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Human NOTCH4 is a key target of RUNX1 in megakaryocytic differentiation

Yueying Li, Chen Jin, Hao Bai, Yongxing Gao, Shu Sun, Lei Chen, Lei Qin, Paul P. Liu, Linzhao Cheng and Qian-Fei Wang

Key points

  • NOTCH4 is a RUNX1 direct target whose expression is negatively regulated by RUNX1 during human megakaryopoiesis.

  • Inhibition of NOTCH4 by genetic approach or chemical inhibitors enhances MK production from human iPSCs and cord-blood CD34+ cells.

Abstract

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 utilized 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 two gene sets whose transcription 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.

  • Submitted April 19, 2017.
  • Accepted October 13, 2017.