Project Summary Erythropoiesis, or the process of red cell formation, is continuous throughout life. Its study helps elucidate erythroid disorders, most notably anemia, which accounts for 8.8% of all disability globally. It is also an accessible model for studying fundamental questions in developmental biology. This proposal is based on recent finding that a key erythroid cell fate decision is associated with dramatic shortening of S phase. Cell fate decisions in some other developmental systems are similarly associated with a faster S phase. A faster S phase might be accomplished at the cost of genomic instability, as in oncogene- induced replicative stress. However, studies of the relationships between a physiologically faster S phase and the DNA damage response in normal development are lacking. This project’s goal is to determine whether the unusually fast S phase of the erythroid developmental switch entails altered DNA replication fidelity and/or alterations in the DNA damage response. Early erythroid progenitors, termed ‘colony-forming-unit-erythroid’ (CFU-e), undergo several self-renewal cell divisions before transitioning into Erythroid Terminal Differentiation (ETD), where they begin to express red cell genes. The transition from self-renewing CFU-e progenitors to maturing ETD erythroblasts is a rapid transcriptional switch that is synchronized with, and dependent on, a single cell cycle S phase. Strikingly, the S-phase of the CFU-e/ETD switch is of uniquely short duration, lasting only 4 hr, compared with 7 hr in preceding CFU-e cycles, as a result of a global, 50% increase in the speed of replication forks. These changes in S phase speed are required for the CFU-e/ETD switch; the slower S phase of CFU-e progenitors promotes their self-renewal, while the fast S phase of early ETD promotes erythroid gene induction. It might be expected that the fast S phase of early ETD erythroblasts would exact a ‘cost’ of increased replication fork stalling events (‘replication stress’) and increased genomic instability. our experimental AIMS test two opposing but not necessarily mutually exclusive hypotheses: Hypothesis 1: The faster S phase of early ETD is achieved at a cost of lower quality replication. Hypothesis 2: The faster S phase of early ETD reflects “supercharged” replication-coupled DNA repair. AIM 1 will analyze the quality of DNA replication in fast-cycling early ETD erythroblasts. AIM 2 will determine how the DNA damage response of fast-cycling ETD erythroblasts differs from that of their slower-cycling CFU- e precursors. AIM 3 will determine whether the faster S phase and the altered DNA damage response of early ETD are genetically separable.