Oxidative stress mechanisms regulating g-globin gene transcription in sickle cell disease Abstract: Sickle cell disease (SCD) is the most common inherited monogenic disorder affecting the β-globin gene, leading to the production of sickled-shaped red blood cells (RBCs). Patients with SCD suffer from severe anemia and painful vasoocclusive crises, which are both exacerbated by increased oxidative stress. Induction of normal, but developmentally silenced γ-globin gene of fetal hemoglobin (HbF) expression reduces RBC sickling-mediated vasoocclusion, and anemia, consequently ameliorating clinical severity of SCD. Reducing oxidative stress also improves SCD phenotypic severity. Yet effective treatment options remain limited. Understanding the pathogenesis of the erythroid mechanisms regulating oxidative stress and factors engaged in silencing γ-globin gene, is of substantial value to SCD patients. Our data uncovered an unanticipated role for mediators of oxidative stress in RBC sickling, and in regulating the transcriptional regulatory machinery that represses γ-globin genes as well as epigenetic enzyme components associated with γ-globin to β-globin gene switch in erythroid progenitors. Here, to extend our studies, we will in vitro and in vivo genetically and/or chemically manipulate these mediators of oxidative stress and the regulated pathways using a SCD mouse model, and determine their effects on γ-globin gene regulation, and thus sickling, and their mechanism of action on the transcriptional regulatory machinery that silences γ-globin gene during sickle RBC production. We will also determine the contribution of these mediators in epigenetic DNA and histone modifications associated with γ-globin to β-globin gene switch during sickle RBC production. Because stress erythropoiesis compensates for anemia caused by oxidative damage to the RBCs, we will further validate the effects of these mediators of oxidative stress on stress erythropoiesis in splenic hematopoietic tissue and examine their role in chronic erythroid stress-response, specifically in erythroid terminal maturation and enucleation. We strongly believe that our studies will provide novel and unprecedented insights into the exact mechanisms regulating γ-globin gene silencing and γ-globin to β-globin gene switch in SCD, as well as ineffective erythroid maturation and enucleation. Our long- term goal is to identify remediable sickle erythroid abnormalities to improve SCD pathophysiology. In addition, our studies will lay the foundation for more rational approaches to therapies that better alleviate SCD clinical symptoms.