3017 – MECHANISMS OF FLOW-DRIVEN TRANSCRIPTIONAL CONTROL OF HEMATOPOIETIC STEM CELL DEVELOPMENT VIA YAP/TAZ REGULATION

Abstract

Hematopoietic stem cells (HSCs) are born from endothelial cells in the ventral dorsal aorta (VDA) that adopt hematopoietic cell fate during development. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in the VDA are required to generate HSCs, but the mechanisms by which these forces are sensed and converted into a “stemness” regulatory program remain unknown. We recently showed that CS from blood flow stimulates activity of the YAP transcription factor (TF) in vitro in human induced pluripotent stem cell-derived CD34+ hemogenic endothelial cells (HE) and in vivo in zebrafish embryos to promote HSC production. YAP, and its paralogous TF TAZ/WWTR1, are members of the Hippo signaling pathway, initiate transcriptional responses downstream of mechanical stimuli, and require DNA binding cofactors to direct activation or repression of target genes. Via gene expression analysis and epistasis experiments modulating Notch signaling in zebrafish, we revealed YAP is responsible for maintenance, not initiation, of the hematopoietic program in newly specified HE. Notably, YAP loss-of-function mimics the phenotype of embryos deficient for the hematopoietic master regulator Runx1, with upregulation of the Notch ligand dll4. YAP/WWTR1 and RUNX TFs can physically interact in certain contexts. Surprisingly, using luciferase-based YAP- and RUNX-responsive reporter assays in HEK293 cells, we saw a potent synergistic effect of TAZ/RUNX1, but not YAP/RUNX1, in transcriptional regulation at RUNX binding elements. These findings position YAP signaling as a key transcriptional regulator of the endothelial-to-hematopoietic transition that produces HSCs from HE, and suggest a previously uninvestigated relationship between alternate utilization of the Hippo TFs and Runx1 that could underly the requirement of hemodynamic forces in this process. Hematopoietic stem cells (HSCs) are born from endothelial cells in the ventral dorsal aorta (VDA) that adopt hematopoietic cell fate during development. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in the VDA are required to generate HSCs, but the mechanisms by which these forces are sensed and converted into a “stemness” regulatory program remain unknown. We recently showed that CS from blood flow stimulates activity of the YAP transcription factor (TF) in vitro in human induced pluripotent stem cell-derived CD34+ hemogenic endothelial cells (HE) and in vivo in zebrafish embryos to promote HSC production. YAP, and its paralogous TF TAZ/WWTR1, are members of the Hippo signaling pathway, initiate transcriptional responses downstream of mechanical stimuli, and require DNA binding cofactors to direct activation or repression of target genes. Via gene expression analysis and epistasis experiments modulating Notch signaling in zebrafish, we revealed YAP is responsible for maintenance, not initiation, of the hematopoietic program in newly specified HE. Notably, YAP loss-of-function mimics the phenotype of embryos deficient for the hematopoietic master regulator Runx1, with upregulation of the Notch ligand dll4. YAP/WWTR1 and RUNX TFs can physically interact in certain contexts. Surprisingly, using luciferase-based YAP- and RUNX-responsive reporter assays in HEK293 cells, we saw a potent synergistic effect of TAZ/RUNX1, but not YAP/RUNX1, in transcriptional regulation at RUNX binding elements. These findings position YAP signaling as a key transcriptional regulator of the endothelial-to-hematopoietic transition that produces HSCs from HE, and suggest a previously uninvestigated relationship between alternate utilization of the Hippo TFs and Runx1 that could underly the requirement of hemodynamic forces in this process.