P53: Emerging Roles in Stem Cells, Development and Beyond Abhinav K

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

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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). ΔNp73), which can exert dominant-negative effects on p53, p63 Furthermore, p53-mutant zebrafish have abnormal gut and neuronal and p73 (Bénard et al., 2003; Courtois et al., 2002). In addition, an development, whereas p53 inhibition in the salamander prevents internal promoter in intron 4 of TP53 results in an N-terminal limb formation; in planaria (which lack p63/p73), p53 loss disrupts truncated isoform of p53 (Δ133p53) that is devoid of the stem cell-dependent regeneration (reviewed by Levine and Berger, transactivation and proline-rich domains (Bourdon et al., 2005). 2017). As mentioned above, p53, p63 and p73 exhibit similarity N-terminal variants of TP53 are also expressed in a cell-type with regard to their amino acid sequence and domain structure specific manner and can activate or repress the transactivation of (Fig. 1), and possess the ability to bind the same consensus DNA- specific p53-target genes (Engelmann and Putzer, 2014). For binding sites to varying degrees at overlapping sets of target genes example, Δ40p53 is highly expressed in mouse ESCs (mESCs), as (Levrero et al., 2000). Thus, in mammals, p63 and p73 may largely the major isoform during early stages of mouse embryogenesis, and compensate for lack of p53 during development. Indeed, p53 wild- haploinsufficiency of Δ40p53 causes loss of pluripotency and type mice that are null for either Trp63 or Trp73 have much more acquisition of a more somatic cell cycle (Ungewitter and Scrable, severe developmental phenotypes compared with Trp53-null mice 2010). These truncated isoforms of the p53 family have been (Table 1). Mice null for Trp63 are born alive but display severe detected in late-stage tumors and metastases, in correlation with developmental defects, such as absent or truncated limbs, due to the cancer cell survival and tumor growth (reviewed by Wei et al., aberrant relaying of signals at the apical ectodermal ridge of the limb 2012). Isoform-specific functions of p53 family have been (Mills et al., 1999; Yang et al., 1999). Trp73-null mice are viable at delineated using elegant mouse models (Table 1); however, the birth but exhibit runting and a high mortality rate within the first specific or interactive contributions of p53, p63 or p73 family 2 months (Yang et al., 2000). Together, these results from model members and their respective isoforms to malignant transformation organisms that lack multiple p53 isoforms or p53 family members remain elusive, adding to the complexity of p53-mediated tumor support the idea that p53 family members partially compensate for suppression. each other and have isoform-specific and shared target genes (Danilova et al., 2008; Levine et al., 2011). However, using The Trp53-null mouse compound knockout mouse models, it was recently reported that the The major phenotype of Trp53-null mice is tumor development and combined loss of p53 and p63 in mouse embryos does not appear to resilience to radiation-induced apoptosis (Clarke et al., 1993; significantly compromise mouse development beyond simple p63 Donehower et al., 1992; Lowe et al., 1993). Given its importance in or p53 deficiency, and that Trp63−/−; Trp73−/− embryos show no tumor suppression, the ability of the Trp53-null mouse to gastrulate, dramatic developmental defects beyond those observed in single develop and apparently thrive, until succumbing to a strain- knockout embryos. These observations suggest that p53 family DEVELOPMENT

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Table 1. Developmental roles of p53 protein family members Mouse mutant Mutation Development Mouse phenotypes References p53−/− Absence of both copies of Trp53 in 129 ± Susceptibility to cancer Armstrong et al. (1995); Donehower background mice Exencephaly et al. (1992); Hu et al. (2007); Rotter Implantation defects et al. (1993) Giant-cell degenerative syndrome due to multinucleated giant cells in testes p53−/− Absence of both copies of Trp53 in C57BL/6 ± Susceptibility to cancer Armstrong et al. (1995); Hu et al. background mice Exencephaly (2007); Rinon et al. (2011); Sah Implantation defects et al. (1995); Saifudeen et al. Renal hypoplasia (2009); Tateossian et al. (2015) Cleft palate Abnormal lung architecture Craniofacial defects in skeletal, neuronal and muscle tissues p53(25,26,53,54)/+ Single, heterozygous mutant alleles that ++ Late-gestational embryonic Van Nostrand et al. (2014) encode a transcriptionally inactive variant of lethality due to CHARGE p53, in which residues 25, 26, 53 and 54 in syndrome the TA domain are mutated TAp63−/− Homozygous null Trp63 mutation +++ Absent or defective limbs Mills et al. (1999); Yang et al. (1999) Absent hair follicles teeth and mammary glands Impaired skin development p63−/−;ΔNp63 Transgenic expression of ΔNTrp63 under the ± Epidermal development Candi et al. (2006) K5 promoter in Trp63−/− background ΔNp63gfp/gfp ΔNTrp63 knock-in mice, in which ΔNTrp63 +++ Post natal lethality Romano et al. (2012) specific exon is replaced by GFP Truncated limbs Craniofacial malformations Lack of mature epidermis TAp73−/− Homozygous null Trp73 mutation +++ Runting and high mortality Yang et al. (2000) Neural and inflammatory defects ΔNp73−/− Homozygous deletion Trp73 mutation, in which +++ Neural defects Wilhelm et al. (2010) exon 3 is missing. ΔN, N-terminal truncated; TA domain, transcription activation domain; CHARGE, coloboma of the eye, heart defects, atresia of the choanae, retardation of growth and development, and ear abnormalities and deafness. ±, ++ and +++ indicate the severity of major phenotypes observed in mutant mouse. members may play redundant roles in specific development consequences of p53 activation to safeguard our genome. An ever- processes (Van Nostrand et al., 2017). This species and isoform growing body of evidence suggests that, in addition to regulating specific disparity in p53 activities is most likely due to the complex cell cycle arrest and apoptosis, p53 controls ‘non-canonical’ layers of p53 modulation and the array of biological processes programs such as autophagy, metabolism, repression of regulated by p53. pluripotency and cellular plasticity, and ferroptosis, all of which contribute to its tumor suppressor functions (Fig. 2). Consistent with p53: its roles, regulation and molecular mechanisms of this notion, mice deficient for p53 target genes responsible for cell action cycle arrest and apoptosis, such as p21 (Cdkn1a), Puma (Bbc3) and In response to a variety of cellular stresses, including DNA damage Noxa (Pmaip1) (i.e. p21−/−Puma−/−Noxa−/− mice) do not develop and replication stress often produced by deregulated oncogenes, p53 lymphomas or other malignancies, as observed in Trp53-null mice, protein is stabilized. Through its DNA-binding ability, p53 governs suggesting that p53-mediated cell cycle arrest and apoptosis are not a complex anti-proliferative transcriptional program, corresponding sufficient for tumor suppression (Valente et al., 2013). to an array of biological responses (Fig. 2). Mechanisms leading to This diversity in p53 functions depends on several factors, the stabilization and activation of p53 are mostly stimulus specific. including cell or tissue type, epigenetic state, differentiation state, DNA-damage promotes post-translational modifications (PTMs) on stress conditions, collaborating environment signals, specific-PTMs p53, such as phosphorylation, acetylation or methylation (Dai and on p53 and associated transcription co-regulatory factors that dictate Gu, 2010), blocking MDM2-mediated degradation, whereas the choice of target genes. Moreover, the crosstalk between p53- signaling as a result of oncogenic stress activates the ARF tumor PTMs also suggests that one PTM may enhance the acquisition of suppressor to inhibit MDM2 (Zhang et al., 1998). The tumor another, unlocking additional layers of p53 stability and biasing p53 suppressor activities of p53 are largely attributed to its ability to binding to DNA on selected target genes. It is fair to speculate that promote cell cycle arrest and apoptosis depending on cell type and the tumor suppressive activities of p53 are not limited to DNA- stimulus, a context specificity not completely understood. However, damage response in differentiated cells. Consistent with this notion, this historic view of the effects of widespread TP53 mutations in based on recent genome-wide analyses comparing p53-DNA tumors is changing with recent revelations of broadly diverse associations with gene expression profiles, a better picture of the DEVELOPMENT

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Fig. 2. Diverse p53-regulated pathways Somatic cells likely impinge upon a common outcome of tumor suppression. p53 controls common - Cell death and distinct biological processes in somatic - Proliferative arrest (top) and stem (bottom) cells. p53-regulated Ferroptosis Autophagy biological processes in blue boxes are SLC7A11 - Repression of oncogenic signaling (e.g. ) (e.g. ATG7) specific to somatic cells, those in red boxes - Dampening of metabolic reprogramming are specific to stem cells and those in white boxes are common to both cell types. The Inhibition of Cell cycle Inflammation outcomes of these activities in somatic and Apoptosis Metabolism DNA repair mobility and arrest and/or TME stem cells are indicated. (e.g. PUMA) (e.g. GLUT1) (e.g. ATM) invasiveness (e.g. CDKN1A) (e.g. THBS1) (e.g. miR-200c) Stem cells Repression of Promotion of pluripotency differentiation - Restraint of stemness (e.g. Nanog, (e.g. HOXA1) - Block of reprogramming LncPRESS1) - Maintenance of homeostasis

functions of p53 in somatic cells versus stem cells is now emerging (reviewed by Krizhanovsky and Lowe, 2009). As the generation of (Fig. 2): in somatic cells under conditions of stress, p53 regulates a iPSCs and the malignant transformation of somatic cells share many plethora of genes that lead to a variety of cellular outcomes, common characteristics, such as unlimited proliferation, similar including cell cycle arrest and apoptosis; conversely, p53 in stem metabolic status and transcriptional activity of pluripotency factors cells regulates pathways that target the pluripotency network Oct4 and Myc (Semi et al., 2013), it is not surprising that p53 acts as (described below), all of which likely contribute to tumor a barrier to reprogramming. However, the constant suppression of suppression. p53 during reprogramming allows widespread genomic instability to occur in the resulting iPSCs (Marion et al., 2009), whereas its p53 in cell differentiation and cell fate reprogramming reactivation during reprogramming disrupts the formation of iPSCs The possibility that p53 functions in early development and cell and induces their differentiation once formed (Yi et al., 2012). differentiation arose from the discovery that, unlike in somatic cells, Several pathways have been implicated in the ability of p53 to p53 is expressed at relatively high levels in all cells of day 8.5 post counteract the reprogramming of somatic cells. These include the coitum (p.c.) and day 10.5 p.c. mouse embryos, with expression p53 gene targets miR-34a, which represses several pluripotency declining in terminally differentiated cells (Schmid et al., 1991). genes [including Sox2, Myc and Nanog (Choi et al., 2011), and Multiple studies have since shown that p53 is implicated in cell CDKN1A, which attenuates cell division (Hanna et al., 2009). In differentiation but that cellular context plays a major and, as yet, not addition, p53 restricts mesenchymal-to-epithelial transition (MET) fully defined role in this p53 function. For example, p53 negatively during early reprogramming, which is primarily mediated by the regulates the proliferation and self-renewal of neural stem cells and ability of p53 to inhibit the activation of specific epithelial genes hematopoietic stem cells to maintain their quiescent state (Liu et al., (Brosh et al., 2013). Collectively, these studies suggest that p53 2009; Meletis et al., 2006), whereas, under the influence of specific governs the transition between cell states and limits the ability of hormonal or chemical inducers, p53 promotes the differentiation of somatic cells to undergo epigenetic reprogramming into iPSCs to both mouse and human ESCs (Akdemir et al., 2014; Jain et al., play a direct role in regulating cellular plasticity. 2012, 2016; Li et al., 2012; Lin et al., 2005) (Fig. 3). The transcriptional activity of p53 is also crucial for regulating the status The multiple functions of p53 in pluripotent stem cells of both ESCs and adult stem cells (discussed in detail below), ESCs, which are derived from the inner cell mass of mouse facilitating specific differentiation programs while inhibiting others embryos, exhibit self-renewal, an unlimited potential to proliferate (Aylon and Oren, 2016; Spike and Wahl, 2011). ESCs possess in vitro and pluripotency, the capacity to develop into all the cell robust mechanisms to preserve their genomic integrity and to avoid types of the embryo proper (Thomson et al., 1998). Several core the propagation of genetic aberrations to their descendent somatic transcription factors (including Oct4, Sox2, Nanog and Klf4) act in cells (Hong et al., 2007). Given that pluripotent and self-renewing an intricate gene regulatory circuitry that maintains the pluripotent ESCs share some but not all of the cellular and molecular status of ESCs by regulating specific transcriptional programs phenotypes of aggressive, p53-mutant cancers [e.g. an ESC-like (Boyer et al., 2005; Jaenisch and Young, 2008). Deregulation of the gene signature is observed in p53 mutant breast cancer (Mizuno core pluripotency network, coupled with alterations to the et al., 2010)], p53 might function to promote differentiation epigenetic status of ESCs, then contributes to their transitioning pathways of ESCs via mechanisms that likewise safeguard from pluripotency to differentiation. In recent years, a number of genomic stability and DNA fidelity to prevent cancer development. studies have shown that p53 plays multiple functions, and hence Numerous studies have reported that inactivation of p53, or must be tightly regulated, in regulating the pluripotent state and the disruption of the pathways that activate p53, can increase the transition to differentiation in both ESCs and iPSCs. efficiency with which mature somatic cells can be reprogrammed to Several early studies have reported that a measurable decrease in pluripotency to generate induced pluripotent stem cells (iPSCs) p53 RNA and protein levels occurs during mESC differentiation (Hong et al., 2009; Kawamura et al., 2009; Marion et al., 2009; and mouse development in vivo (Lin et al., 2005; Rogel et al., 1985; Utikal et al., 2009). These findings further underscore the ability of Sabapathy et al., 1997). Furthermore, the re-expression of p53 in p53 to prevent ‘backsliding’ or the dedifferentiation of somatic cells undifferentiated Trp53-null mESCs drives them towards a more DEVELOPMENT

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Human Mouse Activation of differentiation SIRT1 Development associated Aurora A (e.g. HOX, LHX, genes (e.g. Gata4, Sox17) DLX and PAX)

MDM2 MDM2 p53 Lineage-specific lncRNAs p53 TRIM24 (e.g. HOTAIRM1) TRIM24

Retinoic Mesoendodermal genes* DNA Retinoic acid acid CBP/p300 (e.g. Wnt3 and Fzd1) damage CDK

p53 Cell cycle arrest p53 (e.g. p21)

Repression of pluripotency miRNAs (mir-34a, mir-145) Stem cell factors (e.g. Oct4, Sox2, Nanog) Pluripotency- specific lncRNAs (e.g. LncPRESS1, TUNA)

Key Acetylation Phosphorylation Inhibition Activation p53 modification

Fig. 3. Functions of p53 in the differentiation of human and mouse ESCs. A schematic of p53 signaling and functions in human (left) and mouse (right) ESCs is shown. In response to retinoic acid (RA) in human ESCs, and in response to either RA or DNA damage in mouse ESCs, p53 becomes stabilized by post-translational modifications (such as acetylation and phosphorylation). Aurora kinase-mediated phosphorylation and inactivation of p53 (at Ser212 and Ser312) is specific to mouse ESCs, whereas the inactivation of p53 in human ESCs is mediated by SIRT1, an NAD+-dependent deacetylase that deacetylates p53. By contrast, the RA-induced acetylation of p53 by CBP/p300 in human ESCs, and the phosphorylation of p53 by CDK in mouse ESCs, leads to p53 activation. Once stabilized, p53 directly transcriptionally activates its target genes, which encode a variety of developmental genes and transcription factors. In parallel, p53 either directly represses the expression of pluripotency genes (such as Nanog in mouse ESCs) or activates genes that encode non-coding RNAs (such as miRNAs and lncRNAs in human ESCs), which fine-tune the activity of p53 to achieve sustained repression of pluripotency in ESCs. All members of the p53 family (*) are involved in the activation of mesendodermal genes. HOTAIRM1, Hox transcript antisense RNA, myeloid-specific 1; LIF, leukemia inhibitory factor; LncPRESS1, p53-regulated and ESC-associated 1; TUNA, Tcl1 upstream neuron-associated. differentiated state (Komarov et al., 1999; Sabapathy et al., 1997). Ser312, resulting in the inactivation and inhibition of p53-directed One possible mechanism for this is the direct repression of Nanog ectodermal and mesodermal gene expression (Lee et al., 2012). This expression by p53, which is sufficient to drive mESCs towards phosphorylation of p53 impairs p53-induced ESC differentiation differentiation (Fig. 3) (Lin et al., 2005). Nanog is essential for and p53-mediated suppression of iPSC reprogramming. Thus, maintaining the self-renewal and pluripotency of ESCs, and its aurora kinase-p53 signaling regulates self-renewal, differentiation expression is controlled by members of the core ESC circuitry and somatic cell reprogramming (Fig. 3). The treatment of mESCs (including Oct4 and Sox2) (Silva et al., 2009; Young, 2011), which containing a humanized p53 allele knock-in (p53hki) with retinoic are displaced from the Nanog promoter by p53 during acid (RA) stabilizes p53 as a result of Ser315 phosphorylation by differentiation. In order to directly repress Nanog, p53 binds a cyclin-dependent kinase (CDK), resulting in differentiation. consensus regulatory element (p53RE) on the Nanog promoter and Accordingly, a S315A missense mutation into the endogenous recruits the mSin3a-HDAC complex to bring about histone p53hki allele in these mESCs results in loss of S315 phosphorylation deacetylation and chromatin repression (Lin et al., 2005). and abrogation of p53 transcriptional activity, including Nanog Recently, using an in-depth genome-wide analysis of p53 repression, during RA-induced ESC differentiation (Lin et al., chromatin-interactions and gene expression, it was shown that p53 2005). On the other hand, the DNA damage-induced both activates differentiation-associated genes and represses stem phosphorylation of Ser18 stabilizes and activates p53 to elicit its cell-specific genes in response to DNA damage in mESCs (Fig. 3) effect in antagonizing pluripotency and promoting differentiation of (Li et al., 2012). However, as discussed below, these p53-regulated mESCs (Li et al., 2012). Phosphorylation at Ser18 by ATM kinase is outcomes in mESCs are in contrast to those observed in human a prerequisite for p53 activation during DNA damage and cellular ESCs (hESCs), where p53-regulated chromatin interactions and protection. A knock-in mouse model of Trp53 harboring a mutant gene expression changes in response to DNA damage versus Ser18 that cannot be phosphorylated suffers from several differentiation are distinct. malignancies, including fibrosarcoma, leukemia, leiomyosarcoma One way in which p53 activity is held in check by iPSCs and ESCs and myxosarcoma, which are unusual in p53 mutant mice, is via post-translational modifications. These modifications include suggesting that the phosphorylation of p53 at Ser18 contributes to the ubiquitylation, acetylation, phosphorylation, methylation or tumor suppression in vivo (Armata et al., 2007). Together, these sumoylation of specific residues of p53 (Jain and Barton, 2010). For studies indicate that the post-translational modifications example, aurora A kinase phosphorylates mouse p53 at Ser212 and incorporated into p53 during the in vitro differentiation of ESCs DEVELOPMENT

5 PRIMER Development (2018) 145, dev158360. doi:10.1242/dev.158360 play an essential role in enforcing the tumor suppressive activities of recruiting UTX, a histone H3K27me3-specific demethylase, to the p53 in vivo. promoters of these developmental genes. This, along with Unlike mESCs, which express relatively high levels of (mostly alterations in the genomic profiles of H3K4me3 and H3K27me3 cytoplasmic) p53 protein (Han et al., 2008; Sabapathy et al., 1997), on p53-regulated gene targets, suggests that p53, by recruiting human ESCs have comparatively low levels of p53 due to its control specific epigenetic regulators, plays a role in modifying chromatin by the negative regulators, MDM2 and TRIM24, which ubiquitylate structure to activate a developmental transcriptional program. p53 and trigger its degradation, potentially acting together with Recently, a role for the p53-mediated regulation of long non- other E3 ligases (Jain et al., 2012; Setoguchi et al., 2016). Increasing coding RNAs (lncRNAs) during ESC differentiation and has also the levels of p53 in human ESCs, either by using a small molecule been uncovered. Non-coding RNA transcripts that are longer than inhibitor of MDM2 (Nutlin) (Maimets et al., 2008) or by stimulating 200 nucleotides, often poly-adenylated and devoid of evident open DNA damage (Qin et al., 2007), induces apoptosis and reading frames (ORFs) are called lncRNAs, and these RNAs are differentiation. It has also been shown that p53 protein in hESCs generally poorly conserved among species (Fatica and Bozzoni, is located in the nucleus but is inactive, owing to a lack of 2014). LncRNAs are involved in diverse biological processes and acetylation at lysine residue K373 (Jain et al., 2012). This reduced often interface with epigenetic machinery as effector molecules that acetylation of p53 in hESCs occurs as a result of the OCT4- fine-tune transcriptional regulation (Rinn, 2014). Their expression mediated transcriptional activation of SIRT1, an NAD+-dependent is highly cell- and tissue-specific and regulated by mechanisms that deacetylase that deacetylates p53 (Zhang et al., 2014). This inactive continue to be uncovered (Perry and Ulitsky, 2016). LncRNAs can form of p53 is vulnerable to degradation by its negative regulators function as ligands of proteins and can guide lncRNA-containing MDM2 and TRIM24. The induction of hESC differentiation by the RNA/protein complexes to specific RNA and DNA sites (Guttman addition of RA leads to the acetylation of K373 on p53 by the CBP/ and Rinn, 2012; Wang and Chang, 2011). Thus, lncRNAs can p300 histone acetyl transferases and to the dissociation of p53 from operate through different modes, as signals, scaffolds for protein- its negative regulators, resulting in its stabilization and activation protein interactions, molecular decoys or guides to target elements (Jain et al., 2012). In order to achieve sustained differentiation, in the genome (Wang and Chang, 2011). Recently, by integrating transcriptionally active p53 activates the transcription of CDKN1A genome-wide expression data with p53 chromatin-enrichment (p21), which impedes cellular proliferation, and two small non- profiles in hESCs undergoing differentiation, a high-confidence coding RNAs, miRNA-34a and miRNA-145, which negatively signature of 40 p53-regulated lncRNAs that are regulated by p53- regulate a set of transcription factors (Oct4, Sox2, Lin28a and Klf4) binding to their promoters during hESC differentiation was that favor stem cell maintenance (Fig. 3) (Jain et al., 2012). In line identified (Fatica and Bozzoni, 2014; Jain et al., 2016). This with these findings, it has been shown that the exogenous included differentiation-specific lncRNAs, such as HOTAIRM1, expression of p53 in hESCs induces their spontaneous and several pluripotency-specific transcripts that were named differentiation, while the expression of a DNA-binding-deficient lncPRESS (p53-regulated and ESC-associated) transcripts, the p53 mutant does not (Jain et al., 2012). Overall, these findings expression of which correlated with their histone modification indicate that p53 transcriptionally regulates hESC differentiation, in profiles and with p53 regulation (Fig. 3). LncRNA transcripts that part by activating both protein-coding and non-coding genes. are highly expressed in pluripotent hESCs and repressed by p53 Genome-wide comparisons of p53 chromatin interactions in during hESC differentiation play a modulatory but significant role mESCs and hESCs (via ChIPseq) show that many p53 target genes in stem-cell maintenance. For example, lncPRESS1, a human- are evolutionarily conserved, although their inductive signaling, specific lncRNA, acts as a ‘molecular decoy’ that sequesters SIRT6, developmental timing and dominant pathways may differ (Akdemir a NAD+-dependent deacetylase that specifically deacetylates et al., 2014). Recently, p53 ChIPseq and global gene expression H3K56ac and H3K9ac in the chromatin of ESCs (Jain et al., analyses of pluripotent, RA-treated and doxorubicin (adriamycin, 2016). Acetylation of H3K56 is linked to the core pluripotency Adr)-treated hESCs were performed to compare p53-transcriptional circuitry and to the transcriptional activation of pluripotency genes activity during differentiation and DNA damage (Akdemir et al., in hESCs (Xie et al., 2009). In addition, LncPRESS4, another p53- 2014). This analysis revealed that the RA-mediated transcriptional repressed lncRNA identified in hESCs (Jain et al., 2016), also outcomes of p53 activity are quite distinct from its stress-responsive known as TUNA (Tcl1 upstream neuron-associated or linc86023), is (DNA damage) regulation in hESCs. In particular, the results required for the maintenance of pluripotency (Lin et al., 2014). showed that p53 promotes hESC differentiation by activating the Thus, p53 in ESCs has multiple functions (summarized in Fig. 3): expression of developmental transcription factor genes that are (1) it represses pluripotency by transcriptionally activating non- involved in patterning, morphogenesis and organ development coding RNAs (both miRNAs and lncRNAs); (2) it promotes (Fig. 3). This activated cascade of transcription factors amplifies the differentiation by directly activating developmental genes and outcomes of p53 induction beyond the transient time period when transcription factors; and (3) it provides surveillance for secure, p53 protein is elevated (Jain et al., 2012). RA-induced, genome-stable differentiation (Hamilton and Brickman, 2017). differentiation-specific p53 gene targets in hESCs include those encoding homeodomain proteins such as HOX (homeobox), LHX p53 and its role in mesendoderm development (LIM homeobox), DLX (distal-less-like) and PAX (paired box) One issue concerning p53 and its family members in promoting (Gudas and Wagner, 2011; Hobert and Westphal, 2000), and ESC differentiation is whether p53 plays a role in specifying specific forkhead (Lehmann et al., 2003), SOX (Sry-related HMG particular germ layers, i.e. endoderm, mesoderm or ectoderm. box) (Schepers et al., 2002) and TBX (T-box) family members Indeed, although the gain- or loss-of-function of p53 in various (Showell et al., 2004). In addition, p53 targets CBX2 (chromobox) in vitro models support the notion that p53 regulates specific genes and CBX4, which are part of the polycomb group multi-protein in many differentiation processes, support is strongest for roles for PRC1-like transcription repression complex and are crucial for cell p53 in mesenchymal differentiation programs (reviewed by fate determination (Morey and Helin, 2010). One mechanism by Molchadsky et al., 2010). For example, the exogenous expression which p53 achieves differentiation-specific gene activation is by of p53 in an undifferentiated pre-B cell line induces differentiation DEVELOPMENT

6 PRIMER Development (2018) 145, dev158360. doi:10.1242/dev.158360 to B cells (Aloni-Grinstein et al., 1993). Likewise, p53 is essential How the physiological and developmental functions of p53 intersect for skeletal muscle differentiation and for the osteogenic with the cancer-associated phenotype of p53 loss is quite intriguing. re-programming of skeletal muscle-committed cells (Molchadsky Malignant transformation proceeds by evading terminal et al., 2008). In addition, monolayer cultures of p53-null mESCs, differentiation, and p53 loss is likely one route to abate this innate when subjected to LIF withdrawal, fail to undergo mesodermal barrier to tumorigenesis. Consistent with this notion, striking differentiation (Shigeta et al., 2013). Finally, by leveraging methods similarities between the gene expression signatures of aggressive that enable hESCs to be differentiated in the direction of specific breast cancer tumors that contain TP53 mutations and those of lineages (Gifford et al., 2013) specifically toward definitive pluripotent ESCs have been observed (Kim and Orkin, 2011; ectoderm, endoderm and mesoderm, it was shown that p53 Mizuno et al., 2010). In addition, it has been reported that mutations preferentially activates lineage-identity genes, including lncRNAs, of TP53 facilitate the expansion of hematopoietic stem cell (HSC) to promote a mesendodermal state, i.e. a bipotential state that can clones in otherwise healthy individuals, sometimes taking over the give rise to both mesoderm and endoderm (Jain et al., 2016). entire hematopoietic system and resulting in hematological Previous in vivo studies have established that synergy between malignancies (Xie et al., 2014), while the expansion of p53- Wnt and Nodal-related TGF-β signaling within the mouse primitive mutant HSC clones by genotoxic chemotherapy results in therapy- streak is involved in the formation of mesendoderm (Conlon et al., related acute myeloid leukemia (t-AML) (Wong et al., 2015). Loss 1991). The only direct link previously established between p53 and of p53 can also facilitate lineage switching as a mechanism of mesoderm induction was made in Xenopus laevis embryos: p53- resistance to anti-androgen therapy in prostate cancer (Mu et al., depleted Xenopus embryos fail to gastrulate and the overexpression 2017). Thus, by promoting cellular homeostasis and regulating of p53 induces mesoderm specification (Cordenonsi et al., 2007). proliferation, p53 activity can lead to tumor suppression. This study revealed that the regulation of mesoderm gene expression The regulation of differentiation or de-differentiation by p53 in Xenopus involves cooperation between TGFβ and p53 (reprogramming) by p53, either by employing its canonical signaling. In particular, it was shown that the RTK/Ras/MAPK functions of controlling cell cycle arrest and apoptosis or by (mitogen-activated protein kinase)-mediated phosphorylation of directly regulating various lineage-specific programs, challenges p53 during development promotes p53 interaction with TGFβ- the notion that p53 has no role in development. As aberrations in activated Smads to regulate a specific class of p53-dependent differentiation and de-differentiation programs can promote cell Nodal/Smad gene targets that play roles in mesoderm formation transformation, the abrogation of p53 activity, through deficiency or (Cordenonsi et al., 2007). However, the mechanism by which p53 mutation, might result in the accumulation of oncogenic events and/ regulates mesoderm specification in other contexts and organisms is or in the arrest of progenitor/stem cell differentiation, both of which unclear. are associated with tumor formation. Thus, by promoting directed How functional redundancy between p53 family members affects differentiation and development, and by regulating cellular state, lineage specific differentiation is also not fully understood, although p53 contributes to the repertoire of functions that protect healthy a recent study has provided some insight into this issue (Wang et al., cells and achieve tumor suppression. 2017). This work, which used a compound triple-knockout (TKO) of all three p53 family members in mouse and human ESCs, showed Conclusions that all three p53 family members are required for the induction of In recent years, a growing body of evidence, aided by the Nodal-responsive genes and for mesendoderm specification, and development of new technologies, has revealed that the functions that this requirement is conserved between mouse and human ESCs of p53 extend beyond cellular surveillance and apoptosis to include (Wang et al., 2017). Members of the p53 family control a gene the regulation of cellular homeostasis and metabolism (Gottlieb and regulatory network that integrates Wnt and TGFβ nodal inputs for Vousden, 2010), inflammation (Cooks et al., 2014), reproduction mesendoderm specification (Wang et al., 2017). This network is (Hu et al., 2007; Levine et al., 2011), ageing (Tyner et al., 2002a), composed of multiple layers of regulation: the p53 family activates regeneration (Pomerantz and Blau, 2013) and more (Vousden and Wnt signaling by directly activating Wnt3 and Fzd1 as cells exit Prives, 2009). The broad involvement of p53 in promoting from pluripotency, and Wnt3 and Fzd1 are absolutely required for programs of differentiation and in antagonizing the de- the induction of a class of Smad2/3-responsive, mesendoderm- differentiation of somatic cells relies on its fundamental role in specific genes (Fig. 3) (Wang et al., 2017). These studies using regulating the maintenance of the cellular state, leading to the recent compound knockout ESC models confirm that p53 family members renaming of p53 as the ‘guardian of homeostasis’ (Aylon and Oren, are functionally redundant with each other during mesendoderm 2016). Findings from numerous in vivo and in vitro studies now specification, and that this redundancy might also mask their roles in suggest that p53 likely plays a gatekeeper function to ensure high- gastrulation in single or double knockout mice. fidelity development. This ‘gate-keeping’ role, which is essential for normal development, connects to the better understood roles of Linking p53 roles in development and cancer p53 as a tumor suppressor. However, despite decades of studies on As we have highlighted above, a closer look at the consequences of the significance of p53 in tumor suppression, the identification of Trp53 loss indicate that p53 plays important roles in embryonic p53 mutations in a wide range of tumors and the determination of development. A growing body of evidence also now shows that p53 pathways that negatively regulate p53 in tumors, much remains to be has additional functions in regulating tissue homeostasis. For learned about the roles and regulation of p53. example, p53 restricts the self-renewal of various stem and Exploiting our knowledge of p53 pathways in translation to progenitor cells when subjected to oncogenic stress, probably by clinical applications also remains a challenge. Targeting proteins activating differentiation-inducing checkpoints (Tschaharganeh that negatively regulate p53 seems to be an ideal choice for drug et al., 2014; Wang et al., 2012; Zhao et al., 2010). The self- design. However, although many small molecules that disrupt the renewal capacity of stem cells and the existence of stem-like cells in interactions of p53 with its negative regulator MDM2 have been cancers (cancer stem cells) suggest that these cell types are likely designed (Vassilev et al., 2004), progress beyond the research bench candidates to initiate tumor formation and/or promote metastases. has been slow; clinical outcomes may require wild-type p53 to DEVELOPMENT

7 PRIMER Development (2018) 145, dev158360. doi:10.1242/dev.158360 respond to such inhibitors. Furthermore, although molecules that Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, target mutant p53, either by changing its confirmation to wild-type M. L. and Wyllie, A. H. (1993). Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 362, 849-852. p53 or promoting its degradation, show some promise (Parrales and Conlon, F. L., Barth, K. S. and Robertson, E. J. (1991). A novel retrovirally induced Iwakuma, 2015), none of these has yet been approved for the embryonic lethal mutation in the mouse: assessment of the developmental fate of treatment of cancer. It should also be noted that not all mutant forms embryonic stem cells homozygous for the 413.d proviral integration. Development of p53 are equivalent, hence each may require distinct approaches 111, 969-981. Cooks, T., Harris, C. C. and Oren, M. (2014). Caught in the cross fire: p53 in for targeting. Considering the complexities of p53 regulation and inflammation. Carcinogenesis 35, 1680-1690. the multiple biological outcomes of p53 activation and inactivation, Cordenonsi, M., Montagner, M., Adorno, M., Zacchigna, L., Martello, G., drug discovery to regulate this pathway thus remains complex. Mamidi, A., Soligo, S., Dupont, S. and Piccolo, S. (2007). Integration of TGF- Future work will undoubtedly reveal gene targets (both coding and beta and Ras/MAPK signaling through p53 phosphorylation. Science 315, 840-843. non-coding) and additional pathways that are controlled by p53 to Courtois, S., Verhaegh, G., North, S., Luciani, M.-G., Lassus, P., Hibner, U., fine-tune cellular protection. New technologies, together with an Oren, M. and Hainaut, P. (2002). DeltaN-p53, a natural isoform of p53 lacking the increased understanding of the complexities of p53 action in normal first transactivation domain, counteracts growth suppression by wild-type p53. developmental, homeostatic and pathological contexts, will Oncogene 21, 6722-6728. Dai, C. and Gu, W. (2010). p53 post-translational modification: deregulated in hopefully set the stage for clinical advances and drug discovery to tumorigenesis. Trends Mol. Med. 16, 528-536. combat p53-dependent cancers. Danilova, N., Sakamoto, K. M. and Lin, S. (2008). p53 family in development. Mech. Dev. 125, 919-931. Acknowledgements de Oca Luna, R. M., Wagner, D. S. and Lozano, G. (1995). Rescue of early We thank all current and former members of Barton laboratory for helpful embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, discussions. We regret not being able to cite all the work related to this Primer due to 206-208. space limitations. Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Jr, Butel, J. S. and Bradley, A. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215-221. Competing interests Eischen, C. M. (2016). Genome stability requires p53. Cold Spring Harb. Perspect. The authors declare no competing or financial interests. Med. 6, a026096. El-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W. and Vogelstein, B. References (1992). Definition of a consensus binding site for p53. Nat. Genet. 1, 45-49. Akdemir, K. C., Jain, A. K., Allton, K., Aronow, B., Xu, X., Cooney, A. J., Li, W. Engelmann, D. and Putzer, B. M. (2014). Emerging from the shade of p53 mutants: and Barton, M. C. (2014). Genome-wide profiling reveals stimulus-specific N-terminally truncated variants of the p53 family in EMT signaling and cancer functions of p53 during differentiation and DNA damage of human embryonic stem progression. Sci. Signal. 7,re9. cells. Nucleic Acids Res. 42, 205-223. Fatica, A. and Bozzoni, I. (2014). Long non-coding RNAs: new players in cell Aloni-Grinstein, R., Zan-Bar, I., Alboum, I., Goldfinger, N. and Rotter, V. (1993). differentiation and development. Nat. Rev. Genet. 15, 7-21. Wild type p53 functions as a control protein in the differentiation pathway of the B- Gifford, C. A., Ziller, M. J., Gu, H., Trapnell, C., Donaghey, J., Tsankov, A., cell lineage. Oncogene 8, 3297-3305. Shalek, A. K., Kelley, D. R., Shishkin, A. A., Issner, R. et al. (2013). Armata, H. L., Garlick, D. S. and Sluss, H. K. (2007). The ataxia telangiectasia- Transcriptional and epigenetic dynamics during specification of human embryonic mutated target site Ser18 is required for p53-mediated tumor suppression. Cancer stem cells. Cell 153, 1149-1163. Res. 67, 11696-11703. Gottlieb, E. and Vousden, K. H. (2010). p53 regulation of metabolic pathways. Cold Armstrong, J. F., Kaufman, M. H., Harrison, D. J. and Clarke, A. R. (1995). High- Spring Harb. Perspect. Biol. 2, a001040. frequency developmental abnormalities in p53-deficient mice. Curr. Biol. 5, Gudas, L. J. and Wagner, J. A. (2011). Retinoids regulate stem cell differentiation. 931-936. J. Cell. Physiol. 226, 322-330. Aylon, Y. and Oren, M. (2016). The paradox of p53: what, how, and why? Cold Guttman, M. and Rinn, J. L. (2012). Modular regulatory principles of large non- Spring Harb. Perspect. Med. 6. coding RNAs. Nature 482, 339-346. Baatout, S., Jacquet, P., Michaux, A., Buset, J., Vankerkom, J., Derradji, H., Hamilton, W. B. and Brickman, J. M. (2017). Surveillance for Secure Yan, J., von Suchodoletz, H., de Saint-Georges, L., Desaintes, C. et al. (2002). Differentiation. Cell Stem Cell 20, 3-5. Developmental abnormalities induced by X-irradiation in p53 deficient mice. Han, M.-K., Song, E.-K., Guo, Y., Ou, X., Mantel, C. and Broxmeyer, H. E. (2008). In Vivo 16, 215-221. SIRT1 regulates apoptosis and Nanog expression in mouse embryonic stem cells Belyi, V. A., Ak, P., Markert, E., Wang, H., Hu, W., Puzio-Kuter, A. and Levine, by controlling p53 subcellular localization. Cell Stem Cell 2, 241-251. A. J. (2010). The origins and evolution of the p53 family of genes. Cold Spring Hanna, J., Saha, K., Pando, B., van Zon, J., Lengner, C. J., Creyghton, M. P., van Harb. Perspect. Biol. 2, a001198. Oudenaarden, A. and Jaenisch, R. (2009). Direct cell reprogramming is a Bénard, J., Douc-Rasy, S. and Ahomadegbe, J.-C. (2003). TP53 family members stochastic process amenable to acceleration. Nature 462, 595-601. and human cancers. Hum. Mutat. 21, 182-191. Hobert, O. and Westphal, H. (2000). Functions of LIM-homeobox genes. Trends Bieging, K. T., Mello, S. S. and Attardi, L. D. (2014). Unravelling mechanisms of Genet. 16, 75-83. p53-mediated tumour suppression. Nat. Rev. Cancer 14, 359-370. Hong, Y., Cervantes, R. B., Tichy, E., Tischfield, J. A. and Stambrook, P. J. Bouaoun, L., Sonkin, D., Ardin, M., Hollstein, M., Byrnes, G., Zavadil, J. and (2007). Protecting genomic integrity in somatic cells and embryonic stem cells. Olivier, M. (2016). TP53 variations in human cancers: new lessons from the IARC Mutat. Res. 614, 48-55. TP53 database and genomics data. Hum. Mutat. 37, 865-876. Hong, H., Takahashi, K., Ichisaka, T., Aoi, T., Kanagawa, O., Nakagawa, M., Bourdon, J.-C., Fernandes, K., Murray-Zmijewski, F., Liu, G., Diot, A., Okita, K. and Yamanaka, S. (2009). Suppression of induced pluripotent stem cell Xirodimas, D. P., Saville, M. K. and Lane, D. P. (2005). p53 isoforms can generation by the p53-p21 pathway. Nature 460, 1132-1135. regulate p53 transcriptional activity. Genes Dev. 19, 2122-2137. Hu, W., Feng, Z., Teresky, A. K. and Levine, A. J. (2007). p53 regulates maternal Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P., reproduction through LIF. Nature 450, 721-724. Guenther, M. G., Kumar, R. M., Murray, H. L., Jenner, R. G. et al. (2005). Core Jacks, T., Remington, L., Williams, B. O., Schmitt, E. M., Halachmi, S., Bronson, transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, R. T. and Weinberg, R. A. (1994). Tumor spectrum analysis in p53-mutant mice. 947-956. Curr. Biol. 4, 1-7. Brosh, R., Assia-Alroy, Y., Molchadsky, A., Bornstein, C., Dekel, E., Madar, S., Jaenisch, R. and Young, R. (2008). Stem cells, the molecular circuitry of Shetzer, Y., Rivlin, N., Goldfinger, N., Sarig, R. et al. (2013). p53 counteracts pluripotency and nuclear reprogramming. Cell 132, 567-582. reprogramming by inhibiting mesenchymal-to-epithelial transition. Cell Death Jain, A. K. and Barton, M. C. (2010). Making sense of ubiquitin ligases that regulate Differ. 20, 312-320. p53. Cancer Biol. Ther. 10, 665-672. Candi, E., Rufini, A., Terrinoni, A., Dinsdale, D., Ranalli, M., Paradisi, A., De Jain, A. K., Allton, K., Iacovino, M., Mahen, E., Milczarek, R. J., Zwaka, T. P., Laurenzi, V., Spagnoli, L. G., Catani, M. V., Ramadan, S. et al. (2006). Kyba, M. and Barton, M. C. (2012). p53 regulates cell cycle and microRNAs to Differential roles of p63 isoforms in epidermal development: selective genetic promote differentiation of human embryonic stem cells. PLoS Biol. 10, e1001268. complementation in p63 null mice. Cell Death Differ. 13, 1037-1047. Jain, A. K., Xi, Y., McCarthy, R., Allton, K., Akdemir, K. C., Patel, L. R., Aronow, Choi, Y. J., Lin, C. P., Ho, J. J., He, X., Okada, N., Bu, P., Zhong, Y., Kim, S. Y., B., Lin, C., Li, W., Yang, L. et al. (2016). LncPRESS1 is a p53-regulated LncRNA Bennett, M. J., Chen, C. et al. (2011). miR-34 miRNAs provide a barrier for that safeguards pluripotency by disrupting SIRT6-mediated de-acetylation of

somatic cell reprogramming. Nat. Cell Biol. 13, 1353-1360. histone H3K56. Mol. Cell 64, 967-981. DEVELOPMENT

8 PRIMER Development (2018) 145, dev158360. doi:10.1242/dev.158360

Jeffrey, P. D., Gorina, S. and Pavletich, N. P. (1995). Crystal structure of the role in mesenchymal differentiation programs, in a cell fate dependent manner. tetramerization domain of the p53 tumor suppressor at 1.7angstroms. Science PLoS ONE 3, e3707. 267, 1498-1502. Molchadsky, A., Rivlin, N., Brosh, R., Rotter, V. and Sarig, R. (2010). p53 is Jones, S. N., Roe, A. E., Donehower, L. A. and Bradley, A. (1995). Rescue of balancing development, differentiation and de-differentiation to assure cancer embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, prevention. Carcinogenesis 31, 1501-1508. 206-208. Morey, L. and Helin, K. (2010). Polycomb group protein-mediated repression of Kaufman, M. H., Kaufman, D. B., Brune, R. M., Stark, M., Armstrong, J. F. and transcription. Trends Biochem. Sci. 35, 323-332. Clarke, A. R. (1997). Analysis of fused maxillary incisor dentition in p53-deficient Mu, P., Zhang, Z., Benelli, M., Karthaus, W. R., Hoover, E., Chen, C.-C., exencephalic mice. J. Anat. 191, 57-64. Wongvipat, J., Ku, S.-Y., Gao, D., Cao, Z. et al. (2017). SOX2 promotes lineage Kawamura, T., Suzuki, J., Wang, Y. V., Menendez, S., Morera, L. B., Raya, A., plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate Wahl, G. M. and Belmonte, J. C. I. (2009). Linking the p53 tumour suppressor cancer. Science 355, 84-88. pathway to somatic cell reprogramming. Nature 460, 1140-1144. Pant, V. and Lozano, G. (2014). Limiting the power of p53 through the ubiquitin Kim, J. and Orkin, S. H. (2011). Embryonic stem cell-specific signatures in cancer: proteasome pathway. Genes Dev. 28, 1739-1751. insights into genomic regulatory networks and implications for medicine. Genome Parrales, A. and Iwakuma, T. (2015). Targeting oncogenic mutant p53 for cancer Med. 3, 75. therapy. Front. Oncol. 5, 288. Komarov, P. G., Komarova, E. A., Kondratov, R. V., Christov-Tselkov, K., Coon, Perry, R. B.-T. and Ulitsky, I. (2016). The functions of long noncoding RNAs in J. S., Chernov, M. V. and Gudkov, A. V. (1999). A chemical inhibitor of p53 that development and stem cells. Development 143, 3882-3894. protects mice from the side effects of cancer therapy. Science 285, 1733-1737. Pomerantz, J. H. and Blau, H. M. (2013). Tumor suppressors: enhancers or Krizhanovsky, V. and Lowe, S. W. (2009). Stem cells: the promises and perils of suppressors of regeneration? Development 140, 2502-2512. p53. Nature 460, 1085-1086. Qin, H., Yu, T., Qing, T., Liu, Y., Zhao, Y., Cai, J., Li, J., Song, Z., Qu, X., Zhou, P. Lane, D. P., Madhumalar, A., Lee, A. P., Tay, B.-H., Verma, C., Brenner, S. and et al. (2007). Regulation of apoptosis and differentiation by p53 in human Venkatesh, B. (2011). Conservation of all three p53 family members and Mdm2 embryonic stem cells. J. Biol. Chem. 282, 5842-5852. and Mdm4 in the cartilaginous fish. Cell Cycle 10, 4272-4279. Rinn, J. L. (2014). lncRNAs: linking RNA to chromatin. Cold Spring Harb. Perspect. Lee, D.-F., Su, J., Ang, Y.-S., Carvajal-Vergara, X., Mulero-Navarro, S., Pereira, Biol. 6. C. F., Gingold, J., Wang, H.-L., Zhao, R., Sevilla, A. et al. (2012). Regulation of Rinon, A., Molchadsky, A., Nathan, E., Yovel, G., Rotter, V., Sarig, R. and embryonic and induced pluripotency by aurora kinase-p53 signaling. Cell Stem Tzahor, E. (2011). p53 coordinates cranial neural crest cell growth and epithelial- Cell 11, 179-194. mesenchymal transition/delamination processes. Development 138, 1827-1838. Lehmann, O. J., Sowden, J. C., Carlsson, P., Jordan, T. and Bhattacharya, S. S. Rogel, A., Popliker, M., Webb, C. G. and Oren, M. (1985). p53 cellular tumor (2003). Fox’s in development and disease. Trends Genet. 19, 339-344. antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors. Levine, A. J. and Berger, S. L. (2017). The interplay between epigenetic changes Mol. Cell. Biol. 5, 2851-2855. and the p53 protein in stem cells. Genes Dev. 31, 1195-1201. Romano, R.-A., Smalley, K., Magraw, C., Serna, V. A., Kurita, T., Raghavan, S. Levine, A. J., Tomasini, R., McKeon, F. D., Mak, T. W. and Melino, G. (2011). The and Sinha, S. (2012). DeltaNp63 knockout mice reveal its indispensable role as a p53 family: guardians of maternal reproduction. Nat. Rev. Mol. Cell Biol. 12, master regulator of epithelial development and differentiation. Development 139, 772-782. 259-265. Rotter, V., Schwartz, D., Almon, E., Goldfinger, N., Kapon, A., Meshorer, A., Levrero, M., De Laurenzi, V., Costanzo, A., Gong, J., Wang, J. Y. and Melino, G. Donehower, L. A. and Levine, A. J. (1993). Mice with reduced levels of p53 (2000). The p53/p63/p73 family of transcription factors: overlapping and distinct protein exhibit the testicular giant-cell degenerative syndrome. Proc. Natl. Acad. functions. J. Cell Sci. 113, 1661-1670. Sci. USA 90, 9075-9079. Li, M., He, Y., Dubois, W., Wu, X., Shi, J. and Huang, J. (2012). Distinct regulatory Sabapathy, K., Klemm, M., Jaenisch, R. and Wagner, E. F. (1997). Regulation of mechanisms and functions for p53-activated and p53-repressed DNA damage ES cell differentiation by functional and conformational modulation of p53. EMBO response genes in embryonic stem cells. Mol. Cell 46, 30-42. J. 16, 6217-6229. Lin, J., Chen, J., Elenbaas, B. and Levine, A. J. (1994). Several hydrophobic Sah, V. P., Attardi, L. D., Mulligan, G. J., Williams, B. O., Bronson, R. T. and amino acids in the p53 amino-terminal domain are required for transcriptional Jacks, T. (1995). A subset of p53-deficient embryos exhibit exencephaly. Nat. activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein. Genes Dev. Genet. 10, 175-180. 8, 1235-1246. Saifudeen, Z., Dipp, S., Stefkova, J., Yao, X., Lookabaugh, S. and El-Dahr, S. S. Lin, T., Chao, C., Saito, S., Mazur, S. J., Murphy, M. E., Appella, E. and Xu, Y. (2009). p53 regulates metanephric development. J. Am. Soc. Nephrol. 20, (2005). p53 induces differentiation of mouse embryonic stem cells by suppressing 2328-2337. Nanog expression. Nat. Cell Biol. 7, 165-171. Schepers, G. E., Teasdale, R. D. and Koopman, P. (2002). Twenty pairs of sox: Lin, N., Chang, K.-Y., Li, Z., Gates, K., Rana, Z. A., Dang, J., Zhang, D., Han, T., extent, homology, and nomenclature of the mouse and human sox transcription Yang, C.-S., Cunningham, T. J. et al. (2014). An evolutionarily conserved long factor gene families. Dev. Cell 3, 167-170. noncoding RNA TUNA controls pluripotency and neural lineage commitment. Mol. Schmid, P., Lorenz, A., Hameister, H. and Montenarh, M. (1991). Expression of Cell 53, 1005-1019. p53 during mouse embryogenesis. Development 113, 857-865. Liu, Y., Elf, S. E., Miyata, Y., Sashida, G., Huang, G., Di Giandomenico, S., Lee, Semi, K., Matsuda, Y., Ohnishi, K. and Yamada, Y. (2013). Cellular J. M., Deblasio, A., Menendez, S., Antipin, J. et al. (2009). p53 regulates reprogramming and cancer development. Int. J. Cancer 132, 1240-1248. hematopoietic stem cell quiescence. Cell Stem Cell 4, 37-48. Setoguchi, K., TeSlaa, T., Koehler, C. M. and Teitell, M. A. (2016). P53 regulates Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A. and Jacks, T. (1993). rapid apoptosis in human pluripotent stem cells. J. Mol. Biol. 428, 1465-1475. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362, Shigeta, M., Ohtsuka, S., Nishikawa-Torikai, S., Yamane, M., Fujii, S., 847-849. Murakami, K. and Niwa, H. (2013). Maintenance of pluripotency in mouse ES Maimets, T., Neganova, I., Armstrong, L. and Lako, M. (2008). Activation of p53 cells without Trp53. Sci. Rep. 3, 2944. by nutlin leads to rapid differentiation of human embryonic stem cells. Oncogene Shin, M. H., He, Y. and Huang, J. (2013). Embryonic stem cells shed new light on 27, 5277-5287. the developmental roles of p53. Cell Biosci. 3, 42. Marion, R. M., Strati, K., Li, H., Murga, M., Blanco, R., Ortega, S., Fernandez- Showell, C., Binder, O. and Conlon, F. L. (2004). T-box genes in early Capetillo, O., Serrano, M. and Blasco, M. A. (2009). A p53-mediated DNA embryogenesis. Dev. Dyn. 229, 201-218. damage response limits reprogramming to ensure iPS cell genomic integrity. Silva, J., Nichols, J., Theunissen, T. W., Guo, G., van Oosten, A. L., Barrandon, Nature 460, 1149-1153. O., Wray, J., Yamanaka, S., Chambers, I. and Smith, A. (2009). Nanog is the McGowan, K. A., Li, J. Z., Park, C. Y., Beaudry, V., Tabor, H. K., Sabnis, A. J., gateway to the pluripotent ground state. Cell 138, 722-737. Zhang, W., Fuchs, H., de Angelis, M. H., Myers, R. M. et al. (2008). Ribosomal Spike, B. T. and Wahl, G. M. (2011). p53, stem cells, and reprogramming: tumor mutations cause p53-mediated dark skin and pleiotropic effects. Nat. Genet. 40, suppression beyond guarding the genome. Genes Cancer 2, 404-419. 963-970. Stiewe, T. (2007). The p53 family in differentiation and tumorigenesis. Nat. Rev. Meletis, K., Wirta, V., Hede, S. M., Nister, M., Lundeberg, J. and Frisen, J. (2006). Cancer 7, 165-167. p53 suppresses the self-renewal of adult neural stem cells. Development 133, Tateossian, H., Morse, S., Simon, M. M., Dean, C. H. and Brown, S. D. M. (2015). 363-369. Interactions between the otitis media gene, Fbxo11, and p53 in the mouse Mills, A. A., Zheng, B., Wang, X.-J., Vogel, H., Roop, D. R. and Bradley, A. (1999). embryonic lung. Dis. Model Mech. 8, 1531-1542. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, 398, 708-713. J. J., Marshall, V. S. and Jones, J. M. (1998). Embryonic stem cell lines derived Mizuno, H., Spike, B. T., Wahl, G. M. and Levine, A. J. (2010). Inactivation of p53 in from human blastocysts. Science 282, 1145-1147. breast cancers correlates with stem cell transcriptional signatures. Proc. Natl. Tschaharganeh, D. F., Xue, W., Calvisi, D. F., Evert, M., Michurina, T. V., Dow, Acad. Sci. USA 107, 22745-22750. L. E., Banito, A., Katz, S. F., Kastenhuber, E. R., Weissmueller, S. et al. (2014). Molchadsky, A., Shats, I., Goldfinger, N., Pevsner-Fischer, M., Olson, M., p53-dependent Nestin regulation links tumor suppression to cellular plasticity in

Rinon, A., Tzahor, E., Lozano, G., Zipori, D., Sarig, R. et al. (2008). p53 plays a liver cancer. Cell 158, 579-592. DEVELOPMENT

9 PRIMER Development (2018) 145, dev158360. doi:10.1242/dev.158360

Tyner, S. D., Venkatachalam, S., Choi, J., Jones, S., Ghebranious, N., Igelmann, Inputs in Mesendodermal Differentiation of Embryonic Stem Cells. Cell Stem Cell H., Lu, X., Soron, G., Cooper, B., Brayton, C. et al. (2002a). p53 mutant mice 20, 70-86. that display early ageing-associated phenotypes. Nature 415, 45-53. Wei, J., Zaika, E. and Zaika, A. (2012). p53 family: role of protein isoforms in human Tyner, S. D., Venkatachalam, S., Choi, J., Jones, S., Ghebranious, N., Igelmann, cancer. J. Nucleic. Acids 2012, 687359. H., Lu, X., Soron, G., Cooper, B., Brayton, C. et al. (2002b). p53 mutant mice Wilhelm, M. T., Rufini, A., Wetzel, M. K., Tsuchihara, K., Inoue, S., Tomasini, R., that display early ageing-associated phenotypes. Nature 415, 45-53. Itie-Youten, A., Wakeham, A., Arsenian-Henriksson, M., Melino, G. et al. Ungewitter, E. and Scrable, H. (2010). Delta40p53 controls the switch from (2010). Isoform-specific p73 knockout mice reveal a novel role for delta Np73 in pluripotency to differentiation by regulating IGF signaling in ESCs. Genes Dev. 24, the DNA damage response pathway. Genes Dev. 24, 549-560. 2408-2419. Wong, T. N., Ramsingh, G., Young, A. L., Miller, C. A., Touma, W., Welch, J. S., Utikal, J., Polo, J. M., Stadtfeld, M., Maherali, N., Kulalert, W., Walsh, R. M., Lamprecht, T. L., Shen, D., Hundal, J., Fulton, R. S. et al. (2015). Role of TP53 Khalil, A., Rheinwald, J. G. and Hochedlinger, K. (2009). Immortalization mutations in the origin and evolution of therapy-related acute myeloid leukaemia. eliminates a roadblock during cellular reprogramming into iPS cells. Nature 460, Nature 518, 552-555. 1145-1148. Xie, W., Song, C., Young, N. L., Sperling, A. S., Xu, F., Sridharan, R., Conway, Valente, L. J., Gray, D. H., Michalak, E. M., Pinon-Hofbauer, J., Egle, A., Scott, A. E., Garcia, B. A., Plath, K., Clark, A. T. et al. (2009). Histone h3 lysine 56 C. L., Janic, A. and Strasser, A. (2013). p53 efficiently suppresses tumor acetylation is linked to the core transcriptional network in human embryonic stem development in the complete absence of its cell-cycle inhibitory and proapoptotic cells. Mol. Cell 33, 417-427. effectors p21, Puma, and Noxa. Cell Rep. 3, 1339-1345. Xie, M., Lu, C., Wang, J., McLellan, M. D., Johnson, K. J., Wendl, M. C., Van Nostrand, J. L., Brady, C. A., Jung, H., Fuentes, D. R., Kozak, M. M., McMichael, J. F., Schmidt, H. K., Yellapantula, V., Miller, C. A. et al. (2014). Johnson, T. M., Lin, C.-Y., Lin, C.-J., Swiderski, D. L., Vogel, H. et al. (2014). Age-related mutations associated with clonal hematopoietic expansion and Inappropriate p53 activation during development induces features of CHARGE malignancies. Nat. Med. 20, 1472-1478. syndrome. Nature 514, 228-232. Yang, A., Schweitzer, R., Sun, D., Kaghad, M., Walker, N., Bronson, R. T., Tabin, Van Nostrand, J. L., Bowen, M. E., Vogel, H., Barna, M. and Attardi, L. D. (2017). C., Sharpe, A., Caput, D., Crum, C. et al. (1999). p63 is essential for regenerative The p53 family members have distinct roles during mammalian embryonic proliferation in limb, craniofacial and epithelial development. Nature 398, 714-718. development. Cell Death Differ. 24, 575-579. Yang, A., Walker, N., Bronson, R., Kaghad, M., Oosterwegel, M., Bonnin, J., Vassilev, L. T., Vu, B. T., Graves, B., Carvajal, D., Podlaski, F., Filipovic, Z., Vagner, C., Bonnet, H., Dikkes, P., Sharpe, A. et al. (2000). p73-deficient mice Kong, N., Kammlott, U., Lukacs, C., Klein, C. et al. (2004). In vivo activation of have neurological, pheromonal and inflammatory defects but lack spontaneous the p53 pathway by small-molecule antagonists of MDM2. Science 303, 844-848. tumours. Nature 404, 99-103. Vousden, K. H. and Prives, C. (2009). Blinded by the light: the growing complexity Yang, A., Kaghad, M., Caput, D. and McKeon, F. (2002). On the shoulders of of p53. Cell 137, 413-431. giants: p63, p73 and the rise of p53. Trends Genet. 18, 90-95. Walker, K. K. and Levine, A. J. (1996). Identification of a novel p53 functional Yi, L., Lu, C., Hu, W., Sun, Y. and Levine, A. J. (2012). Multiple roles of p53-related domain that is necessary for efficient growth suppression. Proc. Natl. Acad. Sci. pathways in somatic cell reprogramming and stem cell differentiation. Cancer Res. USA 93, 15335-15340. 72, 5635-5645. Wallingford, J. B., Seufert, D. W., Virta, V. C. and Vize, P. D. (1997). p53 activity is Young, R. A. (2011). Control of the embryonic stem cell state. Cell 144, 940-954. essential for normal development in Xenopus. Curr. Biol. 7, 747-757. Zhang, Y., Xiong, Y. and Yarbrough, W. G. (1998). ARF promotes MDM2 Wang, K. C. and Chang, H. Y. (2011). Molecular mechanisms of long noncoding degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb RNAs. Mol. Cell 43, 904-914. and p53 tumor suppression pathways. Cell 92, 725-734. Wang, J., Sun, Q., Morita, Y., Jiang, H., Gross, A., Lechel, A., Hildner, K., Zhang, Z.-N., Chung, S.-K., Xu, Z. and Xu, Y. (2014). Oct4 maintains the Guachalla, L. M., Gompf, A., Hartmann, D. et al. (2012). A differentiation pluripotency of human embryonic stem cells by inactivating p53 through Sirt1- checkpoint limits hematopoietic stem cell self-renewal in response to DNA mediated deacetylation. Stem Cells 32, 157-165. damage. Cell 148, 1001-1014. Zhao, Z., Zuber, J., Diaz-Flores, E., Lintault, L., Kogan, S. C., Shannon, K. and Wang, Q., Zou, Y., Nowotschin, S., Kim, S. Y., Li, Q. V., Soh, C.-L., Su, J., Zhang, Lowe, S. W. (2010). p53 loss promotes acute myeloid leukemia by enabling C., Shu, W., Xi, Q. et al. (2017). The p53 Family Coordinates Wnt and Nodal aberrant self-renewal. Genes Dev. 24, 1389-1402. DEVELOPMENT