Identification and functional characterization of an intragenic DNA binding site for the spumaretroviral trans-activator in the human p57Kip2 gene.
Expression of the human cyclin-dependent protein kinase inhibitor p57(Kip2) gene was previously shown to be specifically and strongly activated by the retroviral trans-activator Bel1 of human foamy virus by means of expression profiling, Northern, and Western blot analysis. Here we report that Bel1-mediated trans-activation was conferred by a Bel1 response element (BRE) located in the second exon of p57(Kip2). The intragenic Kip2-BRE was capable of trans-activating the luciferase reporter gene upon cotransfection with Bel1. In electrophoretic mobility shift assays using 293T nuclear extracts or a purified glutathione S-transferase (GST) small middle dotBel1 fusion protein, we identified the 55-nucleotide-long Kip2-BRE site that mainly consists of three direct repeats of 14-mers partially homologous to a functionally active BRE in the viral internal promoter. The specificity of the transactivator-DNA binding was shown by using mutated and shortened Kip2-BRE oligodeoxynucleotides in competition experiments with the authentic viral internal promoter and by Bel1-specific antibody that led to a supershift of the nuclear protein small middle dotKip2-BRE and GST small middle dotBel1 small middle dotKip2-BRE complex. The data indicate that Bel1 can directly bind to BRE sites. The cellular Kip2-BRE can be used to predict those human genes that are directly or indirectly activated by the Bel1 trans-activator.[1]References
- Identification and functional characterization of an intragenic DNA binding site for the spumaretroviral trans-activator in the human p57Kip2 gene. Kido, K., Doerks, A., Lochelt, M., Flügel, R.M. J. Biol. Chem. (2002) [Pubmed]
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