Alberto Inga

p53 mutants can often transactivate promoters containing a p21 but not Bax or PIG3 responsive elements.

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fishers exact test), more rarely mutated (P<0.006, Fishers exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fishers exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fishers exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.

Noncanonical DNA motifs as transactivation targets by wild type and mutant p53.

Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network.

p53 mutants exhibiting enhanced transcriptional activation and altered promoter selectivity are revealed using a sensitive, yeast-based functional assay.

Changes in promoter specificity and binding affinity that may be associated with p53 mutations or post-translational modifications are useful in understanding p53 structure/function relationships and categorizing tumor mutations. We have exploited variable expression of human p53 in yeast to identify mutants with novel phenotypes that would correspond to altered promoter selectivity and affinity. The p53 cDNA regions coding for the DNA binding and tetramerization domains were subjected to random PCR mutagenesis and were cloned directly by recombination in yeast into a vector with a GAL1 promoter whose level of expression could be easily varied. p53 variants exhibiting higher than wild type levels of transactivation (supertrans) for the RGC responsive element were identified at low level of p53 protein expression. All the p53 mutants obtained with this screen were located in the DNA binding domain. Two out of 17 supertrans mutants have been found in tumors. Six mutations were in the L1 loop region between amino acids 115 and 124. The transactivation potential of a panel of supertrans p53 mutants on different promoters was evaluated using the p53 responsive elements, RGC, PIG3, p21 and bax. Although all mutants retained some activity with all promoters, we found different patterns of induction based on strength and promoter specificity. In particular none of the mutants was supertrans for the p21 responsive element. Interestingly, further analysis in yeast showed that the transactivation function could be retained even in the presence of dominant-negative p53 tumor mutations that could inhibit wild type p53. Five mutants were also characterized in human cells in terms of growth suppression and transactivation of various promoters. These novel supertrans p53 mutants may be useful in studies aimed at dissecting p53 downstream pathways, understanding specific interactions between p53 and the DNA, and could replace wild type p53 in cancer gene therapy protocols. The approach may also prove useful in identifying p53 tumor mutations.

Changing the p53 master regulatory network: ELEMENTary, my dear Mr Watson.

The p53 master regulatory network provides for the stress-responsive direct control of a vast number of genes in humans that can be grouped into several biological categories including cell-cycle control, apoptosis and DNA repair. Similar to other sequence-specific master regulators, there is a matrix of key components, which provide for variation within the p53 master regulatory network that include p53 itself, target response element sequences (REs) that provide for p53 regulation of target genes, chromatin, accessory proteins and transcription machinery. Changes in any of these can impact the expression of individual genes, groups of genes and the eventual biological responses. The many REs represent the core of the master regulatory network. Since defects or altered expression of p53 are associated with over 50% of all cancers and greater than 90% of p53 mutations are in the sequence-specific DNA-binding domain, it is important to understand the relationship between wild-type or mutant p53 proteins and the target response elements. In the words of the legendary detective Sherlock Holmes, it is ‘Elementary, my dear Mr. Watson.’

Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code

Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein–DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122→L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc−/−) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122→L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122→L allele in p53−/− mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122→L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122→L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122→L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122→L mutant allele in the Xpc−/− background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.

Expression of PTCH1b tumor suppressor gene is controlled by different 5’-untranslated region cis-regulatory elements

Background The PTCH1 tumor suppressor gene encodes for a 12-pass transmembrane receptor with a negative regulatory role in Hedgehog signaling pathway. The PTCH1 germline mutations cause Gorlin syndrome, disorder characterized by developmental abnormalities and tumor susceptibility. Most of the malformations are caused by PTCH1 haploinsufficiency, indicative of fine-tuning needed to properly regulate the activity of pathway which is involved in pathogenesis of various tumors. Since in patients with different tumors and healthy controls we identified 5 to 8 CGG repeats located 4 bases upstream of a translation initiation site, in this study we wanted to examine how 5’ untranslated region (5’UTR) regulates the expression of PTCH1 transcript 1b. Material and methods Various in silico tools and databases were used to reveal all potential cis-regulatory elements in the PTCH1b 5’UTR. Since PTCH1b transcript has two different-sized 5’UTRs, we built pGL3-P-based plasmids by inserting upstream of firefly luciferase gene either 188- or 300bp-long 5’UTR, each harboring 5 to 8 CGG repeats. As the last 76bp of 5’UTR were predicted as an internal ribosome entry site (IRES), we constructed bicistronic pRuF vectors by cloning each PTCH1b 5’UTR between Renilla and firefly luciferase gene. Reporter gene assays and qPCR were performed in transfected MCF-7, HCT 116 and HEK 293T cells. Results Dual luciferase assays showed that shorter 5’UTR significantly increased reporter activity, with a subtle reduction with increased number of repeats. Longer 5’UTR led to much reduced reporter activity, without a difference among repeats. Luciferase mRNA quantification showed that both 5’UTR lengths significantly increased transcription. Site-directed mutagenesis proved hypothesis that 2 potential upstream open reading frames, contained in first 112bp of longer 5’UTR, might account for this severe reduction in reporter activity. Both 5’UTR lengths significantly increased firefly luciferase activity of pRuF vectors (proved by PCR this is not due to alternative splicing), with no difference among repeats. Firefly luciferase activity was significantly reduced when predicted IRES motif was removed from pRuF plasmid. Firefly/Renilla luciferase mRNA ratios were the same as for empty vector, indicating that observed higher firefly luciferase activity with equal mRNA levels should be due to a post-transcriptional level of regulation, i.e., cap-independent translation of firefly luciferase gene. Conclusions All our results point to the exceptionally complex and so far unexplored role of 5’UTR in the regulation of PTCH1b expression, whilst the existence of an IRES motif would enable Ptch1 protein to be synthesized under conditions when the general level of protein synthesis is reduced, such as in hypoxia, which is known activator of Hedgehog signaling pathway.

The p53 Master Regulator and Rules of Engagement with Target Sequences

Publisher Summary This chapter addresses the concept of the response element sequences (REs) that provide for p53 recruitment to target promoters, focusing on the intrinsic potential of wild-type and mutant p53 to transactivate from various REs as a source of transactivation selectivity. The p53 protein is a master regulator of cellular responses to stress. It functions as a tetrameric, sequence specific transcription factor at the hub of various distinct transcriptional programs. p53 is a prominent tumor suppressor and is impaired in nearly all human tumors, primarily by somatically acquired p53 mutations. Through a combination of transcription and DNA binding assays in yeast and human cell systems it has been possible to address intrinsic functional properties of p53 interactions with target REs. There is a wide opportunity for transactivation specificity, suggesting that changes in RE sequences can be a source of inter-individual or evolutionary diversification in p53 mediated responses. Along this line, results from functional as well as DNA binding assays have led to the development of in silico approaches for the identification of single nucleotide polymorphisms (SNPs) at p53 REs that are predicted to be functionally significant. In a broader sense, the growing knowledge of the multiple layers and players contributing to transactivation selectivity by p53, along with a more detailed understanding of which subsets of p53 induced responses are most effective in tumor suppression, can generate new targets and strategies for therapeutic intervention.