In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation.
The tumor suppressor protein p53 has a transcriptional activation activity thought to mediate its biologic function including G1 arrest and perhaps apoptosis. To learn more about p53's transactivator function in vivo, we performed genomic footprinting experiments examining p53-DNA interactions in the regulatory regions of the p53-regulated genes p21, GADD45, and MDM2. Using ionizing radiation to induce DNA damage in human ML-1 myeloblastic leukemia cells, the promoter and intronic regions of these genes containing p53-consensus binding sites were examined for in vivo footprints. There was a uniform and sustained expression of p53 protein as well as a strong induction of p21, GADD45, and MDM2 mRNA following irradiation. At the two p53 consensus binding sites in the p21 promoter, reduced DNaseI cleavage was observed in irradiated cells beginning 1 to 2h after irradiation, being most pronounced after 2 h and diminishing after 8 h. A partial in vivo footprint was also observed in the third intron of the GADD45 gene beginning 2 h after irradiation. No in vivo footprints were seen at the two p53 binding sites in the MDM2 gene. Our study provides direct evidence that the DNA damage-induced activity of p53 is mediated by its consensus DNA binding sites in the p21 and GADD45 genes. We suggest that the transient nature and relative instability of p53-DNA interactions in vivo may make the p53 protein more accessible to a rapid turnover pathway which might be impaired under conditions when the protein is stably bound to DNA.
Chin
P L
PL
City of Hope National Medical Center, Department of Biology, Duarte, California 91010, USA.
Momand
J
J
Pfeifer
G P
GP
D001665:Binding Sites; D016384:Consensus Sequence; D004249:DNA Damage; D018983:DNA Footprinting; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D015470:Leukemia, Myeloid, Acute; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D011506:Proteins / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051736:Proto-Oncogene Proteins c-mdm2; D016283:Proto-Oncogene Proteins p21(ras) / Q000528:radiation effects*; D011839:Radiation, Ionizing; D013997:Time Factors; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism / Q000528:radiation effects*; D000097642:GADD45 Proteins
D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D016283:Proto-Oncogene Proteins p21(ras); D016159:Tumor Suppressor Protein p53; D000097642:GADD45 Proteins; C497206:MDM2 protein, human; C501009:HRAS protein, human