E2f-2 Regulates Caspase-3 Expression and Mitotic Checkpoint Control during End-Stage Erythroid Maturation.

E2f-2 is a member of the E2F superfamily of transcription factors that plays multiple roles in regulating cell cycle progression, cell cycle exit and checkpoint control [1]. While over-expression studies have ascribed a role for E2f-2 as a transcriptional activator of cell cycle genes, our loss of function studies performed in vivo have revealed a novel role for E2f-2 in repressing cellular proliferation and mediating differentiation. Our expression analyses demonstrate that E2f-2 is up-regulated in hematopoietic tissues, and is most strongly expressed in differentiating erythroblasts. Furthermore, E2f-2 is the major pRb-associated E2F in hematopoietic tissues, suggesting E2f-2 can function as a co-repressor of cell cycle genes during end-stage differentiation. Compound Rb−/−;E2f2−/− mice were generated to understand how deregulated E2f-2 activity contributes to the erythroid defects in Rb null mice. Loss of E2f-2 restored end-stage erythroid differentiation and enucleation to Rb null erythroblasts and extended survival of Rb null mice. Loss of E2f-2 was sufficient to bypass the mitotic arrest observed in Rb null erythroblasts [2] and was associated with reduced expression of known E2f target genes involved in M phase checkpoint control, such as Aurora B and Survivin. In addition, we observed a significant increase in caspase-3 expression in E2f-2 null erythroblasts and demonstrate, for the first time, direct regulation of caspase-3 expression by E2f-2. Given known roles for caspases in promoting erythroid differentiation and enucleation, we have over-expressed caspase-3 in Rb null erythroblasts and are currently analyzing its effects on cell cycle and maturation. Thus, our work identifies caspase-3 as a key downstream target of E2f-2 during end-stage erythroid differentiation whose expression may alleviate enucleation and mitotic defects in Rb null erythroblasts. Future work aims to understand which E2f-2 target genes ensure mitotic checkpoint control, and the molecular mechanism by which caspase-3 coordinates cell cycle control with erythroid differentiation.