HOPX: a novel regulator of hemogenic endothelium identified by chromatin dynamics

NJ Palpant

Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Rd, Brisbane, QLD, Australia

Gene Expression and epigenetic changes during differentiation provide insights into mechanisms underlying cell fate decisions during development. Human embryonic stem cell (hESC)-derived endothelial cells were analyzed for chromatin dynamics and transcriptional profiling. An unbiased algorithm was designed to hierarchically rank genes modulated at the level of chromatin to identify novel regulators of mesoderm. HOPX, a non-DNA binding homeodomain protein, was identified as a novel candidate regulator of hemogenic endothelial cells (HECs). HOPX reporter hESCs engineered by CRISPR/Cas9 showed dynamic activation of HOPX in the specification of hemogenic endothelium. Expression analysis of 1,205 KDR+ cells isolated from E6.5-7.75 (epiblast to head fold stage) during mouse development in vivo showed that HOPX is not expressed in uncommitted mesoderm but markedly activated in endothelium, blood progenitors, and primitive erythrocytes. Morpholino knockdown and CRIPSR KO of HOPX during zebrafish development showed markedly reduced hematopoiesis by whole mount o-dianisidine staining that could be partially rescued by co-injection with HOPX RNA. Analysis of blood formation in HOPX KO hESCs showed reduced primitive hematopoiesis based on production of CD43+/CD235a+ cells and CFU analysis with no effect of HOPX KO on definitive hematopoiesis. Wnt signaling, a key pathway that represses primitive hematopoiesis, was increased in HOPX KO HECs implicating HOPX as a negative regulator of Wnt signaling. Addition of an exogenous Wnt inhibitor XAV-939 during differentiation partially rescued primitive hematopoiesis in HOPX KO HECs. Lastly, we show that SCL, the master transcription factor of hemato-endothelial lineage specification, functionally binds the HOPX locus by ChIP-seq and expression analysis of differentiated WT and SCL KO KDR+ cells shows that SCL is functionally required for HOPX expression. Taken together, we show that HOPX is a novel regulator of hemato-endothelial fate specification in vitro and in vivo that functionally regulates Wnt signaling to modulate primitive hematopoiesis in an SCL-dependent manner.