Interrogating the differential roles of Ezh1/2 in hematopoietic stem and progenitor cell formation from endothelial precursors

Abstract

Understanding how hematopoietic stem and progenitor cells (HSPCs) are specified from endothelial precursors is essential to the goal of generating patient-specific HSPCs for therapeutic use. Coordinated action of cell extrinsic signaling and intrinsic transcription factor function result in the production of HSPCs from hemogenic endothelium in anatomically select arterial niches during embryonic development through a process termed endothelial-to-hematopoietic transition (EHT). Despite increasing efforts to recapitulate this process in vitro, current differentiation protocols largely fail to produce long-lived multipotent progenitors from human induced pluripotent stem cell (iPSC) sources. Here, we identify the mechanism by which the Polycomb group protein, Enhancer of zeste-homolog 1 (Ezh1), controls vertebrate HSPC production in vivo via regulation of hemogenic competence. Specifically, loss of ezh1 favored commitment towards hemogenic (gata2b) and HSPC (runx1) fate at the expense of arterial identity (ephrinb2a, dll4). Furthermore, Ezh1 and its related PRC2 subunit, Ezh2, were found to exert sequential, non-redundant effects during de novo HSPC formation. As Notch Signaling has been implicated in both arterial specification and HSPC emergence, we observed differential regulation of Notch ligands and receptor with Ezh1/2 modulation. In addition to Notch, numerous pathways cooperate to specify HSPCs, including inflammatory signaling. To that end, we independently demonstrated IL1activity can be modulated by activation of the NLRP3 inflammasome complex in response to sterile developmental stressors, such as alterations in in vivo metabolic signals during embryogenesis. Interestingly, Gene Set Enrichment Analysis (GSEA) from our prior in vitro studies revealed inflammatory signaling pathways, known to regulate HSPC formation/expansion, were significantly altered following EZH1 knockdown; in particular, a role for TNF via NFB activity, necessary for inflammasome priming, was highlighted. Previous reports have implicated TNF and NFB in regulating HSPC production from hemogenic endothelium through Notch signaling. We similarly observed an upregulation of the ligand, tnf, in ezh1 morphants, consistent with alterations in Notch activity and a hypothesized inflammatory interaction following Ezh1/2 modulation. Together, the work presented in this thesis functionally integrates the role of cell signaling and epigenetic regulation of key transcription factors in the specification and maintenance of developmental HSPC formation in the hemogenic niche.