Self-renewal is the process by which a cell divides to generate daughter cells that have the same developmental potential as the parent cell. The overall goal of our collaborative research project is to gain a mechanistic understanding of erythroid progenitor (EP) self-renewal, both in vitro and in vivo. Red blood cells (RBCs) make up more than 80% of all cells in the human body, but have a limited lifespan, necessitating a massively expansive and exquisitely responsive production system. This production is regulated predominantly at the late progenitor phase of erythropoiesis, since downstream erythroid precursors undergo a fixed number of maturational cell divisions. In vitro, erythroid progenitor self-renewal is limited, which is a major stumbling block to production of specialized units of blood. The Palis lab discovered that erythroblasts derived from the yolk sac of murine embryos were capable of essentially unlimited self-renewal. These cells had increased expression of the Polychrome Repressive Complex (PRC1) members Bmi1 and Ring1b, as well as cholesterol metabolism genes. Importantly, overexpression of BMI1, a chromatin modulator normally expressed in erythroid progenitors, can also expand human erythroid cells in vitro, termed BMI1-induced self-renewing erythroblasts (iSREs). Collaborative studies with the Steiner lab provide evidence that BMI1 alters the self- renewal capacity of human erythroblasts both through known (e.g., Ink/Arf locus), and novel (e.g., cholesterol homeostasis) pathways. Our data further suggest that BMI alters the activity of RING1B, which monoubiquitinates histone H2A lysine 119, and also has essential roles in regulating higher order chromatin interactions. In Aim 1 we will test the hypothesis that BMI1/Ring1b regulate human SRE self-renewal through epigenetic control of transcription and cholesterol homeostasis. Expansion of erythroid progenitors is essential to maintain steady state erythropoiesis and for the response to acute anemic or hypoxic stress. In preliminary studies we have defined 4 progressive stages of murine erythroid progenitors, termed EP1 to EP4, analogous to those recently defined in humans, and determined that late-stage EP3/4 progenitors preferentially expand in the marrow of mice in response to acute anemia. In Aim 2 we will test the hypothesis that Bmi1/Ring1b and its downstream targets regulate the expansion of late-stage (EP3/EP4) erythroid progenitors both at steady-state and in response to acute anemia. Together, these studies will provide mechanistic understanding of erythroid progenitor self-renewal and bring a renewable source of human erythroblasts closer to the clinic to meet specialized transfusion needs.