Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements

The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein-DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process.     

Wild-type but not mutant p53 activates the hepatocyte growth factor/scatter factor promoter.

p53 transactivates the expression of a variety of genes by binding to specific DNA sequences within the promoter. We have investigated the ability of wild-type p53 and a non-DNA binding p53 mutant to activate the hepatocyte growth factor/scatter factor (HGF/SF) promoter using chloramphenicol acetyltransferase reporter constructs. We also used deletion sequences of the HGF/SF promoter to identify which regions, if any, were responsible for p53 binding. Our results show that wild-type but not mutant p53 activates the HGF/SF promoter when using -3000 and -755 bp upstream of the HGF/SF gene. This activation is lost when promoter sequences covering -365 and -239 bp are used. Analysis of the DNA sequence between -365 and -755 bp shows one putative p53 half-site with 80% homology to the consensus sequence and another half-site 3 bases downstream of this with 100% homology to the consensus sequence. In contrast to previously identified p53 binding DNA sequences, the downstream half-site is inverted. We propose that the HGF/SF promoter can be activated by wild-type p53 in vivo and that this could be as a result of a novel form of sequence-specific DNA binding.     

p300 binding by E1A cosegregates with p53 induction but is dispensable for apoptosis.

E1A expression during adenovirus infection induces apoptosis. E1A expression causes accumulation of the p53 tumor suppressor protein, and E1A-induced apoptosis is p53 mediated in primary rodent cells, implying that p53 induction may be linked to apoptosis induction by E1A. Adenoviruses containing mutations in the E1A gene were tested for the ability to trigger both p53 accumulation and the appearance of enhanced cytopathy (cyt phenotype) and degradation of DNA (deg phenotype), indicative of apoptosis in infected HeLa cells. The adenoviruses had mutations which disrupted the pRb- and/or p300-binding activities of E1A so that the relationship between p53 induction and apoptosis and binding to these cellular proteins by E1A could be determined. An E1A mutation that specifically disrupted the p300-binding activity failed to induce p53 accumulation, whereas mutations in E1A which affected pRb binding induced p53 accumulation. Thus, p300 binding was required and pRb binding was dispensable for E1A-mediated accumulation of p53 in HeLa cells. All the E1A mutant viruses, regardless of the ability to induce p53 accumulation, induced the cyt and deg phenotypes, suggesting that p53 induction in infected HeLa cells was not essential for apoptosis, nor was binding of E1A to the pRb and/or p300 protein. The possibility that E1A induced a p53-independent apoptosis pathway was tested by analyzing the appearance of the cyt and deg phenotypes in Saos-2 cells, which were null for both alleles of p53, upon adenovirus infection. An adenovirus expressing wild-type 12S E1A induced both the cyt and deg phenotypes in Saos-2 cells, as did all the E1A mutant viruses. Thus, E1A expression during infection of human cells may trigger redundant p53-independent and -dependent apoptotic pathways.