FUNCTIONAL AND MECHANISTIC STUDY OF DOT1L IN MOUSE EMBRYONIC HEMATOPOIESIS

DOT1 is the histone 3 lysine 79 methyltransferase with both unique structure and substrate specificity. It plays critical role in telomere silencing maintenance, transcription regulation, DNA repair, and cell cycle regulation. DOT1L in mammals is required for embryonic development. In the absence of DOT1L, mouse embryos die by E13.5. Severe anemia was observed in Dot1L mutant embryos at E10.5. In order to study a potential role for DOT1L in embryonic hematopoiesis, colony forming assays were performed using both primitive and definitive yolk sac progenitors. We found that DOT1L is required for both primitive and definitive hematopoietic development in the mouse. Interestingly, there is a specific defect in erythroid lineage development. Using flow cytometric analysis, we showed that Dot1L deficiency does not affect the emergence of the progenitor population. When Dot1L mutant progenitor cells differentiate into the erythroid lineage, they fail to progress normally through the cell cycle and undergo increased apoptosis when compared to wild type progenitors. In order to investigate the molecular mechanism of DOT1L in hematopoiesis, the expression of genes that are critical in hematopoiesis was analyzed in Dot1L mutant progenitors. Although Gata-2 expression appears to be regulated by DOT1L, the reduction of Gata-2 in yolk sac progenitors alone does not lead to an observable hematopoietic defect. Trpc6, which plays a role in EPO-mediated calcium influx, was down regulated significantly in Dot1L mutant progenitors. Based on our data and previous studies demonstrating a role for TRPC proteins in mediating EPO induced calcium influx, I hypothesize that Dot1L deficiency results in an abnormal EPO response in hematopoietic progenitor cells. This response is characterized by significantly increased calcium influx induced by EPO. This abnormal intracellular calcium levels in turn result in the observed defective erythropoiesis. In support of this hypothesis, calcium imaging analysis demonstrated that the intracellular calcium level was much higher in Dot1L mutant progenitor cells after EPO treatment in comparison with wild type cells. My study explores the cellular and biological function of Dot1L and provides more insight into the complex regulation of embryonic hematopoiesis