Functional Analysis of p53 Binding under Differential Stresses

Abstract

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.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank Lawrence Heisler and Sandy Der at the University of Toronto for providing access to the CpG Island Microarray Database, Bert Vogelstein at Johns Hopkins University for providing the HCT116-derived cell lines and the pG13 and pM15 luciferase vectors, Jennifer Pietenpol at Vanderbilt University for permission to use the pCEP4-p53 expression plasmid, Janoz Demeter of the Stanford Microarray Database for expert help with creating CGI gene list files, and Ali Salim for expert assistance with microarray hybridization techniques. We also thank Denise Chan, Patrick Sutphin, and Sacha Krieg for critical reading of the manuscript.

This work was supported by an NIH grant (CA 88480) awarded to A.J.G.