PROJECT SUMMARY/ABSTRACT Hematopoiesis is a cellular developmental process that is controlled by a cell-intrinsic developmental program and cell-extrinsic factors from the microenvironment. The vascular niche present in the bone marrow and other hematopoietic organs provides the physical space and signals necessary for the proper regulation of this process. Hematopoietic stem cell (HSC) phylogenetic and functional diversity are restricted in patients who receive chemotherapy or are recipients of hematopoietic stem cell transplantation (HSCT), leading to clonal hematopoiesis and cytopenias which are significant sources of morbidity and mortality in these patients. At present, very little is known about the role of the microenvironment in regulating HSC diversity. We hypothesized that mechanisms exist within the vascular niche that can be modulated to allow it to support a more phylogenetically and functionally diverse population of HSCs. We performed a genetic screen in barcoded GESTALT zebrafish and found that dysregulated expression of prkcda, the zebrafish PKC-delta homolog, increased the number of HSC clones contributing to hematopoiesis by more than 50%. Phenotypic analysis using single cell RNA-seq demonstrated the presence of a novel population of immature neutrophils and expansion of erythroid precursor cells. Single cell ATAC-seq analysis showed that chromatin accessibility at the promoter of the native prkcda locus is reduced specifically in vascular niche cell populations. These data led us to hypothesize the existence of a specific epigenetic program within the vascular niche that regulates expression of factors supporting hematopoiesis and thereby maintains the capacity of the niche to support HSC phylogenetic and functional diversity. In Aim 1, we will use Cut and Run analysis for specific epigenetic factors to characterize the vascular niche in human endothelial cell cultures. Multimodal single cell RNA-seq/ATAC-seq will be used in adult and embryonic zebrafish to identify niche cell populations and cis-acting DNA elements near genes important for supporting hematopoiesis. In Aim 2, a CRISPR screen will be used to identify specific transcription factor binding sites in human vascular niche cells lying proximal to and controlling transcription of key genes involved in niche function, including CXCL12, ANG1, and KITLG. A panel of zebrafish CRISPR mutants will be made to study the function of selected enhancer elements in vivo. Changes in niche function will be assayed in vitro by co-culture with HSPCs and in vivo by GESTALT barcoding and scRNA-seq. This work will provide new mechanistic understanding of how the capacity of the hematopoietic niche to support a diverse population of HSCs is regulated and may lead to new therapies that improve hematopoietic recovery after chemotherapy or transplantation.