Insights Into Terminal Erythropoiesis Influenced By Human Genetic Variation

Red blood cells (RBCs) carry hemoglobin, enabling delivery of oxygen to all tissues of the body. They are the products of a highly specialized differentiation process that begins with a hematopoietic stem cell and results in an enucleated, biconcave RBC. This thesis is focused on the use of human genetic studies to gain a better understanding of the molecular processes occurring during terminal erythroid differentiation. We studied the regulation and roles of two erythroid-restricted genes, Trim58 and Hemoglobin Gamma Chain (HBG1 and HBG2, γ-globin), by using a combination of loss-of-function techniques, including RNA-interference-mediated gene suppression, a mutant mouse model, and CRISPR/Cas9 mediated genome editing. Previous genome-wide association studies implicated variation in TRIM58 in RBC development and function. Our experiments with Trim58 revealed a direct interaction with the molecular motor dynein and enzymatic function as an E3 ubiquitin ligase in promoting its proteasomal degradation. This interaction is necessary for enucleation in knockdown studies in vitro, but genetic studies in the mouse show Trim58 is not required for erythropoiesis or enucleation. In the second part of this thesis, we used CRISPR/Cas9 to recreate a known mutation associated with hereditary persistence of fetal hemoglobin, a benign condition that ameliorates co-inherited sickle cell disease (SCD). Genome editing in human hematopoietic stem and progenitor cells reversed the hemoglobin switch at levels sufficient in vitro to correct pathological morphologies in SCD patient-derived RBCs. We identify a cis-regulatory element in the γ-globin promoter as a potential target for genome-editing therapy for SCD. Together, these findings underscore the importance of utilizing both common and rare genetic variants to uncover new aspects of erythroid biology.