The DNA-binding sites of the ETS2 promoter that are involved in ETS2 transcriptional regulation have previously been identified, as well as a novel gene that recognizes one of those sites. The protein was expressed in insect cells and purified to homogeneity with ion exchange and affinity chromatography. This protein was used to raise antibodies in mice. In addition, another cDNA was isolated that encodes for a protein that interacts with another motif on the ETS2 promoter. The gene is localized on human chromosome 19 and is transcribed to about 2.8 kb mRNA, which encodes for a 528 amino acid protein. This protein is a novel member of the ets family of genes and harbors the DNA recognition domain of the amino terminus of the protein. Cotransfection experiments indicate that this protein regulates ETS2 transcription. The role of the triple-helix DNA structure in the ETS2 promoter was also studied. These structures do not affect the DNA replication, but the mRNA transcription. However, they do not account for the full activation of the ETS2 promoter, indicating that there are additional elements involved in ETS2 regulation. Preliminary data indicating the interaction of proteins in this region supports the involvement of these triple-helix DNA structures in ETS2 transcriptional regulation. Finally, a method for the muta-genesis of the ETS1 binding domain was developed and used to determine the amino acids that are vital for the interaction of the protein with its DNA target.
