Nucleic Acids Research, 1994, Vol. 22, No. 4 613-618
© 1994
MOLECULAR BIOLOGY |
One of two Ets-binding sites in the cytokeratin EndoA enhancer is essential for enhancer activity and binds to Ets-2 related proteins
Department of Microbial Genetics, Research Institute for Microbial Diseases Osaka University, 3–1 Yamadaoka, Suita, Osaka 565, Japan
*To whom correspondence should be addressed
Received November 3, 1993. Accepted January 25, 1994.
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.coll interacts with EBS1 but not with EBS2. Using co-transfection assays, we showed that Ets-2 can frans-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.
+Present addresses: La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Road, La Jolla, CA 92037
Hitachi Co. Ltd, Saitama 350-03, Japan
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