The p53 tumour suppressor is modified through mutation or changes in expression in most cancers, leading to the altered regulation of hundreds of genes that are directly influenced by this sequence-specific transcription factor. Central to the p53 master regulatory network are the target response element (RE) sequences. The extent of p53 transactivation and transcriptional repression is influenced by many factors, including p53 levels, cofactors and the specific RE sequences, all of which contribute to the role that p53 has in the aetiology of cancer. This Review describes the identification and functionality of REs and highlights the inclusion of non-canonical REs that expand the universe of genes and regulation mechanisms in the p53 tumour suppressor network.

Despite the utility of CRISPR-Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes1,2. We developed a yeast-based assay to identify optimized Streptococcus pyogenes Cas9 (SpCas9) variants that enables simultaneous evaluation of on- and off-target activity. We screened a library of SpCas9 variants carrying random mutations in the REC3 domain and identified mutations that increased editing accuracy whilst maintaining editing efficiency. We combined four beneficial mutations to generate evoCas9, a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants3,4 (4-fold average improvement), while maintaining near wild-type on-target editing efficiency (90% median residual activity). Evaluating evoCas9 on endogenous genomic loci, we demonstrated a substantial improved specificity and observed no off-target sites for 4 of the 8 sgRNAs tested. Finally, we showed that following long-term expression (40 days), evoCas9 strongly limited the unspecific cleavage of a difficult-to-discriminate off-target site and fully abrogated the cleavage of two additional off-targets.

Little is known about the mechanisms that regulate differential transactivation by p53. We developed a system in the yeast Saccharomyces cerevisiae that addresses p53 transactivation capacity from 26 different p53 response elements (REs) under conditions where all other factors, such as chromatin, are kept constant. The system relies on a tightly regulated promoter (rheostatable) that can provide for a broad range of p53 expression. The p53 transactivation capacity toward each 20-to 22-bp-long RE could be ranked by using a simple phenotypic assay. Surprisingly, there was as much as a 1,000-fold difference in transactivation. There was no correlation between the functional rank and statistical predictions of binding energy of the REs. Instead we found that the central sequence element in an RE greatly affects p53 transactivation capacity, possibly because of DNA structural properties. Our results suggest that intrinsic DNA binding affinity and p53 protein levels are important contributors to p53-induced differential transactivation. These results are also relevant to understanding the regulation by other families of transcription factors that recognize several sequence-related response elements and/or have tightly regulated expression. We found that p53 had weak activity towards half the apoptotic REs. In addition, p53 alleles associated with familial breast cancer, previously classified as wild type, showed subtle differences in transactivation capacity towards several REs.

The tumor suppressor TP53 is the most frequently mutated gene product in human cancer. Close to half of all solid tumors carry inactivating mutations in the TP53 gene, while in the remaining cases, TP53 activity is abrogated by other oncogenic events, such as hyperactivation of its endogenous repressors MDM2 or MDM4. Despite identification of hundreds of genes regulated by this transcription factor, it remains unclear which direct target genes and downstream pathways are essential for the tumor suppressive function of TP53. We set out to address this problem by generating multiple genomic data sets for three different cancer cell lines, allowing the identification of distinct sets of TP53-regulated genes, from early transcriptional targets through to late targets controlled at the translational level. We found that although TP53 elicits vastly divergent signaling cascades across cell lines, it directly activates a core transcriptional program of ∼100 genes with diverse biological functions, regardless of cell type or cellular response to TP53 activation. This core program is associated with high-occupancy TP53 enhancers, high levels of paused RNA polymerases, and accessible chromatin. Interestingly, two different shRNA screens failed to identify a single TP53 target gene required for the anti-proliferative effects of TP53 during pharmacological activation in vitro. Furthermore, bioinformatics analysis of thousands of cancer genomes revealed that none of these core target genes are frequently inactivated in tumors expressing wild-type TP53. These results support the hypothesis that TP53 activates a genetically robust transcriptional program with highly distributed tumor suppressive functions acting in diverse cellular contexts.

There are many sources of genetic diversity, ranging from programmed mutagenesis in antibody genes to random mutagenesis during species evolution or development of cancer. We propose that mutations in DNA sequence-specific transcription factors that target response elements (REs) in many genes can also provide for rapid and broad phenotypic diversity, if the mutations lead to altered binding affinities at individual REs. To test this concept, we examined the in vivo transactivation capacity of wild-type human and murine p53 and 25 partial function mutants. The p53s were expressed in yeast from a rheostatable promoter, and the transactivation capacities toward >15 promoter REs upstream of a reporter gene were measured. Surprisingly, there was wide variation in transactivation by the mutant p53s toward the various REs. This is the first study to address directly the impact of mutations in a sequence-specific transcription factor on transactivation from a wide array of REs. We propose a master gene hypothesis for phenotypic diversity where the master gene is a single transcriptional activator (or repressor) that regulates many genes through different REs. Mutations of the master gene can lead to a variety of simultaneous changes in both the selection of targets and the extent of transcriptional modulation at the individual targets, resulting in a vast number of potential phenotypes that can be created with minimal mutational changes without altering existing protein-protein interactions.transactivation ͉ evolution ͉ networks ͉ mutation ͉ promoter

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.

A novel resource centre for TP53 mutations and mutants has been developed (http://p53.fr). TP53 gene dysfunction can be found in the majority of human cancer types. The potential use of TP53 mutation as a biomarker for clinical studies or exposome analysis has led to the publication of thousands of reports describing the TP53 gene status in >10 000 tumours. The UMD TP53 mutation database was created in 1990 and has been regularly updated. The 2012 release of the database has been carefully curated, and all suspicious reports have been eliminated. It is available either as a flat file that can be easily manipulated or as novel multi-platform analytical software that has been designed to analyse various aspects of TP53 mutations. Several tools to ascertain TP53 mutations are also available for download. We have developed TP53MULTLoad, a manually curated database providing comprehensive details on the properties of 2549 missense TP53 mutants. More than 100 000 entries have been arranged in 39 different activity fields, such as change of transactivation on various promoters, apoptosis or growth arrest. For several hot spot mutants, multiple gain of function activities are also included. The database can be easily browsed via a graphical user interface.

Highlights d LncRNA MEG3 is a tumor suppressor that stimulates the p53 pathway d The p53-stimulating core of MEG3 comprises of two conserved, structured domains d Two distal motifs in the MEG3 core form pseudoknot interactions (''kissing loops'') d Mutations in these pseudoknots disrupt MEG3 architecture and impair its function