Identification of NOTCH4in28 cis-regulatory element with enhancer activity. (A) A region in the 28th intron of NOTCH4 containing SOX17 binding sites is conserved throughout mammals. The upper right corner image shows ATAC-seq and SOX17 ChIP-seq peaks in D4 HE from DOX-treated and untreated iSOX17 hPSC differentiation cultures¹⁸ and ATAC-seq peaks from human fetal liver samples.³¹ (B) Schematic diagram of ChIP PCR. ChIP P1 and P2 are screening primers. Quantitative ChIP-qPCR analysis of SOX17, H3K27ac, and H3K4me1 at intron 28 of NOTCH4 in D4 HE from iSOX17 hPSCs (means ± standard deviations [SDs], n = 6 experiments). ∗∗∗∗P < .0001, 2-way analysis of variance (ANOVA), Sidak multiple comparisons test. (C) iSOX17 hPSCs were transfected with Luc reporter constructs containing the intact NOTCH4in28 or NOTCH4in28 with a deleted 295-bp fragment including SOX17 motifs. After transfection, cells were treated 2 μg/mL DOX. Luc values were normalized to Renilla Luc control reporter values (means ± SDs, n = 3 experiments). ∗∗∗∗P < .0001, 1-way ANOVA, Tukey multiple comparisons test. HSPC, hematopoietic stem and progenitor cell; IgG, immunoglobulin G.