The GATA-1 and Spi-1 transcriptional factors bind to a GATA/EBS dual element in the Fli-1 exon 1

Fli-1 is a proto-oncogene which is rearranged in tumors induced by three di fferent retroviruses, Cas-Br-E, F-MuLV, and 10A1. This gene is a member of the Ets gene family, a class of transcription factors that recognize and bi nd to a DNA motif known as the Ets binding site (EBS). Our laboratory has p reviously cloned and characterized the promoter region of both human and mo use Fli-1 genes. We had then identified several regulatory elements conserv ed between the two species. Two of them, an exon 1 GATA/EBS dual element an d an EBS element located in the 5' end of intron 1, were analysed in the pr esent study. EMSA analysis performed with nuclear extracts from different c ell lines showed that the EBS element in intron 1 (EBSi) was bound by one p otential Ets-related ubiquitous factor. The GATA/ EBS element was bound by several factors that seemed Ets-related, one of which was found to be speci fically expressed in hematopoietic cells. the GATA/EBS dual element was thu s chosen for further analysis. A human Fli-1-derived genomic fragment conta ining the GATA/ EBS led to enhanced transcription when positioned upstream of the SV40 promoter in the erythroleukemic HEL cell line. In addition, an increasing number of GATA/EBS oligonucleotides upstream of this same promot er resulted in a copy number-dependent increase in luciferase activity whic h was greatly reduced when the EBS consensus sequence was mutated. One of t he factors binding to the GATA/EBS region was identified to be Spi-1 by sup ershift analysis and was also shown to bind to the EBS element of the human Ets-2 gene. Supershift analysis also demonstrated the binding of the GATA- 1 factor to the GATA/EBS dual element. Our results suggest that Spi-1 and G ATA-1 might play a key role in the regulation of Fli-1.