RESUMEN
During self-renewal, cell-type-defining features are drastically perturbed in mitosis and must be faithfully reestablished upon G1 entry, a process that remains largely elusive. Here, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. We captured distinct waves of transcriptional reactivation with rapid induction of stem cell genes and transient activation of lineage-specific genes. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed partial coordination with transcriptional resetting. Globally, rapid transcriptional and architectural resetting associated with mitotic retention of H3K27 acetylation, supporting a bookmarking function. Indeed, mitotic depletion of H3K27ac impaired the early reactivation of bookmarked, stem-cell-associated genes. However, 3D chromatin reorganization remained largely unaffected, suggesting that these processes are driven by distinct forces upon mitotic exit. This study uncovers principles and mediators of PSC molecular resetting during self-renewal.
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Cromatina/genética , Código de Histonas/genética , Histonas/genética , Mitosis/genética , Células Madre Pluripotentes/fisiología , Acetilación , Animales , Línea Celular , Drosophila/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Transcripción Genética/genética , Activación Transcripcional/genéticaRESUMEN
Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.
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Alelos , Proteínas de Unión al Calcio/genética , Metilación de ADN/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Animales , Cromosomas/genética , Impresión Genómica/genética , Yoduro Peroxidasa/genética , Ratones , Regiones Promotoras Genéticas/genética , ARN Largo no Codificante/genéticaRESUMEN
Higher-order chromatin structure is tightly linked to gene expression and therefore cell identity. In recent years, the chromatin landscape of pluripotent stem cells has become better characterized, and unique features at various architectural levels have been revealed. However, the mechanisms that govern establishment and maintenance of these topological characteristics and the temporal and functional relationships with transcriptional or epigenetic features are still areas of intense study. Here, we will discuss progress and limitations of our current understanding regarding how the 3D chromatin topology of pluripotent stem cells is established during somatic cell reprogramming and maintained during cell division. We will also discuss evidence and theories about the driving forces of topological reorganization and the functional links with key features and properties of pluripotent stem cell identity.
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División Celular , Reprogramación Celular , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Humanos , Células Madre Pluripotentes/citologíaRESUMEN
TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP-seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.
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Metilación de ADN , Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/metabolismo , Oxigenasas de Función Mixta/fisiología , Regiones Promotoras Genéticas , Animales , Diferenciación Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Dioxigenasas/fisiología , Células Madre Embrionarias/citología , Humanos , Ratones , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Mutación , Placa Neural/citología , Factor de Transcripción PAX6/biosíntesis , Factor de Transcripción PAX6/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiologíaRESUMEN
In the version of this article initially published, in the Methods, the Gene Expression Omnibus accession code for H3K36me3 ChIP-seq data was incorrectly given as GSM1003585 instead of GSM733725. The error has been corrected in the HTML, PDF and print versions of the article.
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The version of the Supplementary Text and Figures file initially posted was missing Supplementary Tables 1-6 and the Supplementary Note and used incorrect versions of the supplementary figures.
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During mitosis, transcription is halted and many chromatin features are lost, posing a challenge for the continuity of cell identity, particularly in fast cycling stem cells, which constantly balance self-renewal with differentiation. Here we show that, in pluripotent stem cells, certain histone marks and stem cell regulators remain associated with specific genomic regions of mitotic chromatin, a phenomenon known as mitotic bookmarking. Enhancers of stem cell-related genes are bookmarked by both H3K27ac and the master regulators OCT4, SOX2, and KLF4, while promoters of housekeeping genes retain high levels of mitotic H3K27ac in a cell-type invariant manner. Temporal degradation of OCT4 during mitotic exit compromises its ability both to maintain and induce pluripotency, suggesting that its regulatory function partly depends on its bookmarking activity. Together, our data document a widespread yet specific bookmarking by histone modifications and transcription factors promoting faithful and efficient propagation of stemness after cell division.
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Código de Histonas , Mitosis , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Cromatina/metabolismo , Histonas/metabolismo , Humanos , Factor 4 Similar a Kruppel , Lisina/metabolismo , ProteolisisRESUMEN
Membrane-less organelles are intracellular compartments specialized to carry out specific cellular functions. There is growing evidence supporting the possibility that such organelles form as a new phase, separating from cytoplasm or nucleoplasm. However, a main challenge to such phase separation models is that the initial assembly, or nucleation, of the new phase is typically a highly stochastic process and does not allow for the spatiotemporal precision observed in biological systems. Here, we investigate the initial assembly of the nucleolus, a membrane-less organelle involved in different cellular functions including ribosomal biogenesis. We demonstrate that the nucleolus formation is precisely timed in D. melanogaster embryos and follows the transcription of rRNA. We provide evidence that transcription of rRNA is necessary for overcoming the highly stochastic nucleation step in the formation of the nucleolus, through a seeding mechanism. In the absence of rDNA, the nucleolar proteins studied are able to form high-concentration assemblies. However, unlike the nucleolus, these assemblies are highly variable in number, location, and time at which they form. In addition, quantitative study of the changes in the nucleoplasmic concentration and distribution of these nucleolar proteins in the wild-type embryos is consistent with the role of rRNA in seeding the nucleolus formation.