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HumanNOTCH4is a key target of RUNX1 in megakaryocytic differentiation
Blood ( IF 21.0 ) Pub Date : 2018-01-11 , DOI: 10.1182/blood-2017-04-780379
Yueying Li 1 , Chen Jin 1, 2 , Hao Bai 3, 4 , Yongxing Gao 3, 4 , Shu Sun 1, 2 , Lei Chen 1, 2 , Lei Qin 1, 2 , Paul P Liu 5 , Linzhao Cheng 3, 4 , Qian-Fei Wang 1, 2
Affiliation  

Megakaryocytes (MKs) in adult marrow produce platelets that play important roles in blood coagulation and hemostasis. Monoallelic mutations of the master transcription factor gene RUNX1 lead to familial platelet disorder (FPD) characterized by defective MK and platelet development. However, the molecular mechanisms of FPD remain unclear. Previously, we generated human induced pluripotent stem cells (iPSCs) from patients with FPD containing a RUNX1 nonsense mutation. Production of MKs from the FPD-iPSCs was reduced, and targeted correction of the RUNX1 mutation restored MK production. In this study, we used isogenic pairs of FPD-iPSCs and the MK differentiation system to identify RUNX1 target genes. Using integrative genomic analysis of hematopoietic progenitor cells generated from FPD-iPSCs, and mutation-corrected isogenic controls, we identified 2 gene sets the transcription of which is either up- or downregulated by RUNX1 in mutation-corrected iPSCs. Notably, NOTCH4 expression was negatively controlled by RUNX1 via a novel regulatory DNA element within the locus, and we examined its involvement in MK generation. Specific inactivation of NOTCH4 by an improved CRISPR-Cas9 system in human iPSCs enhanced megakaryopoiesis. Moreover, small molecules known to inhibit Notch signaling promoted MK generation from both normal human iPSCs and postnatal CD34+ hematopoietic stem and progenitor cells. Our study newly identified NOTCH4 as a RUNX1 target gene and revealed a previously unappreciated role of NOTCH4 signaling in promoting human megakaryopoiesis. Our work suggests that human iPSCs with monogenic mutations have the potential to serve as an invaluable resource for discovery of novel druggable targets.

中文翻译:

HumanNOTCH4是RUNX1在巨核细胞分化中的关键靶标

成人骨髓中的巨核细胞(MK)产生血小板,在凝血和止血中发挥重要作用。主转录因子基因 RUNX1 的单等位基因突变会导致家族性血小板疾病 (FPD),其特征是 MK 和血小板发育缺陷。然而,FPD 的分子机制仍不清楚。此前,我们从含有 RUNX1 无义突变的 FPD 患者中产生了人类诱导多能干细胞 (iPSC)。FPD-iPSC 产生的 MK 减少,而对 RUNX1 突变的针对性纠正恢复了 MK 的产生。在本研究中,我们使用 FPD-iPSC 的同基因对和 MK 分化系统来鉴定 RUNX1 靶基因。通过对 FPD-iPSC 生成的造血祖细胞和突变校正的同基因对照进行整合基因组分析,我们鉴定了 2 个基因组,其转录在突变校正的 iPSC 中被 RUNX1 上调或下调。值得注意的是,NOTCH4 的表达受到 RUNX1 通过基因座内的新型调控 DNA 元件的负控制,我们检查了它在 MK 生成中的参与。改进的 CRISPR-Cas9 系统在人 iPSC 中特异性灭活 NOTCH4,增强了巨核细胞生成。此外,已知抑制Notch信号传导的小分子可促进正常人iPSC和出生后CD34+造血干细胞和祖细胞的MK生成。我们的研究新确定了 NOTCH4 作为 RUNX1 靶基因,并揭示了 NOTCH4 信号在促进人类巨核细胞生成中以前未被认识到的作用。我们的工作表明,具有单基因突变的人类 iPSC 有潜力成为发现新型药物靶标的宝贵资源。
更新日期:2018-01-11
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