来自原代人类巨核细胞-红系祖细胞 (MEP) 的巨核细胞 (Mk) 或红系 (E) 谱系的规范对于造血稳态至关重要，但调节命运规范的潜在机制仍然难以捉摸。在这项研究中，我们将 RUNX1 确定为 MEP 命运规范期间基因表达的关键调节剂。原代人 MEP 中 RUNX1 的过度表达促进 Mk 特化，而泛 RUNX 抑制则有利于 E 特化。尽管 Mk 祖细胞 (MkP) 和 E 祖细胞 (ErP) 之间的总 RUNX1 水平没有差异，但 MkP 中丝氨酸磷酸化 RUNX1 的水平高于 ErP，并且具有磷酸丝氨酸/苏氨酸模拟突变的突变体 RUNX1 (RUNX1-4D)显着增强 RUNX1 的功能功效。为了模拟 RUNX1 变体的影响，我们采用表达野生型 (WT)、拟磷酸化 (RUNX1-4D) 和非磷酸化 (RUNX1-4A) 突变体的人红白血病 (HEL) 细胞系，表明 RUNX1 的三种形式存在差异调节 2,625 个基因的表达。 WT 和 RUNX1-4D 变体均增加了 40% 的表达，并减少了另外 40% 的表达，其中 RUNX1-4A 的影响较小。我们发现表达 WT 和 RUNX1-4D 的 HEL 细胞中上调的基因与原代人 MkP 与 MEP 中上调的基因之间存在显着重叠。虽然抑制已知的 RUNX1 丝氨酸/苏氨酸激酶不会影响原代 MEP 中的磷酸丝氨酸 RUNX1 水平，但特异性抑制 MEP 中的 CDK9 会导致 RUNX1 磷酸化降低和红细胞定向增加。总的来说，我们的研究结果表明，RUNX1 的丝氨酸/苏氨酸磷酸化可促进 Mk 命运规范，并为 RUNX1 引入一种新型激酶，将基本转录机制与细胞类型特异性转录因子的激活联系起来。版权所有 © 2024 美国血液学会。

The specification of megakaryocytic (Mk) or erythroid (E) lineages from primary human megakaryocytic-erythroid progenitors (MEP) is crucial for hematopoietic homeostasis, yet the underlying mechanisms regulating fate specification remain elusive. In this study, we identify RUNX1 as a key modulator of gene expression during MEP fate specification. Overexpression of RUNX1 in primary human MEP promotes Mk specification, while pan-RUNX inhibition favors E specification. Although total RUNX1 levels do not differ between Mk progenitors (MkP) and E progenitors (ErP), there are higher levels of serine-phosphorylated RUNX1 in MkP than ErP, and mutant RUNX1 with phospho-serine/threonine mimetic mutations (RUNX1-4D) significantly enhances the functional efficacy of RUNX1. To model the effects of RUNX1 variants, we employ human erythroleukemia (HEL) cell lines expressing wild-type (WT), phosphomimetic (RUNX1-4D), and non-phosphorylatable (RUNX1-4A) mutants showing that the three forms of RUNX1 differentially regulate expression of 2,625 genes. Both WT and RUNX1-4D variants increase expression in 40%, and decrease expression in another 40%, with lesser effects of RUNX1-4A. We find a significant overlap between the upregulated genes in WT and RUNX1-4D-expressing HEL cells and those upregulated in primary human MkP versus MEP. While inhibition of known RUNX1 serine/threonine kinases does not affect phosphoserine RUNX1 levels in primary MEP, specific inhibition of CDK9 in MEP leads to both decreased RUNX1 phosphorylation and increased erythroid commitment. Collectively, our findings show that serine/threonine phosphorylation of RUNX1 promotes Mk fate specification and introduce a novel kinase for RUNX1 linking the fundamental transcriptional machinery with activation of a cell-type specific transcription factor.Copyright © 2024 American Society of Hematology.