Friend virus-induced erythroleukemia involves two members of the ETS family of transcriptional regulators, both activated via proviral insertion in the corresponding loci. Spi-1/PU.1 is expressed in the disease induced by the original Friend virus SFFV(F-MuLV) complex in adult mice. In contrast, FLI-1 is overexpressed in about 75% of the erythroleukemias induced by the F-MuLV helper virus in newborn mice. To analyse the consequences of the enforced expression of FLI-1 on erythroblast differentiation and proliferation and to compare its activity to that of PU.1/Spi-1, we used a heterologous system of avian primary erythroblasts previously described to study the cooperation between Spi-1/PU.1 and the other molecular alterations observed in SFFV-induced disease. FLI-1 was found: (i) to inhibit the apoptotic cell death program normally activated in erythroblasts following Epo deprivation; (ii) to inhibit the terminal differentiation program induced in these cells in response to Epo and; (iii) to induce their proliferation. However, in contrast to Spi-1/PU.1, the effects of FLI-1 on erythroblast, differentiation and proliferation did not require its cooperation with an abnormally activated form of the EpoR. Enhanced survival of FLI-1 expressing erythroblasts correlated with the upregulation of bcl2 expression. FLI-1 also prevented the rapid downregulation of cyclin D2 and D3 expression normally observed during Epo-induced differentiation and delayed the downregulation of several other genes involved in cell cycle or cell proliferation control. Our results show that overexpression of FLI-1 profoundly deregulates the normal balance between differentiation and proliferation in primary erythroblasts. Thus, the activation of FLI-1 expression observed at the onset of F-MuLV-induced erythroleukemia may provide a proliferative advantage to virus infected cells that would otherwise undergo terminal differentiation or cell death.

contributed equally to this work Rearrangement of the FLI-1 locus with ensuing overexpression of FLI-1 is an early event in Friend murine leukemia virus-induced disease. When overexpressed in primary erythroblasts, FLI-1 blocks erythropoeitin (Epo)-induced terminal differentiation and inhibits apoptosis normally induced in response to Epo withdrawal. We show here that the survival-inducing property of FLI-1 is associated with increased transcription of BCL-2. We further show that FLI-1 binds BCL-2 promoter sequences in transformed erythroblasts, and in vitro studies identify speci®c FLI-1-binding sites essential for the transactivation of the BCL-2 promoter by FLI-1. Analysis of FLI-1 mutants showed a correlation between the ability of FLI-1 to transactivate BCL-2 promoter sequences and their ability to inhibit apoptosis in the absence of Epo. Moreover, inhibitor studies con®rmed the essential role of BCL-2 for FLI-1-transformed erythroblast survival. Finally, enforced expression of BCL-2 was suf®cient to promote survival and terminal differentiation of erythroblasts in the absence of Epo. These results show that BCL-2 is an in vivo target of FLI-1 in FLI-1-transformed erythroblasts and that its deregulated expression is instrumental in the survival of these cells.

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