NOTCH4 is directly and negatively regulated by RUNX1 in hematopoiesis. (A) Venn diagram showing the overlap between RUNX1 binding genes in RUNX1 mutation-corrected CD34⁺ cells and differentially expressed genes, either upregulated or downregulated genes, in RUNX1 mutation-corrected (RUNX1^+/+) CD34⁺ cells compared with RUNX1^+/− CD34⁺ cells. (B) GO enrichment analysis of RUNX1 binding genes that were downregulated in RUNX1 mutation-corrected CD34⁺ cells. (C) Heat map depiction of gene expression of RUNX1 binding genes that were downregulated in RUNX1^+/+ CD34⁺ cells compared with RUNX1^+/− CD34⁺ cells. The genes are classified as signaling, transcription factor, enzyme, and others. (D) Bar graph depicting fold changes of the cell numbers of HSPCs, MKPs, and MKs on day 14 in hematopoietic cells derived from FPD (RUNX1⁺⁻)-iPSCs, with addition of 10 μM GSIs (RO4929097 or DAPT) or DMSO (control). Data are shown as means ± standard error of the mean (SEM), n = 3. *P < .05; **P < .01. n.s., not significant. (E) RUNX1 binding region in intron 29 of NOTCH4. The 3ʹ region of the human NOTCH4 gene is enlarged. The genomic location of the RUNX1 binding region determined by ChIP-Seq is marked in the gene structure as a horizontal black line, and the RUNX1 consensus binding site is marked as a vertical red line. (F) RT-qPCR analysis of NOTCH4 mRNA level during differentiation from NOTCH4 intron 29 KO and WT BC1-iPSCs cells. β-Actin was used as an internal control. Data are shown as means ± SEM, n = 3. **P < .01; ***P < .001. (G) Bar graph depicting the cell numbers of HSPCs, MKPs, and MKs on day 14 derived from NOTCH4 intron 29 KO cells. Data are shown as means ± SEM, n = 3. *P < .05.