RESUMO
N6-methyladenosine (m6A) is the most common mRNA modification. Recent studies have revealed that depletion of m6A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the m6A 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was m6A modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that m6A-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role m6A serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.
Assuntos
Adenosina/análogos & derivados , Interações Hospedeiro-Patógeno/genética , Imunidade Inata/genética , Interferon Tipo I/genética , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Citomegalovirus/imunologia , Modelos Animais de Doenças , Feminino , Fibroblastos , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Interferon Tipo I/imunologia , Masculino , Metilação , Metiltransferases/genética , Metiltransferases/imunologia , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Muromegalovirus/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/metabolismoRESUMO
In the version of this article initially published, the penultimate sentence of the abstract included a typographical error ('cxgenes'). The correct word is 'genes'. The error has been corrected in the HTML and PDF version of the article.
RESUMO
The N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous m6A-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers' expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers' role during early development that is Ythdf1/2/3 gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.
Assuntos
Mecanismo Genético de Compensação de Dose , Gametogênese/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias , Fertilidade/genética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos KnockoutRESUMO
Understanding the biologic role of N6-methyladenosine (m6A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m6A in exons but very rarely in introns. The m6A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m6A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only â¼10% of m6As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m6A in wild-type mouse stem cells is spliced the same in cells lacking the major m6A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m6As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m6A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m6A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.
Assuntos
Citoplasma/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Animais , Cromatina/metabolismo , Células-Tronco Embrionárias , Éxons/genética , Técnicas de Inativação de Genes , Células HeLa , Humanos , Íntrons/genética , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , CamundongosRESUMO
Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.
Assuntos
Reprogramação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Biológicos , Animais , Linhagem Celular , Células Cultivadas , Reprogramação Celular/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Fatores de Transcrição/genéticaRESUMO
Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3ß signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Animais , Blastocisto/citologia , Reprogramação Celular , Quimera/embriologia , Cromatina/metabolismo , Metilação de DNA , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Feminino , Camadas Germinativas/citologia , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Masculino , Camundongos , Mórula/citologia , Organogênese , Regiões Promotoras Genéticas/genética , Medicina Regenerativa , Reprodutibilidade dos Testes , Transdução de Sinais , Inativação do Cromossomo XRESUMO
Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by ectopic expression of different transcription factors, classically Oct4 (also known as Pou5f1), Sox2, Klf4 and Myc (abbreviated as OSKM). This process is accompanied by genome-wide epigenetic changes, but how these chromatin modifications are biochemically determined requires further investigation. Here we show in mice and humans that the histone H3 methylated Lys 27 (H3K27) demethylase Utx (also known as Kdm6a) regulates the efficient induction, rather than maintenance, of pluripotency. Murine embryonic stem cells lacking Utx can execute lineage commitment and contribute to adult chimaeric animals; however, somatic cells lacking Utx fail to robustly reprogram back to the ground state of pluripotency. Utx directly partners with OSK reprogramming factors and uses its histone demethylase catalytic activity to facilitate iPSC formation. Genomic analysis indicates that Utx depletion results in aberrant dynamics of H3K27me3 repressive chromatin demethylation in somatic cells undergoing reprogramming. The latter directly hampers the derepression of potent pluripotency promoting gene modules (including Sall1, Sall4 and Utf1), which can cooperatively substitute for exogenous OSK supplementation in iPSC formation. Remarkably, Utx safeguards the timely execution of H3K27me3 demethylation observed in embryonic day 10.5-11 primordial germ cells (PGCs), and Utx-deficient PGCs show cell-autonomous aberrant epigenetic reprogramming dynamics during their embryonic maturation in vivo. Subsequently, this disrupts PGC development by embryonic day 12.5, and leads to diminished germline transmission in mouse chimaeras generated from Utx-knockout pluripotent cells. Thus, we identify Utx as a novel mediator with distinct functions during the re-establishment of pluripotency and germ cell development. Furthermore, our findings highlight the principle that molecular regulators mediating loss of repressive chromatin during in vivo germ cell reprogramming can be co-opted during in vitro reprogramming towards ground state pluripotency.
Assuntos
Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Células Germinativas/metabolismo , Histona Desmetilases/metabolismo , Proteínas Nucleares/metabolismo , Alelos , Animais , Biocatálise , Linhagem da Célula , Quimera , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Feminino , Fibroblastos , Técnicas de Silenciamento de Genes , Células Germinativas/enzimologia , Células HEK293 , Histona Desmetilases/deficiência , Histona Desmetilases/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Transgenes/genéticaRESUMO
The outer mitochondrial membrane protein, the voltage-dependent anion channel (VDAC), is increasingly implicated in the control of apoptosis. Oligomeric assembly of VDAC1 was shown to be coupled to apoptosis induction, with oligomerization increasing substantially upon apoptosis induction and inhibited by apoptosis blockers. In this study, structure- and computation-based selection of the predicated VDAC1 dimerization site, in combination with site-directed mutagenesis, cysteine replacement, and chemical cross-linking, were employed to identify contact sites between VDAC1 molecules in dimers and higher oligomers. The predicted weakly stable ß-strands were experimentally found to represent the interfaces between VDAC1 monomers composing the oligomer. Replacing hydrophobic amino acids with charged residues in ß-strands 1, 2, and 19 interfered with VDAC1 oligomerization. The proximity of ß-strands 1, 2, and 19 within the VDAC1 dimer and the existence of other association sites involving ß-strand 16 were confirmed when a cysteine was introduced at defined positions in cysteineless VDAC1 mutants, together with the use of cysteine-specific cross-linker bis(maleimido)ethane. Moreover, the results suggest that VDAC1 also exists as a dimer that upon apoptosis induction undergoes conformational changes and that its oligomerization proceeds through a series of interactions involving two distinct interfaces. Dissection of VDAC1 dimerization/oligomerization as presented here provides structural insight into the oligomeric status of cellular VDAC1 under physiological and apoptotic conditions.
Assuntos
Apoptose/fisiologia , Multimerização Proteica/fisiologia , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Ratos , Relação Estrutura-Atividade , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
Structural studies place the VDAC1 (voltage-dependent anion channel 1) N-terminal region within the channel pore. Biochemical and functional studies, however, reveal that the N-terminal domain is cytoplasmically exposed. In the present study, the location and translocation of the VDAC1 N-terminal domain, and its role in voltage-gating and as a target for anti-apoptotic proteins, were addressed. Site-directed mutagenesis and cysteine residue substitution, together with a thiol-specific cross-linker, served to show that the VDAC1 N-terminal region exists in a dynamic equilibrium, located within the pore or exposed outside the ß-barrel. Using a single cysteine-residue-bearing VDAC1, we demonstrate that the N-terminal region lies inside the pore. However, the same region can be exposed outside the pore, where it dimerizes with the N-terminal domain of a second VDAC1 molecule. When the N-terminal region α-helix structure was perturbed, intra-molecular cross-linking was abolished and dimerization was enhanced. This mutant also displays reduced voltage-gating and reduced binding to hexokinase, but not to the anti-apoptotic proteins Bcl-2 and Bcl-xL. Replacing glycine residues in the N-terminal domain GRS (glycine-rich sequence) yielded less intra-molecular cross-linked product but more dimerization, suggesting that GRS provides the flexibility needed for N-terminal translocation from the internal pore to the channel face. N-terminal mobility may thus contribute to channel gating and interaction with anti-apoptotic proteins.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Ativação do Canal Iônico/fisiologia , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Cisteína/química , Células HEK293 , Humanos , Dados de Sequência Molecular , Transporte Proteico/genética , Ratos , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
The VDAC (voltage-dependent anion channel) is proposed to control metabolic cross-talk between mitochondria and the cytosol, as well as apoptotic cell death. It has been suggested that apoptosis is modulated by the oxidation state of VDAC. Since cysteine residues are the major target for oxidation/reduction, we verified whether one or both VDAC1 cysteine residues are involved in VDAC1-mediated transport or apoptosis activities. To assess the function of VDAC1 cysteine residues in channel activity and to probe cysteine topology with respect to facing the pore or the bilayer, we used thiol-modifying agents, namely membrane-permeable NEM (N-ethylmaleimide), bulky charged 5-FM (fluorescein-5-maleimide) and the cross-linking reagent BMOE [bis(maleimido)ethane]. Bilayer-reconstituted VDAC conductance was decreased by 5-FM, but not by NEM, whereas 5-FM had no effect on NEM-labelled VDAC conductance. BMOE caused the formation of dimeric VDAC1, suggesting that one of the two VDAC1 cysteine residues is exposed and available for cross-linking. The results thus suggest that one of the VDAC1 cysteine residues faces the VDAC pore, whereas the second is oriented towards the lipid bilayer. Mutated rat VDAC1 in which the two cysteine residues, Cys127 and Cys232, were replaced by alanine residues showed channel activity like native VDAC1 and, when expressed in cells, was localized to mitochondria. Human VDAC1-shRNA (small hairpin RNA)- or -siRNA (small interfering RNA)-treated cells, expressing low levels of endogenous human VDAC1 together with native or cysteine-less rat VDAC1, undergo apoptosis as induced by overexpression of these VDAC1 or upon treatment with reactive oxygen species-producing agents, H2O2, As2O3 or selenite, suggesting that the two cysteine residues are not required for apoptosis or VDAC1 oligomerization.
Assuntos
Apoptose/fisiologia , Cisteína/fisiologia , Membranas Mitocondriais/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Trióxido de Arsênio , Arsenicais/farmacologia , Bovinos , Cisteína/química , Cisteína/genética , Eletroforese em Gel de Poliacrilamida , Etilmaleimida/farmacologia , Fluoresceínas/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Immunoblotting , Bicamadas Lipídicas/química , Camundongos , Membranas Mitocondriais/efeitos dos fármacos , Óxidos/farmacologia , RNA Interferente Pequeno , Coelhos , Ratos , Selenito de Sódio/farmacologia , Relação Estrutura-Atividade , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/ß-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.
Assuntos
Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Embrião de Mamíferos , Humanos , Camundongos , Transdução de Sinais , TrofoblastosRESUMO
Haematopoietic stem cells (HSCs) maintain balanced self-renewal and differentiation, but how these functions are precisely regulated is not fully understood. N6-methyladenosine (m6A) messenger RNA methylation has emerged as an important mode of epitranscriptional gene expression regulation affecting many biological processes. We show that deletion of the m6A methyltransferase Mettl3 from the adult haematopoietic system led to an accumulation of HSCs in the bone marrow and a marked reduction of reconstitution potential due to a blockage of HSC differentiation. Interestingly, deleting Mettl3 from myeloid cells using Lysm-cre did not impact myeloid cell number or function. RNA sequencing revealed 2,073 genes with significant m6A modifications in HSCs. Myc was identified as a direct target of m6A in HSCs. Mettl3-deficient HSCs failed to upregulate MYC expression following stimulation to differentiate and enforced expression of Myc rescued differentiation defects of Mettl3-deficient HSCs. Our results reveal a key role of m6A in governing HSC differentiation.
Assuntos
Adenosina/análogos & derivados , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Adenosina/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Hematopoéticas/metabolismo , Metilação , Camundongos , RNA Mensageiro/genética , Análise de Sequência de RNARESUMO
N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed m6A/m, in the stress response in the adult brain in vivo is currently unknown. Here, we provide a detailed analysis of the stress epitranscriptome using m6A/m-seq, global and gene-specific m6A/m measurements. We show that stress exposure and glucocorticoids region and time specifically alter m6A/m and its regulatory network. We demonstrate that deletion of the methyltransferase Mettl3 or the demethylase Fto in adult neurons alters the m6A/m epitranscriptome, increases fear memory, and changes the transcriptome response to fear and synaptic plasticity. Moreover, we report that regulation of m6A/m is impaired in major depressive disorder patients following glucocorticoid stimulation. Our findings indicate that brain m6A/m represents a novel layer of complexity in gene expression regulation after stress and that dysregulation of the m6A/m response may contribute to the pathophysiology of stress-related psychiatric disorders.
Assuntos
Adenosina/análogos & derivados , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Psicológico/genética , Estresse Psicológico/metabolismo , Adenosina/genética , Adenosina/metabolismo , Adulto , Animais , Linhagem Celular Transformada , Humanos , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estresse Psicológico/psicologiaRESUMO
Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição GATA/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Feminino , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos TransgênicosRESUMO
Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.
Assuntos
Transdiferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Feminino , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Camundongos TransgênicosRESUMO
Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.