One of two 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-2 cells and that Ets-2 protein produced by E. coli interacts with EBS1 but not with EBS2. Using co-transfection assays, we showed that Ets-2 can trans-activate the enhancer in PYS-2 cells. Mutations that impair Ets-2 binding abolished 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.
Fujimura
Y
Y
Department of Microbial Genetics, Osaka University, Japan.
Yamamoto
H
H
Hamazato
F
F
Nozaki
M
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D007633:Keratins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494070:Ets2 protein, mouse; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D007633:Keratins; D015500:Chloramphenicol O-Acetyltransferase