RESUMO
The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurônios/fisiologia , RNA , Anêmonas-do-Mar/fisiologia , Actinas/química , Motivos de Aminoácidos , Animais , Cromatina/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Genoma , Genômica , Filogenia , Anêmonas-do-Mar/genética , Análise de Sequência de RNA , Transcriptoma , Tubulina (Proteína)/químicaRESUMO
The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.
Assuntos
Elementos Facilitadores Genéticos , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Extremidades/fisiologia , Proteínas de Homeodomínio , Humanos , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , XenopusRESUMO
Somatic structural variations (SVs) in cancer can shuffle DNA content in the genome, relocate regulatory elements, and alter genome organization. Enhancer hijacking occurs when SVs relocate distal enhancers to activate proto-oncogenes. However, most enhancer hijacking studies have only focused on protein-coding genes. Here, we develop a computational algorithm 'HYENA' to identify candidate oncogenes (both protein-coding and non-coding) activated by enhancer hijacking based on tumor whole-genome and transcriptome sequencing data. HYENA detects genes whose elevated expression is associated with somatic SVs by using a rank-based regression model. We systematically analyze 1146 tumors across 25 types of adult tumors and identify a total of 108 candidate oncogenes including many non-coding genes. A long non-coding RNA TOB1-AS1 is activated by various types of SVs in 10% of pancreatic cancers through altered 3-dimensional genome structure. We find that high expression of TOB1-AS1 can promote cell invasion and metastasis. Our study highlights the contribution of genetic alterations in non-coding regions to tumorigenesis and tumor progression.
RESUMO
In the originally published version of this Letter, ref. 43 was erroneously provided twice. In the 'Estimation of relative cell-type-specific composition of AML samples' section in the Methods, the citation to ref. 43 after the GEO dataset GSE24759 is correct. However, in the 'Mice' section of the Methods, the citation to ref. 43 after 'TAMERE' should have been associated with a new reference1. The original Letter has been corrected online (with the new reference included as ref. 49).
RESUMO
The transcription factor Myc is essential for the regulation of haematopoietic stem cells and progenitors and has a critical function in haematopoietic malignancies. Here we show that an evolutionarily conserved region located 1.7 megabases downstream of the Myc gene that has previously been labelled as a 'super-enhancer' is essential for the regulation of Myc expression levels in both normal haematopoietic and leukaemic stem cell hierarchies in mice and humans. Deletion of this region in mice leads to a complete loss of Myc expression in haematopoietic stem cells and progenitors. This caused an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, mimicking the phenotype caused by Mx1-Cre-mediated conditional deletion of the Myc gene in haematopoietic stem cells. This super-enhancer comprises multiple enhancer modules with selective activity that recruits a compendium of transcription factors, including GFI1b, RUNX1 and MYB. Analysis of mice carrying deletions of individual enhancer modules suggests that specific Myc expression levels throughout most of the haematopoietic hierarchy are controlled by the combinatorial and additive activity of individual enhancer modules, which collectively function as a 'blood enhancer cluster' (BENC). We show that BENC is also essential for the maintenance of MLL-AF9-driven leukaemia in mice. Furthermore, a BENC module, which controls Myc expression in mouse haematopoietic stem cells and progenitors, shows increased chromatin accessibility in human acute myeloid leukaemia stem cells compared to blasts. This difference correlates with MYC expression and patient outcome. We propose that clusters of enhancers, such as BENC, form highly combinatorial systems that allow precise control of gene expression across normal cellular hierarchies and which also can be hijacked in malignancies.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Genes myc/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Leucemia/genética , Leucemia/patologia , Família Multigênica/genética , Animais , Linfócitos B/citologia , Diferenciação Celular , Linhagem da Célula/genética , Cromatina/genética , Cromatina/metabolismo , Regulação para Baixo , Feminino , Deleção de Genes , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes/citologia , Células Mieloides/citologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Deleção de Sequência , Análise de Sobrevida , Fatores de Transcrição/metabolismoRESUMO
Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.
Assuntos
Músculos Oculomotores/embriologia , Músculos Oculomotores/crescimento & desenvolvimento , Tretinoína/metabolismo , Animais , Tecido Conjuntivo/fisiologia , Desenvolvimento Embrionário , Olho , Imageamento Tridimensional/métodos , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Morfogênese , Transdução de Sinais , Tendões/fisiologia , Tretinoína/fisiologiaRESUMO
Imaging and chromosome conformation capture studies have revealed several layers of chromosome organization, including segregation into megabase-sized active and inactive compartments, and partitioning into sub-megabase domains (TADs). It remains unclear, however, how these layers of organization form, interact with one another and influence genome function. Here we show that deletion of the cohesin-loading factor Nipbl in mouse liver leads to a marked reorganization of chromosomal folding. TADs and associated Hi-C peaks vanish globally, even in the absence of transcriptional changes. By contrast, compartmental segregation is preserved and even reinforced. Strikingly, the disappearance of TADs unmasks a finer compartment structure that accurately reflects the underlying epigenetic landscape. These observations demonstrate that the three-dimensional organization of the genome results from the interplay of two independent mechanisms: cohesin-independent segregation of the genome into fine-scale compartments, defined by chromatin state; and cohesin-dependent formation of TADs, possibly by loop extrusion, which helps to guide distant enhancers to their target genes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Posicionamento Cromossômico , Animais , Cromatina/química , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Fígado/metabolismo , Camundongos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , CoesinasRESUMO
Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Variações do Número de Cópias de DNA/genética , Doença/genética , Duplicação Gênica/genética , Animais , DNA/genética , Fácies , Feminino , Fibroblastos , Dedos/anormalidades , Deformidades Congênitas do Pé/genética , Expressão Gênica , Genômica , Deformidades Congênitas da Mão/genética , Masculino , Camundongos , Fenótipo , Fatores de Transcrição SOX9/genéticaRESUMO
Genomic structural variants have long been implicated in phenotypic diversity and human disease, but dissecting the mechanisms by which they exert their functional impact has proven elusive. Recently however, developments in high-throughput DNA sequencing and chromosomal engineering technology have facilitated the analysis of structural variants in human populations and model systems in unprecedented detail. In this Review, we describe how structural variants can affect molecular and cellular processes, leading to complex organismal phenotypes, including human disease. We further present advances in delineating disease-causing elements that are affected by structural variants, and we discuss future directions for research on the functional consequences of structural variants.
Assuntos
Doença/genética , Estudos de Associação Genética , Variação Estrutural do Genoma , Animais , Biologia Computacional , Modelos Animais de Doenças , Epistasia Genética , Dosagem de Genes , Redes Reguladoras de Genes , Doenças Genéticas Inatas/genética , Humanos , Camundongos , Modelos Genéticos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Large-scale identification of elements associated with gene expression revealed that many of them are located extremely far from gene transcriptional start sites. We review here the growing evidence that show that distal cis-acting elements provide key instructions to genes, as genetic variation affecting them is growingly identified as an importance source of phenotypic diversity and disease. We discuss the different mechanisms that allow these elements to exert their regulatory functions, in a robust and specific manner, despite the large genomic distances separating them from their target genes. We particularly focus on the role of the structural organization of the genome in guiding such regulatory interactions.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Animais , Redes Reguladoras de Genes , Genoma , Humanos , Modelos GenéticosRESUMO
Developmental progression is driven by specific spatiotemporal domains of gene expression, which give rise to stereotypically patterned embryos even in the presence of environmental and genetic variation. Views of how transcription factors regulate gene expression are changing owing to recent genome-wide studies of transcription factor binding and RNA expression. Such studies reveal patterns that, at first glance, seem to contrast with the robustness of the developmental processes they encode. Here, we review our current knowledge of transcription factor function from genomic and genetic studies and discuss how different strategies, including extensive cooperative regulation (both direct and indirect), progressive priming of regulatory elements, and the integration of activities from multiple enhancers, confer specificity and robustness to transcriptional regulation during development.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Cromatina/metabolismo , Humanos , Camundongos , Regiões Promotoras Genéticas , Ratos , Transcrição GênicaRESUMO
Despite the well-documented role of remote enhancers in controlling developmental gene expression, the mechanisms that allocate enhancers to genes are poorly characterized. Here, we investigate the cis-regulatory organization of the locus containing the Tfap2c and Bmp7 genes in vivo, using a series of engineered chromosomal rearrangements. While these genes lie adjacent to one another, we demonstrate that they are independently regulated by distinct sets of enhancers, which in turn define non-overlapping regulatory domains. Chromosome conformation capture experiments reveal a corresponding partition of the locus in two distinct structural entities, demarcated by a discrete transition zone. The impact of engineered chromosomal rearrangements on the topology of the locus and the resultant gene expression changes indicate that this transition zone functionally organizes the structural partition of the locus, thereby defining enhancer-target gene allocation. This partition is, however, not absolute: we show that it allows competing interactions across it that may be non-productive for the competing gene, but modulate expression of the competed one. Altogether, these data highlight the prime role of the topological organization of the genome in long-distance regulation of gene expression.
Assuntos
Proteína Morfogenética Óssea 7/genética , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos , Coração/crescimento & desenvolvimento , Fator de Transcrição AP-2/genética , Animais , Blastocisto/citologia , Proteína Morfogenética Óssea 7/biossíntese , Cromossomos/genética , Elementos de DNA Transponíveis/genética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Camundongos , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição AP-2/biossínteseRESUMO
Copy number variations on human chromosome 15q11-q13 have been implicated in several neurodevelopmental disorders. A paternal loss or duplication of the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region confers a risk of obesity, although the mechanism remains a mystery due to a lack of an animal model that accurately recreates the obesity phenotype. We performed detailed analyses of mice with duplication of PWS/AS locus (6 Mb) generated by chromosome engineering and found that animals with a paternal duplication of this region (patDp/+) show late-onset obesity, high sensitivity for high-fat diet, high levels of blood leptin and insulin without an increase in food intake. We show that prior to becoming obese, young patDp/+ mice already had enlarged white adipocytes. Transcriptome analysis of adipose tissue revealed an up-regulation of Secreted frizzled-related protein 5 (Sfrp5), known to promote adipogenesis. We additionally generated a new mouse model of paternal duplication focusing on a 3 Mb region (3 Mb patDp/+) within the PWS/AS locus. These mice recapitulate the obese phenotypes including expansion of visceral adipose tissue. Our results suggest paternally expressed genes in PWS/AS locus play a significant role for the obesity and identify new potential targets for future research and treatment of obesity.
Assuntos
Duplicação Cromossômica , Cromossomos Humanos Par 15/genética , Loci Gênicos , Metabolismo dos Lipídeos/genética , Obesidade , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , SíndromeRESUMO
Long-range regulatory interactions play an important role in shaping gene-expression programs. However, the genomic features that organize these activities are still poorly characterized. We conducted a large operational analysis to chart the distribution of gene regulatory activities along the mouse genome, using hundreds of insertions of a regulatory sensor. We found that enhancers distribute their activities along broad regions and not in a gene-centric manner, defining large regulatory domains. Remarkably, these domains correlate strongly with the recently described TADs, which partition the genome into distinct self-interacting blocks. Different features, including specific repeats and CTCF-binding sites, correlate with the transition zones separating regulatory domains, and may help to further organize promiscuously distributed regulatory influences within large domains. These findings support a model of genomic organization where TADs confine regulatory activities to specific but large regulatory domains, contributing to the establishment of specific gene expression profiles.
Assuntos
Sítios de Ligação , Elementos Facilitadores Genéticos , Animais , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos , Genoma , Camundongos , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Proteínas Repressoras/metabolismo , CoesinasRESUMO
Established transgenesis methods for fish model systems allow efficient genomic integration of transgenes. However, thus far a way of controlling copy number and integration sites has not been available, leading to variable transgene expression caused by position effects. The integration of transgenes at predefined genomic positions enables the direct comparison of different transgenes, thereby improving time and cost efficiency. Here, we report an efficient PhiC31-based site-specific transgenesis system for medaka. This system includes features that allow the pre-selection of successfully targeted integrations early on in the injected generation. Pre-selected embryos transmit the correctly integrated transgene through the germline with high efficiency. The landing site design enables a variety of applications, such as reporter and enhancer switch, in addition to the integration of any insert. Importantly, this allows assaying of enhancer activity in a site-specific manner without requiring germline transmission, thus speeding up large-scale analyses of regulatory elements.
Assuntos
Proteínas de Choque Térmico HSP70/genética , Integrases/genética , Oryzias/genética , Animais , Animais Geneticamente Modificados , DNA/genética , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Integrases/metabolismo , Oryzias/metabolismo , Regiões Promotoras Genéticas , Transgenes/genética , Peixe-Zebra/genéticaRESUMO
More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5' untranslated region (5'UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5'UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.
Assuntos
Células-Tronco Embrionárias/metabolismo , Retrovirus Endógenos/genética , Inativação Gênica , Genes de Partícula A Intracisternal/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Regiões 5' não Traduzidas/genética , Acetilação , Animais , Metilação de DNA , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/virologia , Células-Tronco Embrionárias/virologia , Fibroblastos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/fisiologia , Lisina/metabolismo , Metilação , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Proteína 28 com Motivo TripartidoRESUMO
The developmental regulation of vertebrate Hox gene transcription relies on the interplay between local and long-range controls. To study this complex genomic organization, we designed a strategy combining meiotic and targeted recombinations to induce large chromosomal rearrangements in vivo without manipulating embryonic stem cells. With this simple approach (called STRING), we engineered a large 7-cM inversion, which split the Hoxd cluster into two independent pieces. Expression analyses showed a partition of global enhancers, allowing for their precise topographic allocation on either side of the cluster. Such a functional organization probably contributed to keeping these genes clustered in the course of vertebrate evolution. This approach can be used to study the relationship between genome architecture and gene expression, such as the effects of genome rearrangements in human diseases or during evolution.
Assuntos
Inversão Cromossômica , Regulação da Expressão Gênica/genética , Genes Homeobox , Família Multigênica , Animais , Evolução Molecular , Hibridização in Situ Fluorescente , CamundongosRESUMO
Somatic structural variations (SVs) in cancer can shuffle DNA content in the genome, relocate regulatory elements, and alter genome organization. Enhancer hijacking occurs when SVs relocate distal enhancers to activate proto-oncogenes. However, most enhancer hijacking studies have only focused on protein-coding genes. Here, we develop a computational algorithm "HYENA" to identify candidate oncogenes (both protein-coding and non-coding) activated by enhancer hijacking based on tumor whole-genome and transcriptome sequencing data. HYENA detects genes whose elevated expression is associated with somatic SVs by using a rank-based regression model. We systematically analyze 1,146 tumors across 25 types of adult tumors and identify a total of 108 candidate oncogenes including many non-coding genes. A long non-coding RNA TOB1-AS1 is activated by various types of SVs in 10% of pancreatic cancers through altered 3-dimensional genome structure. We find that high expression of TOB1-AS1 can promote cell invasion and metastasis. Our study highlights the contribution of genetic alterations in non-coding regions to tumorigenesis and tumor progression.
RESUMO
MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.