RUNX1B expression distinguishes megakaryocytic and erythroid lineage fate in adult hematopoiesis

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny, which 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 model, we demonstrate here 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 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 the level 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 provide the opportunity to further investigate and define the molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Moreover, comparison of a RUNX1C null (KO) PreMegE to its WT counterpart demonstrated considerably enhanced erythroid specification at the expense of megakaryopoiesis in the absence of P1-specified RUNX1C expression. mRNA profiles of wild type (WT), Runx1 P2-hCD4+ (P2+), Runx1 P2-hCD4- (P2-) and RUNX1C knockout (KO) bone marrow Pre-Megakaryocyte/Erythroid (PreMegE) progenitors were generated from young adult (12-16 weeks) mice by deep sequencing, in triplicate, using Illumina NextSeq 500.核心结合因子（Core Binding Factor, CBF）蛋白RUNX1是定型造血的主控调控因子，在个体发育过程中对造血干细胞（hematopoietic stem cell, HSC）的产生至关重要，同时在成年小鼠体内也发挥着关键作用，调控众多血液谱系的正确特化。与其他哺乳动物Runx基因类似，Runx1拥有两个启动子P1（远端启动子）和P2（近端启动子），二者可编码产生不同的蛋白质同工型。这两个启动子在成年造血过程中的活性与具体功能仍有待全面阐明。本研究借助双报告基因模型，证实远端P1启动子在成年造血干祖细胞（hematopoietic stem and progenitor cell, HSPC）群体中广泛活跃。与之相反，近端P2启动子的活性更为局限；在未成熟谱系阴性Sca1高表达c-Kit高表达（LSK）以及双潜能巨核细胞/红细胞祖细胞（PreMegE）群体中，P2启动子的上调与红细胞谱系特化能力丧失同步发生。据此，可基于Runx1 P2的活性将PreMegE群体前瞻性地分为"促红细胞系"与"促巨核细胞系"两个亚群。对Runx1 P2阳性与P2阴性群体的比较基因表达分析显示，在野生型（wild type, WT）骨髓（bone marrow, BM）中，CD34的表达水平可替代P2活性，用于区分这两个细胞群体。对这两个亚群的前瞻性分离，将为进一步研究并阐明巨核细胞/红细胞（Mk/Ery）细胞命运决定相关的分子机制提供契机。此外，将RUNX1C敲除（RUNX1C null, KO）PreMegE细胞与其野生型对应群体进行比较后发现，在缺乏P1调控表达的RUNX1C蛋白时，红细胞谱系特化能力显著增强，而巨核细胞生成则受到抑制。本研究通过Illumina NextSeq 500测序平台，对来自12~16周龄年轻成年小鼠的野生型、Runx1 P2-hCD4阳性（P2+）、Runx1 P2-hCD4阴性（P2-）以及RUNX1C敲除（KO）骨髓双潜能巨核细胞/红细胞祖细胞的mRNA表达谱进行了三次生物学重复的深度测序。