EXCISION OF ETS BY AN INDUCIBLE SITE-SPECIFIC RECOMBINASE CAUSES DIFFERENTIATION OF MYB-ETS-TRANSFORMED HEMATOPOIETIC PROGENITORS

Background: The Myb-Ets protein encoded by the E26 acute avian leukemi a virus is a paradigm for the function of fused transcriptional activa tor oncoproteins. Myb-Ets transforms hematopoietic progenitor cells (M yb-Ets progenitors, MEPs) that can be induced to differentiate into eo sinophilic and myeloid cells by the activation of pathways involving R as and/or protein kinase C. The Ets portion of the fusion protein seem s to be required to maintain the multipotency of MEPs:MEPs transformed with a temperature-sensitive E26 mutant with a lesion in Ets (ts1.1) and shifted to the non-permissive temperature predominantly form eryth roid cells, but also form eosinophilic and myeloid cells, This interpr etation IS complicated, however, by the observation that ts1.1-transfo rmed MEPs differ from MEPs transformed with wild-type E26 in that they express erythroid ana eosinophil markers even at the permissive tempe rature, Results: in oder ia alleviate the problems associated with the use of temperature-sensitive mutants we have designed a vector that a llows the inducible deletion of the Ets domain. To this end, we introd uced FLP recombinase target sites into the E26 virus on the 5' and 3' sides of es and included within the same retroviral vector sequences e ncoding an estrogen dependent FLP recombinase. This construct, termed FRV-3, is capable of transforming cells to pro-duce a phenotype indist inguishable from that of MEPs obtained with wild-type virus.: Hormone treatment of MEPs transformed with FRV-9 induced erythroid differentia tion ina subpopulation of the coils, this subpopulation was found to h ave completely excised ets. However, in contrast to previous results o btained with ts1.1-transformed MEPs, no differentiation along the eosi nophilic and myeloid lineages-was seen in hormone-treated FRV-3-transf ormed MEPs. Conclusions: Our results demonstrate the feasibility of us ing a site-specific recombinase to excise a fused oncoprotein domain e ncoded by a retrovirus. More specifically, they Show that the Ets port ion of the Myb-Ets protein selectively inhibits differentiation of MEP s along the erythroid lineage, and suggest that Ets is also required t or their differentiation along the eosinophil and, possibly, myeloid l ineages.