Excision of Ets by an inducible site-specific recombinase causes differentiation of Myb–Ets-transformed hematopoietic progenitors

Abstract

Background: The Myb–Ets protein encoded by the E26 acute avian leukemia virus is a paradigm for the function of fused transcriptional activator oncoproteins. Myb–Ets transforms hematopoietic progenitor cells (Myb–Ets progenitors, MEPs) that can be induced to differentiate into eosinophilic and myeloid cells by the activation of pathways involving Ras and/or protein kinase C. The Ets portion of the fusion protein seems 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 erythroid cells, but also form eosinophilic and myeloid cells. This interpretation is complicated, however, by the observation that ts1.1-transformed MEPs differ from MEPs transformed with wild-type E26 in that they express erythroid and eosinophil markers even at the permissive temperature.Results In order to alleviate the problems associated with the use of temperature-sensitive mutants we have designed a vector that allows the inducible deletion of the Ets domain. To this end, we introduced FLP recombinase target sites into the E26 virus on the 5′ and 3′ sides of ets and included within the same retroviral vector sequences encoding an estrogen-dependent FLP recombinase. This construct, termed FRV-3, is capable of transforming cells to produce a phenotype indistinguishable from that of MEPs obtained with wild-type virus. Hormone treatment of MEPs transformed with FRV-3 induced erythroid differentiation in a subpopulation of the cells; this subpopulation was found to have completely excised ets. However, in contrast to previous results obtained with ts1.1-transformed MEPs, no differentiation along the eosinophilic and myeloid lineages was seen in hormone-treated FRV-3-transformed MEPs.Conclusion Our results demonstrate the feasibility of using a site-specific recombinase to excise a fused oncoprotein domain encoded by a retrovirus. More specifically, they show that the Ets portion of the Myb–Ets protein selectively inhibits differentiation of MEPs along the erythroid lineage, and suggest that Ets is also required for their differentiation along the eosinophil and, possibly, myeloid lineages.