P53 Family Proteins Research Articles

TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

Dysregulated mitochondrial fusion and fission has been implicated in the pathogenesis of numerous diseases. We have identified a novel function of the p53 family protein TAp73 in regulating mitochondrial dynamics. TAp73 regulates the expression of Optic Atrophy 1 (OPA1), a protein responsible for controlling mitochondrial fusion, cristae biogenesis and electron transport chain function. Disruption of this axis results in a fragmented mitochondrial network and an impaired capacity for energy production via oxidative phosphorylation. Owing to the role of OPA1 in modulating cytochrome c release, TAp73−/− cells display an increased sensitivity to apoptotic cell death, e.g., via BH3-mimetics. We additionally show that the TAp73/OPA1 axis has functional relevance in the upper airway, where TAp73 expression is essential for multiciliated cell differentiation and function. Consistently, ciliated epithelial cells of Trp73−/− (global p73 knock-out) mice display decreased expression of OPA1 and perturbations of the mitochondrial network, which may drive multiciliated cell loss. In support of this, Trp73 and OPA1 gene expression is decreased in chronic obstructive pulmonary disease (COPD) patients, a disease characterised by alterations in mitochondrial dynamics. We therefore highlight a potential mechanism involving the loss of p73 in COPD pathogenesis. Our findings also add to the growing body of evidence for growth-promoting roles of TAp73 isoforms.

P53 and BCL-2 family proteins PUMA and NOXA define competitive fitness in pluripotent cell competition.

Cell Competition is a process by which neighboring cells compare their fitness. As a result, viable but suboptimal cells are selectively eliminated in the presence of fitter cells. In the early mammalian embryo, epiblast pluripotent cells undergo extensive Cell Competition, which prevents suboptimal cells from contributing to the newly forming organism. While competitive ability is regulated by MYC in the epiblast, the mechanisms that contribute to competitive fitness in this context are largely unknown. Here, we report that P53 and its pro-apoptotic targets PUMA and NOXA regulate apoptosis susceptibility and competitive fitness in pluripotent cells. PUMA is widely expressed specifically in pluripotent cells in vitro and in vivo. We found that P53 regulates MYC levels in pluripotent cells, which connects these two Cell Competition pathways, however, MYC and PUMA/NOXA levels are independently regulated by P53. We propose a model that integrates a bifurcated P53 pathway regulating both MYC and PUMA/NOXA levels and determines competitive fitness.

Neuronal IL-17 controls Caenorhabditis elegans developmental diapause through CEP-1/p53

During metazoan development, how cell division and metabolic programs are coordinated with nutrient availability remains unclear. Here, we show that nutrient availability signaled by the neuronal cytokine, ILC-17.1, switches Caenorhabditis elegans development between reproductive growth and dormancy by controlling the activity of the tumor suppressor p53 ortholog, CEP-1. Specifically, upon food availability, ILC-17.1 signaling by amphid neurons promotes glucose utilization and suppresses CEP-1/p53 to allow growth. In the absence of ILC-17.1, CEP-1/p53 is activated, up-regulates cell-cycle inhibitors, decreases phosphofructokinase and cytochrome C expression, and causes larvae to arrest as stress-resistant, quiescent dauers. We propose a model whereby ILC-17.1 signaling links nutrient availability and energy metabolism to cell cycle progression through CEP-1/p53. These studies describe ancestral functions of IL-17 s and the p53 family of proteins and are relevant to our understanding of neuroimmune mechanisms in cancer. They also reveal a DNA damage-independent function of CEP-1/p53 in invertebrate development and support the existence of a previously undescribed C. elegans dauer pathway.

The presence of a G-quadruplex prone sequence upstream of a minimal promoter increases transcriptional activity in the yeast Saccharomyces cerevisiae.

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast Saccharomyces cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides basal reporter activity, the P53 RE enables LUC1 transactivation under the control of P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family protein-dependent expression was measured after shifting cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter increased its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this feature, when present near the target binding site of P53 family transcription factors, can be exploited to regulate the transcriptional activity of P53, P63 and P73 proteins.

Transactivation by partial function P53 family mutants is increased by the presence of G-quadruplexes at a promoter site

The effect of mutations in the P53 family of transcription factors on their biological functions, including partial or complete loss of transcriptional activity, has been confirmed several times. At present, P53 family proteins showing partial loss of activity appear to be promising potential candidates for the development of novel therapeutic strategies which could restore their transcriptional activity. In this context, it is important to employ tools to precisely monitor their activity; in relation to this, non-canonical DNA secondary structures in promoters including G-quadruplexes (G4s) were shown to influence the activity of transcription factors. Here, we used a defined yeast assay to evaluate the impact of differently modeled G4 forming sequences on a panel of partial function P53 family mutant proteins. Specifically, a 22-mer G4 prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate characteristic guanine stretches were placed upstream of a minimal promoter, adjacent to a P53 response element in otherwise isogenic yeast luciferase reporter strains. The transactivation ability of cancer-associated P53 (TA-P53α: A161T, R213L, N235S, V272L, R282W, R283C, R337C, R337H, and G360V) or Ectodermal Dyplasia syndromes-related P63 mutant proteins (ΔN-P63α: G134D, G134V and inR155) were tested. Our results show that the presence of G4 forming sequences can increase the transactivation ability of partial function P53 family proteins. These observations are pointing to the importance of DNA structural characteristics for accurate classification of P53 family proteins functionality in the context of the wide variety of TP53 and TP63 germline and somatic mutations.

Deciphering the Role of p53 and TAp73 in Neuroblastoma: From Pathogenesis to Treatment.

Neuroblastoma (NB) is an embryonic cancer that develops from neural crest stem cells, being one of the most common malignancies in children. The clinical manifestation of this disease is highly variable, ranging from spontaneous regression to increased aggressiveness, which makes it a major therapeutic challenge in pediatric oncology. The p53 family proteins p53 and TAp73 play a key role in protecting cells against genomic instability and malignant transformation. However, in NB, their activities are commonly inhibited by interacting proteins such as murine double minute (MDM)2 and MDMX, mutant p53, ΔNp73, Itch, and Aurora kinase A. The interplay between the p53/TAp73 pathway and N-MYC, a known biomarker of poor prognosis and drug resistance in NB, also proves to be decisive in the pathogenesis of this tumor. More recently, a strong crosstalk between microRNAs (miRNAs) and p53/TAp73 has been established, which has been the focused of great attention because of its potential for developing new therapeutic strategies. Collectively, this review provides an updated overview about the critical role of the p53/TAp73 pathway in the pathogenesis of NB, highlighting encouraging clues for the advance of alternative NB targeted therapies.

The p53 Family Members p63 and p73 Roles in the Metastatic Dissemination: Interactions with microRNAs and TGFβ Pathway

TP53 (TP53), p73 (TP73), and p63 (TP63) are members of the p53 transcription factor family, which has many activities spanning from embryonic development through to tumor suppression. The utilization of two promoters and alternative mRNA splicing has been shown to yield numerous isoforms in p53, p63, and p73. TAp73 is thought to mediate apoptosis as a result of nuclear accumulation following chemotherapy-induced DNA damage, according to a number of studies. Overexpression of the nuclear ΔNp63 and ΔNp73 isoforms, on the other hand, suppresses TAp73's pro-apoptotic activity in human malignancies, potentially leading to metastatic spread or inhibition. Another well-known pathway that has been associated to metastatic spread is the TGF pathway. TGFs are a family of structurally related polypeptide growth factors that regulate a variety of cellular functions including cell proliferation, lineage determination, differentiation, motility, adhesion, and cell death, making them significant players in development, homeostasis, and wound repair. Various studies have already identified several interactions between the p53 protein family and the TGFb pathway in the context of tumor growth and metastatic spread, beginning to shed light on this enigmatic intricacy.

STAT3 and p63 in the Regulation of Cancer Stemness.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor with many important functions in normal and transformed cells. STAT3 regulatory activities are highly complex as they are involved in various signaling pathways in different cell types under different conditions. Biologically, STAT3 is a regulative factor for normal and cancer stem cells (CSCs). Tumor protein p63 (p63), a member of the p53 protein family, is involved in these biological processes and is also physically and functionally associated with STAT3. STAT3 activation occurs during various aspects of carcinogenesis, including regulation of CSCs properties. In combination with p63, STAT3 is a possible biological marker of CSCs and a major regulator of maintenance of stemness in CSCs. We summarized the STAT3 functions and regulation and its role in CSC properties and highlight how these are affected by its associations with p63.

FOXL2 and FOXA1 cooperatively assemble on the TP53 promoter in alternative dimer configurations.

Although both the p53 and forkhead box (FOX) family proteins are key transcription factors associated with cancer progression, their direct relationship is unknown. Here, we found that FOX family proteins bind to the non-canonical homotypic cluster of the p53 promoter region (TP53). Analysis of crystal structures of FOX proteins (FOXL2 and FOXA1) bound to the p53 homotypic cluster indicated that they interact with a 2:1 stoichiometry accommodated by FOX-induced DNA allostery. In particular, FOX proteins exhibited distinct dimerization patterns in recognition of the same p53-DNA; dimer formation of FOXA1 involved protein–protein interaction, but FOXL2 did not. Biochemical and biological functional analyses confirmed the cooperative binding of FOX proteins to the TP53 promoter for the transcriptional activation of TP53. In addition, up-regulation of TP53 was necessary for FOX proteins to exhibit anti-proliferative activity in cancer cells. These analyses reveal the presence of a discrete characteristic within FOX family proteins in which FOX proteins regulate the transcription activity of the p53 tumor suppressor via cooperative binding to the TP53 promoter in alternative dimer configurations.

The Therapeutic Potential of the Restoration of the p53 Protein Family Members in the EGFR-Mutated Lung Cancer.

Despite the recent development of precision medicine and targeted therapies, lung cancer remains the top cause of cancer-related mortality worldwide. The patients diagnosed with metastatic disease have a five-year survival rate lower than 6%. In metastatic disease, EGFR is the most common driver of mutation, with the most common co-driver hitting TP53. EGFR-positive patients are offered the frontline treatment with tyrosine kinase inhibitors, yet the development of resistance and the lack of alternative therapies make this group of patients only fit for clinical trial participation. Since mutant p53 is the most common co-driver in the metastatic setting, therapies reactivating the p53 pathway might serve as a promising alternative therapeutic approach in patients who have developed a resistance to tyrosine kinase inhibitors. This review focuses on the molecular background of EGFR-mutated lung cancer and discusses novel therapeutic options converging on the reactivation of p53 tumor suppressor pathways.

Designed Ankyrin Repeat Proteins as a tool box for analyzing p63

The function of the p53 transcription factor family is dependent on several folded domains. In addition to a DNA-binding domain, members of this family contain an oligomerization domain. p63 and p73 also contain a C-terminal Sterile α-motif domain. Inhibition of most transcription factors is difficult as most of them lack deep pockets that can be targeted by small organic molecules. Genetic knock-out procedures are powerful in identifying the overall function of a protein, but they do not easily allow one to investigate roles of individual domains. Here we describe the characterization of Designed Ankyrin Repeat Proteins (DARPins) that were selected as tight binders against all folded domains of p63. We determine binding affinities as well as specificities within the p53 protein family and show that DARPins can be used as intracellular inhibitors for the modulation of transcriptional activity. By selectively inhibiting DNA binding of the ΔNp63α isoform that competes with p53 for the same promoter sites, we show that p53 can be reactivated. We further show that inhibiting the DNA binding activity stabilizes p63, thus providing evidence for a transcriptionally regulated negative feedback loop. Furthermore, the ability of DARPins to bind to the DNA-binding domain and the Sterile α-motif domain within the dimeric-only and DNA-binding incompetent conformation of TAp63α suggests a high structural plasticity within this special conformation. In addition, the developed DARPins can also be used to specifically detect p63 in cell culture and in primary tissue and thus constitute a very versatile research tool for studying the function of p63.

Hepatic p63 regulates glucose metabolism by repressing SIRT1

Objectivep63 is a transcription factor within the p53 protein family that has key roles in development, differentiation and prevention of senescence, but its metabolic actions remain largely unknown. Herein, we...

Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins

The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain–containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that FIH catalyzes the unprecedented post-translational hydroxylation of both asparaginyl residues in “VNVN” and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function.

The Role of E3 Ligase Pirh2 in Disease.

The p53-dependent ubiquitin ligase Pirh2 regulates a number of proteins involved in different cancer-associated processes. Targeting the p53 family proteins, Chk2, p27Kip1, Twist1 and others, Pirh2 participates in such cellular processes as proliferation, cell cycle regulation, apoptosis and cellular migration. Thus, it is not surprising that Pirh2 takes part in the initiation and progression of different diseases and pathologies including but not limited to cancer. In this review, we aimed to summarize the available data on Pirh2 regulation, its protein targets and its role in various diseases and pathological processes, thus making the Pirh2 protein a promising therapeutic target.

Reciprocal regulation of p21 and Chk1 controls the cyclin D1-RB pathway to mediate senescence onset after G2 arrest.

Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.

Noncanonical roles of p53 in cancer stemness and their implications in sarcomas.

Impairment of the prominent tumor suppressor p53, well known for its canonical role as the "guardian of the genome", is found in almost half of human cancers. More recently, p53 has been suggested to be a crucial regulator of stemness, orchestrating the differentiation of embryonal and adult stem cells, suppressing reprogramming into induced pluripotent stem cells, or inhibiting cancer stemness (i.e., cancer stem cells, CSCs), which underlies the development of therapy-resistant tumors. This review addresses these noncanonical roles of p53 and their implications in sarcoma initiation and progression. Indeed, dysregulation of p53 family proteins is a common event in sarcomas and is associated with poor survival. Additionally, emerging studies have demonstrated that loss of wild-type p53 activity hinders the terminal differentiation of mesenchymal stem cells and leads to the development of aggressive sarcomas. This review summarizes recent findings on the roles of aberrant p53 in sarcoma development and stemness and further describes therapeutic approaches to restore normal p53 activity as a promising anti-CSC strategy to treat refractory sarcomas.

The p53 Family: A Role in Lipid and Iron Metabolism.

The p53 family of tumor suppressors, which includes p53, p63, and p73, has a critical role in many biological processes, such as cell cycle arrest, apoptosis, and differentiation. In addition to tumor suppression, the p53 family proteins also participate in development, multiciliogenesis, and fertility, indicating these proteins have diverse roles. In this review, we strive to cover the relevant studies that demonstrate the roles of p53, p63, and p73 in lipid and iron metabolism.

Diosgenin exerts anti-tumor effects through inactivation of cAMP/PKA/CREB signaling pathway in colorectal cancer

Colorectal cancer (CRC) is the most fatal gastrointestinal tumor and it is urge to explore powerful drugs for the treatment. Diosgenin (DSG) as a new steroidal had been reported exerts anti-tumor activity in multiple cancers, including CRC. However, the potential mechanism of DSG suppresses CRC remains further to be revealed. Here, we reported that DSG inhibited proliferation of CRC cells in dose- and time-dependent manner, induced apoptosis by modulating p53 and Bcl-2 family proteins expression to mediate mitochondrial apoptosis pathway, suppressed migration and invasion by reducing MMP-9 (matrix metalloproteinase) and decreased aerobic glycolysis by mediating glucose transporter (GLUT) like GLUT3 and GLUT4, and pyruvate carboxylase PC downregulation. Intriguingly, mechanistic study suggests those phenotypes involved DSG inhibited cAMP/PKA/CREB pathway in CRC cells, and result to inhibit the phosphorylation of CREB to regulate the transcription of genes above-mentioned. Finally, nude mice xenograft tumor model further indicated that DSG could be a great agent to suppress the growth of CRC cells in vivo and have no obvious side effects. Taken together, we revealed a unique mechanism that DSG suppresses CRC cells through cAMP/PKA/CREB pathway and DSG is a promising candidate drug for CRC treatment.

The Pharmacological Blockade of Phosphodiesterase IV Potentiates the Anti-Tumor Effects of Arsenic Trioxide (ATO) in Acute Myelogenous Leukemia Cells through Multiple Signaling Pathways

Preclinical in vitro and in vivo studies showed that Arsenic Trioxide (ATO) has antileukemic effects both as a single agent and in combination with conventional therapies or with other molecularly targeted agents . Furthermore, analogs of cAMP have been shown to enhance the therapeutic effects of ATRA and ATO in APL cells. Because cAMP pathway is modulated by various Inhibitiors of phosphodiesterase IV (PDEIV), some of which are already in the clinics for the treatment of asthma, the aim of this study was to investigate whether the pharmacological inhibition of PDEIV potentiates the cytodifferentiating and/or proapoptotic activities of ATO in acute myelogenous leukemia (AML) cells.We found that PDEIV inhibitors induced the upregulation of the myeloid markers (CD38 and CD11b), and potentiated the cytodifferentiating action of ATO in APL cells via induction of cAMP; the cytodifferentiating effects of the PDEIV inhibitors /ATO combination in leukemic cells was also confirmed by nitro blue tetrazolium reductase activity assay. Accordingly, we also found that the inhibition of PDEIV accelerated the ATO-induced degradation of PML-retinoic acid receptor α (PML-RARα) oncoprotein in NB4 cells, thus removing the PML-RARα-induced maturation blocks.Furthermore we also demonstrated that PDEIV inhibitors synergized with ATO to induce apoptosis in both APL NB4 and non-APL HL-60, MOLM-13 and OCI-AML-3 AML cell lines and significantly (P<.01) potentiated the ATO-induced cell death of fresh purified leukemic blasts in 8 out of 10 AML patients analyzed with different genetic abnormalities; in contrast, no significant cytotoxicity in peripheral blood mononuclear cells from 3 healthy volunteers was observed after PDEIV inhibitors/ATO treatment. We consistently found that the inhibition of PDEIV strongly potentiated the ATO-induced activation of caspase-3 and PARP fragmentation in all the tested cell lines as well as in primary AML blasts from patients, and, by using a peptide inhibitor approach, we demonstrated that caspase activity was indispensable for PDEIV inhibitors / ATO-induced apoptosis. Furthermore, siRNA knockdown of endogenous PDE4A and PDE4B gene expression recapitulated the ability of PDEIV inhibitors to sensitize leukemic cells to ATO, thereby confirming the significant role of these enzymes in mediating ATO sensitization of leukemic cells to PDEIV inhibitors /ATO-induced apoptosis. Interestingly, by using cAMP analogs and cAMP competitors we demonstrated that PDEIV inhibitors /ATO -induced apoptosis was cAMP independent.In order to investigate the molecular effectors involved in PDEIV inhibitors /ATO-induced apoptosis we first evaluated the effects of the combined treatment on Bcl-2 and p53 family proteins and their biologic relevance in the synergism observed between PDEIV inhibitors and ATO. By western blot analysis we demonstrated that co-treatment with PDEIV inhibitors and ATO increased protein expression of pro-apoptotic of Bax, Bak and/or Puma and decrease anti-apoptotic Mcl-1, A1/Bfl1 and/or Bcl-2; in agreement with the biochemical findings indicating the activation of the mitochondrial apoptotic pathway, we found that combined treatment with PDEIV inhibitors and ATO strongly potentiated mitochondrial depolarization induced by ATO alone in all the tested cell lines. Preliminary data indicate the involvement of p73 pathway which is consistent with the observed upregulation of Bax, Bak and/or Puma in AML cells; ongoing experiment are in progress to evaluate whether p53 pathway is activated by the combined treatment in MOLM-13 and OCI-AML-3 AML cell lines as well as in fresh purified leukemic blasts.Additionally, we found that co-treatment with PDEIV inhibitors and ATO also induced prosurvival signals by increasing ERK and Bad phosphorylation and the expression of anti-apoptotic Bcl-xL, and the pharmacological or genetic disruption of ERK or Bcl-xL consistently and significantly potentiated the cytotoxic effects of the combination PDEIV inhibitors /ATO. Altogether these findings suggest to extend the study to more patients and strongly support the rationale for testing these promising combinations in clinically relevant in vivo mouse models of drug-resistant leukemia. DisclosuresNo relevant conflicts of interest to declare.

Deciphering the Nature of Trp73 Isoforms in Mouse Embryonic Stem Cell Models: Generation of Isoform-Specific Deficient Cell Lines Using the CRISPR/Cas9 Gene Editing System

Simple SummaryThe Trp73 gene is involved in the regulation of multiple biological processes such as response to stress, differentiation and tissue architecture. This gene gives rise to structurally different N and C-terminal isoforms which lead to differences in its biological activity in a cell type dependent manner. However, there is a current lack of physiological models to study these isoforms. The aim of this study was to generate specific p73-isoform-deficient mouse embryonic stem cell lines using the CRISPR/Cas9 system. Their special features, self-renewal and pluripotency, make embryonic stem cells a useful research tool that allows the generation of cells from any of the three germ layers carrying specific inactivation of p73-isoforms. Characterization of the generated cell lines indicates that while the individual elimination of TA- or DN-p73 isoform is compatible with pluripotency, it results in alterations of the transcriptional profiles and the pluripotent state of the embryonic stem cells in an isoform-specific manner.The p53 family has been widely studied for its role in various physiological and pathological processes. Imbalance of p53 family proteins may contribute to developmental abnormalities and pathologies in humans. This family exerts its functions through a profusion of isoforms that are generated by different promoter usage and alternative splicing in a cell type dependent manner. In particular, the Trp73 gene gives rise to TA and DN-p73 isoforms that confer p73 a dual nature. The biological relevance of p73 does not only rely on its tumor suppression effects, but on its pivotal role in several developmental processes. Therefore, the generation of cellular models that allow the study of the individual isoforms in a physiological context is of great biomedical relevance. We generated specific TA and DN-p73-deficient mouse embryonic stem cell lines using the CRISPR/Cas9 gene editing system and validated them as physiological bona fide p73-isoform knockout models. Global gene expression analysis revealed isoform-specific alterations of distinctive transcriptional networks. Elimination of TA or DN-p73 is compatible with pluripotency but prompts naïve pluripotent stem cell transition into the primed state, compromising adequate lineage differentiation, thus suggesting that differential expression of p73 isoforms acts as a rheostat during early cell fate determination.