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.
Kido
Kenji
K
Division of Retroviral Gene Expression, Research Program Applied Tumor Virology, German Cancer Research Center, Im Neuenheimer Feld 242, 69009 Heidelberg, Germany.
Doerks
Anja
A
Lochelt
Martin
M
Flügel
Rolf M
RM
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D050761:Cyclin-Dependent Kinase Inhibitor p57; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005091:Exons; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012191:Retroviridae Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
C493964:CDKN1C protein, human; D050761:Cyclin-Dependent Kinase Inhibitor p57; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012191:Retroviridae Proteins; D015534:Trans-Activators; C074061:bel1 protein, Human foamy virus; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase