We have previously identified the DNA binding sites of the ETS2 promoter that are involved in the ETS2 transcriptional regulation. We used these motifs to screen cDNA expression libraries, and we were able to isolate a cDNA clone that encodes for a protein which specifically interacts with ETS2 promoter sequences. The 2.4 kb cDNA clone was sequenced and a 328 amino acid open reading frame was determined. The predicted amino acid sequence contains several motifs found in other transcription factors as a leucine zipper, a helix-loop-helix, an amphiphatic region and two acidic domains. In addition, several putative phosphorylation sites can be detected at the carboxy-terminus of the protein. Somatic cell hybrids, Southern analysis and in situ hybridization indicate that the gene is localized on chromosome Xpll.2-pll.3. Northern analysis indicates that the gene is transcribed to a single 3 kb mRNA splice which can be detected in a variety of tissues and cell lines. The protein binds DNA as a dimer and the leucine zipper motif is required for this dimerization. We used protein expressed in insect cells via a baculovirus system to determine the optimal binding sequence by random selection and PCR--CAC/t G/A T/A G. We obtained cell line clones that express the gene under an inducible promoter after transfection of the appropriate constructs and G418 selection. These cell lines were morphologically altered and the transformation was serum and induction dependent. These cell lines could not grow in semisolid media, but did form solid tumors after injection in athymic mice.
