RESUMO
Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.
Diffuse midline gliomas (DMGs) are among the most aggressive and fatal brain cancers in children. They are often associated with changes in histones, the proteins that control gene activity and give chromosomes their structure. Most children with DMGs, for example, share the same anomaly in their histone H3 protein (referred to as the H3-K27M mutation). This change affects how small chemical tags called methyl and acetyl groups can be added onto histone 3, which in turn alters the way the protein can switch genes on and off. As a result, tumours start to develop. One potential therapeutic strategy against DMGs is to use histone deacetylase inhibitors (HDACi), a promising type of drugs which inhibits the enzymes that remove acetyl groups from histones. Patients can develop resistance to HDACi, however, highlighting the need to explore other approaches. One possibility is to treat patients with several types of drugs, each usually targeting a distinct biological process that contributes to the emergence of cancer. This combined approach can have multiple benefits; the drugs potentially amplify each other's effect, for example, and it is also less likely for cells to become resistant to more than one compound at the time. In addition, each drug in the combination can be used in a lower dose to reduce side effects and benefit patients. DMG tumour cells often feature higher activity levels of a protein known as MYC, which can contribute to the growth of the tumour. Algranati, Oren et al. therefore set out to test whether combining an HDACi known as Vorinostat with a drug that blocks MYC activity (Sulfopin) can act as an effective treatment for this cancer. Tumour samples from eight DMG patients were treated with either Sulfopin alone, or Sulfopin in association with Vorinostat. Cells exposed to both drugs were less likely to survive, and additional genetic experiments showed that the combined treatment had resulted in pathways that promote tumour development being blocked. When both Sulfopin and Vorinostat were administered to mice made to grow human DMG tumors, the animals showed a greater reduction in tumor growth. Treatment options for DMG are usually limited, with chemotherapy often being ineffective and surgery impossible. The work by Algranati, Oren et al. suggests that combining HDACi and drugs targeting the MYC pathway is a strategy that should be examined further to determine whether clinical applications are possible.
Assuntos
Glioma , Inibidores de Histona Desacetilases , Histona Desacetilases , Histonas , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Humanos , Animais , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Histonas/metabolismo , Histonas/genética , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Vorinostat/farmacologia , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Criança , Modelos Animais de Doenças , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) in multiple tumors is associated with a poor prognosis partly because of the metabolic diversion of cytosolic aspartate for pyrimidine synthesis, supporting proliferation and mutagenesis owing to nucleotide imbalance. Here, we find that prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts from subjects with citrullinemia type I. Following acute induction of DNA damage with doxorubicin, ASS1 expression is elevated in the cytosol and the nucleus with at least a partial dependency on p53; ASS1 metabolically restrains cell cycle progression in the cytosol by restricting nucleotide synthesis. In the nucleus, ASS1 and ASL generate fumarate for the succination of SMARCC1, destabilizing the chromatin-remodeling complex SMARCC1-SNF5 to decrease gene transcription, specifically in a subset of the p53-regulated cell cycle genes. Thus, following DNA damage, ASS1 is part of the p53 network that pauses cell cycle progression, enabling genome maintenance and survival. Loss of ASS1 contributes to DNA damage and promotes cell cycle progression, likely contributing to cancer mutagenesis and, hence, adaptability potential.
Assuntos
Argininossuccinato Sintase , Núcleo Celular , Citosol , Dano ao DNA , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Citosol/metabolismo , Argininossuccinato Sintase/metabolismo , Argininossuccinato Sintase/genética , Núcleo Celular/metabolismo , Ciclo Celular/genéticaRESUMO
The analysis of cell-free tumor DNA (ctDNA) and proteins in the blood of cancer patients potentiates a new generation of non-invasive diagnostics and treatment monitoring approaches. However, confident detection of these tumor-originating markers is challenging, especially in the context of brain tumors, in which extremely low amounts of these analytes circulate in the patient's plasma. Here, we applied a sensitive single-molecule technology to profile multiple histone modifications on millions of individual nucleosomes from the plasma of Diffuse Midline Glioma (DMG) patients. The system reveals epigenetic patterns that are unique to DMG, significantly differentiating this group of patients from healthy subjects or individuals diagnosed with other cancer types. We further develop a method to directly capture and quantify the tumor-originating oncoproteins, H3-K27M and mutant p53, from the plasma of children diagnosed with DMG. This single-molecule system allows for accurate molecular classification of patients, utilizing less than 1ml of liquid-biopsy material. Furthermore, we show that our simple and rapid detection strategy correlates with MRI measurements and droplet-digital PCR (ddPCR) measurements of ctDNA, highlighting the utility of this approach for non-invasive treatment monitoring of DMG patients. This work underscores the clinical potential of single-molecule-based, multi-parametric assays for DMG diagnosis and treatment monitoring.
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Prolonged metabolic stress can lead to severe pathologies. In metabolically challenged primary fibroblasts, we assigned a novel role for the poorly characterized miR-4734 in restricting ATF4 and IRE1-mediated upregulation of a set of proinflammatory cytokines and endoplasmic reticulum stress-associated genes. Conversely, inhibition of this miRNA augmented the expression of those genes. Mechanistically, miR-4734 was found to restrict the expression of the transcriptional activator NF-kappa-B inhibitor zeta (NFKBIZ), which is required for optimal expression of the proinflammatory genes and whose mRNA is targeted directly by miR-4734. Concordantly, overexpression of NFKBIZ compromised the effects of miR-4734, underscoring the importance of this direct targeting. As the effects of miR-4734 were evident under stress but not under basal conditions, it may possess therapeutic utility towards alleviating stress-induced pathologies.
Assuntos
MicroRNAs , Citocinas/genética , Citocinas/metabolismo , Estresse do Retículo Endoplasmático/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , HumanosRESUMO
The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.
Assuntos
Ácidos Nucleicos Livres , Neoplasias , Nucleossomos , Humanos , Biomarcadores Tumorais , Ácidos Nucleicos Livres/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Proteínas de Neoplasias/genética , Neoplasias/diagnóstico , Neoplasias/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Imagem Individual de MoléculaRESUMO
Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a "basal-like" state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed "basal-like" genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/genética , Genes Reguladores , Proteínas Serina-Treonina Quinases/genética , Mama , Repressão Psicológica , Correpressor 1 de Receptor Nuclear/genéticaRESUMO
Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.
Assuntos
Neoplasias do Tronco Encefálico , Glioma , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/metabolismo , Neoplasias do Tronco Encefálico/patologia , Cromatina/genética , Epigênese Genética , Glioma/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , MutaçãoRESUMO
Cancer-associated mutations in genes encoding histones dramatically reshape chromatin and support tumorigenesis. Lysine to methionine substitution of residue 27 on histone H3 (K27M) is a driver mutation in high-grade pediatric gliomas, known to abrogate polycomb repressive complex 2 (PRC2) activity. We applied single-molecule systems to image individual nucleosomes and delineate the combinatorial epigenetic patterns associated with H3-K27M expression. We found that chromatin marks on H3-K27M-mutant nucleosomes are dictated both by their incorporation preferences and by intrinsic properties of the mutation. Mutant nucleosomes not only preferentially bind PRC2 but also directly interact with MLL1, leading to genome-wide redistribution of H3K4me3. H3-K27M-mediated deregulation of repressive and active chromatin marks leads to unbalanced "bivalent" chromatin, which may support a poorly differentiated cellular state. This study provides evidence for a direct effect of H3-K27M oncohistone on the MLL1-H3K4me3 pathway and highlights the capability of single-molecule tools to reveal mechanisms of chromatin deregulation in cancer.
Assuntos
Neoplasias Encefálicas , Glioma , Histona-Lisina N-Metiltransferase , Proteína de Leucina Linfoide-Mieloide , Nucleossomos , Neoplasias Encefálicas/genética , Criança , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Glioma/genética , Glioma/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismoRESUMO
Genome regulation is governed by the dynamics of chromatin modifications. The extensive and diverse array of DNA and histone modifications allow multiple elements to act combinatorically and direct tissue-specific and cell-specific outcomes. Yet, our ability to elucidate these complex combinations and link them to normal genome regulation, as well as understand their deregulation in cancer, has been hindered by the lack of suitable technologies. Here, we describe recent findings indicating the importance of the combinatorial epigenome, and novel methodologies to measure and characterize these combinations. These complementary methods span multiple disciplines, providing a means to decode epigenetic combinations and link them to biological outcomes. Finally, we discuss the promise of harnessing the rich combinatorial epigenetic information to improve cancer diagnostics and monitoring.
Assuntos
Epigenoma , Neoplasias , Cromatina/genética , Metilação de DNA/genética , Epigênese Genética/genética , Epigenoma/genética , Epigenômica , Genoma , Código das Histonas/genética , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMO
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/imunologia , Imagem Individual de Molécula/métodos , Proteínas Virais/genética , Anticorpos Antivirais/sangue , Sequência de Bases , COVID-19/sangue , COVID-19/imunologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/normas , Teste Sorológico para COVID-19/normas , Ensaio de Imunoadsorção Enzimática , Humanos , Soros Imunes/química , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Nasofaringe/virologia , Poliproteínas/sangue , Poliproteínas/genética , RNA Viral/sangue , RNA Viral/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade , Imagem Individual de Molécula/instrumentação , Proteínas Virais/sangueRESUMO
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
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Deregulated activity of LArge Tumor Suppressor (LATS) tumor suppressors has broad implications on cellular and tissue homeostasis. We examined the consequences of down-regulation of either LATS1 or LATS2 in breast cancer. Consistent with their proposed tumor suppressive roles, expression of both paralogs was significantly down-regulated in human breast cancer, and loss of either paralog accelerated mammary tumorigenesis in mice. However, each paralog had a distinct impact on breast cancer. Thus, LATS2 depletion in luminal B tumors resulted in metabolic rewiring, with increased glycolysis and reduced peroxisome proliferator-activated receptor γ (PPARγ) signaling. Furthermore, pharmacological activation of PPARγ elicited LATS2-dependent death in luminal B-derived cells. In contrast, LATS1 depletion augmented cancer cell plasticity, skewing luminal B tumors towards increased expression of basal-like features, in association with increased resistance to hormone therapy. Hence, these two closely related paralogs play distinct roles in protection against breast cancer; tumors with reduced expression of either LATS1 or LATS2 may rewire signaling networks differently and thus respond differently to anticancer treatments.
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Within the tumor microenvironment, cancer cells coexist with noncancerous adjacent cells that constitute the tumor microenvironment and impact tumor growth through diverse mechanisms. In particular, cancer-associated fibroblasts (CAFs) promote tumor progression in multiple ways. Earlier studies have revealed that in normal fibroblasts (NFs), p53 plays a cell nonautonomous tumor-suppressive role to restrict tumor growth. We now wished to investigate the role of p53 in CAFs. Remarkably, we found that the transcriptional program supported by p53 is altered substantially in CAFs relative to NFs. In agreement, the p53-dependent secretome is also altered in CAFs. This transcriptional rewiring renders p53 a significant contributor to the distinct intrinsic features of CAFs, as well as promotes tumor cell migration and invasion in culture. Concordantly, the ability of CAFs to promote tumor growth in mice is greatly compromised by depletion of their endogenous p53. Furthermore, cocultivation of NFs with cancer cells renders their p53-dependent transcriptome partially more similar to that of CAFs. Our findings raise the intriguing possibility that tumor progression may entail a nonmutational conversion ("education") of stromal p53, from tumor suppressive to tumor supportive.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Fibroblastos Associados a Câncer/patologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Técnicas de Cocultura/métodos , Progressão da Doença , Feminino , Células HEK293 , Humanos , Camundongos , Pessoa de Meia-Idade , Neoplasias/patologia , Transcrição Gênica/fisiologia , Transcriptoma/fisiologia , Microambiente Tumoral/fisiologiaRESUMO
The three p53 family members, p53, p63 and p73, are structurally similar and share many biochemical activities. Yet, along with their common fundamental role in protecting genomic fidelity, each has acquired distinct functions related to diverse cell autonomous and non-autonomous processes. Similar to the p53 family, the Hippo signaling pathway impacts a multitude of cellular processes, spanning from cell cycle and metabolism to development and tumor suppression. The core Hippo module consists of the tumor-suppressive MST-LATS kinases and oncogenic transcriptional co-effectors YAP and TAZ. A wealth of accumulated data suggests a complex and delicate regulatory network connecting the p53 and Hippo pathways, in a highly context-specific manner. This generates multiple layers of interaction, ranging from interdependent and collaborative signaling to apparent antagonistic activity. Furthermore, genetic and epigenetic alterations can disrupt this homeostatic network, paving the way to genomic instability and cancer. This strengthens the need to better understand the nuances that control the molecular function of each component and the cross-talk between the different components. Here, we review interactions between the p53 and Hippo pathways within a subset of physiological contexts, focusing on normal stem cells and development, as well as regulation of apoptosis, senescence and metabolism in transformed cells.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Carcinogênese , Senescência Celular , Humanos , Células-Tronco Neoplásicas/metabolismo , Ploidias , Transdução de Sinais , Células-Tronco/metabolismoRESUMO
Crosstalk between growth factors (GFs) and steroid hormones recurs in embryogenesis and is co-opted in pathology, but underlying mechanisms remain elusive. Our data from mammary cells imply that the crosstalk between the epidermal GF and glucocorticoids (GCs) involves transcription factors like p53 and NF-κB, along with reduced pausing and traveling of RNA polymerase II (RNAPII) at both promoters and bodies of GF-inducible genes. Essentially, GCs inhibit positive feedback loops activated by GFs and stimulate the reciprocal inhibitory loops. As expected, no alterations in DNA methylation accompany the transcriptional events instigated by either stimulus, but forced demethylation of regulatory regions broadened the repertoire of GF-inducible genes. We report that enhancers, like some promoters, are poised for activation by GFs and GCs. In addition, within the cooperative interface of the crosstalk, GFs enhance binding of the GC receptor to DNA and, in synergy with GCs, promote productive RNAPII elongation. Reciprocally, within the antagonistic interface GFs hyper-acetylate chromatin at unmethylated promoters and enhancers of genes involved in motility, but GCs hypoacetylate the corresponding regions. In conclusion, unmethylated genomic regions that encode feedback regulatory modules and differentially recruit RNAPII and acetylases/deacetylases underlie the crosstalk between GFs and a steroid hormone.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Regiões Promotoras Genéticas/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Metilação de DNA , Dexametasona/farmacologia , Humanos , NF-kappa B/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Proper cellular functionality and homeostasis are maintained by the convergent integration of various signaling cascades, which enable cells to respond to internal and external changes. The Dbf2-related kinases LATS1 and LATS2 (LATS) have emerged as central regulators of cell fate, by modulating the functions of numerous oncogenic or tumor suppressive effectors, including the canonical Hippo effectors YAP/TAZ, the Aurora mitotic kinase family, estrogen signaling and the tumor suppressive transcription factor p53. While the basic functions of the LATS kinase module are strongly conserved over evolution, the genomic duplication event leading to the emergence of two closely related kinases in higher organisms has increased the complexity of this signaling network. Here, we review the LATS1 and LATS2 intrinsic features as well as their reported cellular activities, emphasizing unique characteristics of each kinase. While differential activities between the two paralogous kinases have been reported, many converge to similar pathways and outcomes. Interestingly, the regulatory networks controlling the mRNA expression pattern of LATS1 and LATS2 differ strongly, and may contribute to the differences in protein binding partners of each kinase and in the subcellular locations in which each kinase exerts its functions.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Fosforilação/genética , Fosforilação/fisiologia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naïve mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53-/-) mESCs is the result of augmented overall DNA methylation as well as increased methylation landscape heterogeneity. In differentiating p53-/- mESCs, elevated methylation persists, albeit more mildly. Importantly, concomitant with DNA methylation heterogeneity, p53-/- mESCs display increased cellular heterogeneity both in the "naïve" state and upon induced differentiation. This impact of p53 loss on 5-methylcytosine (5mC) heterogeneity was also evident in human ESCs and mouse embryos in vivo. Hence, p53 helps maintain DNA methylation homeostasis and clonal homogeneity, a function that may contribute to its tumor suppressor activity.