RESUMO
The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/genética , Deleção de Genes , Edição de Genes/métodos , Mutagênese Insercional , Proteína 9 Associada à CRISPR/metabolismo , Proliferação de Células , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Células HEK293 , Células Hep G2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Motivos de Nucleotídeos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Epigenetic regulators are often hijacked by cancer cells to sustain malignant phenotypes. How cells repurpose key regulators of cell identity as tumour-promoting factors is unclear. The antithetic role of the Polycomb component EZH2 in normal brain and glioma provides a paradigm to dissect how wild-type chromatin modifiers gain a pathological function in cancer. Here, we show that oncogenic signalling induces redistribution of EZH2 across the genome, and through misregulation of homeotic genes corrupts the identity of neural cells. Characterisation of EZH2 targets in de novo transformed cells, combined with analysis of glioma patient datasets and cell lines, reveals that acquisition of tumorigenic potential is accompanied by a transcriptional switch involving de-repression of spinal cord-specifying HOX genes and concomitant silencing of the empty spiracles homologue EMX2, a critical regulator of neurogenesis in the forebrain. Maintenance of tumorigenic potential by glioblastoma cells requires EMX2 repression, since forced EMX2 expression prevents tumour formation. Thus, by redistributing EZH2 across the genome, cancer cells subvert developmental transcriptional programmes that specify normal cell identity and remove physiological breaks that restrain cell proliferation.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Glioma/patologia , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromatina/metabolismo , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Genes Homeobox , Glioma/genética , Humanos , Masculino , Camundongos Endogâmicos NOD , Modelos Biológicos , Fenótipo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Epigenetic aberrations have been recognized as important contributors to cancer onset and development, and increasing evidence suggests that linker histone H1 variants may serve as biomarkers useful for patient stratification, as well as play an important role as drivers in cancer. Although traditionally histone H1 levels have been studied using antibody-based methods and RNA expression, these approaches suffer from limitations. Mass spectrometry (MS)-based proteomics represents the ideal tool to accurately quantify relative changes in protein abundance within complex samples. In this study, we used a label-free quantification approach to simultaneously analyze all somatic histone H1 variants in clinical samples and verified its applicability to laser micro-dissected tissue areas containing as low as 1000 cells. We then applied it to breast cancer patient samples, identifying differences in linker histone variants patters in primary triple-negative breast tumors with and without relapse after chemotherapy. This study highlights how label-free quantitation by MS is a valuable option to accurately quantitate histone H1 levels in different types of clinical samples, including very low-abundance patient tissues.
Assuntos
Histonas/genética , Recidiva Local de Neoplasia/genética , Proteômica , Neoplasias de Mama Triplo Negativas/genética , Biomarcadores Tumorais/genética , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/patologia , Processamento de Proteína Pós-Traducional/genética , Espectrometria de Massas em Tandem , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Chromatin-related proteins have emerged as important players in the initiation and maintenance of several types of cancer. In addition to the established role of histone-modifying enzymes and chromatin remodelers in promoting and sustaining malignant phenotypes, recent findings suggest that the basic components of chromatin, the histone proteins, also suffer severe alterations in cancer and may contribute to the disease. Histopathological examination of clinical samples, characterization of the mutational landscape of various types of cancer and functional studies in cancer cell lines have highlighted the linker histone H1 both as a potential biomarker and a driver in cancer. This review summarizes H1 abnormalities in cancer identified by various approaches and critically discusses functional implications of such alterations, as well as potential mechanisms through which they may contribute to the disease.
Assuntos
Histonas/fisiologia , Neoplasias/etiologia , Animais , Linhagem Celular Tumoral , Histonas/genética , Humanos , Mutação , Neoplasias/metabolismoRESUMO
Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery. SIGNIFICANCE: Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution.
Assuntos
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/patologia , Epigenômica , Medicina de Precisão , Microambiente Tumoral/genéticaRESUMO
Dissecting mechanisms driving subclone expansion in primary cancers has been challenging. Here, we present a protocol to systematically disrupt entire gene networks and assess the functional impact of this perturbation on cancer cell fitness. By combining arrayed CRISPR libraries and high-content microscopy, we describe steps to identify classes of genes whose inactivation promotes resistance to environmental challenges faced by cancer cells during tumor growth or upon therapy. A proof-of-principle interrogation of the epigenetic regulatory network is described. For complete details on the use and execution of this protocol, please refer to Loukas et al. (2022).1.
Assuntos
Neoplasias , Humanos , Mutação/genética , Neoplasias/genética , Epigenômica , Redes Reguladoras de GenesRESUMO
The evolution of established cancers is driven by selection of cells with enhanced fitness. Subclonal mutations in numerous epigenetic regulator genes are common across cancer types, yet their functional impact has been unclear. Here, we show that disruption of the epigenetic regulatory network increases the tolerance of cancer cells to unfavorable environments experienced within growing tumors by promoting the emergence of stress-resistant subpopulations. Disruption of epigenetic control does not promote selection of genetically defined subclones or favor a phenotypic switch in response to environmental changes. Instead, it prevents cells from mounting an efficient stress response via modulation of global transcriptional activity. This "transcriptional numbness" lowers the probability of cell death at early stages, increasing the chance of long-term adaptation at the population level. Our findings provide a mechanistic explanation for the widespread selection of subclonal epigenetic-related mutations in cancer and uncover phenotypic inertia as a cellular trait that drives subclone expansion.
Assuntos
Neoplasias , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , FenótipoRESUMO
Mammalian genomes harbor abundant transposable elements (TEs) and their remnants, with numerous epigenetic repression mechanisms enacted to silence TE transcription. However, TEs are upregulated during early development, neuronal lineage, and cancers, although the epigenetic factors contributing to the transcription of TEs have yet to be fully elucidated. Here, we demonstrate that the male-specific lethal (MSL)-complex-mediated histone H4 acetylation at lysine 16 (H4K16ac) is enriched at TEs in human embryonic stem cells (hESCs) and cancer cells. This in turn activates transcription of subsets of full-length long interspersed nuclear elements (LINE1s, L1s) and endogenous retrovirus (ERV) long terminal repeats (LTRs). Furthermore, we show that the H4K16ac-marked L1 and LTR subfamilies display enhancer-like functions and are enriched in genomic locations with chromatin features associated with active enhancers. Importantly, such regions often reside at boundaries of topologically associated domains and loop with genes. CRISPR-based epigenetic perturbation and genetic deletion of L1s reveal that H4K16ac-marked L1s and LTRs regulate the expression of genes in cis. Overall, TEs enriched with H4K16ac contribute to the cis-regulatory landscape at specific genomic locations by maintaining an active chromatin landscape at TEs.
Assuntos
Elementos de DNA Transponíveis , Retrovirus Endógenos , Animais , Humanos , Masculino , Elementos de DNA Transponíveis/genética , Cromatina/genética , Sequências Reguladoras de Ácido Nucleico/genética , Retrovirus Endógenos/genética , Genômica , Mamíferos/genéticaRESUMO
Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease caused by a spontaneous point mutation in lamin A (encoded by LMNA), one of the major architectural elements of the mammalian cell nucleus. The HGPS mutation activates an aberrant cryptic splice site in LMNA pre-mRNA, leading to synthesis of a truncated lamin A protein and concomitant reduction in wild-type lamin A. Fibroblasts from individuals with HGPS have severe morphological abnormalities in nuclear envelope structure. Here we show that the cellular disease phenotype is reversible in cells from individuals with HGPS. Introduction of wild-type lamin A protein does not rescue the cellular disease symptoms. The mutant LMNA mRNA and lamin A protein can be efficiently eliminated by correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site. Upon splicing correction, HGPS fibroblasts assume normal nuclear morphology, the aberrant nuclear distribution and cellular levels of lamina-associated proteins are rescued, defects in heterochromatin-specific histone modifications are corrected and proper expression of several misregulated genes is reestablished. Our results establish proof of principle for the correction of the premature aging phenotype in individuals with HGPS.
Assuntos
Lamina Tipo A/metabolismo , Membrana Nuclear/patologia , Progéria/patologia , Processamento Alternativo , Linhagem Celular , Núcleo Celular , Regulação da Expressão Gênica , Humanos , Fenótipo , Progéria/metabolismo , Síndrome , TransfecçãoRESUMO
Rewiring of cellular programmes in malignant cells generates cancer-specific vulnerabilities. Here, using an unbiased screening strategy aimed at identifying non-essential genes required by tumour cells to sustain unlimited proliferative capacity, we identify the male-specific lethal (MSL) acetyltransferase complex as a vulnerability of genetically unstable cancers. We find that disruption of the MSL complex and consequent loss of the associated H4K16ac mark do not substantially alter transcriptional programmes but compromise chromosome integrity and promote chromosomal instability (CIN) that progressively exhausts the proliferative potential of cancer cells through a p53-independent mechanism. This effect is dependent on pre-existing genomic instability, and normal cells are insensitive to MSL disruption. Using cell- and patient-derived xenografts from multiple cancer types, we show that excessive CIN induced by MSL disruption inhibits tumour maintenance. Our findings suggest that targeting MSL may be a valuable means to increase CIN beyond the level tolerated by cancer cells without inducing severe adverse effects in normal tissues.
Assuntos
Proliferação de Células/genética , Instabilidade Cromossômica/genética , Complexos Multiproteicos/genética , Neoplasias/genética , Animais , Linhagem Celular Tumoral , Reprogramação Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Xenoenxertos , Histona Acetiltransferases/genética , Humanos , Camundongos , Neoplasias/patologia , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. In the last decade, several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure. In addition, we studied at the protein level the expression of these genes in cancer samples and performed functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. Remarkably, we found that 20% of lung cancer patients show ARID2 protein loss, partially explained by the presence of ARID2 mutations. In addition, we showed that ARID2 deficiency provokes profound chromatin structural changes altering cell transcriptional programs, which bolsters the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we demonstrated that ARID2 deficiency impairs DNA repair, enhancing the sensitivity of the cells to DNA-damaging agents. Our findings support that ARID2 is a bona fide tumor suppressor gene in lung cancer that may be exploited therapeutically.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição/deficiência , Células A549 , Animais , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Taxa de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Continuous cancer growth is driven by subsets of self-renewing malignant cells. Targeting of uncontrolled self-renewal through inhibition of stem cell-related signaling pathways has proven challenging. Here, we show that cancer cells can be selectively deprived of self-renewal ability by interfering with their epigenetic state. Re-expression of histone H1.0, a tumor-suppressive factor that inhibits cancer cell self-renewal in many cancer types, can be broadly induced by the clinically well-tolerated compound Quisinostat. Through H1.0, Quisinostat inhibits cancer cell self-renewal and halts tumor maintenance without affecting normal stem cell function. Quisinostat also hinders expansion of cells surviving targeted therapy, independently of the cancer types and the resistance mechanism, and inhibits disease relapse in mouse models of lung cancer. Our results identify H1.0 as a major mediator of Quisinostat's antitumor effect and suggest that sequential administration of targeted therapy and Quisinostat may be a broadly applicable strategy to induce a prolonged response in patients.
Assuntos
Autorrenovação Celular , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Neoplasias/genética , RecidivaRESUMO
Genetic diseases often reveal the physiological roles of the affected proteins. The identification of mutations in the nuclear envelope proteins lamin A and lamin C as the cause of a diverse group of human diseases has expanded our understanding of the lamin proteins from being merely structural elements of the cell nucleus and has implicated them in novel cellular functions including signal transduction and gene expression. However, it now appears that the physiological relevance of one of the lamin proteins in organismal function has been overestimated. In this issue of the JCI, Fong et al. demonstrate that lamin A-deficient mice are phenotypically normal (see the related article beginning on page 743). The good news is these findings open the door to a new strategy for the therapeutic treatment of diseases caused by mutations in lamin A, such as muscular dystrophies and some types of premature aging syndromes.
Assuntos
Lamina Tipo A/deficiência , Lamina Tipo A/genética , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Membrana Nuclear/genética , Animais , Lamina Tipo A/fisiologia , Doenças Metabólicas/patologia , Camundongos , Camundongos Knockout , Membrana Nuclear/fisiologiaRESUMO
The identification of cancer-promoting genetic alterations is challenging particularly in highly unstable and heterogeneous cancers, such as esophageal adenocarcinoma (EAC). Here we describe a machine learning algorithm to identify cancer genes in individual patients considering all types of damaging alterations simultaneously. Analysing 261 EACs from the OCCAMS Consortium, we discover helper genes that, alongside well-known drivers, promote cancer. We confirm the robustness of our approach in 107 additional EACs. Unlike recurrent alterations of known drivers, these cancer helper genes are rare or patient-specific. However, they converge towards perturbations of well-known cancer processes. Recurrence of the same process perturbations, rather than individual genes, divides EACs into six clusters differing in their molecular and clinical features. Experimentally mimicking the alterations of predicted helper genes in cancer and pre-cancer cells validates their contribution to disease progression, while reverting their alterations reveals EAC acquired dependencies that can be exploited in therapy.
Assuntos
Adenocarcinoma/genética , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias Esofágicas/genética , Perfilação da Expressão Gênica/métodos , Medicina de Precisão/métodos , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Progressão da Doença , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Instabilidade Genômica , Humanos , Aprendizado de Máquina , Modelos Genéticos , Família Multigênica/efeitos dos fármacos , Taxa de Mutação , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Genome structure and gene expression depend on a multitude of chromatin-binding proteins. The binding properties of these proteins to native chromatin in intact cells are largely unknown. Here, we describe an approach based on combined in vivo photobleaching microscopy and kinetic modeling to analyze globally the dynamics of binding of chromatin-associated proteins in living cells. We have quantitatively determined basic biophysical properties, such as off rate constants, residence time, and bound fraction, of a wide range of chromatin proteins of diverse functions in vivo. We demonstrate that most chromatin proteins have a high turnover on chromatin with a residence time on the order of seconds, that the major fraction of each protein is bound to chromatin at steady state, and that transient binding is a common property of chromatin-associated proteins. Our results indicate that chromatin-binding proteins find their binding sites by three-dimensional scanning of the genome space and our data are consistent with a model in which chromatin-associated proteins form dynamic interaction networks in vivo. We suggest that these properties are crucial for generating high plasticity in genome expression.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genoma , Modelos Biológicos , Animais , Linhagem Celular , Recuperação de Fluorescência Após Fotodegradação , Humanos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoAssuntos
Reparo do DNA/fisiologia , Instabilidade Genômica , Lamina Tipo A/genética , Progéria/genética , Animais , Lamina Tipo A/metabolismo , Lipoproteínas/genética , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Metaloproteases/genética , Camundongos , Camundongos Mutantes , MutaçãoRESUMO
High mobility group box (HMGB) 1 and 2 are two abundant nonhistone nuclear proteins that have been found in association with chromatin. Previous studies based on immunofluorescence analysis indicated that HMGB1 dissociates from chromosomes during mitosis. In the present work, HMGB1 and 2 subcellular localization was reinvestigated in living cells by using enhanced green fluorescent protein- and Discosome sp. red fluorescent protein-tagged proteins. Contrary to previous reports, HMGB1 and 2 were shown to be present under two forms in mitotic cells, i.e., free and associated with the condensed chromatin, which rapidly exchange. A detailed analysis of HMGB2 interaction with mitotic chromosomes indicated that two sites encompassing HMG-box A and B are responsible for binding. Importantly, this interaction was rapidly inactivated when cells were permeabilized or exposed to chemical fixatives that are widely used in immunodetection techniques. A comparable behavior was also observed for two proteins of the HMG-nucleosome binding (HMGN) group, namely, HMGN1 and HMGN2.
Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Proteína HMGB1/metabolismo , Proteína HMGB2/metabolismo , Células 3T3 , Animais , Membrana Celular/metabolismo , Células Cultivadas , Cromatina/fisiologia , Proteínas de Fluorescência Verde , Proteínas HMGN/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes , Camundongos , Mitose/fisiologia , Permeabilidade , Proteínas Recombinantes , Proteína Vermelha FluorescenteRESUMO
Epigenetic mechanisms have emerged as key players in cancer development which affect cellular states at multiple stages of the disease. During carcinogenesis, alterations in chromatin and DNA methylation resulting from genetic lesions unleash cellular plasticity and favor oncogenic cellular reprogramming. At later stages, during cancer growth and progression, additional epigenetic changes triggered by interaction with the microenvironment modulate cancer cell phenotypes and properties, and shape tumor architecture. We review here recent advances highlighting the interplay between epigenetics, genetics, and cell-to-cell signaling in cancer, with particular emphasis on mechanisms relevant for cancer stem cell formation (CSC) and function.
Assuntos
Plasticidade Celular/genética , Reprogramação Celular/genética , Epigênese Genética/genética , Células-Tronco Neoplásicas/metabolismo , Humanos , Células-Tronco Neoplásicas/patologiaRESUMO
The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions.
Assuntos
Sistemas CRISPR-Cas , Epigênese Genética , Biblioteca Gênica , RNA Guia de Cinetoplastídeos/genética , Animais , Clonagem Molecular/métodos , Vetores Genéticos/genética , Células Hep G2 , Humanos , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation status, patient survival, and, at the single-cell level, cancer stem cell markers. Silencing of H1.0 promotes maintenance of self-renewing cells by inducing derepression of megabase-sized gene domains harboring downstream effectors of oncogenic pathways. Self-renewing epigenetic states are not stable, and reexpression of H1.0 in subsets of tumor cells establishes transcriptional programs that restrict cancer cells' long-term proliferative potential and drive their differentiation. Our results uncover epigenetic determinants of tumor-maintaining cells.