PROJECT ABSTRACT Until recently it was believed that hematopoiesis is driven by hematopoietic stem cells (HSCs) in the normal setting. However, studies using elegant methods for clonal tracking demonstrate that steady-state hematopoiesis in the unperturbed mouse is largely sustained by long-lived progenitors. Under the stress of bone marrow transplantation, these progenitors appear short-lived. In addition, transplantation of single, purified HSCs has revealed marked lineage bias, such that HSCs may be “balanced”, lymphoid-, myeloid, or platelet-biased in their outcome. This project addresses the molecular mechanisms underlying clonal dynamics and lineage bias. We have developed an experimental platform to score lineage bias of HSCs and performed preliminary single-cell gene expression analyses that identify transcripts relatively increased or decreased in myeloid- vs lymphoid-biased HSCs. Among candidate epigenetic regulators are histone deacetylases (HDACs). In collaboration with other investigators in this program project, we will apply clonal tracking in the mouse by transposon tagging (Camargo) and rainbow fluorescent marker tagging (Scadden) to dissect the contributions of individual HDACs to lineage bias in vivo as compared with ex vivo and determine pathways through which they function. In addition, we will assess other candidate epigenetic regulators and specifically explore the role of polycomb repressive complex 2 (PRC2) heterozygosity in clonal dominance of HSCs. Results will be intersected with findings of the Zon group in the Zebrafish. In a parallel aim, we will establish a high-throughput genetic approach to identifying epigenetic regulators of HSC self-renewal through ex vivo gene modification and in vivo phenotypic readout by transplantation. We have created a new mouse strain in which the Cas9 enzyme is inducible. This strain will facilitate gene editing of HSCs and progenitors with the CRISPR/Cas9 platform. We will establish the parameters for CRISPR/Cas9 library screening of HSCs and progenitors, and determine the critical epigenetic regulators from among ~300+ known factors. In parallel, we will compare our findings with those of the Zon and Scadden groups, and test candidate regulators (including RNAs) identified in the project of the Tenen laboratory. Through this integrative genetic approach to HSC and progenitor function, we seek to improve methods for expansion of HSCs for both experimental and therapeutic purposes.