The ets gene family has at least 12 unique members that were cloned by different approaches. All the ets family proteins have a common ets- binding domain. The ets proteins are transcription factors that stimulate transcription by binding to purine-rich sequences. However, different members have distinct affinities and specificities for the DNA binding and transactivation activity. EndoA is a type II keratin, and with EndoB (type I keratin), forms intermediate filaments in various simple epithelial tissues. EndoA is developmentally regulated and has an enhancer that is located at the 3'-end of the gene. This enhancer contains 12 Ets binding sites in the form of six tandem repeats. So far, no other promoter or enhancer has been shown to contain as many potential clustered ets-binding sites. To study the transcriptional regulation of EndoA by ets family proteins, the EndoA enhancer fragment from mouse genomic DNA was amplified by PCR and cloned into the pBLCAT2 vector upstream of the CAT reporter gene. Several pBLCAT-ENDOA clones were sequenced to verify the presence of all the ets-binding sites and PCR- generated mutations. Two clones that did not show any point mutations were chosen to study the transcription regulation by ETS1 and ETS2 gene products. EMSA results indicated that the ETS1 and ETS2 gene products bind to the enhancer sequence, and DNaseI protection data demonstrated that the ETS1 protein protects all the EBS core sequences. Cotransfection of NIH3T3 or COS cells with pBLCAT-ENDOA construct, and increasing amounts of different ets expression vectors, resulted in significant induction of the CAT reporter gene expression. Thus, results indicate that the EndoA enhancer interacts directly with ets proteins via binding to ets-binding site repeat sequences. Currently, other transcriptional factors, which interact with ets family proteins to regulate the EndoA enhancer, are being investigated.
