生的不同蛋白质可能在驱动这两种不同类型细胞的产生方面具有不同的作用。
The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into “pro-erythroid” and “pro-megakaryocyte” populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment. The transcription factor RUNX1 is considered a master regulator of adult and embryonic blood cell production. Mutations in RUNX1 cause defects in different blood lineages in human patients and mouse models, including leukemia and blood clotting defects due to a shortage of platelet-producing megakaryocytes. Together with the other RUNX genes present in mammals, RUNX1 is expressed from two promoters, which produce several distinct RNA transcripts and protein isoforms. To investigate the timing and localization of the expression of these two promoters (termed distal and proximal), we created a mouse model with reporter genes expressed under the control of the Runx1 promoters. We previously described the activities of the Runx1 promoters at the initiation of blood production in the developing embryo. We now investigate the output from the two promoters in adult organs, including bone marrow, spleen and thymus. We show here that the distal Runx1 promoter is highly expressed but the proximal promoter is more restricted and in particular marks the point in adult blood production where the red blood cell and megakaryocyte pathways separate. The different proteins produced by these two Runx1 promoters may therefore have different roles in driving the production of these two distinct cell types.