THE RETINOBLASTOMA BINDING-FACTOR-1 (RBF-1) SITE IN RB GENE PROMOTER BINDS PREFERENTIALLY E4TF1, A MEMBER OF THE ETS TRANSCRIPTION FACTORS FAMILY

The tumor suppressor retinoblastoma gene product, pRB, is a well known regulator of G1/S cell cycle progression. Moreover, mutational inacti vations within the retinoblastoma gene (RB) are found in many human ma lignant tumors, and thus, believed to be an essential step in tumor fo rmation. The human RB gene is considered as a housekeeping gene with n o characteristic TATA or CAAT elements in its promoter region, but the sequence between 206 and 185 bases upstream of the initiation codon, essential for RB promoter activity, contains putative Sp1 and ATF reco gnition sites. We have previously reported that point mutations in thi s region, causing low penetrance retinoblastomas, completely reduced R B promoter activity, and that a nuclear factor, named RBF-1 (retinobla stoma binding factor 1), could specifically bind to this sequence, ove rlapping Sp1 recognition sequence. We show here, that RBF-1 can recogn ize a specific DNA sequence, 5'-GGCGGAAGT-3', overlapping the Sp1 and ATF sites and corresponding to the consensus DNA binding site for memb ers of Ets transcription factors family. When RBF-1 site was used for sequence specific DNA affinity purification from erythroleukemia cells , reconstitution assays, immunoblotting analysis and peptide mapping s how that the two major co-purified proteins are identical to human E4T F1-60 and -53 proteins. This reveals that E4TF1 can bind to the RBF-1 site of RB gene promoter, which, thus, constitutes a new target for th is member of Ets transcription factors family.