The Ewing family of tumors harbors chromosomal translocations that join the 
N-terminal region of the EWS gene with the C-terminal region of several 
transcription factors of the ETS family, mainly FLI1, resulting in chimeric 
transcription factors that play a pivotal role in the pathogenesis of Ewing 
tumors. To identify downstream targets of the EWS/FLI1 fusion protein, we 
established 293 cells expressing constitutively either the chimeric EWS/FLI1 or 
wild type FLI1 proteins and used cDNA arrays to identify genes differentially 
regulated by EWS/FLI1. DAX1 (NR0B1), an unusual orphan nuclear receptor involved 
in gonadal development, sex determination and steroidogenesis, showed a 
consistent up-regulation by EWS/FLI1 oncoprotein, but not by wild type FLI1. 
Specific induction of DAX1 by EWS/FLI1 was confirmed in two independent cell 
systems with inducible expression of EWS/FLI1. We also analyzed the expression 
of DAX1 in Ewing tumors and derived cell lines, as well as in other nonrelated 
small round cell tumors. DAX1 was expressed in all Ewing tumor specimens 
analyzed, and in seven out of eight Ewing tumor cell lines, but not in any 
neuroblastoma or embryonal rhabdomyosarcoma. Furthermore, silencing of EWS/FLI1 
by RNA interference in a Ewing tumor cell line markedly reduced the levels of 
DAX1 mRNA and protein, confirming that DAX1 up-regulation is dependent upon 
EWS/FLI1 expression. The high levels of DAX1 found in Ewing tumors and its 
potent transcriptional repressor activity suggest that the oncogenic effect of 
EWS/FLI1 may be mediated, at least in part, by the up-regulation of DAX1 
expression.