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BACKGROUND: c-Jun is a proto-oncogene functioning as a transcription factor to activate gene expression under many physiological and pathological conditions, particularly in somatic cells. However, its role in early embryonic development remains unknown. RESULTS: Here, we show that c-Jun acts as a one-way valve to preserve the primed state and impair reversion to the naïve state. c-Jun is induced during the naive to primed transition, and it works to stabilize the chromatin structure and inhibit the reverse transition. Loss of c-Jun has surprisingly little effect on the naïve to primed transition, and no phenotypic effect on primed cells, however, in primed cells the loss of c-Jun leads to a failure to correctly close naïve-specific enhancers. When the primed cells are induced to reprogram to a naïve state, these enhancers are more rapidly activated when c-Jun is lost or impaired, and the conversion is more efficient. CONCLUSIONS: The results of this study indicate that c-Jun can function as a chromatin stabilizer in primed EpiSCs, to maintain the epigenetic cell type state and act as a one-way valve for cell fate conversions.
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Somatic cell reprogramming and oncogenic transformation share surprisingly similar features, yet transformed cells are resistant to reprogramming. Epigenetic barriers must block transformed cells from reprogramming, but the nature of those barriers is unclear. In this study, we generated a systematic panel of transformed mouse embryonic fibroblasts (MEFs) using oncogenic transgenes and discovered transformed cell lines compatible with reprogramming when transfected with Oct4/Sox2/Klf4/Myc. By comparing the reprogramming-capable and incapable transformed lines we identified multiple stages of failure in the reprogramming process. Some transformed lines failed at an early stage, whilst other lines seemed to progress through a conventional reprogramming process. Finally, we show that MEK inhibition overcomes one critical reprogramming barrier by indirectly suppressing a hyperacetylated active epigenetic state. This study reveals that diverse epigenetic barriers underly resistance to reprogramming of transformed cells.
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Around 60% of in vitro fertilized (IVF) human embryos irreversibly arrest before compaction between the 3- to 8-cell stage, posing a significant clinical problem. The mechanisms behind this arrest are unclear. Here, we show that the arrested embryos enter a senescent-like state, marked by cell cycle arrest, the down-regulation of ribosomes and histones and down-regulation of MYC and p53 activity. The arrested embryos can be divided into 3 types. Type I embryos fail to complete the maternal-zygotic transition, and Type II/III embryos have low levels of glycolysis and either high (Type II) or low (Type III) levels of oxidative phosphorylation. Treatment with the SIRT agonist resveratrol or nicotinamide riboside (NR) can partially rescue the arrested phenotype, which is accompanied by changes in metabolic activity. Overall, our data suggests metabolic and epigenetic dysfunctions underlie the arrest of human embryos.
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Embrión de Mamíferos , Fertilización In Vitro , Embrión de Mamíferos/metabolismo , Epigénesis Genética , Histonas/metabolismo , Humanos , Cigoto/metabolismoRESUMEN
Transposable elements (TEs) occupy nearly 40% of mammalian genomes and, whilst most are fragmentary and no longer capable of transposition, they can nevertheless contribute to cell function. TEs within genes transcribed by RNA polymerase II can be copied as parts of primary transcripts; however, their full contribution to mature transcript sequences remains unresolved. Here, using long and short read (LR and SR) RNA sequencing data, we show that 26% of coding and 65% of noncoding transcripts in human pluripotent stem cells (hPSCs) contain TE-derived sequences. Different TE families are incorporated into RNAs in unique patterns, with consequences to transcript structure and function. The presence of TE sequences within a transcript is correlated with TE-type specific changes in its subcellular distribution, alterations in steady-state levels and half-life, and differential association with RNA Binding Proteins (RBPs). We identify hPSC-specific incorporation of endogenous retroviruses (ERVs) and LINE:L1 into protein-coding mRNAs, which generate TE sequence-derived peptides. Finally, single cell RNA-seq reveals that hPSCs express ERV-containing transcripts, whilst differentiating subpopulations lack ERVs and express SINE and LINE-containing transcripts. Overall, our comprehensive analysis demonstrates that the incorporation of TE sequences into the RNAs of hPSCs is more widespread and has a greater impact than previously appreciated.
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Retrovirus Endógenos/genética , Elementos de Nucleótido Esparcido Largo/genética , Células Madre Pluripotentes/metabolismo , Transcriptoma , Línea Celular , Humanos , ARN no Traducido/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
A major event in embryonic development is the rearrangement of epigenetic information as the somatic genome is reprogrammed for a new round of organismal development. Epigenetic data are held in chemical modifications on DNA and histones, and there are dramatic and dynamic changes in these marks during embryogenesis. However, the mechanisms behind this intricate process and how it is regulating and responding to embryonic development remain unclear. As embryos develop from totipotency to pluripotency, they pass through several distinct stages that can be captured permanently or transiently in vitro. Pluripotent naïve cells resemble the early epiblast, primed cells resemble the late epiblast, and blastomere-like cells have been isolated, although fully totipotent cells remain elusive. Experiments using these in vitro model systems have led to insights into chromatin changes in embryonic development, which has informed exploration of pre-implantation embryos. Intriguingly, human and mouse cells rely on different signaling and epigenetic pathways, and it remains a mystery why this variation exists. In this review, we will summarize the chromatin rearrangements in early embryonic development, drawing from genomic data from in vitro cell lines, and human and mouse embryos.
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Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that ß-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by ß-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.
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SUMMARY: Cells are generally resistant to cell type conversions, but can be converted by the application of growth factors, chemical inhibitors and ectopic expression of genes. However, it remains difficult to accurately identify the destination cell type or differentiation bias when these techniques are used to alter cell type. Consequently, there is demand for computational techniques that can help researchers understand both the cell type and differentiation bias. While advanced tools identifying cell types exist for single cell data and the deconvolution of mixed cell populations, the problem of exploring partially differentiated cells of indeterminate transcriptional identity has not been addressed. To fill this gap, we developed driver-predictor, which relies on scoring per gene transcriptional similarity between RNA-Seq datasets to reveal directional bias of differentiation. By comparing against large cell type transcriptome libraries or a desired target expression profile, the tool enables the user to visualize both the changes in transcriptional identity as well as the genes accounting for the cell type changes. This software will be a powerful tool for researchers to explore in vitro experiments that involve cell type conversions. AVAILABILITY AND IMPLEMENTATION: Source code is open source under the MIT license and is freely available on https://github.com/LoaloaF/DPre. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Programas Informáticos , Transcriptoma , Diferenciación Celular , Biología ComputacionalRESUMEN
The majority of mammalian genomes are devoted to transposable elements (TEs). Whilst TEs are increasingly recognized for their important biological functions, they are a potential danger to genomic stability and are carefully regulated by the epigenetic system. However, the full complexity of this regulatory system is not understood. Here, using mouse embryonic stem cells, we show that TEs are suppressed by heterochromatic marks like H3K9me3, and are also labelled by all major types of chromatin modification in complex patterns, including bivalent activatory and repressive marks. We identified 29 epigenetic modifiers that significantly deregulated at least one type of TE. The loss of Setdb1, Ncor2, Rnf2, Kat5, Prmt5, Uhrf1, and Rrp8 caused widespread changes in TE expression and chromatin accessibility. These effects were context-specific, with different chromatin modifiers regulating the expression and chromatin accessibility of specific subsets of TEs. Our work reveals the complex patterns of epigenetic regulation of TEs.
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Cromatina/metabolismo , Elementos Transponibles de ADN/genética , Epigénesis Genética , Histonas/metabolismo , Animales , Línea Celular , Cromatina/genética , Metilación de ADN/genética , Técnicas de Silenciamiento del Gen , Código de Histonas , Histonas/genética , Ratones , Células Madre Embrionarias de RatonesRESUMEN
In the version of this Article originally published, in Fig. 2c, the '+' sign and 'OSKM' were superimposed in the label '+OSKM'. In Fig. 4e, in the labels, all instances of 'Ant' should have been 'Anti-'. And, in Fig. 7a, the label '0.0' was misplaced; it should have been on the colour scale bar. These figures have now been corrected in the online versions.
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Somatic cell reprogramming by exogenous factors requires cooperation with transcriptional co-activators and co-repressors to effectively remodel the epigenetic environment. How this interplay is regulated remains poorly understood. Here, we demonstrate that NCoR/SMRT co-repressors bind to pluripotency loci to create a barrier to reprogramming with the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC), and consequently, suppressing NCoR/SMRT significantly enhances reprogramming efficiency and kinetics. The core epigenetic subunit of the NCoR/SMRT complex, histone deacetylase 3 (HDAC3), contributes to the effects of NCoR/SMRT by inducing histone deacetylation at pluripotency loci. Among the Yamanaka factors, recruitment of NCoR/SMRT-HDAC3 to genomic loci is mostly facilitated by c-MYC. Hence, we describe how c-MYC is beneficial for the early phase of reprogramming but deleterious later. Overall, we uncover a role for NCoR/SMRT co-repressors in reprogramming and propose a dual function for c-MYC in this process.
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Reprogramación Celular , Epigénesis Genética , Células Madre Embrionarias de Ratones/metabolismo , Co-Represor 1 de Receptor Nuclear/metabolismo , Co-Represor 2 de Receptor Nuclear/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Acetilación , Animales , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos ICR , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
The current classification of cells in an organism is largely based on their anatomic and developmental origin. Cells types and tissues are traditionally classified into those that arise from the three embryonic germ layers, the ectoderm, mesoderm and endoderm, but this model does not take into account the organization of cell type-specific patterns of gene expression. Here, we present computational models for cell type and tissue specification derived from a collection of 921 RNA-sequencing samples from 272 distinct mouse cell types or tissues. In an unbiased fashion, this analysis accurately predicts the three known germ layers. Unexpectedly, this analysis also suggests that in total there are eight major domains of cell type-specification, corresponding to the neurectoderm, neural crest, surface ectoderm, endoderm, mesoderm, blood mesoderm, germ cells and the embryonic domain. Further, we identify putative genes responsible for specifying the domain and the cell type. This model has implications for understanding trans-lineage differentiation for stem cells, developmental cell biology and regenerative medicine.
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Linaje de la Célula/genética , Ectodermo/metabolismo , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Mesodermo/metabolismo , Animales , Diferenciación Celular , Ectodermo/citología , Ectodermo/crecimiento & desarrollo , Endodermo/citología , Endodermo/crecimiento & desarrollo , Ontología de Genes , Mesodermo/citología , Mesodermo/crecimiento & desarrollo , Ratones , Anotación de Secuencia Molecular , Especificidad de Órganos , Análisis de Componente Principal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Organisms are made of a limited number of cell types that combine to form higher order tissues and organs. Cell types have traditionally been defined by their morphologies or biological activity, yet the underlying molecular controls of cell type remain unclear. The onset of single cell technologies, and more recently genomics (particularly single cell genomics), has substantially increased the understanding of the concept of cell type, but has also increased the complexity of this understanding. These new technologies have added a new genome wide molecular dimension to the description of cell type, with genome-wide expression and epigenetic data acting as a cell type 'fingerprint' to describe the cell state. Using these genomic fingerprints cell types are being increasingly defined based on specific genomic and molecular criteria, without necessarily a distinct biological function. In this review, we will discuss the molecular definitions of cell types and cell type control, and particularly how endogenous and exogenous transcription factors can control cell types and cell type conversions.