Erythroid terminal differentiation is comprised of 3-4 rapid terminal cell divisions also known as ?differentiation divisions?, which are coupled with morphological changes such as a dramatic decrease in cell and nuclear size. The switch from self-renewal to terminal divisions in erythroid cells are peculiarly characterized by short G1 and S phases, and fast DNA replication. We do not yet understand the processes that regulate the timing, integrity and the numbers of these rapid terminal divisions. When these divisions go awry, it leads to severe anemias such as Congenital Dyserythropoietic Anemia (CDA), which arise due to failures in DNA replication and/or cytokinesis, and are characterized by binucleate erythroblasts in the bone marrow and in some cases chromatin bridges between erythroblasts. EKLF/KLF1 is one the genes when mutated causes a type of CDA, CDA type IV. Although previous genome wide studies have alluded to its roles in DNA replication/repair and cytokinesis, this has not been functionally investigated. Interestingly, EKLF-/- erythroid cultures during terminal differentiation have increased proportions of binucleate cells and chromatin bridges; similar to what has been observed in CDA disorders. Based on our preliminary data, we hypothesize that EKLF transcriptionally upregulates the genes involved in the maintenance of DNA replication fidelity (Aim1) and cytokinesis (Aim2) to accommodate the rapid pace of terminal erythroid cell divisions; impairment of this regulation in EKLF-/- erythroblasts results in replication stress, cytokinesis failure and the formation of binucleate erythroblasts. I will study the role of EKLF in the maintenance of DNA replication fidelity by quantifying the levels of DNA damage and replication stress, replication dynamics, and the extent to which unresolved DNA damage perturbs cytokinesis. I will investigate the role of EKLF in cytokinesis by studying the formation, structure, and the function of the midbody organelle, which forms between two daughter cells and is essential for abscission. Finally, I will also examine the extent to which EKLF regulated candidate genes contribute to the observed defects in EKLF-/- erythroblasts. These studies will reveal a specialized transcriptional regulation in erythroid cells to ensure that the cell cycle machinery is able to accommodate the rapid pace of the terminal cell divisions. They will also provide insights on the pathogenesis of severe anemias such as CDA, some of whose etiology is unknown. The studies proposed here along with career development plan described in my application, will enable me to benefit from the mentorship of Dr. James Bieker, who discovered EKLF and has contributed immensely to the field of erythropoiesis, forge collaborations to expand my expertise, and gain guidance on career and scientific progression from my advisory committee. Overall, this will pave the way for my successful transition to independence.
These proposed studies will reveal a specialized transcriptional regulation in erythroid cells to ensure that the cell cycle machinery is able to accommodate the rapid pace of the terminal cell divisions. In addition, these studies will shed light on the pathogenesis of severe anemias such as CDAs that arise due to a failure in terminal cell divisions, including CDA type IV (caused by a hypomorphic mutation of EKLF), and other CDAs whose etiology is unknown.
