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.The tumor microenvironment is a major factor influencing tumor growth, metastatic potential, and response to chemotherapeutic drugs and radiation therapy (7). The hypoxia-inducible factor (HIF) family of transcription factors regulates the expression of key enzymes in anaerobic glycolysis and proangiogenic factors (i.e., vascular endothelial growth factor), aiding cells in adapting to a low-oxygen environment (15). Thus, a tumor adapts to hypoxia first by sustaining itself via anaerobic metabolism and then by stimulating angiogenesis to acquire more nutrients and oxygen. However, the resulting vasculature tends to have aberrant structures with irregular blood flow and leaky walls, paradoxically creating regions of transient hypoxia and reoxygenation (76). Reoxygenation after hypoxia causes DNA damage, exacerbating genomic instability (25). The porous structure of the tumor vasculature may also provide an escape route for metastasizing cells. Repeated cycles of hypoxia, vascular formation, and reoxygenation select for cells that are more likely to survive in a restrictive environment. The consequences of this process are cancer cells that are more aggressive, more likely to metastasize, and more likely to be resistant to radiation and chemotherapy. Intimately tied to this process is the selection for cells that have lost the expression of functional p53 (19).A variety of cellular stresses, including DNA damage, oncogenic transformation, and hypoxia, stabilize the p53 protein by activating a host of stress-inducible kinases that phosphorylate p53 and MDM2, resulting in increased levels of p53 (17). Stabilized ...

Chromosomal imbalances and partial uniparental disomies in primary central nervous system lymphoma

“…48 In 4q12, a 200-kb MCR of deletion involving the SCFD2 gene, a potential p53 target gene, was detected. 49 In 4q35.2, the FAT tumor suppressor gene was affected by deletion. Another small MCR of deletion affected the tumor suppressor gene FAS gene at 10q23.…”

Hypoxia-induced and stress-specific changes in chromatin structure and function

“…mSin3A is generally found as a member of HDAC-protein complexes and functions as a transcriptional corepressor by mediating interactions between HDACs and transcription regulatory proteins [64][65][66]. Recent global analysis of p53-interactions with chromatin in response to stress, including hypoxia, showed little change in promoter-association of p53 although gene expression patterns change markedly [67]. These and other studies underscore the importance of corepressor or coactivator association with chromatin-bound, transcription factors to effect alteration of chromatin structure and gene expression.…”

DNA polymerase zeta (pol ζ) in higher eukaryotes

“…It has been proposed as a possible regulatory element [27], but no functional effect of the hairpin loop has yet emerged [30]. Upstream of the REV3L promoter there is a response element to which p53 binds, as identified by chromatin immunoprecipitation and CpG island microarray hybridization [32]. REV3L transcription is inducible by treatment with the DNA-damaging agent adriamycin [32] and by N-methyl-N′-nitro-N-nitrosoguanidine [33], but not by hypoxic conditions [32].…”

“…Upstream of the REV3L promoter there is a response element to which p53 binds, as identified by chromatin immunoprecipitation and CpG island microarray hybridization [32]. REV3L transcription is inducible by treatment with the DNA-damaging agent adriamycin [32] and by N-methyl-N′-nitro-N-nitrosoguanidine [33], but not by hypoxic conditions [32]. Murine Rev3L was first identified as a gene induced by treatment of primary cultured cerebral cortical cells with the seizure-inducing agent pentylentetrazol [34].…”