Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site

• Published in: Oncogene Vol. 19; no. 14; pp. 1735 - 1743
• Main Authors: TAKIMOTO, R, EL-DEIRY, W. S
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing 30.03.2000; Nature Publishing Group
• Subjects: Ageing, cell death; Apoptosis; Binding Sites; Biological and medical sciences; Cancer; Cell cycle; Cell death; Cell physiology; Chemotherapy; Cytochrome; Cytotoxicity; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA Damage; Doxorubicin; Doxorubicin - pharmacology; DR5 gene; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Reporter; Genetic aspects; Genomics; Health aspects; Human papillomavirus; Humans; Introns; KILLER gene; Ligands; Medicine; Molecular and cellular biology; Mutagenesis; Mutants; Nucleotide sequence; Oncogene Proteins, Viral - pharmacology; Oncology; p53 Protein; Physiological aspects; Promoter Regions, Genetic; Protein binding; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor - genetics; Reporter gene; TRAIL protein; Transcription; Transcriptional Activation - drug effects; Tumor Cells, Cultured; Tumor necrosis factor; Tumor necrosis factor-TNF; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; United States; United States > US; Pennsylvania; Human; Regulation(control); Cell line; Intron; p53 Protein; Molecular interaction; Binding site; Gene expression; Carcinogenesis; Tumor suppressor gene; Apoptosis
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/sj.onc.1203489

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.