title: Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site.
abstract: KILLER/DR5, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor gene, has been shown to be induced by DNA damaging agents and radiation in a p53-dependent manner. Although TRAIL is a potential therapeutic agent for cancer, the induction mechanism of its receptors is poorly understood. Here we show the identification of three p53 DNA-binding sites in the KILLER/DR5 genomic locus located upstream (BS1; -0.82 Kb) of the ATG site, within Intron 1 (BS2; +0.25 Kb downstream of the ATG) and within Intron 2 (BS3; +1.25 Kb downstream of the ATG). A modified p53-binding and immunoselection protocol using a wild-type p53-expressing adenovirus vector (Ad-p53) was used to identify the binding sites and to show that each binding site can bind specifically to wild-type p53 protein (wt-p53). A reporter assay revealed that only BS2 could enhance luciferase expression driven by a basal promoter. We constructed a reporter plasmid carrying the genomic regulatory region of KILLER/DR5 including the three p53 DNA-binding sites but no additional basal promoter. The genomic fragment showed basal transcriptional activity which was induced by wt-p53 but not by mutant p53, and human papilloma virus E6 inhibited the p53-dependent activation. Mutation of BS2 abrogated not only the binding activity of wt-p53 but also the induction of the KILLER/DR5 genomic promoter-reporter gene, indicating that BS2 is responsible for the p53-dependent transactivation of KILLER/ DR5. In p53-wild-type but not -mutant or -null cell lines, doxorubicin treatment stabilized p53 protein, and increased specific binding to BS2 as revealed by EMSA, and upregulated the KILLER/DR5 promoter-luciferase reporter gene. These results suggest that the transactivation of KILLER/DR5 is directly regulated by exogenous or endogenous wt-p53 and establishes KILLER/DR5 as a p53 target gene that can signal apoptotic death.
authors.affiliation: Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
authors.forename: R
authors.initials: R
authors.lastname: Takimoto
authors.forename: W S
authors.initials: WS
authors.lastname: El-Deiry
mesh_terms: D017209:Apoptosis; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004317:Doxorubicin / Q000494:pharmacology; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D016296:Mutagenesis; D009856:Oncogene Proteins, Viral / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: C118860:E6 protein, Human papillomavirus type 6; D009856:Oncogene Proteins, Viral; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; C505639:TNFRSF10B protein, human; D016159:Tumor Suppressor Protein p53; D004317:Doxorubicin; D004247:DNA