RESUMO
A remarkable epigenetic remodelling process occurs shortly after fertilization, which restores totipotency to the zygote. This involves global DNA demethylation, chromatin remodelling, genome spatial reorganization and substantial transcriptional changes. Key to these changes is the transition from the maternal environment of the oocyte to an embryonic-driven developmental expression programme, a process termed the maternal-to-zygotic transition (MZT). Zygotic genome activation occurs predominantly at the two-cell stage in mice and the eight-cell stage in humans, yet the dynamics of its control are still mostly obscure. In recent years, partly due to single-cell and low-cell number epigenomic studies, our understanding of the epigenetic and chromatin landscape of preimplantation development has improved considerably. In this Review, we discuss the latest advances in the study of the MZT, focusing on DNA methylation, histone post-translational modifications, local chromatin structure and higher-order genome organization. We also discuss key mechanistic studies that investigate the mode of action of chromatin regulators, transcription factors and non-coding RNAs during preimplantation development. Finally, we highlight areas requiring additional research, as well as new technological advances that could assist in eventually completing our understanding of the MZT.
Assuntos
Epigênese Genética/genética , Zigoto/fisiologia , Animais , Cromatina/genética , Metilação de DNA/genética , Epigenômica/métodos , Humanos , Processamento de Proteína Pós-Traducional/genéticaRESUMO
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.
Assuntos
Diferenciação Celular , Impressão Genômica , Células Germinativas/metabolismo , Células Germinativas/fisiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Feminino , Humanos , MasculinoRESUMO
Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.
Assuntos
Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Técnicas Citológicas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Mitocôndrias/metabolismo , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues1-3. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development4-6 and to advance stem-cell-based regenerative approaches7. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.
Assuntos
Callithrix , Gastrulação , Útero , Animais , Callithrix/embriologia , Diferenciação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Endoderma/citologia , Endoderma/embriologia , Feminino , Perfilação da Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
DNA methylation is a key layer of epigenetic regulation. The deposition of methylation marks relies on the catalytic activity of DNA methyltransferases (DNMTs), and their active removal relies on the activity of ten-eleven translocation (TET) enzymes. Paradoxically, in important biological contexts these antagonistic factors are co-expressed and target overlapping genomic regions. The ensuing cyclic biochemistry of cytosine modifications gives rise to a continuous, out-of-thermal equilibrium transition through different methylation states. But what is the purpose of this intriguing turnover of DNA methylation? Recent evidence demonstrates that methylation turnover is enriched at gene distal regulatory elements, including enhancers, and can give rise to large-scale oscillatory dynamics. We discuss this phenomenon and propose that DNA methylation turnover might facilitate key lineage decisions.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linhagem da Célula , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Dioxigenases , Humanos , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
The molecular regulation of zygotic genome activation (ZGA) in mammals remains an exciting area of research. Primed mouse embryonic stem cells contain a rare subset of "2C-like" cells that are epigenetically and transcriptionally similar to the two-cell embryo and thus represent an in vitro approximation for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA in either a Dux-dependent or Dux-independent manner. Here we performed a candidate-based overexpression screen, identifying, among others, developmental pluripotency-associated 2 (Dppa2) and Dppa4 as positive regulators of 2C-like cells and transcription of ZGA genes. In the germline, promoter DNA demethylation coincides with expression of Dppa2 and Dppa4, which remain expressed until embryonic day 7.5 (E7.5), when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during induced pluripotent stem cell (iPSC) reprogramming at the time that 2C-like transcription transiently peaks. Through a combination of overexpression, knockdown, knockout, and rescue experiments together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we teased apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilized. Dppa2 and Dppa4 require Dux to initiate 2C-like transcription, suggesting that they act upstream by directly regulating Dux. Supporting this, ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild-type cells but, in contrast to Dux, can no longer do so in Dppa2/4 double-knockout cells, suggesting that it may act to stabilize rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux up-regulation and activation of the 2C-like transcriptional program, which is subsequently reinforced by Zscan4c.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Zigoto , Animais , Cromatina/metabolismo , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias , Epigênese Genética/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Camundongos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genéticaRESUMO
Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Atenção à Saúde/métodos , Atenção à Saúde/tendências , Medicina/métodos , Medicina/tendências , Patologia , Análise de Célula Única , Inteligência Artificial , Atenção à Saúde/ética , Atenção à Saúde/normas , Diagnóstico Precoce , Educação Médica , Europa (Continente) , Feminino , Saúde , Humanos , Legislação Médica , Masculino , Medicina/normasRESUMO
The preconceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of assisted reproductive technologies (ART; â¼25%), additives and adjuvants, such as glucocorticoids, are used to improve the success rate. Considering the dynamic developmental events that occur during this window, these exposures may alter blastocyst formation at a molecular level, and as such, affect not only the viability of the embryo and the ability of the blastocyst to implant, but also the developmental trajectory of the first three cell lineages, ultimately influencing the physiology of the embryo. In this study, we present a comprehensive single-cell transcriptome, methylome, and small RNA atlas in the day 7 human embryo. We show that, despite no change in morphology and developmental features, preimplantation glucocorticoid exposure reprograms the molecular profile of the TE lineage, and these changes are associated with an altered metabolic and inflammatory response. Our data also suggest that glucocorticoids can precociously mature the TE sublineages, supported by the presence of extravillous trophoblast markers in the polar sublineage and presence of X Chromosome dosage compensation. Further, we have elucidated that epigenetic regulation-DNA methylation and microRNAs (miRNAs)-likely underlies the transcriptional changes observed. This study suggests that exposures to exogenous compounds during preimplantation may unintentionally reprogram the human embryo, possibly leading to suboptimal development and longer-term health outcomes.
RESUMO
Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.
Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Gastrulação , Organogênese , Análise de Célula Única , Animais , Linhagem da Célula/genética , Quimera/embriologia , Quimera/genética , Quimera/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Endotélio/citologia , Endotélio/embriologia , Endotélio/metabolismo , Feminino , Gastrulação/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Mutação/genética , Células Mieloides/citologia , Organogênese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/deficiência , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genéticaRESUMO
Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
Assuntos
Metilação de DNA , Epigênese Genética , Gástrula/citologia , Gástrula/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA/genética , Análise de Célula Única , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Cromatina/metabolismo , Desmetilação , Corpos Embrioides/citologia , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Elementos Facilitadores Genéticos/genética , Epigenoma/genética , Eritropoese , Análise Fatorial , Gástrula/embriologia , Gastrulação/fisiologia , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA/análise , Fatores de Tempo , Dedos de ZincoRESUMO
Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as CDKN1A We developed an innovative approach that integrates single-cell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16INK4a, p21CIP1, and p15INK4b, preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes.
Assuntos
Reprogramação Celular , Senescência Celular , Perfilação da Expressão Gênica , RNA Interferente Pequeno , Análise de Sequência de RNA , Serina-Treonina Quinases TOR/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Análise de Célula Única , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
Zygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.
Assuntos
Cromatina/genética , Desenvolvimento Embrionário/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Ativação Transcricional/genética , Zigoto/crescimento & desenvolvimentoRESUMO
RNA is not only a messenger operating between DNA and protein. Transcription of essentially the entire eukaryotic genome generates a myriad of non-protein-coding RNA species that show complex overlapping patterns of expression and regulation. Although long noncoding RNAs (lncRNAs) are among the least well-understood of these transcript species, they cannot all be dismissed as merely transcriptional "noise." Here, we review the evolution of lncRNAs and their roles in transcriptional regulation, epigenetic gene regulation, and disease.
Assuntos
Evolução Molecular , Regulação da Expressão Gênica , RNA não Traduzido/metabolismo , Animais , Humanos , RNA não Traduzido/genéticaRESUMO
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
Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.
Assuntos
Blastômeros/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Blastocisto/citologia , Blastômeros/metabolismo , Linhagem da Célula , Células Cultivadas , Quimera , Embrião de Mamíferos/citologia , Endoderma/citologia , Epigênese Genética , Epigenômica , Feminino , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Placenta/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Gravidez , Análise de Célula Única , Transcriptoma , Trofoblastos/citologiaRESUMO
Adult skeletal muscles are maintained during homeostasis and regenerated upon injury by muscle stem cells (MuSCs). A heterogeneity in self-renewal, differentiation and regeneration properties has been reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the molecular determinants that govern these differences remain undefined. Here we show that EOM and limb MuSCs have distinct DNA methylation signatures associated with enhancers of location-specific genes, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle environment. Notably, EOM MuSCs expressed host-site specific positional Hox codes after engraftment and self-renewal within the host muscle. However, about 10% of EOM-specific genes showed engraftment-resistant expression, pointing to cell-intrinsic molecular determinants of the higher engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC populations and molecularly define their plasticity in response to microenvironmental cues. These findings provide insights into strategies designed to improve the functional capacity of MuSCs in the context of regenerative medicine.
Assuntos
Plasticidade Celular/genética , Epigenoma/genética , Transplante de Células-Tronco , Transcriptoma/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células/genética , Extremidades/crescimento & desenvolvimento , Variação Genética/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Musculares/citologia , Fibras Musculares Esqueléticas , Músculo Esquelético/citologia , Mioblastos/citologia , Regeneração/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
TET3 is a member of the ten-eleven translocation (TET) family of enzymes which oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Tet3 is highly expressed in the brain, where 5hmC levels are most abundant. In adult mice, we observed that TET3 is present in mature neurons and oligodendrocytes but is absent in astrocytes. To investigate the function of TET3 in adult postmitotic neurons, we crossed Tet3 floxed mice with a neuronal Cre-expressing mouse line, Camk2a-CreERT2, obtaining a Tet3 conditional KO (cKO) mouse line. Ablation of Tet3 in adult mature neurons resulted in increased anxiety-like behavior with concomitant hypercorticalism, and impaired hippocampal-dependent spatial orientation. Transcriptome and gene-specific expression analysis of the hippocampus showed dysregulation of genes involved in glucocorticoid signaling pathway (HPA axis) in the ventral hippocampus, whereas upregulation of immediate early genes was observed in both dorsal and ventral hippocampal areas. In addition, Tet3 cKO mice exhibit increased dendritic spine maturation in the ventral CA1 hippocampal subregion. Based on these observations, we suggest that TET3 is involved in molecular alterations that govern hippocampal-dependent functions. These results reveal a critical role for epigenetic modifications in modulating brain functions, opening new insights into the molecular basis of neurological disorders.
Assuntos
Ansiedade , Cognição , Dioxigenases , Neurônios , Animais , Ansiedade/genética , Encéfalo/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Camundongos , Neurônios/metabolismo , Sistema Hipófise-Suprarrenal/metabolismoRESUMO
Constitutive heterochromatin is typically defined by high levels of DNA methylation and H3 lysine 9 trimethylation (H3K9Me3), whereas facultative heterochromatin displays DNA hypomethylation and high H3 lysine 27 trimethylation (H3K27Me3). The two chromatin types generally do not coexist at the same loci, suggesting mutual exclusivity. During development or in cancer, pericentromeric regions can adopt either epigenetic state, but the switching mechanism is unknown. We used a quantitative locus purification method to characterize changes in pericentromeric chromatin-associated proteins in mouse embryonic stem cells deficient for either the methyltransferases required for DNA methylation or H3K9Me3. DNA methylation controls heterochromatin architecture and inhibits Polycomb recruitment. BEND3, a protein enriched on pericentromeric chromatin in the absence of DNA methylation or H3K9Me3, allows Polycomb recruitment and H3K27Me3, resulting in a redundant pathway to generate repressive chromatin. This suggests that BEND3 is a key factor in mediating a switch from constitutive to facultative heterochromatin.