RESUMO
Life on Earth has been through numerous challenges over eons and, one way or another, has always triumphed. From mass extinctions to more daily plights to find food, unpredictability is everywhere. The adaptability of life-forms to ever-changing environments is the key that confers life's robustness. Adaptability has become synonymous with Darwinian evolution mediated by heritable genetic changes. The extreme gene-centric view, while being of central significance, at times has clouded our appreciation of the cell as a self-regulating entity informed of, and informing, the genetic data. An essential element that powers adaptability is the ability to regulate cell growth. In this review, we provide an extensive overview of growth regulation spanning species, tissues, and regulatory mechanisms. We aim to highlight the commonalities, as well as differences, of these phenomena and their molecular regulators. Finally, we curate open questions and areas for further exploration.
RESUMO
Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.
Assuntos
Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Autorrenovação Celular , Imunoprecipitação da Cromatina , Retrovirus Endógenos/genética , Feminino , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Oligorribonucleotídeos Antissenso/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Interferência de RNA , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteína 28 com Motivo Tripartido/antagonistas & inibidores , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo , Regulação para Cima , NucleolinaRESUMO
Dormancy is a key feature of stem cell function in adult tissues as well as in embryonic cells in the context of diapause. The establishment of dormancy is an active process that involves extensive transcriptional, epigenetic, and metabolic rewiring. How these processes are coordinated to successfully transition cells to the resting dormant state remains unclear. Here we show that microRNA activity, which is otherwise dispensable for preimplantation development, is essential for the adaptation of early mouse embryos to the dormant state of diapause. In particular, the pluripotent epiblast depends on miRNA activity, the absence of which results in the loss of pluripotent cells. Through the integration of high-sensitivity small RNA expression profiling of individual embryos and protein expression of miRNA targets with public data of protein-protein interactions, we constructed the miRNA-mediated regulatory network of mouse early embryos specific to diapause. We find that individual miRNAs contribute to the combinatorial regulation by the network, and the perturbation of the network compromises embryo survival in diapause. We further identified the nutrient-sensitive transcription factor TFE3 as an upstream regulator of diapause-specific miRNAs, linking cytoplasmic MTOR activity to nuclear miRNA biogenesis. Our results place miRNAs as a critical regulatory layer for the molecular rewiring of early embryos to establish dormancy.
Assuntos
Proliferação de Células , MicroRNAs , Células-Tronco Pluripotentes , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Camundongos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Desenvolvimento Embrionário/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Blastocisto/metabolismo , Blastocisto/citologia , FemininoRESUMO
Organisms across species differ in the relative size and complexity of their tissues to serve the specific purposes of the host. Correct timing is a crucial ingredient in the development of tissues, as reaching the right size and complexity requires a careful balance between cellular proliferation and differentiation. Premature or delayed differentiation, for instance, can result in tissue imbalance, malformation or malfunction. Despite seemingly rigid constraints on development, however, there is flexibility in both the timing and differentiation trajectories within and between species. In this Spotlight, we discuss how time is measured and regulated in development, and question whether developmental timing is in fact different between species.
Assuntos
Evolução Biológica , Fatores de TempoRESUMO
The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development.
Assuntos
Endoderma , Linha Primitiva , Animais , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias , Endoderma/metabolismo , Hipóxia/metabolismo , MamíferosRESUMO
DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.
Assuntos
Núcleo Celular/metabolismo , Período de Replicação do DNA , Proteínas de Ligação a Telômeros/metabolismo , Animais , Proliferação de Células , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Ilhas de CpG/genética , Fase G1 , Deleção de Genes , Regulação da Expressão Gênica , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a Telômeros/química , Sítio de Iniciação de TranscriçãoRESUMO
Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2-3 days around the time of blastocyst implantation. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent-able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.
Assuntos
Blastocisto/citologia , Células-Tronco Pluripotentes/citologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Células Cultivadas , Regulação para Baixo , Desenvolvimento Embrionário , Feminino , Camadas Germinativas/citologia , Histonas/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Biossíntese de Proteínas , Serina-Treonina Quinases TOR/metabolismo , Transcrição GênicaRESUMO
Heterochromatin is required to restrict aberrant expression of retrotransposons, but it remains poorly defined due to the underlying repeat-rich sequences. We dissected Suv39h-dependent histone H3 lysine 9 trimethylation (H3K9me3) by genome-wide ChIP sequencing in mouse embryonic stem cells (ESCs). Refined bioinformatic analyses of repeat subfamilies indicated selective accumulation of Suv39h-dependent H3K9me3 at interspersed repetitive elements that cover â¼5% of the ESC epigenome. The majority of the â¼8,150 intact long interspersed nuclear elements (LINEs) and endogenous retroviruses (ERVs), but only a minor fraction of the >1.8 million degenerate and truncated LINEs/ERVs, are enriched for Suv39h-dependent H3K9me3. Transcriptional repression of intact LINEs and ERVs is differentially regulated by Suv39h and other chromatin modifiers in ESCs but governed by DNA methylation in committed cells. These data provide a function for Suv39h-dependent H3K9me3 chromatin to specifically repress intact LINE elements in the ESC epigenome.
Assuntos
Células-Tronco Embrionárias/enzimologia , Retrovirus Endógenos/genética , Inativação Gênica , Histona-Lisina N-Metiltransferase/fisiologia , Histonas/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Metiltransferases/fisiologia , Proteínas Repressoras/fisiologia , Animais , Células Cultivadas , Metilação de DNA , Camundongos , Processamento de Proteína Pós-TraducionalRESUMO
The pluripotent mammalian epiblast undergoes unusually fast cell proliferation. This rapid growth is expected to generate a high transcriptional demand, but the underlying mechanisms remain unknown. We show here that the chromatin remodeler Chd1 is required for transcriptional output and development of the mouse epiblast. Chd1(-/-) embryos exhibit proliferation defects and increased apoptosis, are smaller than controls by E5.5 and fail to grow, to become patterned or to gastrulate. Removal of p53 allows progression of Chd1(-/-) mutants only to E7.0-8.0, highlighting the crucial requirement for Chd1 during early post-implantation development. Chd1(-/-) embryonic stem cells (ESCs) have a self-renewal defect and a genome-wide reduction in transcriptional output at both known mRNAs and intergenic transcripts. These transcriptional defects were only uncovered when cell number-normalized approaches were used, and correlate with a lower engagement of RNAP II with transcribed genes in Chd1(-/-) ESCs. We further show that Chd1 directly binds to ribosomal DNA, and that both Chd1(-/-) epiblast cells in vivo and ESCs in vitro express significantly lower levels of ribosomal RNA. In agreement with these findings, mutant cells in vivo and in vitro exhibit smaller and more elongated nucleoli. Thus, the RNA output by both Pol I and II is reduced in Chd1(-/-) cells. Our data indicate that Chd1 promotes a globally elevated transcriptional output required to sustain the distinctly rapid growth of the mouse epiblast.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Camadas Germinativas/crescimento & desenvolvimento , Camadas Germinativas/metabolismo , Transcrição Gênica , Animais , Apoptose/genética , Padronização Corporal/genética , Ciclo Celular/genética , Cruzamentos Genéticos , DNA Ribossômico/genética , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Endogâmicos C57BL , Precursores de RNA/genéticaRESUMO
Embryonic and adult stem cells enable development and regeneration. Embryonic cells, like adult stem cells, can enter dormancy as part of their lifecycle. Recent evidence suggests that this cellular transition to dormancy requires active rewiring of metabolism. The dormancy-induced metabolic switches in embryonic and adult stem cells are explored here.
RESUMO
Mammalian developmental timing is adjustable in vivo by preserving pre-implantation embryos in a dormant state called diapause. Inhibition of the growth regulator mTOR (mTORi) pauses mouse development in vitro, yet how embryonic dormancy is maintained is not known. Here we show that mouse embryos in diapause are sustained by using lipids as primary energy source. In vitro, supplementation of embryos with the metabolite L-carnitine balances lipid consumption, puts the embryos in deeper dormancy and boosts embryo longevity. We identify FOXO1 as an essential regulator of the energy balance in dormant embryos and propose, through meta-analyses of dormant cell signatures, that it may be a common regulator of dormancy across adult tissues. Our results lift a constraint on in vitro embryo survival and suggest that lipid metabolism may be a critical metabolic transition relevant for longevity and stem cell function across tissues.
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Embrião de Mamíferos , Metabolismo dos Lipídeos , Animais , Camundongos , Desenvolvimento Embrionário/fisiologia , Metabolismo Energético , MamíferosRESUMO
Dormancy is an essential biological process for the propagation of many life forms through generations and stressful conditions. Early embryos of many mammals are preservable for weeks to months within the uterus in a dormant state called diapause, which can be induced in vitro through mTOR inhibition. Cellular strategies that safeguard original cell identity within the silent genomic landscape of dormancy are not known. Here we show that the protection of cis-regulatory elements from silencing is key to maintaining pluripotency in the dormant state. We reveal a TET-transcription factor axis, in which TET-mediated DNA demethylation and recruitment of methylation-sensitive transcription factor TFE3 drive transcriptionally inert chromatin adaptations during dormancy transition. Perturbation of TET activity compromises pluripotency and survival of mouse embryos under dormancy, whereas its enhancement improves survival rates. Our results reveal an essential mechanism for propagating the cellular identity of dormant cells, with implications for regeneration and disease.
RESUMO
The energetically costly mammalian investment in gestation and lactation requires plentiful nutritional sources and thus links the environmental conditions to reproductive success. Flexibility in adjusting developmental timing enhances chances of survival in adverse conditions. Over 130 mammalian species can reversibly pause early embryonic development by switching to a near dormant state that can be sustained for months, a phenomenon called embryonic diapause. Lineage-specific cells are retained during diapause, and they proliferate and differentiate upon activation. Studying diapause thus reveals principles of pluripotency and dormancy and is not only relevant for development, but also for regeneration and cancer. In this review, we focus on the molecular regulation of diapause in early mammalian embryos and relate it to maintenance of potency in stem cells in vitro. Diapause is established and maintained by active rewiring of the embryonic metabolome, epigenome, and gene expression in communication with maternal tissues. Herein, we particularly discuss factors required at distinct stages of diapause to induce, maintain, and terminate dormancy.
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Stem and progenitor cells undergo a global elevation of nascent transcription, or hypertranscription, during key developmental transitions involving rapid cell proliferation. The chromatin remodeler Chd1 mediates hypertranscription in pluripotent cells but its mechanism of action remains poorly understood. Here we report a novel role for Chd1 in protecting genome integrity at promoter regions by preventing DNA double-stranded break (DSB) accumulation in ES cells. Chd1 interacts with several DNA repair factors including Atm, Parp1, Kap1 and Topoisomerase 2ß and its absence leads to an accumulation of DSBs at Chd1-bound Pol II-transcribed genes and rDNA. Genes prone to DNA breaks in Chd1 KO ES cells are longer genes with GC-rich promoters, a more labile nucleosomal structure and roles in chromatin regulation, transcription and signaling. These results reveal a vulnerability of hypertranscribing stem cells to accumulation of endogenous DNA breaks, with important implications for developmental and cancer biology.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Transdução de Sinais , Sítio de Iniciação de TranscriçãoRESUMO
A permissive chromatin environment coupled to hypertranscription drives the rapid proliferation of embryonic stem cells (ESCs) and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ESCs. The results revealed that cellular growth pathways, most prominently translation, perpetuate the euchromatic state and hypertranscription of ESCs. Acute inhibition of translation rapidly depletes euchromatic marks in mouse ESCs and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Translation inhibition promotes rewiring of chromatin accessibility, which decreases at a subset of active developmental enhancers and increases at histone genes and transposable elements. Proteome-scale analyses revealed that several euchromatin regulators are unstable proteins and continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription, and translation sets the pace of proliferation at peri-implantation and may be employed by other stem/progenitor cells.
Assuntos
Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Biossíntese de Proteínas , Transcrição Gênica , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular , Elementos de DNA Transponíveis/genética , Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos/genética , Eucromatina/metabolismo , Feminino , Genoma , Código das Histonas , Masculino , Camundongos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Serina-Treonina Quinases TOR/metabolismoRESUMO
Cells can globally upregulate their transcriptome during specific transitions, a phenomenon called hypertranscription. Evidence for hypertranscription dates back over 70 years but has gone largely ignored in the genomics era until recently. We discuss data supporting the notion that hypertranscription is a unifying theme in embryonic development, stem cell biology, regeneration, and cell competition. We review the history, methods for analysis, underlying mechanisms, and biological significance of hypertranscription.
Assuntos
Desenvolvimento Embrionário/genética , Regeneração/genética , Células-Tronco/metabolismo , Transcrição Gênica , Animais , Humanos , Ativação Linfocitária/genética , Zigoto/metabolismoRESUMO
Heterochromatin is important for genome integrity and stabilization of gene-expression programs. We have identified the transcription factors Pax3 and Pax9 as redundant regulators of mouse heterochromatin, as they repress RNA output from major satellite repeats by associating with DNA within pericentric heterochromatin. Simultaneous depletion of Pax3 and Pax9 resulted in dramatic derepression of major satellite transcripts, persistent impairment of heterochromatic marks and defects in chromosome segregation. Genome-wide analyses of methylated histone H3 at Lys9 showed enrichment at intergenic major satellite repeats only when these sequences retained intact binding sites for Pax and other transcription factors. Additionally, bioinformatic interrogation of all histone methyltransferase Suv39h-dependent heterochromatic repeat regions in the mouse genome revealed a high concordance with the presence of transcription factor binding sites. These data define a general model in which reiterated arrangement of transcription factor binding sites within repeat sequences is an intrinsic mechanism of the formation of heterochromatin.