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Mutant p53 proteins not only lose their tumour suppressive ability, but also gain new properties that promote tumorigenesis. What are these properties and what are the clinical implications? Key Points The tumour suppressor p53 (encoded by TP53 in humans) functions primarily as a transcription factor, which, upon cellular stress signals, regulates a plethora of genes that promote cell cycle arrest, senescence, apoptosis, differentiation, DNA repair and other processes. TP53 is somatically mutated in the majority of sporadic human cancers, and mutations in TP53 are also associated with Li–Fraumeni Syndrome, a familial cancer predisposition syndrome. The majority of cancer-associated mutations in TP53 are missense mutations in its DNA-binding domain. These mutations usually lead to the formation of a full-length mutant protein (mutant p53) incapable of activating p53 target genes and suppressing tumorigenesis. Besides losing their wild-type activities, many p53 mutants also function as dominant-negative proteins that inactivate wild-type p53 expressed from the remaining wild-type allele. Moreover, some mutant p53 forms also acquire new oncogenic properties that are independent of wild-type p53, known as 'gain-of-function' properties. In the past three decades ample data were collected in support of the importance of mutant p53 gain-of-function properties for tumorigenesis. These data include cell culture studies that demonstrated the capability of mutant p53 to impinge on pivotal cellular regulatory networks, mouse models that established the ability of mutant p53 to increase tumour aggressiveness and metastatic potential, as well as clinical studies that revealed associations between TP53 mutations and poor clinical outcome in a variety of malignancies. This Review describes recent advances in the research field of mutant p53, with an emphasis on the transcriptional effects of mutant p53, the expression signatures associated with TP53 mutations in vitro and in vivo and the diagnostic, prognostic and predictive value of TP53 mutations in human cancer. Ample data indicate that mutant p53 proteins not only lose their tumour suppressive functions, but also gain new abilities that promote tumorigenesis. Moreover, recent studies have modified our view of mutant p53 proteins, portraying them not as inert mutants, but rather as regulated proteins that influence the cancer cell transcriptome and phenotype. This influence is clinically manifested as association of TP53 mutations with poor prognosis and drug resistance in a growing array of malignancies. Here, we review recent studies on mutant p53 regulation, gain-of-function mechanisms, transcriptional effects and prognostic association, with a focus on the clinical implications of these findings.

PRDM14 is a crucial regulator of mouse primordial germ cells (mPGCs), epigenetic reprogramming and pluripotency, but its role in the evolutionarily divergent regulatory network of human PGCs (hPGCs) remains unclear. Besides, a previous knockdown study indicated that PRDM14 might be dispensable for human germ cell fate. Here, we decided to use inducible degrons for a more rapid and comprehensive PRDM14 depletion. We show that PRDM14 loss results in significantly reduced specification efficiency and an aberrant transcriptome of hPGC-like cells (hPGCLCs) obtained in vitro from human embryonic stem cells (hESCs). Chromatin immunoprecipitation and transcriptomic analyses suggest that PRDM14 cooperates with TFAP2C and BLIMP1 to upregulate germ cell and pluripotency genes, while repressing WNT signalling and somatic markers. Notably, PRDM14 targets are not conserved between mouse and human, emphasising the divergent molecular mechanisms of PGC specification. The effectiveness of degrons for acute protein depletion is widely applicable in various developmental contexts. PRDM14 is a critical transcription factor for mouse primordial germ cell specification, but its role in human remains unclear. Here, PRDM14 protein depletion using auxin-inducible degron uncovers a critical role for human germ cell specification, but regulation of a different set of target genes than in mouse.

Amos Tanay and colleagues characterize DNA methylation polymorphism within cell populations and track immortalized fibroblasts in culture for over 300 generations to show that formation of differentially methylated regions occurs through a stochastic process and nearly deterministic epigenetic remodeling. DNA methylation has been comprehensively profiled in normal and cancer cells, but the dynamics that form, maintain and reprogram differentially methylated regions remain enigmatic. Here, we show that methylation patterns within populations of cells from individual somatic tissues are heterogeneous and polymorphic. Using in vitro evolution of immortalized fibroblasts for over 300 generations, we track the dynamics of polymorphic methylation at regions developing significant differential methylation on average. The data indicate that changes in population-averaged methylation occur through a stochastic process that generates a stream of local and uncorrelated methylation aberrations. Despite the stochastic nature of the process, nearly deterministic epigenetic remodeling emerges on average at loci that lose or gain resistance to methylation accumulation. Changes in the susceptibility to methylation accumulation are correlated with changes in histone modification and CTCF occupancy. Characterizing epigenomic polymorphism within cell populations is therefore critical to understanding methylation dynamics in normal and cancer cells.

Loss-of-function studies are fundamental for dissecting gene function. Yet, methods to rapidly and effectively perturb genes in mammalian cells, and particularly in stem cells, are scarce. Here we present a system for simultaneous conditional regulation of two different proteins in the same mammalian cell. This system harnesses the plant auxin and jasmonate hormone-induced degradation pathways, and is deliverable with only two lentiviral vectors. It combines RNAi-mediated silencing of two endogenous proteins with the expression of two exogenous proteins whose degradation is induced by external ligands in a rapid, reversible, titratable and independent manner. By engineering molecular tuners for NANOG, CHK1, p53 and NOTCH1 in mammalian stem cells, we have validated the applicability of the system and demonstrated its potential to unravel complex biological processes. Loss-of-function approaches are fundamental for dissecting the roles played by genes but methods to simultaneously perturb several proteins in the same mammalian cell are scarce. Here the authors harness the plant auxin and jasmonate hormone-degradation pathways and RNAi technology, to control the levels of two proteins and validate its application in stem cells.

Prostate cancer is the most common non-dermatologic malignancy in men in the Western world. Recently, a frequent chromosomal aberration fusing androgen regulated TMPRSS2 promoter and the ERG gene (TMPRSS2/ERG) was discovered in prostate cancer. Several studies demonstrated cooperation between TMPRSS2/ERG and other defective pathways in cancer progression. However, the unveiling of more specific pathways in which TMPRSS2/ERG takes part, requires further investigation. Using immortalized prostate epithelial cells we were able to show that TMPRSS2/ERG over-expressing cells undergo an Epithelial to Mesenchymal Transition (EMT), manifested by acquisition of mesenchymal morphology and markers as well as migration and invasion capabilities. These findings were corroborated in vivo, where the control cells gave rise to discrete nodules while the TMPRSS2/ERG-expressing cells formed malignant tumors, which expressed EMT markers. To further investigate the general transcription scheme induced by TMPRSS2/ERG, cells were subjected to a microarray analysis that revealed a distinct EMT expression program, including up-regulation of the EMT facilitators, ZEB1 and ZEB2, and down-regulation of the epithelial marker CDH1(E-Cadherin). A chromatin immunoprecipitation assay revealed direct binding of TMPRSS2/ERG to the promoter of ZEB1 but not ZEB2. However, TMPRSS2/ERG was able to bind the promoters of the ZEB2 modulators, IL1R2 and SPINT1. This set of experiments further illuminates the mechanism by which the TMPRSS2/ERG fusion affects prostate cancer progression and might assist in targeting TMPRSS2/ERG and its downstream targets in future drug design efforts.

Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild‐type p53. These miRNAs are repressed by p53 in an E2F1‐mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi‐gene transcriptional and post‐transcriptional feed‐forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network. Synopsis Precise regulation of gene expression is crucial for maintaining homeostasis in healthy tissues and for the execution of cellular programs such as proliferation, differentiation and cell death. In the last decade, microRNAs (miRNAs) have been uncovered as an expanding family of gene expression regulators. These short non‐coding RNAs regulate gene expression at the post‐transcriptional level by promoting translational inhibition or mRNA degradation (Bartel, 2004 ). Similar to protein‐coding genes, the expression of miRNAs is also regulated by transcription factors (TFs), and induction or repression of miRNAs has been demonstrated to play a role in physiological processes such as immune response (Thai et al , 2007 ) and apoptosis (Chang et al , 2007 ; Raver‐Shapira et al , 2007 ). Accordingly, deregulation of miRNAs is associated with diverse types of diseases, including a variety of cancers (Esquela‐Kerscher and Slack, 2006 ; Volinia et al , 2006 ). In an earlier computational study, we predicted the presence of several types of regulatory network motifs that involve TFs and miRNAs (Shalgi et al , 2007 ), and may provide a mechanism for fine‐tuned coordination between transcriptional and post‐transcriptional regulation of gene expression. Here, we describe and experimentally demonstrate one such regulatory motif, termed feed‐forward loop (FFL), which involves the TF E2F1, a set of miRNAs, and their common targets (Figure 8 ). In this FFL, E2F1, a key regulator of cell‐cycle progression, transcriptionally activates a family of 15 miRNAs that are organized in three paralogous polycistrons on three different chromosomes. These miRNAs silence a group of antiproliferative regulators including the pocket proteins pRb and p130 and the CDK inhibitors p21 and p57. Importantly, these genes are themselves transcriptional targets of E2F1. Thus, a TF activates a set of genes as well as a set of miRNAs, which in turn post‐transcriptionally regulate that set of genes. Increasing the complexity of this regulatory FFL, many of the shared targets of E2F1 and the miRNAs function as regulators of the cell cycle; some negatively regulate E2F itself. For example, the pocket proteins pRB and p130 are the major components that regulate the activity of E2F family members throughout the phases of the cell cycle through direct protein–protein interaction. The TF p53 is regarded as one of the key proteins that prevent malignant transformation (Ryan et al , 2001 ), and deactivating mutations of this tumor suppressor are highly common in a wide variety of tumors (Hussain and Harris, 1999 ). A hallmark activity of p53 is the inhibition of proliferation and the induction of cellular senescence on diverse types of stress signals with oncogenic potential, including DNA damage, telomere shortening and oncogene activation. There are several known mechanisms by which p53 negatively regulates proliferation, the key one being the transcriptional activation of the CDK inhibitor p21, which indirectly inhibits the activity of E2F family members. Another recently discovered mechanism for inhibiting proliferation by p53 is the induction of miRNAs from the miR‐34 family, which also modulate the E2F pathway (He et al , 2007 ; Tarasov et al , 2007 ; Tazawa et al , 2007 ; Kumamoto et al , 2008 ). Additionally, direct and indirect transcriptional repression by p53 is considered important for its ability to inhibit proliferation (Ho and Benchimol, 2003 ). Using miRNA microarrays, we discovered that p53 activation during cellular senescence in primary human fibroblasts leads to a decrease in the expression of the above‐mentioned family of miRNAs, including members of the miR‐17‐92, miR‐106b/93/25 and miR‐106a‐92 polycistronic miRNA clusters. A similar decrease in miRNA expression was observed in human breast cancer specimens that harbor wild‐type p53 as compared with those that harbor mutant forms of p53. We further investigated the mechanism by which p53 represses the expression of this group of miRNAs, and found that activation of p53 leads to a dramatic reduction of E2F1 mRNA, protein and activity levels, which in turn leads to a decrease in the E2F1‐dependent transcriptional activation of these miRNAs. To study the consequence of deregulation of this FFL and importance of its inhibition by p53, we ectopically expressed representative members from the set of p53‐repressed miRNAs, namely the miR‐106b/93/25 polycistron, in primary human fibroblasts. Consequently, these cells acquired an enhanced proliferative phenotype manifested by increased growth rate, increased colony formation efficiency and delayed entry into replicative senescence. These results position the repression of this set of miRNAs as a novel mechanism by which p53 inhibits proliferation and controls cell fate. Here we identified a group of 15 co‐regulated paralogous miRNAs which are transcriptionally activated by E2F1. This group includes the miR‐17‐92, miR‐106a‐92 and miR‐106b/93/25 polycistronic miRNAs. These miRNAs silence anti‐proliferative genes, which themselves are E2F1 targets and function as negative regulators of proliferation. Thus, E2F1 and this group of microRNAs cooperate in a feed‐forward loop that involves transcriptional and post‐transcriptional modes of regulation. The key tumor suppressor p53 disrupts this feed‐forward loop by inactivating E2F1 in senescent cells and in human cancers. This inhibition serves as another arm of p53's tight control of proliferation.

Partial gain of chromosome arm 17q is an abundant aberrancy in various cancer types such as lung and prostate cancer with a prominent occurrence and prognostic significance in neuroblastoma--one of the most common embryonic tumors. The specific genetic element/s in 17q responsible for the cancer-promoting effect of these aberrancies is yet to be defined although many genes located in 17q have been proposed to play a role in malignancy. We report here the characterization of a naturally-occurring, non-reciprocal translocation der(X)t(X;17) in human lung embryonal-derived cells following continuous culturing. This aberrancy was strongly correlated with an increased proliferative capacity and with an acquired ability to form colonies in vitro. The breakpoint region was mapped by fluorescence in situ hybridization (FISH) to the 17q24.3 locus. Further characterization by a custom-made comparative genome hybridization array (CGH) localized the breakpoint within the Bromodomain PHD finger Transcription Factor gene (BPTF), a gene involved in transcriptional regulation and chromatin remodeling. Interestingly, this translocation led to elevation in the mRNA levels of the endogenous BPTF. Knock-down of BPTF restricted proliferation suggesting a role for BPTF in promoting cellular growth. Furthermore, the BPTF chromosomal region was found to be amplified in various human tumors, especially in neuroblastomas and lung cancers in which 55% and 27% of the samples showed gain of 17q24.3, respectively. Additionally, 42% percent of the cancer cell lines comprising the NCI-60 had an abnormal BPTF locus copy number. We suggest that deregulation of BPTF resulting from the translocation may confer the cells with the observed cancer-promoting phenotype and that our cellular model can serve to establish causality between 17q aberrations and carcinogenesis.

Transcription factors (TF) regulate expression by binding to specific DNA sequences. A binding event is functional when it affects gene expression. Functionality of a binding site is reflected in conservation of the binding sequence during evolution and in over represented binding in gene groups with coherent biological functions. Functionality is governed by several parameters such as the TF-DNA binding strength, distance of the binding site from the transcription start site (TSS), DNA packing, and more. Understanding how these parameters control functionality of different TFs in different biological contexts is a must for identifying functional TF binding sites and for understanding regulation of transcription. We introduce a novel method to screen the promoters of a set of genes with shared biological function (obtained from the functional Gene Ontology (GO) classification) against a precompiled library of motifs, and find those motifs which are statistically over-represented in the gene set. More than 8,000 human (and 23,000 mouse) genes, were assigned to one of 134 GO sets. Their promoters were searched (from 200 bp downstream to 1,000 bp upstream the TSS) for 414 known DNA motifs. We optimized the sequence similarity score threshold, independently for every location window, taking into account nucleotide heterogeneity along the promoters of the target genes. The method, combined with binding sequence and location conservation between human and mouse, identifies with high probability functional binding sites for groups of functionally-related genes. We found many location-sensitive functional binding events and showed that they clustered close to the TSS. Our method and findings were tested experimentally. We identified reliably functional TF binding sites. This is an essential step towards constructing regulatory networks. The promoter region proximal to the TSS is of central importance for regulation of transcription in human and mouse, just as it is in bacteria and yeast.

Pervasive transcriptional activity is observed across diverse species. The genomes of extant organisms have undergone billions of years of evolution, making it unclear whether these genomic activities represent effects of selection or 'noise'14. Characterizing default genome states could help understand whether pervasive transcriptional activity has biological meaning. Here we addressed this question by introducing a synthetic 101-kb locus into the genomes of Saccharomyces cereuisiae and Mus musculus and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including its flanking regions, thus retaining basic features of the natural sequence but ablating evolved coding or regulatory information. We observed widespread activity of both reversed and native HPRT1 loci in yeast, despite the lack of evolved yeast promoters. By contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, and instead exhibited repressive chromatin signatures. The repressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless, this variant was also transcriptionally inactive. These results show that synthetic genomic sequences that lack coding information are active in yeast, but inactive in mouse embryonic stem cells, consistent with a major difference in 'default genomic states' between these two divergent eukaryotic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information and the birth of new genes.

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the ‘anthropomorphous apes’ 1 – 3 , with a proposed role in contributing to human bipedalism 4 – 6 . Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element—inserted into an intron of the TBXT gene 7 – 9 —pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt , mimicking the expression pattern of its hominoid orthologue TBXT . Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans 10 . Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today. An insertion of an Alu element into an intron of the TBXT gene is identified as a genetic mechanism of tail-loss evolution in humans and apes, with implications for human health today.