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

Oncogene. 2000 Mar 30;19(14):1735-43. doi: 10.1038/sj.onc.1203489.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Apoptosis*
  • Binding Sites
  • DNA / metabolism
  • DNA Damage
  • Doxorubicin / pharmacology
  • Gene Expression
  • Genes, Reporter
  • Humans
  • Introns*
  • Mutagenesis
  • Oncogene Proteins, Viral / pharmacology
  • Promoter Regions, Genetic
  • Receptors, TNF-Related Apoptosis-Inducing Ligand
  • Receptors, Tumor Necrosis Factor / genetics*
  • Transcriptional Activation* / drug effects
  • Tumor Cells, Cultured
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*

Substances

  • E6 protein, Human papillomavirus type 6
  • Oncogene Proteins, Viral
  • Receptors, TNF-Related Apoptosis-Inducing Ligand
  • Receptors, Tumor Necrosis Factor
  • TNFRSF10B protein, human
  • Tumor Suppressor Protein p53
  • Doxorubicin
  • DNA