Mutant mice lacking alternatively spliced p53 isoforms unveil Ackr4 as a male-specific prognostic factor in Myc-driven B-cell lymphomas.

The Trp53 gene encodes several isoforms of elusive biological significance. Here, we show that mice lacking the Trp53 alternatively spliced (AS) exon, thereby expressing the canonical p53 protein but not isoforms with the AS C-terminus, have unexpectedly lost a male-specific protection against Myc-induced B-cell lymphomas. Lymphomagenesis was delayed in Trp53+/+Eµ-Myc males compared to Trp53ΔAS/ΔAS Eµ-Myc males, but also compared to Trp53+/+Eµ-Myc and Trp53ΔAS/ΔAS Eµ-Myc females. Pre-tumoral splenic cells from Trp53+/+Eµ-Myc males exhibited a higher expression of Ackr4, encoding an atypical chemokine receptor with tumor suppressive effects. We identified Ackr4 as a p53 target gene whose p53-mediated transactivation is inhibited by estrogens, and as a male-specific factor of good prognosis relevant for murine Eµ-Myc-induced and human Burkitt lymphomas. Furthermore, the knockout of ACKR4 increased the chemokine-guided migration of Burkitt lymphoma cells. These data demonstrate the functional relevance of alternatively spliced p53 isoforms and reveal sex disparities in Myc-driven lymphomagenesis.

Human cells divide many times during a lifetime, a process that requires careful regulation to avoid uncontrolled cell division, which can lead to various disorders, including cancer. For example, TP53, which encodes multiple proteins, is the most commonly mutated gene in cancers. TP53 carries the instructions to make a tumor suppressor protein, known as p53, which can stop cancers from forming and spreading. In humans and mice, TP53 (and the mouse analogue Trp53) can also be read by the cell to make several slightly different versions of the p53 protein, known as isoforms. The p53 isoforms are much less studied and their role in an organism is still unclear. To address this, Fajac et al. used genome editing to make mouse strains that were still able to express p53, but were only able to create a specific subset of p53 isoforms. In these mice, part of the Trp53 gene had been mutated to remove the cell's ability to make isoforms with an alternative C-terminal end. Fajac et al. then allowed these mice to breed with mice that were model organisms for a cancer called B-cell lymphoma. This revealed that male offspring that lacked alternative p53 isoforms were more susceptible to B-cell lymphoma and that they had decreased levels of the protein ACKR4, a receptor for signaling proteins that regulate cellular movement. Human datasets showed that having higher levels of ACKR4 could be linked to a better disease prognosis in male patients with Burkitt lymphoma, a rare but aggressive form of B-cell lymphoma. The same effect was not observed in females, suggesting that measuring ACKR4 gene expression in male patients with Burkitt lymphoma might be useful to identify the patients at higher risk. The study from Fajac et al. provides a new perspective on p53 ­ one of the most studied proteins. It highlights specific p53 isoforms and the ACKR4 protein as a potential way to identify male patients at higher risk from a type of B-cell lymphoma.

A systematic approach identifies p53-DREAM pathway target genes associated with blood or brain abnormalities.

p53 (encoded by Trp53) is a tumor suppressor, but mouse models have revealed that increased p53 activity may cause bone marrow failure, likely through dimerization partner, RB-like, E2F4/E2F5 and MuvB (DREAM) complex-mediated gene repression. Here, we designed a systematic approach to identify p53-DREAM pathway targets, the repression of which might contribute to abnormal hematopoiesis. We used Gene Ontology analysis to study transcriptomic changes associated with bone marrow cell differentiation, then chromatin immunoprecipitation-sequencing (ChIP-seq) data to identify DREAM-bound promoters. We next created positional frequency matrices to identify evolutionary conserved sequence elements potentially bound by DREAM. The same approach was developed to find p53-DREAM targets associated with brain abnormalities, also observed in mice with increased p53 activity. Putative DREAM-binding sites were found for 151 candidate target genes, of which 106 are mutated in a blood or brain genetic disorder. Twenty-one DREAM-binding sites were tested and found to impact gene expression in luciferase assays, to notably regulate genes mutated in dyskeratosis congenita (Rtel1), Fanconi anemia (Fanca), Diamond-Blackfan anemia (Tsr2), primary microcephaly [Casc5 (or Knl1), Ncaph and Wdr62] and pontocerebellar hypoplasia (Toe1). These results provide clues on the role of the p53-DREAM pathway in regulating hematopoiesis and brain development, with implications for tumorigenesis.

Germline mutation of MDM4, a major p53 regulator, in a familial syndrome of defective telomere maintenance.

Dyskeratosis congenita is a cancer-prone inherited bone marrow failure syndrome caused by telomere dysfunction. A mouse model recently suggested that p53 regulates telomere metabolism, but the clinical relevance of this finding remained uncertain. Here, a germline missense mutation of MDM4, a negative regulator of p53, was found in a family with features suggestive of dyskeratosis congenita, e.g., bone marrow hypocellularity, short telomeres, tongue squamous cell carcinoma, and acute myeloid leukemia. Using a mouse model, we show that this mutation (p.T454M) leads to increased p53 activity, decreased telomere length, and bone marrow failure. Variations in p53 activity markedly altered the phenotype of Mdm4 mutant mice, suggesting an explanation for the variable expressivity of disease symptoms in the family. Our data indicate that a germline activation of the p53 pathway may cause telomere dysfunction and point to polymorphisms affecting this pathway as potential genetic modifiers of telomere biology and bone marrow function.

Essential role for centromeric factors following p53 loss and oncogenic transformation.

In mammals, centromere definition involves the histone variant CENP-A (centromere protein A), deposited by its chaperone, HJURP (Holliday junction recognition protein). Alterations in this process impair chromosome segregation and genome stability, which are also compromised by p53 inactivation in cancer. Here we found that CENP-A and HJURP are transcriptionally up-regulated in p53-null human tumors. Using an established mouse embryonic fibroblast (MEF) model combining p53 inactivation with E1A or HRas-V12 oncogene expression, we reproduced a similar up-regulation of HJURP and CENP-A. We delineate functional CDE/CHR motifs within the Hjurp and Cenpa promoters and demonstrate their roles in p53-mediated repression. To assess the importance of HJURP up-regulation in transformed murine and human cells, we used a CRISPR/Cas9 approach. Remarkably, depletion of HJURP leads to distinct outcomes depending on their p53 status. Functional p53 elicits a cell cycle arrest response, whereas, in p53-null transformed cells, the absence of arrest enables the loss of HJURP to induce severe aneuploidy and, ultimately, apoptotic cell death. We thus tested the impact of HJURP depletion in pre-established allograft tumors in mice and revealed a major block of tumor progression in vivo. We discuss a model in which an "epigenetic addiction" to the HJURP chaperone represents an Achilles' heel in p53-deficient transformed cells.

Yeast cells reveal the misfolding and the cellular mislocalization of the human BRCA1 protein.

Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast and ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological processes behind these events in yeast remain to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment, and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations.

p53 downregulates the Fanconi anaemia DNA repair pathway.

Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53(Δ31), a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53(Δ31/Δ31) fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53(Δ31/Δ31) fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop.

Mutant mice lacking the p53 C-terminal domain model telomere syndromes.

Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53Δ31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hallmarks of syndromes caused by short telomeres. Indeed, p53Δ31/Δ31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Heterozygous p53+/Δ31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53Δ31/Δ31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism.

Of mice and men: fuzzy tandem repeats and divergent p53 transcriptional repertoires.

The clinical importance of tumor suppressor p53 makes it one of the most studied transcription factors. A comparison of mammalian p53 transcriptional repertoires may help identify fundamental principles in genome evolution and better understand cancer processes. Here we summarize mechanisms underlying the divergence of mammalian p53 transcriptional repertoires, with an emphasis on the rapid evolution of fuzzy tandem repeats containing p53 response elements.

Fuzzy tandem repeats containing p53 response elements may define species-specific p53 target genes.

Evolutionary forces that shape regulatory networks remain poorly understood. In mammals, the Rb pathway is a classic example of species-specific gene regulation, as a germline mutation in one Rb allele promotes retinoblastoma in humans, but not in mice. Here we show that p53 transactivates the Retinoblastoma-like 2 (Rbl2) gene to produce p130 in murine, but not human, cells. We found intronic fuzzy tandem repeats containing perfect p53 response elements to be important for this regulation. We next identified two other murine genes regulated by p53 via fuzzy tandem repeats: Ncoa1 and Klhl26. The repeats are poorly conserved in evolution, and the p53-dependent regulation of the murine genes is lost in humans. Our results indicate a role for the rapid evolution of tandem repeats in shaping differences in p53 regulatory networks between mammalian species.

Assessment of human Nter and Cter BRCA1 mutations using growth and localization assays in yeast.

A large number of missense mutations have been identified within the tumor suppressor gene BRCA1. Most of them, called "variants of unknown significance" (VUS), cannot be classified as pathogenic or neutral by genetic methods, which complicates their cancer risk assessment. Functional assays have been developed to circumvent this uncertainty. They aim to determine how VUS impact the BRCA1 protein structure or function, thereby giving an indication of their potential to cause cancer. So far, three relevant assays have been designed in yeast and used on large sets of variants. However, they are limited to variants mapped in restricted domains of BRCA1. One of them, the small colony phenotype (SCP) assay, monitors the BRCA1-dependent growth of yeast colonies that increases with pathogenic but not neutral mutations positioned in the Cter region. Here, we extend this assay to the Nter part of BRCA1. We also designed a new assay, called the "yeast localization phenotype (YLP) assay," based on the accumulation of BRCA1 in a single inclusion body in the yeast nucleus. This phenotype is altered by variants positioned both in the Nter and Cter regions. Together, these assays provide new perspectives for the functional assessment of BRCA1 mutations in yeast.