Genome-wide screen identifies Runx2 as a novel regulator of hematopoietic stem cell expansion and T-cell commitment

Self-renewing multipotent hematopoietic stem cells (HSCs) are a rare but important cell population which can reconstitute the entire blood and immune system following transplantation. Due to their rarity, it has been difficult to comprehensively study the mechanisms regulating HSC activity. However, recent improvements in hematopoietic stem and progenitor cell (HSPC) culture methods using polyvinyl alcohol (PVA)-based media now facilitate large-scale ex vivo HSC expansion. Here we performed a genome-wide CRISPR knockout (KO) screen in primary mouse HSPCs to discover novel regulators of ex vivo expansion. The screen identified Runx2 as a strong negative regulator of HSC expansion, which we validated using ex vivo and in vivo assays. Loss of Runx2 increased the frequency of immunophenotypic HSCs in HSPC cultures by ~3-fold. Following expansion, Runx2-KO HSCs engrafted at ~5-fold higher levels in competitive transplantation assays. Non-cultured Runx2-KO HSCs also displayed enhanced reconstitution potential, but loss of Runx2 did not alter blood parameters. Notably however, T-cell reconstitution was diminished from Runx2-KO HSCs, and we further validated an additional role for Runx2 in T-cell commitment using ex vivo and in vivo assays. In summary, we have identified a multifaceted role for Runx2 in HSCs, as a negative regulator of HSC self-renewal and as a facilitator of T-cell commitment. These results will contribute understanding transcriptional regulation of hematopoiesis and improve HSC therapies. Overall design: RNA-seq profiling of CD201+CD150+cKit+Sca1+Lineage- sorted HSCs, either from Runx2-flox mice or with CRISPR-mediated Runx2 knockout. CUT&Tag profiling for RUNX2 and H3K4me3 in in CD201+CD150+cKit+Sca1+Lineage- sorted HSCs