Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.DOI: http://dx.doi.org/10.7554/eLife.13909.001

The high percentage of p53 missense mutations found in cancer has been attributed to mutant acquired oncogenic gain of functions. Different aspects of these tumour-promoting functions are caused by repression of the transcriptional activity of p53 family members p63 and p73. A subset of frequently occurring p53 mutations results in thermodynamic destabilisation of the DNA-binding domain (DBD) rendering this domain highly unstable. These conformational mutants (such as p53R175H) have been suggested to directly bind to p63 and p73 via a co-aggregation mechanism mediated by their DBDs. Although the DBDs of p63 and p73 are in fact not sufficient for the interaction as shown previously, we demonstrate here that the transactivation inhibitory (TI) domains within the α-isoform-specific C termini of p63 and p73 are essential for binding to p53R175H. Hence, the closed dimeric conformation of inactive TAp63α that renders the TI domain inaccessible prevents efficient interaction. We further show that binding to p53R175H correlates with an intrinsic aggregation propensity of the tetrameric α-isoforms conferred by an openly accessible TI domain again supporting interaction via a co-aggregation mechanism.

Members of the p53 tumor-suppressor family are expressed as multiple isoforms. Isoforms with an N-terminal transactivation domain are transcriptionally active, while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ΔNp63α inhibits the tumor-suppressor function of TAp73β. This can in principle be due to blocking of the promoter or by direct interaction between both proteins. p63 and p73 can heterooligomerize through their tetramerization domains and a hetero-tetramer consisting of two p63 and two p73 molecules is thermodynamically more stable than both homo-tetramers. Here we show that cells expressing both p63 and p73 exist in mouse epidermis and hair follicle and that hetero-tetramer complexes can be detected by immunoprecipitation in differentiating keratinocytes. Through structure determination of the hetero-tetramer, we reveal why this hetero-tetramer is the thermodynamically preferred species. We have created mutants that exclusively form either hetero-tetramers or homo-tetramers, allowing to investigate the function of these p63/p73 hetero-tetramers. Using these tools, we show that inhibition of TAp73β in squamous cell carcinomas is due to promoter squelching and not direct interaction.

SignificanceThe p63 gene encodes a master regulator of epidermal development and function. Specific mutations in p63 are causative of a life-threatening disorder mainly characterized by severe skin erosions and cleft palate. Little is known about the mechanisms underlying disease pathology and possible treatments. Based on biochemical studies, genetic mouse models, and functional assays, we demonstrate that these mutations cause p63 protein misfolding and aggregation. Protein aggregation lead to reduced DNA binding and impaired transcriptional activity. Importantly, genetic modifications of p63 that abolish aggregation of the mutant proteins rescue its function, revealing that ankyloblepharon-ectodermal defects-cleft lip/palate syndrome is a protein aggregation disorder and opening avenues for therapeutic intervention.

Germ cells are totipotent and, in principle, immortal as they are the source for new germ cells in each generation. This very special role requires tight quality control systems. The p53 protein family constitutes one of the most important quality surveillance systems in cells. Whereas p53 has become famous for its role as the guardian of the genome in its function as the most important somatic tumor suppressor, p63 has been nicknamed 'guardian of the female germ line'. p63 is strongly expressed in resting oocytes and responsible for eliminating those that carry DNA double-strand breaks. The third family member, p73, acts later during oocyte and embryo development by ensuring correct assembly of the spindle assembly checkpoint. In addition to its role in the female germ line, p73 regulates cell-cell contacts between developing sperm cells and supporting somatic cells in the male germ line. Here, we review the involvement of the p53 protein family in the development of germ cells with a focus on quality control in the female germ line and discuss medical implications for cancer patients.

Post-translational modifications are essential for regulating the transcription factor p53 which binds DNA in a highly cooperative manner to control expression of a plethora of tumor suppressive programs. Here we show at the biochemical, cellular, and organismal level that the cooperative nature of DNA binding is reduced by phosphorylation of highly conserved serine residues (human S183/S185, mouse S180) in the DNA binding domain. To explore the role of this inhibitory phosphorylation in vivo, new phosphorylation-deficient p53-S180A knock-in mice were generated.ChIP-seq and RNA-seq studies of S180A knock-in cells demonstrated enhanced DNA binding and increased target gene expression. In vivo, this translated into a tissuespecific vulnerability of the bone marrow that caused depletion of hematopoietic stem cells and impaired proper regeneration of hematopoiesis after DNA damage. Median lifespan was significantly reduced by 20% from 709 days in wild-type to only 568 days in S180A littermates. Importantly, lifespan was reduced by a loss of general fitness and increased susceptibility to age-related diseases, not by increased cancer incidence as often seen in other p53 mutant mouse models. For example, S180A knock-in mice showed markedly reduced spontaneous tumorigenesis and increased resistance to Myc-driven lymphoma and Eml4-Alk-driven lung cancer. Preventing phosphorylation of S183/S185 in human cells boosted p53 activity and allowed tumor cells to be killed more efficiently. Together out data identify p53 DNA binding domain phosphorylation as a druggable mechanism that balances tumorigenesis and aging.Significance: Findings demonstrate that p53 tumor suppressor activity is reduced by DNA binding domain phosphorylation to prevent aging and identify this phosphorylation as a potential target for cancer therapy.Research.

The transcription factor p63 mediates distinct cellular responses, primarily regulating epithelial and oocyte biology. In addition to the two amino terminal isoforms, TAp63 and ΔNp63, the 3’-end of p63 mRNA undergoes tissue-specific alternative splicing that leads to several isoforms, including p63α, p63β and p63γ. To investigate in vivo how the different isoforms fulfil distinct functions at the cellular and developmental levels, we developed a mouse model replacing the p63α with p63β by deletion of exon 13 in the Trp63 gene. Here, we report that whereas in two organs physiologically expressing p63α, such as thymus and skin, no abnormalities are detected, total infertility is evident in heterozygous female mice. A sharp reduction in the number of primary oocytes during the first week after birth occurs as a consequence of the enhanced expression of the pro-apoptotic transcriptional targets Puma and Noxa by the tetrameric, constitutively active, TAp63β isoform. Hence, these mice show a condition of ovary dysfunction, resembling human primary ovary insufficiency. Our results show that the p63 C-terminus is essential in TAp63α-expressing primary oocytes to control cell death in vivo, expanding the current understanding of human primary ovarian insufficiency.

The p53 homolog p63 plays important roles in development of epithelial tissues and quality control in germ cells. These two functions are executed by two distinct isoforms of p63. They are created by different promotors resulting in isoforms having either an N-terminal transactivation domain (TAp63) or a truncated form (ΔNp63). In addition to these two N-terminal isoforms a third one with an even longer N-terminus, named TA*p63, has been found. A fourth N-terminal isoform, GTAp63, that closely resembles TA*p63 was discovered in male germ cells where it is involved in genetic quality control. Here, we characterize TA*p63α and GTAp63α and show that their N-terminal extensions stabilize the closed and only dimeric conformation adopted by the shorter TAp63α protein. Both proteins can be activated by the two kinases Chk2 and CK1 resulting in the open tetrameric state. In this conformation, the N-terminal extension acts as an additional transactivation domain enhancing transcriptional activity. Through this mechanism, the difference in transcriptional activity between the repressed and the active state of the protein gets enhanced relative to TAp63α. Finally, we show by mass spectrometry that TA*p63α is expressed in the breast cancer cell line Sum159 at the protein level together with mutant p53. Upon doxorubicin treatment, TA*p63α gets activated, providing a potential new tool to fight cancer.