Binding of ETS family members is important for the function of the c-sis/platelet-derived growth factor-B TATA neighboring sequence in 12-O-tetradecanoylphorbol-13-acetate-treated K562 cells

The c-sis/platelet-derived growth factor (PDGF)-B TATA neighboring sequence (TNS) is a promoter element that is required for the full induction of this gene in K562 erythroleukemia cells undergoing 12-O-tetradecanoylphorbol-13-acetate-mediated megakaryoblastic differentiation. Nuclear factors from K562 cells can bind to the c-sis/PDGF-B TNS, generating four complexes in electrophoretic mobility shift assays. One of these complexes was previously shown to contain Sp family members. In this work, we provide evidence implicating two of the remaining complexes as belonging to the ETS family of transcription factors. This includes the identification of a novel constitutive TNS-binding complex containing the ETS family member ELK-1. The binding of both ETS-like complexes was disrupted by mutations in a central CCGGAA core within the TNS and, for one of the complexes, could be promoted by bringing the sequences flanking the core closer to a consensus ETS binding site. The molecular weights of these TNS-binding factors were estimated by UV cross-linking analysis and found to be consistent with those of several ETS family transcription factors, including ELK-1. A consensus ELK-1 binding site could compete for the binding of both putative ETS-like factors, and the novel complex could be disrupted by the addition of an antibody raised against ELK-1. Transient transfection analysis using mutant TNS promoter-reporter constructs demonstrated a strong correlation between the binding of the ETS-like factors and the transcriptional activity of the TNS. In contrast, mutations that prevented the binding of Sp family transcription factors had no effect on promoter activity. Thus, ETS family members, such as ELK-1, are not only capable of binding to the TNS but seem to be necessary for the function of this element in differentiating K562 cells.