Project Summary/Abstract Individuals with sickle cell disease (SCD) have severe anemia due to chronic red blood cell hemolysis and increased oxidative stress producing endothelial cell dysfunction and vasculopathy. Strategies to induce efficacious levels of γ-globin gene expression and fetal hemoglobin (HbF) synthesis will alleviate SCD clinical symptoms. In addition, SCD phenotypic severity is ameliorated by reducing oxidative stress. We previously demonstrated that transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2) is activated by dimethyl fumarate as a mechanisms of HbF induction in erythroid progenitors. We also demonstrated an essential role of NRF2 in ameliorating the severity of SCD using a novel NRF2 knockout SCD mouse model established in our laboratory. To expand on these findings, we will determine the molecular mechanism of NRF2-mediated γ-globin gene regulation and erythropoiesis through DNA and histone epigenetic modifications during development. We will further assess the ability of NRF2 activators in alleviating disease severity in SCD mouse models. Our Specific Aim is to determine epigenetic mechanisms of NRF2-mediated globin gene regulation in the SCD mouse model and develop novel HbF inducing agents. In Sub-aim A, we will determine the effects of NRF2 knockout on erythroid differentiation and globin gene regulation. We will examine the effects of NRF2 loss on erythropoiesis, metabolism and gene expression. The effects of NRF2 loss on HBB chromatin structure for DNA and histone epigenetic modifications in splenic hematopoietic tissue will be investigated. In Sub aim B, We will evaluate the FDA-approved NRF2 activating drugs as novel HbF inducers. FDA-approved drugs, including dimethyl fumarate (Tecfidera), Simvastatin (Zocor), and MLN9708 (Ixazomib), all NRF2 activators, will be tested. The effect of NRF2 activators on erythropoiesis, HBB locus chromatin structure and in alleviating disease severity in preclinical SCD mouse model will be determined. The knowledge gained will reveal the critical role of NRF2 in globin gene regulation through epigenetic DNA and histone modifications and validate the design of novel NRF2 activators for the treatment of SCD.