The previously identified novel genes involved in ETS2 regulation, SBF and ERF, were further analyzed in conjunction with ETS2 transcriptional regulation. The SBF gene dissection revealed the existence of two transcriptional activation domains that are required for ETS2 transactivation. Heterodimers appear to have a greater activation potency and are in accordance with the in vivo existence of a alternatively spliced form of SBF that eliminates one of the two activation domains. We identified an alternatively spliced product of the ERF gene (A45) that is producing a protein through a different open reading frame (ORF). This protein is capable of relieving the repression by ERF, probably by affecting the nuclear transportation of ERF. We also analyzed the phosphorylation pattern of the ERF protein in relation to the cell cycle and the involvement of the regulatory kinases CDC2 and ERK2 in the phosphorylation of ERF. We were able to identify a 60 kb genomic locus that contains the ERF gene and analyze its genomic organization. We expressed fragments of the ERF protein in bacteria; then we purified them to homogeneity and are using them to raise monoclonal antibodies as well as to identify proteins with which they may interact.
