Spi-1/PU.1 Is a Positive Regulator of the Fli-1 Gene Involved in Inhibition of Erythroid Differentiation in Friend Erythroleukemic Cell Lines

Abstract

Spi-1/PU.1 and Fli-1 are two members of the ETS family of transcription factors whose expression is deregulated by proviral insertion in most erythroleukemic cell lines induced by the spleen focus-forming virus (SFFV) and Friend murine leukemia virus (F-MuLV) components of the Friend viral complex, respectively. In this study, we present evidence that transcription of the Fli-1 gene is positively regulated by Spi-1/PU.1 in SFFV-transformed cell lines: (i) all SFFV-transformed cell lines expressing Spi-1/PU.1 are characterized by a specific pattern of Fli-1 gene transcripts initiated in the −200 region instead of position −400 as reported for F-MuLV-transformed cell lines; (ii) these Fli-1 transcripts initiated in the −200 region are downregulated in parallel with that of Spi-1/PU.1 during hexamethylenebisacetamide (HMBA) induced differentiation; and (iii) Fli-1 transcription is upregulated in SFFV cells lines following stable transfection of a Spi-1/PU.1 expression vector. Furthermore, we found by transient transfection assays that the −270/−41 region of the Fli-1 gene displays promoter activity which is transactivated by Spi-1/PU.1. This promoter is strictly dependent on the integrity of two highly conserved ETS DNA binding sites that bind the Spi-1/PU.1 protein in vitro. Finally, we show that transfection of constitutive or inducible Fli-1 expression vectors in SFFV-transformed cells inhibits their erythroid differentiation induced by HMBA. Overall, these data indicate that Fli-1 is a target gene of the Spi-1/PU.1 transcription factor in SFFV-transformed cell lines. We further suggest that deregulated synthesis of Fli-1 may trigger a common mechanism contributing to erythroleukemia induced by either SFFV or F-MuLV.

ACKNOWLEDGMENTS

We are very grateful to F. Wending, F. Moreau-Gachelin, and Y. Ben-David for providing Friend erythroleukemic cell lines, to F. Moreau-Gachelin for Spi-1/PU.1 antiserum, to H. Itoh for providing vector pEF-LAC-CAT, and to F. Grignani for providing vector pMTCI. We thank also P. Remy and C. M. Wolff for making available the sequence of the 5′ region of the Xenopus Fli-1 gene, V. Laudet and S. Gisselbrecht for critical reading of the manuscript, O. Gandrillon for helpful discussions, and T. Drynda for help in editing figures.

This work was supported by grants from the Association pour la Recherche contre le Cancer (ARC grant 1508), from the Ligue Nationale contre le Cancer, from the Centre National de la Recherche Scientifique, and from the Université Lyon-1. G.R. and A.S. were supported by NIH grant CA16368.