During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression.

The p53 canonical consensus sequence is a 10-bp repeat of PuPuPuC(A/T)(A/T)GPyPyPy, separated by a spacer with up to 13 bases. C(A/T)(A/T)G is the core sequence and purine (Pu) and pyrimidine (Py) bases comprise the flanking sequence. However, in the p53 noncanonical sequences, there are many variations, such as length of consensus sequence, variance of core sequence or flanking sequence, and variance in number of bases making up the spacer or AT gap composition. In comparison to p53, the p53 family members p63 and p73 have been found to have more tolerance to bind and activate several of these noncanonical sequences. The p53 protein forms monomers, dimers, and tetramers, and its nonspecific binding domain is well-defined; however, those for p63 or p73 are still not fully understood. Study of p63 and p73 structure to determine the monomers, dimers or tetramers to bind and regulate noncanonical sequence is a new challenge which is crucial to obtaining a complete picture of structure and function in order to understand how p63 and p73 regulate genes differently from p53. In this review, we will summarize the rules of p53 family non-canonical sequences, especially focusing on the structure of p53 family members in the regulation of specific target genes. In addition, we will compare different software programs for prediction of p53 family responsive elements containing parameters with canonical or non-canonical sequences.

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.

(1) Background: A premature termination codon (PTC) can be induced by a type of point mutation known as a nonsense mutation, which occurs within the coding region. Approximately 3.8% of human cancer patients have nonsense mutations of p53. However, the non-aminoglycoside drug PTC124 has shown potential to promote PTC readthrough and rescue full-length proteins. The COSMIC database contains 201 types of p53 nonsense mutations in cancers. We built a simple and affordable method to create different nonsense mutation clones of p53 for the study of the PTC readthrough activity of PTC124. (2) Methods: A modified inverse PCR-based site-directed mutagenesis method was used to clone the four nonsense mutations of p53, including W91X, S94X, R306X, and R342X. Each clone was transfected into p53 null H1299 cells and then treated with 50 μM of PTC124. (3) Results: PTC124 induced p53 re-expression in H1299-R306X and H1299-R342X clones but not in H1299-W91X and H1299-S94X clones. (4) Conclusions: Our data showed that PTC124 more effectively rescued the C-terminal of p53 nonsense mutations than the N-terminal of p53 nonsense mutations. We introduced a fast and low-cost site-directed mutagenesis method to clone the different nonsense mutations of p53 for drug screening.

The canonical core sequence of the p53 response element, CATG, has a two-base A/T gap. Previously, we found that p53 can also activate a non-canonical four-base A/T gap CATATG core sequence. In this study, we investigated the possible number of A/T bases used by p53 and showed that a six-base A/T gap CATATATG core sequence was the maximum A/T gap in the p53 response element that could be upregulated by p53 and p63. Canonical and non-canonical p53 response elements also have three-base flanking sequences. A/T bases could be substituted by G/C bases, including CACACG and CGTGTG, but not CGCGCG. We found that the SV40 promoter with functional six- and two-base A/T gap core sequences could be activated by TAp63γ and that TAp63γ could upregulate SV40 small and large T antigens expression in COS7 cells. We also found that the distal region of PUMA promoter with functional two six-base A/T gap core sequences could be activated by TAp63γ in 293T cells. These new findings could provide novel rules for the non-canonical p53 family response element and could extend the entire p53 family regulation network.