ONE OF 2 ETS-BINDING SITES IN THE CYTOKERATIN ENDOA ENHANCER IS ESSENTIAL FOR ENHANCER ACTIVITY AND BINDS TO ETS-2 RELATED PROTEINS

Expression of the mouse cytokeratin EndoA gene is restricted in endodermal and epithelial cells, and is regulated by an enhancer that is located 1 kilobase (kb) 3' downstream from the gene. The enhancer consists of six direct repeats, of which each contains two predicted Ets binding sites (EBS1 and EBS2) containing GGAA as a core. Mutation analysis showed that EBS1 is essential for the enhancer activity and additional effects of EBS2, suggesting that some Ets-related proteins bind and activate the enhancer through EBS1. We also showed that Ets-2 mRNA is expressed in PYS-P cells and that Ets-2 protein produced by E.coli interacts with EBS1 but not with EBSS. Using co-transfection assays, we showed that Ets-2 can trans-activate the enhancer in PYS-2 cells. Mutations that impair Ets-2 binding abolish the activity of the EndoA enhancer. The results obtained from the binding competition assay using an Ets-2 specific antibody, however, suggest that EBS1 binds to an Ets protein which is distinct from Ets-2. These data show that Ets-2 related protein binds and activates the EndoA enhancer in a sequence-specific fashion.