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J. Biol. Chem., Vol. 277, Issue 14, 12032-12039, April 5, 2002

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Identification and Functional Characterization of an Intragenic DNA Binding Site for the Spumaretroviral trans-Activator in the Human p57^Kip2 Gene^*

Kenji Kido, Anja Doerks, Martin Löchelt, and Rolf M. Flügel

From the Division of Retroviral Gene Expression, Research Program Applied Tumor Virology, German Cancer Research Center, Im Neuenheimer Feld 242, 69009 Heidelberg, Germany

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)·Bel1 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·Kip2-BRE and GST·Bel1·Kip2-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.

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