NOTCH4 Intron 28 Regulatory Element Controls Arterial Specification and Lymphoid Development from hPSCs

• Expression of NOTCH4 in endothelium is controlled by NOTCH4in28 cis-regulatory element

• NOTCH4in28 element is regulated by SOX17 and involved in arterial specification and lymphoid development from hPSCs

NOTCH signaling plays a critical role in arterial specification, and the formation of transient definitive lymphomyeloid progenitors and HSCs from hemogenic endothelium (HE). To investigate NOTCH signaling mechanisms critical for arterial and lymphoid specification, we performed single cell multiomics analysis of human pluripotent stem cells (hPSCs) at different stages of hematopoietic differentiation. These studies uncovered the activation of NOTCH signaling and the arterial program, along with dynamic changes in related regulons throughout the HE specification and the endothelial-to-hematopoietic transition. We revealed that expression of NOTCH4 in the arterial endothelium is controlled by a cis-regulatory element within intron 28 (NOTCH4in28) through SOX17 binding. Deletion of NOTCH4in28 in conventional and conditional iSOX17 hPSCs impaired the formation of the early wave DLL4⁺CXCR4^+/- HE with arterial features, as well as hematopoietic progenitors with T and NK lymphoid potential. Collectively, we provide compelling evidence that NOTCH4 and the NOTCH4in28 cis-regulatory element play an important role in arterial specification and lymphoid development in hPSC cultures.