THE ENDOA ENHANCER CONTAINS MULTIPLE ETS BINDING-SITE REPEATS AND IS REGULATED BY ETS PROTEINS

EndoA is a type II keratin and with EndoB (type T keratin), constitute s intermediate filaments in various simple epithelial tissues. EndoA i s developmentally regulated and has an enhancer that is located at the 3' - end of the gene. This enhancer contains two single and five dual Ets binding sites. Thus far, no other promoter or enhancer has been s hown to contain as many potential clustered Ets binding sites. To stud y the transcriptional regulation of EndoA by the ETS family proteins, we amplified the EndoA enhancer fragment from mouse genomic DNA by PCR , and cloned it into the pBLCAT2 vector upstream from the CAT reporter gene. Several pBLCAT-ENDOA clones were squenced to verify the presenc e of all the ETS binding sites. Clones that did not show any point mut ations in the ETS binding sites were chosen to study the transcription regulation by ETS1, ETS2 and ERGB/FLI-1 gene products. EMSA results i ndicated that the ETS1, ETS2 and ERGB/FLI-1 proteins bind to the enhan cer sequence, and DNase I protection data demonstrated that the ETS pr oteins protect all seven EBS core sequences. Cotransfection of the COS cells with the pBLCAT-ENDOA construct, along with increasing amounts of different ETS expression vectors, resulted in a significant inducti on of CAT reporter gene expression. Previously, we have shown that the overexpression of the ETS1 gene transforms NIH3T3, and these transfor med cells (7AQS2.1) produce high levels of ETS1 protein (Seth and Papa s, 1990). In this report, we show that the undifferentiated P19 EC cel ls do not express detectable levels of ETS1; however, an elevated leve l of ETS1 is expressed in differentiated derivatives of these cells. W e therefore used these two cell lines to examine the activity of the E ndoA enhancer with the ETS1 product. Transfection of the pBLCAT-ENDOA construct alone in undifferentiated P19 EC cells results in very low C AT gene expression; however, upon differentiation with retinoic acid t he level of CAT gene activity increases dramatically. Similarly, an in crease in CAT expression from the same construct (pBLCAT-ENDOA) was al so observed in 7AQS2.1 cells. Our results therefore indicate that the EndoA enhancer is regulated by ETS proteins via interaction with multi ple ETS-binding site sequences.