RESUMO
DNA methylation is an essential epigenetic mark that regulates normal mammalian embryonic development. DNA methylation profiles are not always static, especially during germline development. In zygotes, DNA is typically highly methylated but, during preimplantation, DNA methylation is erased globally. Then, at the start of post-implantation development in mouse embryos, DNA again becomes dramatically hypermethylated. Chromatin structure regulates the accessibility of DNA-modifying enzymes to target DNA. Beyond that, however, our understanding of the pathway by which chromatin regulation initiates changes in global DNA methylation during mouse embryonic development remains incomplete. To analyse the relationship between global regulation of DNA methylation and chromatin status, we examined 5-methylcytosine (5mC), modified by the DNA methyltransferase DNMT, and the oxidative derivative 5-hydroxymethylation (5hmC), converted from 5mC by TET-family enzymes, by means of immunofluorescence staining of mitotic chromosomes in mouse embryonic stem cells (ESCs). Our comparison of immunostaining patterns for those epigenetic modifications in wild-type, DNMT-deficient, and TET-deficient ESCs allowed us to visualise cell cycle-mediated DNA methylation changes, especially in euchromatic regions. Our findings suggest that DNA methylation patterns in undifferentiated mouse ESCs are stochastically balanced by the opposing effects of two activities: demethylation by TET and subsequent remethylation by DNMT.
Assuntos
Desenvolvimento Embrionário , Células-Tronco Embrionárias Murinas , 5-Metilcitosina , Animais , DNA , Metilação de DNA , Desmetilação , Feminino , Camundongos , GravidezRESUMO
Large intergenic noncoding RNAs (lincRNAs) in ESCs may play an important role in the maintenance of pluripotency. The identification of stem cell-specific lincRNAs and their interacting partners will deepen our understanding of the maintenance of stem cell pluripotency. We identified a lincRNA, LincQ, which is specifically expressed in ESCs and is regulated by core pluripotent transcription factors. It was rapidly downregulated during the differentiation process. Knockdown of LincQ in ESCs led to differentiation, downregulation of pluripotency-related genes, and upregulation of differentiation-related genes. We found that exon 1 of LincQ can specifically bind to Sox2. The Soxp region in Sox2, rather than the high mobility group domain, is responsible for LincQ binding. Importantly, the interaction between LincQ and Sox2 is required for the maintenance of pluripotency in ESCs and the transcription of pluripotency genes. Esrrb and Tfcp2l1 are key downstream targets of LincQ and Sox2, since overexpression of Esrrb and Tfcp2l1 can restore the loss of ESC pluripotency that is induced by LincQ depletion. In summary, we found that LincQ specifically interacts with Sox2 and contributes to the maintenance of pluripotency, highlighting the critical role of lincRNA in the pluripotency regulatory network.
Assuntos
Células-Tronco Embrionárias Murinas , RNA Longo não Codificante , Animais , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Due to an extreme rarity of 5-carboxylcytosine (5caC) in the mammalian genome, investigation of its role brings a considerable challenge. Methods based on bisulfite sequencing have been proposed for genome-wide 5caC analysis. However, bisulfite-based sequencing of scarcely abundant 5caC demands significant experimental and computational resources, increasing sequencing cost. Here, we present a bisulfite-free approach, caCLEAR, for high-resolution mapping of 5caCGs. The method uses an atypical activity of the methyltransferase eM.SssI to remove a carboxyl group from 5caC, generating unmodified CGs, which are localized by uTOP-seq sequencing. Validation of caCLEAR on model DNA systems and mouse ESCs supports the suitability of caCLEAR for analysis of 5caCGs. The 5caCG profiles of naive and primed pluripotent ESCs reflect their distinct demethylation dynamics and demonstrate an association of 5caC with gene expression. Generally, we demonstrate that caCLEAR is a robust economical approach that could help provide deeper insights into biological roles of 5caC.
Assuntos
Citosina/análogos & derivados , Genoma , Sulfitos/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Citosina/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant heterocyclic aromatic amines (HAAs) found in the human diet, is primarily produced during high-temperature meat or fish cooking. While MeIQx has been investigated as a potential carcinogen, the cytotoxicity and related molecular mechanisms remain unclear. Here, we demonstrate that autophagosome maturation is blocked by MeIQx. Mechanistically, MeIQx inhibits acidification of lysosomes rather than prevents autophagosome-lysosome fusion. Moreover, cellular lipid profiles are altered by MeIQx treatment. Notably, many phospholipids and sphingolipids are significantly upregulated after exposure to MeIQx. Furthermore, MeIQx decreases expression of pluripotency-associated proteins in mouse embryonic stem cells (ESCs). Together, MeIQx blocks autophagosome maturation through inhibiting acidification of lysosomes, alters lipid metabolism, and decreases expression of pluripotent factors. Our studies provide more cytotoxic evidence and elucidate related mechanisms on the risk of HAA exposure and are expected to promote supervision of food safety and human health.
Assuntos
Autofagossomos/efeitos dos fármacos , Lipídeos/química , Quinoxalinas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Autofagossomos/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Transcrição/genéticaRESUMO
Long non-coding RNAs (lncRNAs) are currently recognized as crucial players in nervous system development, function and pathology. In Amyotrophic Lateral Sclerosis (ALS), identification of causative mutations in FUS and TDP-43 or hexanucleotide repeat expansion in C9ORF72 point to the essential role of aberrant RNA metabolism in neurodegeneration. In this study, by taking advantage of an in vitro differentiation system generating mouse motor neurons (MNs) from embryonic stem cells, we identified and characterized the long non-coding transcriptome of MNs. Moreover, by using mutant mouse MNs carrying the equivalent of one of the most severe ALS-associated FUS alleles (P517L), we identified lncRNAs affected by this mutation. Comparative analysis with human MNs derived in vitro from induced pluripotent stem cells indicated that candidate lncRNAs are conserved between mouse and human. Our work provides a global view of the long non-coding transcriptome of MN, as a prerequisite toward the comprehension of the still poorly characterized non-coding side of MN physiopathology.