RESUMO
DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae, share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication.
Assuntos
Replicação do DNA , DNA Fúngico/biossíntese , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Metiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Fúngico/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Metilação , Metiltransferases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.
Assuntos
Guanosina/análogos & derivados , Metiltransferases/genética , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , Células A549 , Sequência de Bases , Bioensaio , Células CACO-2 , Movimento Celular , Proliferação de Células , Guanosina/metabolismo , Células HEK293 , Humanos , Metilação , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Conformação de Ácido NucleicoRESUMO
Using the timely re-activation of WNT signalling in neuralizing human induced pluripotent stem cells (hiPSCs), we have produced neural progenitor cells with a gene expression profile typical of human embryonic dentate gyrus (DG) cells. Notably, in addition to continuous WNT signalling, a specific laminin isoform is crucial to prolonging the neural stem state and to extending progenitor cell proliferation for over 200â days in vitro. Laminin 511 is indeed specifically required to support proliferation and to inhibit differentiation of hippocampal progenitor cells for extended time periods when compared with a number of different laminin isoforms assayed. Global gene expression profiles of these cells suggest that a niche of laminin 511 and WNT signalling is sufficient to maintain their capability to undergo typical hippocampal neurogenesis. Moreover, laminin 511 signalling sustains the expression of a set of genes responsible for the maintenance of a hippocampal neurogenic niche. Finally, xenograft of human DG progenitors into the DG of adult immunosuppressed host mice produces efficient integration of neurons that innervate CA3 layer cells spanning the same area of endogenous hippocampal neuron synapses.
Assuntos
Células-Tronco Pluripotentes Induzidas , Laminina , Animais , Diferenciação Celular/genética , Giro Denteado , Hipocampo/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/metabolismo , Camundongos , Neurogênese/genética , Via de Sinalização WntRESUMO
N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia.
Assuntos
Adenosina/análogos & derivados , Regulação Neoplásica da Expressão Gênica/genética , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Metiltransferases/metabolismo , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , Adenosina/genética , Adenosina/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Cromatina/metabolismo , Feminino , Genes Neoplásicos/genética , Humanos , Leucemia Mieloide Aguda/patologia , Metiltransferases/química , Metiltransferases/deficiência , Metiltransferases/genética , Camundongos , Biossíntese de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
BACKGROUND: A widespread modulation of gene expression occurs in the aging brain, but little is known as to the upstream drivers of these changes. MicroRNAs emerged as fine regulators of gene expression in many biological contexts and they are modulated by age. MicroRNAs may therefore be part of the upstream drivers of the global gene expression modulation correlated with aging and aging-related phenotypes. RESULTS: Here, we show that microRNA-29 (miR-29) is induced during aging in short-lived turquoise killifish brain and genetic antagonism of its function induces a gene-expression signature typical of aging. Mechanicistically, we identified Ireb2 (a master gene for intracellular iron delivery that encodes for IRP2 protein), as a novel miR-29 target. MiR-29 is induced by iron loading and, in turn, it reduces IRP2 expression in vivo, therefore limiting intracellular iron delivery in neurons. Genetically modified fish with neuro-specific miR-29 deficiency exhibit increased levels of IRP2 and transferrin receptor, increased iron content, and oxidative stress. CONCLUSIONS: Our results demonstrate that age-dependent miR-29 upregulation is an adaptive mechanism that counteracts the expression of some aging-related phenotypes and its anti-aging activity is primarily exerted by regulating intracellular iron homeostasis limiting excessive iron-exposure in neurons.
Assuntos
Envelhecimento/genética , Ferro/metabolismo , Peixes Listrados/crescimento & desenvolvimento , Peixes Listrados/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Animais , Sequência de Bases , Encéfalo/metabolismo , Homeostase/genética , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , MicroRNAs/genética , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genética , Peixe-Zebra/genéticaRESUMO
We investigated the effects of bone morphogenetic proteins (BMPs) in determining the positional identity of neurons generated in vitro from mouse embryonic stem cells (ESCs), an aspect that has been neglected thus far. Classical embryological studies in lower vertebrates indicate that BMPs inhibit the default fate of pluripotent embryonic cells, which is both neural and anterior. Moreover, mammalian ESCs generate neurons more efficiently when cultured in a minimal medium containing BMP inhibitors. In this paper, we show that mouse ESCs produce, secrete, and respond to BMPs during in vitro neural differentiation. After neuralization in a minimal medium, differentiated ESCs show a gene expression profile consistent with a midbrain identity, as evaluated by the analysis of a number of markers of anterior-posterior and dorsoventral identity. We found that BMPs endogenously produced during neural differentiation mainly act by inhibiting the expression of a telencephalic gene profile, which was revealed by the treatment with Noggin or with other BMP inhibitors. To better characterize the effect of BMPs on positional fate, we compared the global gene expression profiles of differentiated ESCs with those of embryonic forebrain, midbrain, and hindbrain. Both Noggin and retinoic acid (RA) support neuronal differentiation of ESCs, but they show different effects on their positional identity: whereas RA supports the typical gene expression profile of hindbrain neurons, Noggin induces a profile characteristic of dorsal telencephalic neurons. Our findings show that endogenously produced BMPs affect the positional identity of the neurons that ESCs spontaneously generate when differentiating in vitro in a minimal medium. The data also support the existence of an intrinsic program of neuronal differentiation with dorsal telencephalic identity. Our method of ESC neuralization allows for fast differentiation of neural cells via the same signals found during in vivo embryonic development and for the acquisition of cortical identity by the inhibition of BMP alone.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/citologia , Transdução de Sinais/fisiologia , Animais , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Análise em Microsséries , Neurônios/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tretinoína/metabolismoRESUMO
Long Interspersed Nuclear Elements-1s (L1s) are transposable elements that constitute most of the genome's transcriptional output yet have still largely unknown functions. Here we show that L1s are required for proper mouse brain corticogenesis operating as regulatory long non-coding RNAs. They contribute to the regulation of the balance between neuronal progenitors and differentiation, the migration of post-mitotic neurons and the proportions of different cell types. In cortical cultured neurons, L1 RNAs are mainly associated to chromatin and interact with the Polycomb Repressive Complex 2 (PRC2) protein subunits enhancer of Zeste homolog 2 (Ezh2) and suppressor of zeste 12 (Suz12). L1 RNA silencing influences PRC2's ability to bind a portion of its targets and the deposition of tri-methylated histone H3 (H3K27me3) marks. Our results position L1 RNAs as crucial signalling hubs for genome-wide chromatin remodelling, enabling the fine-tuning of gene expression during brain development and evolution.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , RNA Longo não Codificante , Animais , Camundongos , Elementos Nucleotídeos Longos e Dispersos/genética , Diferenciação Celular , Cromatina/genética , Montagem e Desmontagem da Cromatina , RNA Longo não Codificante/genéticaRESUMO
SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N6-methyladenosine (m6A) modified by METTL3 enzyme. Then, we map m6A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m6A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m6A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m6A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs.
RESUMO
SINEUPs are a novel class of natural and synthetic non-coding antisense RNA molecules able to increase the translation of a target mRNA. They present a modular organization comprising an unstructured antisense target-specific domain, which sets the specificity of each individual SINEUP, and a structured effector domain, which is responsible for the translation enhancement. In order to design a fully functional in vitro transcribed SINEUP for therapeutics applications, SINEUP RNAs were synthesized in vitro with a variety of chemical modifications and screened for their activity on endogenous target mRNA upon transfection. Three combinations of modified ribonucleotides-2'O methyl-ATP (Am), N6 methyl-ATP (m6A), and pseudo-UTP (ψ)-conferred SINEUP activity to naked RNA. The best combination tested in this study was fully modified with m6A and ψ. Aside from functionality, this combination conferred improved stability upon transfection and higher thermal stability. Common structural determinants of activity were identified by circular dichroisms, defining a core functional structure that is achieved with different combinations of modifications.
RESUMO
Post-synthesis modification of biomolecules is an efficient way of regulating and optimizing their functions. The human epitranscriptome includes a variety of more than 100 modifications known to exist in all RNA subtypes. Modifications of non-coding RNAs are particularly interesting since they can directly affect their structure, stability, interaction and function. Indeed, non-coding RNAs such as tRNA and rRNA are the most modified RNA species in eukaryotic cells. In the last 20 years, new functions of non-coding RNAs have been discovered and their involvement in human disease, including cancer, became clear. In this review, we will present the evidence connecting modifications of different non-coding RNA subtypes and their role in cancer.
Assuntos
Epigênese Genética , Neoplasias , Humanos , Neoplasias/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA de TransferênciaRESUMO
Cerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different layers. The mechanisms controlling their expression in distinct cells are only partially known. We investigated the expression and stability of Tbr1, Bcl11b, Fezf2, Satb2, and Cux1 mRNAs in single developing mouse cortical cells. We observe that Satb2 mRNA appears much earlier than its protein and in a set of cells broader than expected, suggesting an initial inhibition of its translation, subsequently released during development. Mechanistically, Satb2 3'UTR modulates protein translation of GFP reporters during mouse corticogenesis. We select miR-541, a eutherian-specific miRNA, and miR-92a/b as the best candidates responsible for SATB2 inhibition, being strongly expressed in early and reduced in late progenitor cells. Their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate RNA interference as a major mechanism in timing cortical cell identities.
Assuntos
Córtex Cerebral/metabolismo , Eutérios/genética , Eutérios/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Regiões 3' não Traduzidas , Animais , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , NeurogêneseRESUMO
RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.
Assuntos
Sequenciamento por Nanoporos/métodos , Nanoporos , RNA/metabolismo , Análise de Sequência de RNA/métodos , Sequência de Bases , Biologia Computacional , Perfilação da Expressão Gênica , Técnicas Genéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA/isolamento & purificação , Processamento Pós-Transcricional do RNA , Software , TranscriptomaRESUMO
The morphogen FGF8 plays a pivotal role in neocortical area patterning through its inhibitory effect on COUP-TFI/Nr2f1 anterior expression, but its mechanism of action is poorly understood. We established an in vitro model of mouse embryonic stem cell corticogenesis in which COUP-TFI protein expression is inhibited by the activation of FGF8 in a time window corresponding to cortical area patterning. Interestingly, overexpression of the COUP-TFI 3'UTR reduces the inhibitory effect of FGF8 on COUP-TFI translation. FGF8 induces the expression of few miRNAs targeting COUP-TFI 3'UTR in silico. We found that the functional inhibition of miR-21 can effectively counteract the inhibitory effect of FGF8 in vitro and regulate COUP-TFI protein levels in vivo. Accordingly, miR-21 expression is complementary to COUP-TFI expression during corticogenesis. These data support a translational control of COUP-TFI gradient expression by FGF8 via miR-21 and contribute to our understanding of how regionalized expression is established during neocortical area mapping.
Assuntos
Fator I de Transcrição COUP/genética , Córtex Cerebral/embriologia , Fator 8 de Crescimento de Fibroblasto/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Padronização Corporal , Diferenciação Celular , Córtex Cerebral/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Biossíntese de ProteínasRESUMO
BACKGROUND: The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality. RESULTS: We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers. CONCLUSIONS: This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , RNA Longo não Codificante/genética , Animais , Sequência de Bases , Cromatina/química , Sequência Conservada , Genoma , Humanos , Camundongos , Neoplasias/genética , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , RNA Longo não Codificante/química , Fatores de Transcrição/genéticaRESUMO
Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.
Assuntos
Anticorpos/genética , Diversidade de Anticorpos/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Anticorpos/imunologia , Diversidade de Anticorpos/imunologia , Análise por Conglomerados , Biologia Computacional/métodos , Simulação por Computador , Humanos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Recombinação V(D)J , Fluxo de TrabalhoAssuntos
Neoplasias Duodenais/metabolismo , Epigênese Genética , Gastrinoma/metabolismo , Redes Reguladoras de Genes , MicroRNAs/metabolismo , Neoplasia Endócrina Múltipla Tipo 1/metabolismo , Tumores Neuroendócrinos/metabolismo , Neoplasias Gástricas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/genética , Humanos , Pâncreas/metabolismoRESUMO
Mutations and genetic variability affect gene expression and lifespan, but the impact of variations in gene expression within individuals on their aging-related mortality is poorly understood. We performed a longitudinal study in the short-lived killifish, Nothobranchius furzeri, and correlated quantitative variations in gene expression during early adult life with lifespan. Shorter- and longer-lived individuals differ in their gene expression before the onset of aging-related mortality; differences in gene expression are more pronounced early in life. We identified mitochondrial respiratory chain complex I as a hub in a module of genes whose expression is negatively correlated with lifespan. Accordingly, partial pharmacological inhibition of complex I by the small molecule rotenone reversed aging-related regulation of gene expression and extended lifespan in N. furzeri by 15%. These results support the use of N. furzeri as a vertebrate model for identifying the protein targets, pharmacological modulators, and individual-to-individual variability associated with aging.
Assuntos
Vertebrados , Animais , Ciprinodontiformes , Estudos Longitudinais , RNA , Análise de Sequência de RNARESUMO
BACKGROUND: Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. RESULTS: We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. CONCLUSIONS: We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.
Assuntos
Carboxipeptidases/genética , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias Murinas , Complexo de Inativação Induzido por RNA/genética , Animais , Diferenciação Celular/genética , Cromatina/genética , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Pluripotentes , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
Embryonic stem (ES) cells are becoming a popular model of in vitro neurogenesis, as they display intrinsic capability to generate neural progenitors that undergo the known steps of in vivo neural development. These include the acquisition of distinct regional fates, which depend on growth factors and signals that are present in the culture medium. The control of the intracellular signaling that is active at different steps of ES cell neuralization, even when cells are cultured in chemically defined medium, is complicated by the endogenous production of growth factors. However, this endogenous production has been poorly investigated so far. To address this point, we performed a high-throughput analysis of the expression of morphogens during mouse ES cell neuralization in minimal medium. We found that during their neuralization, ES cells increased the expression of members of Wnt, Fibroblast Growth Factor (FGF), and BMP families. Conversely, the expression of Activin/Nodal and Shh ligands was low in early steps of neuralization. In this experimental condition, neural progenitors and neurons generated by ES cells expressed a gene expression profile that was consistent with a midbrain identity. We found that endogenous BMP and Wnt signaling, but not FGF signaling, synergistically affected ES cell neural patterning, by turning off a profile of dorsal/telencephalic gene expression. Double BMP and Wnt inhibition allowed neuralized ES cells to sequentially activate key genes of cortical differentiation. Our findings are consistent with a novel synergistic effect of Wnt and BMP endogenous signaling of ES cells in inhibiting a cortical differentiation program.