© 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev158360. doi:10.1242/dev.158360 PRIMER p53: emerging roles in stem cells, development and beyond Abhinav K. Jain*,‡ and Michelle Craig Barton*,‡ ABSTRACT creation of elegant mouse models that expressed mutated forms of Most human cancers harbor mutations in the gene encoding p53. As p53, with or without wild-type p53, revealed that p53 functions at a result, research on p53 in the past few decades has focused multiple stages of embryonic development (Van Nostrand et al., primarily on its role as a tumor suppressor. One consequence of this 2014), as well as in aging (Tyner et al., 2002b). These mice also focus is that the functions of p53 in development have largely been provided models of human genetic diseases and of pathologies ignored. However, recent advances, such as the genomic profiling of associated with defective ribosome biogenesis (McGowan et al., embryonic stem cells, have uncovered the significance and 2008). Together, these studies showed that normal differentiation, mechanisms of p53 functions in mammalian cell differentiation and development and aging require p53 levels to be precisely regulated development. As we review here, these recent findings reveal roles in a spatial and temporal manner. that complement the well-established roles for p53 in tumor Despite these early findings, the precise roles of p53 in suppression. differentiation and development have remained relatively under- studied. However, recent genome-wide profiling studies of KEY WORDS: Development, Differentiation, Embryonic stem cells, embryonic stem cells (ESCs) and adult stem cell populations, lncRNA, p53 together with a more in-depth analysis of the developmental defects in mice devoid of Trp53 (Clarke et al., 1993; Donehower et al., 1992; Lowe et al., 1993), have revealed that p53 functions appear to Introduction be intertwined with stem cell biology and differentiation in the soma The gene TP53 (Trp53 in mice), which encodes the transcription of higher organisms. Here, we review these studies, providing an factor p53, is the most frequently mutated tumor suppressor gene in overview of the modes of action of p53 and its function in human cancers (Bouaoun et al., 2016; Vousden and Prives, 2009). development and stem cells, and highlighting how the Since its discovery more than three decades ago, the molecular developmental roles of p53 relate to its well-known functions in mechanisms involved in the selection and execution of the many tumor suppression. functions of p53, as well as how they culminate in safeguarding genomic stability and suppressing tumor development, continue to An overview of the p53 family unfold. Loss of p53 transcriptional activity, by mutations in TP53 or p53 functions as a regulatory node. It receives signals, which are the activation of pathways that repress p53, are major contributing modulated and relayed in a cell- and context-dependent manner, to factors to malignant transformation. p53 safeguards the genome by direct a variety of downstream outcomes, including cell cycle arrest, restricting chromosomal instability through its ability to eliminate apoptosis, senescence, metabolic regulation and other responses cells at risk of aberrant mitoses (Eischen, 2016). Accordingly, that promote the repair and survival or death and elimination of numerous in vivo and in vitro studies have revealed that the loss of abnormal cells (Vousden and Prives, 2009). Adding complexity to p53 function both facilitates the accumulation and permits the the p53 regulatory network are the potential modulatory roles survival of aneuploid cells. Genomic instability fueled by p53 loss played by other p53 family members, p63 and p73, that are found in also leads to the acquisition of additional cancer driver events with mammals. TP53, TP63 and TP73 arose from a common ancestral the potential to accelerate transformation, metastasis and drug gene, first detected in the evolution of modern-day anemones to resistance (Eischen, 2016). protect the germline from genomic instability (Belyi et al., 2010; In normal cells, p53 expression levels are low, and an initial Yang et al., 2002). TP53 of higher eukaryotes diverged from TP63/ response to stress-induced signaling to p53 is disruption of the TP73 before the appearance of bony fishes (Lane et al., 2011) and activity of E3-ubiquitin ligases, such as MDM2, that maintain low acquired tumor-suppressive activities not shared by TP63 and TP73, levels of p53 by ubiquitylation and protein degradation (recently both of which display clear involvement in embryonic development reviewed by Pant and Lozano, 2014). The consequences of (reviewed by Belyi et al., 2010). All p53 family members have a unchecked p53 activity during embryonic differentiation, and conserved protein domain structure (Fig. 1) that includes: an N- support for tight regulation of p53 during development, were terminal transactivation (TA) domain (Lin et al., 1994); a proline- illustrated by the early embryonic lethality of Mdm2−/− mice at rich (PR) region, which is implicated in apoptosis and protein- implantation, a phenotype that is rescued by deletion of Trp53 protein interactions (Walker and Levine, 1996); a DNA-binding (de Oca Luna et al., 1995; Jones et al., 1995). The subsequent domain (DBD) that recognizes a core sequence motif of 10-base pairs (PuPuPuCA/TA/TPyPyPy, where Pu=purine, Department of Epigenetics and Molecular Carcinogenesis, Center for Stem Cell Py=pyrimidine), repeated with varied nucleotide spacing within and Development Biology, Center for Cancer Epigenetics, The University of Texas p53-regulatory elements of genes (El-Deiry et al., 1992); and an MD Anderson UT Health Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. oligomerization domain (OD) that mediates p53 tetramer formation *These authors contributed equally to this work (Jeffrey et al., 1995). p63 and p73 have an additional sterile α motif ‡ (SAM), which is involved in protein-protein interactions. Authors for correspondence ([email protected]; [email protected]) Further adding to the complexity, it has been shown that p53 A.K.J., 0000-0003-3268-514X; M.C.B., 0000-0002-4042-1374 family members exist as various isoforms. The alternative splicing DEVELOPMENT 1 PRIMER Development (2018) 145, dev158360. doi:10.1242/dev.158360 p53 N TA PR DBD OD C dependent profile of tumors at 3-5 months of age, was surprising and supported a limited role for p53 in stem cell differentiation and Δ40p53 N PR DBD OD C development (Bieging et al., 2014; Jacks et al., 1994). However, a more-detailed analysis reveals that a considerable fraction of female Δ133p53 N DBD OD C Trp53−/− embryos exhibit failure in neuronal tube closure, leading to exencephaly in 23% of mutant embryos on a 129/Ola background, or cranio-facial abnormalities, including ocular p63 N TA PR DBD OD SAM C abnormalities and defects in upper incisor tooth formation (Armstrong et al., 1995; Kaufman et al., 1997; reviewed by Shin ΔNp63 N PR DBD OD SAM C et al., 2013) (Table 1). In addition, p53 deletion in C57BL/6J background mice results in a lower than expected number of p73 N TA PR DBD OD SAM C surviving homozygotes (14.3%) and these animals suffer from severely abnormal lung architecture, cleft palate (Tateossian et al., ΔNp73 N PR DBD OD SAM C 2015), craniofacial defects in skeletal, neuronal and muscle tissues (Rinon et al., 2011), and a spectrum of congenital abnormalities in Fig. 1. Domain architecture of p53 family proteins. The major functional the urinary tract and kidney (Saifudeen et al., 2009). Both sexes of domains of p53 family proteins are shown, including the N-terminal Trp53+/− and Trp53−/− mice show significant dwarfism or under- transactivation domains (TA), the proline-rich domain (PR), the central development (Baatout et al., 2002), and Trp53−/− female mice that sequence-specific DNA-binding domain (DBD) and the oligomerization live to adulthood exhibit low fecundity due to loss of p53-dependent domain (OD). The overall structures of p63 and p73 are similar to that of p53; however, some isoforms of these p53-related proteins also contain a expression of Lif1 (Hu et al., 2007). Knock-in mouse strains that 25,26,53,54 C-terminal sterile α-motif (SAM) domain. The genes encoding p53 family express a transcriptionally dead variant of p53 (p53 ) along proteins, Trp53, Trp63 and Trp73, are often transcribed from alternate with a wild-type Trp53 allele suffer late-gestational lethality promoters, generating N-terminal truncated isoforms (e.g. Δ40p53, ΔNp63 and associated with phenotypes consistent with the human CHARGE ΔNp73) that lack the TA domain and can exert dominant-negative effects. An syndrome (Van Nostrand et al., 2014). Thus, p53 functions clearly internal promoter is also found in intron 4 of TP53 and results in an N-terminal- impinge on normal mouse development and, when perturbed, give truncated isoform of p53 (Δ133p53) that is devoid of both the TA and PR domains. rise to distinct phenotypes, although these are perhaps not as dramatic as expected given the roles of p53 in tumorigenesis. A role for p53 in development has also been demonstrated in of the C-terminal exons of TP63 and TP73 results in at least three studies using other model organisms. For example, Xenopus laevis isoforms of TP63 (α, β, γ) and at least seven isoforms of TP73(α, β, embryos that lack p53 expression have severe gastrulation defects, γ, δ, ε, ζ, η) (Bénard et al., 2003; Bourdon et al., 2005). A conserved in sharp contrast to the mostly normal early stages of development feature of the p53 family is the presence of potential transcription of Trp53-null mice (Wallingford et al., 1997). This species-specific start sites from intronic alternative promoters that generate N- difference may be due to lack of the p53 mammalian family terminal truncated isoforms (ΔNp53 or Δ40p53, ΔNp63 and homologues, p63 and p73, in Xenopus laevis (Stiewe, 2007).