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Epigenetic reprogramming in the germline resets genomic potential and erases epigenetic memory. Three studies by Gkountela et al., Guo et al., and Tang et al. analyze the transcriptional and epigenetic landscape of human primordial germ cells, revealing a unique transcriptional network and progressive and conserved global erasure of DNA methylation.
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Diferenciação Celular , Impressão Genômica , Células Germinativas/metabolismo , Células Germinativas/fisiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Feminino , Humanos , MasculinoRESUMO
Hydroxycarbamide (HC, hydroxyurea) is a cytoreductive drug inducing cell cycle blockade. However, emerging evidence suggests that HC plays a role in the modulation of transcription through the activity of transcription factors and DNA methylation. Examining the global mechanism of action of HC in the context of myeloproliferative neoplasms (MPNs), for which HC is the first-line treatment, will provide a better understanding of its molecular effects. To explore the effects of HC genome-wide, transcriptomic analyses were performed on two clinically relevant cell types at different stages of differentiation treated with HC in a murine MPN model. This study was replicated in MPN patients by profiling genome-wide gene expression and DNA methylation using patient blood samples collected longitudinally, before and following HC exposure. The effects of HC on the transcriptome were not only associated with cell cycle interruption but also with hematopoietic functions. Moreover, a group of genes were restored to normal expression levels in murine hematopoietic stem cells (HSCs) following drug treatment, including the master regulator of hematopoiesis, RUNX1 In humans, HC significantly modifies DNA methylation levels in HSCs at several distal regulatory regions, which we show to be associated with SPI1 binding sites and at the SPI1 locus itself. We have identified novel targets of HC that include pivotal transcription factors involved in hematopoiesis, and for the first time we report abnormal methylation patterns in MPN patients at the master regulator gene SPI1 and its distal binding sites, which HC is able to restore to normal levels.
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Metilação de DNA , Neoplasias , Animais , Hematopoese/genética , Humanos , Hidroxiureia/farmacologia , Camundongos , Neoplasias/genética , TranscriptomaRESUMO
Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos.
Assuntos
Reprogramação Celular , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Histonas/metabolismo , Camundongos , Modelos Genéticos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Tempo , Transfecção , Ubiquitina-Proteína LigasesRESUMO
The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.
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Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Sistemas CRISPR-Cas , Técnicas de Cultura de Células , Ciclo Celular , Técnicas de Cocultura , Meios de Cultura/química , Humanos , Cariotipagem , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ácidos Nucleicos/isolamento & purificação , Animais , DNA/isolamento & purificação , Humanos , Campos Magnéticos , Microesferas , RNA/isolamento & purificaçãoRESUMO
Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We adapted amplicon bisulfite-sequencing protocols to design IMPLICON for ICRs in adult tissues of inbred mice, validating it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.
Assuntos
Ilhas de CpG , Metilação de DNA , Impressão Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Células Cultivadas , Feminino , Fibroblastos , Células-Tronco Embrionárias Humanas , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24â h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.
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Elementos de DNA Transponíveis/genética , Epigênese Genética/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Metilação de DNA/genética , Metilação de DNA/fisiologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Genes Ligados ao Cromossomo X/genética , Humanos , Hibridização in Situ Fluorescente , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Inativação do Cromossomo X/genéticaRESUMO
Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/metabolismo , Animais , Ácido Ascórbico/farmacologia , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Medicina Regenerativa , Tretinoína/farmacologia , Vitamina A/farmacologiaRESUMO
The lateral hypothalamic area is considered the classic 'feeding centre', regulating food intake, arousal and motivated behaviour through the actions of orexin and melanin-concentrating hormone (MCH). These neuropeptides are inhibited in response to feeding-related signals and are released during fasting. However, the molecular mechanisms that regulate and integrate these signals remain poorly understood. Here we show that the forkhead box transcription factor Foxa2, a downstream target of insulin signalling, regulates the expression of orexin and MCH. During fasting, Foxa2 binds to MCH and orexin promoters and stimulates their expression. In fed and in hyperinsulinemic obese mice, insulin signalling leads to nuclear exclusion of Foxa2 and reduced expression of MCH and orexin. Constitutive activation of Foxa2 in the brain (Nes-Cre/+;Foxa2T156A(flox/flox) genotype) results in increased neuronal MCH and orexin expression and increased food consumption, metabolism and insulin sensitivity. Spontaneous physical activity of these animals in the fed state is significantly increased and is similar to that in fasted mice. Conditional activation of Foxa2 through the T156A mutation expression in the brain of obese mice also resulted in improved glucose homeostasis, decreased fat and increased lean body mass. Our results demonstrate that Foxa2 can act as a metabolic sensor in neurons of the lateral hypothalamic area to integrate metabolic signals, adaptive behaviour and physiological responses.
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Adaptação Psicológica/fisiologia , Comportamento Animal/fisiologia , Jejum/fisiologia , Jejum/psicologia , Regulação da Expressão Gênica/fisiologia , Fator 3-beta Nuclear de Hepatócito/metabolismo , Animais , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/metabolismo , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Melaninas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Neuropeptídeos/metabolismo , Orexinas , Hormônios Hipofisários/metabolismo , Regiões Promotoras Genéticas/genéticaRESUMO
BACKGROUND: Single-cell chromatin accessibility assays, such as scATAC-seq, are increasingly employed in individual and joint multi-omic profiling of single cells. As the accumulation of scATAC-seq and multi-omics datasets continue, challenges in analyzing such sparse, noisy, and high-dimensional data become pressing. Specifically, one challenge relates to optimizing the processing of chromatin-level measurements and efficiently extracting information to discern cellular heterogeneity. This is of critical importance, since the identification of cell types is a fundamental step in current single-cell data analysis practices. RESULTS: We benchmark 8 feature engineering pipelines derived from 5 recent methods to assess their ability to discover and discriminate cell types. By using 10 metrics calculated at the cell embedding, shared nearest neighbor graph, or partition levels, we evaluate the performance of each method at different data processing stages. This comprehensive approach allows us to thoroughly understand the strengths and weaknesses of each method and the influence of parameter selection. CONCLUSIONS: Our analysis provides guidelines for choosing analysis methods for different datasets. Overall, feature aggregation, SnapATAC, and SnapATAC2 outperform latent semantic indexing-based methods. For datasets with complex cell-type structures, SnapATAC and SnapATAC2 are preferred. With large datasets, SnapATAC2 and ArchR are most scalable.
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Benchmarking , Cromatina , Análise de Célula Única , Análise de Célula Única/métodos , Cromatina/genética , Cromatina/metabolismo , Humanos , Biologia Computacional/métodosRESUMO
OBJECTIVES: We have previously shown that lactate is an essential metabolite for macrophage polarisation during ischemia-induced muscle regeneration. Recent in vitro work has implicated histone lactylation, a direct derivative of lactate, in macrophage polarisation. Here, we explore the in vivo relevance of histone lactylation for macrophage polarisation after muscle injury. METHODS: To evaluate macrophage dynamics during muscle regeneration, we subjected mice to ischemia-induced muscle damage by ligating the femoral artery. Muscle samples were harvested at 1, 2, 4, and 7 days post injury (dpi). CD45+CD11b+F4/80+CD64+ macrophages were isolated and processed for RNA sequencing, Western Blotting, and CUT&Tag-sequencing to investigate gene expression, histone lactylation levels, and histone lactylation genomic localisation and enrichment, respectively. RESULTS: We show that, over time, macrophages in the injured muscle undergo extensive gene expression changes, which are similar in nature and in timing to those seen after other types of muscle-injuries. We find that the macrophage histone lactylome is modified between 2 and 4 dpi, which is a crucial window for macrophage polarisation. Absolute histone lactylation levels increase, and, although subtly, the genomic enrichment of H3K18la changes. Overall, we find that histone lactylation is important at both promoter and enhancer elements. Lastly, H3K18la genomic profile changes from 2 to 4 dpi were predictive for gene expression changes later in time, rather than being a reflection of prior gene expression changes. CONCLUSIONS: Our results suggest that histone lactylation dynamics are functionally important for the function of macrophages during muscle regeneration.
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Histonas , Isquemia , Macrófagos , Camundongos Endogâmicos C57BL , Músculo Esquelético , Regeneração , Animais , Macrófagos/metabolismo , Camundongos , Histonas/metabolismo , Músculo Esquelético/metabolismo , Isquemia/metabolismo , Masculino , Expressão Gênica/genéticaRESUMO
The intersection of metabolic processes and epigenetic regulation during embryogenesis is crucial yet not fully understood. Through a candidate RNAi screen in Caenorhabditis elegans , we identified metabolic enzymes ALDO-2 and PDHB-1 as potential epigenetic regulators. Mild alteration of the chromatin remodeler LET-418 /Mi2 activity rescues embryonic lethality induced by suppressing aldo-2 or pdhb-1 , suggesting a critical role for glucose and pyruvate metabolism in chromatin remodeling during embryogenesis. Given the conservation of central metabolic pathways and chromatin modifiers across species, our findings lay the foundation for future mechanistic investigations into the interplay between epigenetics and metabolism during development and upon disease.
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The establishment of aging clocks highlighted the strong link between changes in DNA methylation and aging. Yet, it is not known if other epigenetic features could be used to predict age accurately. Furthermore, previous studies have observed a lack of effect of age-related changes in DNA methylation on gene expression, putting the interpretability of DNA methylation-based aging clocks into question. In this study, we explore the use of chromatin accessibility to construct aging clocks. We collected blood from 159 human donors and generated chromatin accessibility, transcriptomic, and cell composition data. We investigated how chromatin accessibility changes during aging and constructed a novel aging clock with a median absolute error of 5.27 years. The changes in chromatin accessibility used by the clock were strongly related to transcriptomic alterations, aiding clock interpretation. We additionally show that our chromatin accessibility clock performs significantly better than a transcriptomic clock trained on matched samples. In conclusion, we demonstrate that the clock relies on cell-intrinsic chromatin accessibility alterations rather than changes in cell composition. Further, we present a new approach to construct epigenetic aging clocks based on chromatin accessibility, which bear a direct link to age-related transcriptional alterations, but which allow for more accurate age predictions than transcriptomic clocks.
Assuntos
Cromatina , Epigênese Genética , Humanos , Cromatina/genética , Envelhecimento/genética , Metilação de DNA , Perfilação da Expressão GênicaRESUMO
To implant in the uterus, mammalian embryos form blastocysts comprising trophectoderm (TE) surrounding an inner cell mass (ICM), confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered TE until they implant in the characteristic thick deciduum, whereas human blastocysts attach via polar TE directly to the uterine wall. Using immunofluorescence (IF) of rapidly isolated ICMs, blockade of RNA and protein synthesis in whole embryos, or 3D visualization of immunostained embryos, we provide evidence of multi-layering in human polar TE before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiate formation of the placenta. Using sequential fluorescent labeling, we demonstrate that the majority of inner TE in human blastocysts arises from existing outer cells, with no evidence of conversion from the ICM in the context of the intact embryo.
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Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival.
Assuntos
Cervos , Diapausa , Animais , Blastocisto , Diferenciação Celular , Polaridade Celular , Diapausa/genéticaRESUMO
Aging is the major risk factor for most human diseases and represents a major socio-economical challenge for modern societies. Despite its importance, the process of aging remains poorly understood. Epigenetic dysregulation has been proposed as a key driver of the aging process. Modifications in transcriptional networks and chromatin structure might be central to age-related functional decline. A prevalent feature described during aging is the overall reduction in heterochromatin, specifically marked by the loss of repressive histone modification, Histone 3 lysine 9 trimethylation (H3K9me3). However, the role of H3K9me3 in aging, especially in mammals, remains unclear. Here we show using a novel mouse strain, (TKOc), carrying a triple knockout of three methyltransferases responsible for H3K9me3 deposition, that the inducible loss of H3K9me3 in adulthood results in premature aging. TKOc mice exhibit reduced lifespan, lower body weight, increased frailty index, multi-organ degeneration, transcriptional changes with significant upregulation of transposable elements, and accelerated epigenetic age. Our data strongly supports the concept that the loss of epigenetic information directly drives the aging process. These findings reveal the importance of epigenetic regulation in aging and suggest that interventions targeting epigenetic modifications could potentially slow down or reverse age-related decline. Understanding the molecular mechanisms underlying the process of aging will be crucial for developing novel therapeutic strategies that can delay the onset of age-associated diseases and preserve human health at old age specially in rapidly aging societies.
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Macrophages play crucial roles in organ-specific functions and homeostasis. In the adrenal gland, macrophages closely associate with sinusoidal capillaries in the aldosterone-producing zona glomerulosa. We demonstrate that macrophages preserve capillary specialization and modulate aldosterone secretion. Using macrophage-specific deletion of VEGF-A, single-cell transcriptomics, and functional phenotyping, we found that the loss of VEGF-A depletes PLVAP+ fenestrated endothelial cells in the zona glomerulosa, leading to increased basement membrane collagen IV deposition and subendothelial fibrosis. This results in increased aldosterone secretion, called "haptosecretagogue" signaling. Human aldosterone-producing adenomas also show capillary rarefaction and basement membrane thickening. Mice with myeloid cell-specific VEGF-A deletion exhibit elevated serum aldosterone, hypokalemia, and hypertension, mimicking primary aldosteronism. These findings underscore macrophage-to-endothelial cell signaling as essential for endothelial cell specialization, adrenal gland function, and blood pressure regulation, with broader implications for other endocrine organs.