Functional analysis of p53 binding under differential stresses.
Hypoxia and DNA damage stabilize the p53 protein, but the subsequent effect that each stress has on transcriptional regulation of known p53 target genes is variable. We have used chromatin immunoprecipitation followed by CpG island (CGI) microarray hybridization to identify promoters bound by p53 under both DNA-damaging and non-DNA-damaging conditions in HCT116 cells. Using gene-specific PCR analysis, we have verified an association with CGIs of the highest enrichment (> 2.5-fold) (REV3L, XPMC2H, HNRPUL1, TOR1AIP1, glutathione peroxidase 1, and SCFD2), with CGIs of intermediate enrichment (> 2.2-fold) (COX7A2L, SYVN1, and JAG2), and with CGIs of low enrichment (> 2.0-fold) (MYC and PCNA). We found little difference in promoter binding when p53 is stabilized by these two distinctly different stresses. However, expression of these genes varies a great deal: while a few genes exhibit classical induction with adriamycin, the majority of the genes are unchanged or are mildly repressed by either hypoxia or adriamycin. Further analysis using p53 mutated in the core DNA binding domain revealed that the interaction of p53 with CGIs may be occurring through both sequence-dependent and -independent mechanisms. Taken together, these experiments describe the identification of novel p53 target genes and the subsequent discovery of distinctly different expression phenomena for p53 target genes under different stress scenarios.
Krieg
Adam J
AJ
Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University, Stanford, CA 94303-5152, USA.
Hammond
Ester M
EM
Giaccia
Amato J
AJ
D001483:Base Sequence; D015687:Cell Hypoxia / Q000502:physiology; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D045325:HCT116 Cells; D006801:Humans; D007700:Kinetics; D046228:Microarray Analysis; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D015203:Reproducibility of Results; D020218:Response Elements / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016159:Tumor Suppressor Protein p53; D004247:DNA; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2