Multiple response elements and differential p53 binding control Perp expression during apoptosis

The p53 tumor suppressor gene responds to cellular stress by activating either cell cycle arrest or apoptosis. A growing number of target genes involved in each of these pathways have been identified. However, the mechanism by which the apoptosis versus arrest decision is made remains to be elucidated. Perp is a proapoptotic target gene of p53 expressed to high levels in apoptotic cells compared with those undergoing cell cycle arrest. This pattern of expression is unusual among p53 target genes, many of which are induced to similar levels during arrest and apoptosis. Here, we describe the regulation of the Perp gene by p53 through at least three response elements in the Perp promoter and first intron. These sites are occupied in vivo in E1A-expressing mouse embryo fibroblasts undergoing apoptosis but not cell cycle arrest, in contrast to the p21 5' response element, which is occupied during both. The apoptosis-deficient p53 point mutant, p53V143A, displays a selective deficit in binding to the Perp elements, demonstrating that p53 can distinguish between Perp and p21 at the level of DNA binding. These results provide mechanistic insight into the selective expression of Perp during apoptosis and may provide a useful model for studying the p53-dependent cell cycle arrest versus apoptosis decision.     

Transcriptional activation of the MUC2 gene by p53

MUC2 is one of the major components of mucins that provide a protective barrier between epithelial surfaces and the gut lumen. We investigated possible alterations of MUC2 gene expression by p53 and p21(Sdi1/Waf1/Cip1) in a human colon cancer cell line, DLD-1, establishing subclones in which a tetracycline-regulatable promoter controls exogenous p53 and p21 expression. MUC2 mRNA more significantly increased in response to p53 than to p21. Unexpectedly, MUC2 expression was also induced in human osteosarcoma cells, U-2OS and Saos-2, by exogenous p53. We next performed a reporter assay to test the direct regulation of MUC2 gene expression by p53. Deletion and mutagenesis of the MUC2 promoter region showed that it contains two sites for transactivation by p53. Furthermore, an electrophoretic mobility shift assay indicated that p53 binds to those elements. We analyzed MUC2 expression in other cell types possessing a functional p53 after exposure to various forms of stress. In MCF7 breast cancer and A427 lung cancer cells, MUC2 expression was increased along with the endogenous p53 level by actinomycin D, UVC, and x-ray, but not in RERF-LC-MS lung cancer cells carrying a mutated p53. These results suggest that p53 directly activates the MUC2 gene in many cell types.     

A half-site of the p53-binding site on the keratin 14 promoter is specifically activated by p63

Three members of p53 family, p53, p63 and p73, can transactivate their specific target genes through a p53 consensus sequence-binding motif which consists with direct repeats of PuPuPuC(T/A)(T/A)GPyPyPy as a whole-site of p53-binding site. p63, an epidermal stem cells marker, can regulate epidermal development and differentiation, but p53 has no similar biological activity. One isoform of p63, TAp63α, can active an epidermal basal cell marker, keratin 14. However, the p53-binding site does not exist as a whole-site in the K14 promoter region, although it contains three putative p53 half-binding sites at -269 to -1 of the K14 promoter. Two of three putative half-sites of the p53-binding site can be bound by p63α by electrophoresis mobility shift assay and DNA affinity purification assay. Only mutation of the p53 half-binding site at -140 to -131, the TAp63α induced K14 promoter activity can be abolished. This half-site was specifically activated by p63, but not by p53. Once we extend this p53 half-site to a whole p53-binding site in K14 promoter, both p53 and p63 expression vectors can activate its activity. Therefore, we propose that the different length of p53-binding site would determinate the gene regulated by different p53 family proteins.