RESUMO
BACKGROUND: The Pharyngeal Endoderm (PE) is an extremely relevant developmental tissue, serving as the progenitor for the esophagus, parathyroids, thyroids, lungs, and thymus. While several studies have highlighted the importance of PE cells, a detailed transcriptional and epigenetic characterization of this important developmental stage is still missing, especially in humans, due to technical and ethical constraints pertaining to its early formation. RESULTS: Here we fill this knowledge gap by developing an in vitro protocol for the derivation of PE-like cells from human Embryonic Stem Cells (hESCs) and by providing an integrated multi-omics characterization. Our PE-like cells robustly express PE markers and are transcriptionally homogenous and similar to in vivo mouse PE cells. In addition, we define their epigenetic landscape and dynamic changes in response to Retinoic Acid by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of multiple high-throughput datasets leads to the identification of new putative regulatory regions and to the inference of a Retinoic Acid-centered transcription factor network orchestrating the development of PE-like cells. CONCLUSIONS: By combining hESCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and the modeling of their developmental defects in genetic syndromes.
Assuntos
Diferenciação Celular , Endoderma , Epigênese Genética , Células-Tronco Embrionárias Humanas , Humanos , Endoderma/citologia , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Faringe/citologia , Faringe/metabolismo , Tretinoína/farmacologia , Tretinoína/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , CamundongosRESUMO
DNA methylation (DNAm) is one of the most reliable biomarkers of aging across mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, DNAm gain is less characterized. Studies have demonstrated that CpGs which gain methylation with age are enriched in Polycomb Repressive Complex 2 (PRC2) targets. However, whole-genome examination of all PRC2 targets as well as determination of the pan-tissue or tissue-specific nature of these associations is lacking. Here, we show that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells (PRC2 LMRs) gain methylation with age in all examined somatic mitotic cells. We estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the "PRC2-AgeIndex," defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells which can distinguish the effect of different anti-aging interventions.
Assuntos
Envelhecimento , Biomarcadores , Metilação de DNA , Epigênese Genética , Complexo Repressor Polycomb 2 , Rejuvenescimento , Animais , Envelhecimento/metabolismo , Envelhecimento/genética , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Rejuvenescimento/fisiologia , Biomarcadores/metabolismo , Humanos , Camundongos , Senescência Celular/genética , Ilhas de CpG , Células-Tronco Embrionárias/metabolismo , Masculino , FemininoRESUMO
Over the past decade, there has been a dramatic increase in efforts to ameliorate aging and the diseases it causes, with transient expression of nuclear reprogramming factors recently emerging as an intriguing approach. Expression of these factors, either systemically or in a tissue-specific manner, has been shown to combat age-related deterioration in mouse and human model systems at the cellular, tissue and organismal level. Here we discuss the current state of epigenetic rejuvenation strategies via partial reprogramming in both mouse and human models. For each classical reprogramming factor, we provide a brief description of its contribution to reprogramming and discuss additional factors or chemical strategies. We discuss what is known regarding chromatin remodeling and the molecular dynamics underlying rejuvenation, and, finally, we consider strategies to improve the practical uses of epigenetic reprogramming to treat aging and age-related diseases, focusing on the open questions and remaining challenges in this emerging field.
Assuntos
Células-Tronco Pluripotentes Induzidas , Rejuvenescimento , Humanos , Animais , Camundongos , Envelhecimento/genética , Reprogramação Celular/genética , Epigênese GenéticaRESUMO
Generating primordial germ cell-like cells (PGCLCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but often entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days. We use our simplified differentiation platform and single-cell RNA-sequencing to achieve further insights into PGCLC specification. Transient WNT activation for 12 h followed by WNT inhibition specified PGCLCs; by contrast, sustained WNT induced primitive streak. Thus, somatic cells (primitive streak) and PGCLCs are related-yet distinct-lineages segregated by temporally-dynamic signaling. Pluripotency factors including NANOG are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCLCs can be easily purified by virtue of their CXCR4+PDGFRA-GARP- surface-marker profile and single-cell RNA-sequencing reveals that they harbor transcriptional similarities with fetal PGCs.
Assuntos
Desenvolvimento Embrionário , Células Germinativas , Humanos , Diferenciação Celular , Feto , RNARESUMO
LncRNAs have been recently implicated in the epigenetic control of muscle differentiation and their functional characterization has traditionally relied upon in vitro models of myogenic differentiation. However, the use of experimental paradigms to specifically target lncRNAs expression in muscle stem cells (MuSCs), also known as satellite cells, represents an important requisite to interrogate their function in more physiological contexts. Since isolation and culture of single myofibers preserves satellite cells within their physiological niche underneath the surrounding basal lamina, this procedure represents the optimal approach to follow satellite cell dynamics ex-vivo, such as activation from quiescence, expansion of committed progenitors, differentiation, and self-renewal. Here, we detail an optimized protocol to isolate viable single myofibers from the extensor digitorum longus (EDL) skeletal muscle of adult mice and to manipulate the expression of lncRNAs by antisense LNA GapmeRs-mediated knock-down (KD). Furthermore, we describe a method of EdU incorporation that, coupled to lncRNA KD and subsequent immunofluorescence analysis of proliferating, differentiating, and satellite cell-specific markers, permits the inference of lncRNAs function on muscle stem cells dynamics. Graphic abstract: Graphical representation of the single myofiber isolation method. Experimental workflow showing the main steps of the protocol procedure: EDL muscle harvesting from the mouse hindlimb; EDL digestion into single myofibers; transfection with antisense oligos and culture for 96h; immunofluorescence protocol and image outcome.
RESUMO
Skeletal muscle possesses an outstanding capacity to regenerate upon injury due to the adult muscle stem cell (MuSC) activity. This ability requires the proper balance between MuSC expansion and differentiation, which is critical for muscle homeostasis and contributes, if deregulated, to muscle diseases. Here, we functionally characterize a novel chromatin-associated long noncoding RNA (lncRNA), Lnc-Rewind, which is expressed in murine MuSCs and conserved in human. We find that, in mouse, Lnc-Rewind acts as an epigenetic regulator of MuSC proliferation and expansion by influencing the expression of skeletal muscle genes and several components of the WNT (Wingless-INT) signalling pathway. Among them, we identified the nearby Wnt7b gene as a direct Lnc-Rewind target. We show that Lnc-Rewind interacts with the G9a histone lysine methyltransferase and mediates the in cis repression of Wnt7b by H3K9me2 deposition. Overall, these findings provide novel insights into the epigenetic regulation of adult muscle stem cells fate by lncRNAs.
Assuntos
Epigênese Genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , RNA Longo não Codificante/genética , Animais , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/crescimento & desenvolvimento , RNA Longo não Codificante/metabolismoRESUMO
Chromatin architect of muscle expression (Charme) is a muscle-restricted long noncoding RNA (lncRNA) that plays an important role in myogenesis. Earlier evidence indicates that the nuclear Charme isoform, named pCharme, acts on the chromatin by assisting the formation of chromatin domains where myogenic transcription occurs. By combining RNA antisense purification (RAP) with mass spectrometry and loss-of-function analyses, we have now identified the proteins that assist these chromatin activities. These proteins-which include a sub-set of splicing regulators, principally PTBP1 and the multifunctional RNA/DNA binding protein MATR3-bind to sequences located within the alternatively spliced intron-1 to form nuclear aggregates. Consistent with the functional importance of pCharme interactome in vivo, a targeted deletion of the intron-1 by a CRISPR-Cas9 approach in mouse causes the release of pCharme from the chromatin and results in cardiac defects similar to what was observed upon knockout of the full-length transcript.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Íntrons/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Humanos , CamundongosRESUMO
Recent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease in vitro. We apply CRISPR/Cas9 genome editing technology to introduce three RBM20 mutations in iPSCs and differentiate them into iPSC-CMs to establish an in vitro model of RBM20 mutant dilated cardiomyopathy (DCM). In iPSC-CMs harboring a known causal RBM20 variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-trans retinoic acid upregulates RBM20 expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal RBM20 mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in RBM20.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Humanos , Regulação para CimaRESUMO
Neuronal differentiation is a timely and spatially regulated process, relying on precisely orchestrated gene expression control. The sequential activation/repression of genes driving cell fate specification is achieved by complex regulatory networks, where transcription factors and noncoding RNAs work in a coordinated manner. Herein, we identify the long noncoding RNA HOTAIRM1 (HOXA Transcript Antisense RNA, Myeloid-Specific 1) as a new player in neuronal differentiation. We demonstrate that the neuronal-enriched HOTAIRM1 isoform epigenetically controls the expression of the proneural transcription factor NEUROGENIN 2 that is key to neuronal fate commitment and critical for brain development. We also show that HOTAIRM1 activity impacts on NEUROGENIN 2 downstream regulatory cascade, thus contributing to the achievement of proper neuronal differentiation timing. Finally, we identify the RNA-binding proteins HNRNPK and FUS as regulators of HOTAIRM1 biogenesis and metabolism. Our findings uncover a new regulatory layer underlying NEUROGENIN 2 transitory expression in neuronal differentiation and reveal a previously unidentified function for the neuronal-induced long noncoding RNA HOTAIRM1.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteômica/métodos , Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Epigênese Genética , Inativação Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , TransfecçãoRESUMO
Myogenesis is a highly regulated process that involves the conversion of progenitor cells into multinucleated myofibers. Besides proteins and miRNAs, long noncoding RNAs (lncRNAs) have been shown to participate in myogenic regulatory circuitries. Here, we characterize a murine chromatin-associated muscle-specific lncRNA, Charme, which contributes to the robustness of the myogenic program in vitro and in vivo In myocytes, Charme depletion triggers the disassembly of a specific chromosomal domain and the downregulation of myogenic genes contained therein. Notably, several Charme-sensitive genes are associated with human cardiomyopathies and Charme depletion in mice results in a peculiar cardiac remodeling phenotype with changes in size, structure, and shape of the heart. Moreover, the existence of an orthologous transcript in human, regulating the same subset of target genes, suggests an important and evolutionarily conserved function for Charme Altogether, these data describe a new example of a chromatin-associated lncRNA regulating the robustness of skeletal and cardiac myogenesis.
Assuntos
Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/metabolismo , Remodelação Ventricular , Animais , Humanos , Camundongos , Fibras Musculares Esqueléticas/patologia , Miócitos Cardíacos/patologia , RNA Longo não Codificante/genéticaRESUMO
The completion of the human genome sequence together with advances in sequencing technologies have shifted the paradigm of the genome, as composed of discrete and hereditable coding entities, and have shown the abundance of functional noncoding DNA. This part of the genome, previously dismissed as "junk" DNA, increases proportionally with organismal complexity and contributes to gene regulation beyond the boundaries of known protein-coding genes. Different classes of functionally relevant nonprotein-coding RNAs are transcribed from noncoding DNA sequences. Among them are the long noncoding RNAs (lncRNAs), which are thought to participate in the basal regulation of protein-coding genes at both transcriptional and post-transcriptional levels. Although knowledge of this field is still limited, the ability of lncRNAs to localize in different cellular compartments, to fold into specific secondary structures and to interact with different molecules (RNA or proteins) endows them with multiple regulatory mechanisms. It is becoming evident that lncRNAs may play a crucial role in most biological processes such as the control of development, differentiation and cell growth. This review places the evolution of the concept of the gene in its historical context, from Darwin's hypothetical mechanism of heredity to the post-genomic era. We discuss how the original idea of protein-coding genes as unique determinants of phenotypic traits has been reconsidered in light of the existence of noncoding RNAs. We summarize the technological developments which have been made in the genome-wide identification and study of lncRNAs and emphasize the methodologies that have aided our understanding of the complexity of lncRNA-protein interactions in recent years.
RESUMO
The human transcriptome contains thousands of long non-coding RNAs (lncRNAs). Characterizing their function is a current challenge. An emerging concept is that lncRNAs serve as protein scaffolds, forming ribonucleoproteins and bringing proteins in proximity. However, only few scaffolding lncRNAs have been characterized and the prevalence of this function is unknown. Here, we propose the first computational approach aimed at predicting scaffolding lncRNAs at large scale. We predicted the largest human lncRNA-protein interaction network to date using the catRAPID omics algorithm. In combination with tissue expression and statistical approaches, we identified 847 lncRNAs (â¼5% of the long non-coding transcriptome) predicted to scaffold half of the known protein complexes and network modules. Lastly, we show that the association of certain lncRNAs to disease may involve their scaffolding ability. Overall, our results suggest for the first time that RNA-mediated scaffolding of protein complexes and modules may be a common mechanism in human cells.
Assuntos
Biologia Computacional/métodos , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Algoritmos , Predisposição Genética para Doença/genética , Humanos , Ligação Proteica , Mapas de Interação de Proteínas , Proteoma/genética , Proteoma/metabolismo , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , TranscriptomaRESUMO
Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.