RESUMEN
Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.
Asunto(s)
Citrobacter rodentium , Infecciones por Enterobacteriaceae , Glucólisis , Proteínas HMGB , Inmunidad Innata , Linfocitos , Ratones Noqueados , Animales , Ratones , Adaptación Fisiológica/inmunología , Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/metabolismo , Hexoquinasa/metabolismo , Hexoquinasa/genética , Interleucina-17/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Transactivadores/metabolismo , Transactivadores/genética , Proteínas HMGB/genética , Proteínas HMGB/inmunología , Proteínas HMGB/metabolismoRESUMEN
In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.
Asunto(s)
Genes Homeobox , Proteínas de Homeodominio , Células Madre Embrionarias de Ratones , Factores de Transcripción , Animales , Ratones , Diferenciación Celular , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Madre Embrionarias de Ratones/metabolismoRESUMEN
Totipotency is the ability of a single cell to develop into a full organism and, in mammals, is strictly associated with the early stages of development following fertilization. This unlimited developmental potential becomes quickly restricted as embryonic cells transition into a pluripotent state. The loss of totipotency seems a consequence of the zygotic genome activation (ZGA), a process that determines the switch from maternal to embryonic transcription, which in mice takes place following the first cleavage. ZGA confers to the totipotent cell a transient transcriptional profile characterized by the expression of stage-specific genes and a set of transposable elements that prepares the embryo for subsequent development. The timely silencing of this transcriptional program during the exit from totipotency is required to ensure proper development. Importantly, the molecular mechanisms regulating the transition from totipotency to pluripotency have remained elusive due to the scarcity of embryonic material. However, the development of new in vitro totipotent-like models together with advances in low-input genome-wide technologies, are providing a better mechanistic understanding of how this important transition is achieved. This review summarizes the current knowledge on the molecular determinants that regulate the exit from totipotency.
Asunto(s)
Embrión de Mamíferos , Cigoto , Ratones , Animales , Cigoto/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Mamíferos/genéticaRESUMEN
The naïve epiblast transitions to a pluripotent primed state during embryo implantation. Despite the relevance of the FGF pathway during this period, little is known about the downstream effectors regulating this signaling. Here, we examined the molecular mechanisms coordinating the naïve to primed transition by using inducible ESC to genetically eliminate all RAS proteins. We show that differentiated RASKO ESC remain trapped in an intermediate state of pluripotency with naïve-associated features. Elimination of the transcription factor ERF overcomes the developmental blockage of RAS-deficient cells by naïve enhancer decommissioning. Mechanistically, ERF regulates NANOG expression and ensures naïve pluripotency by strengthening naïve transcription factor binding at ESC enhancers. Moreover, ERF negatively regulates the expression of the methyltransferase DNMT3B, which participates in the extinction of the naïve transcriptional program. Collectively, we demonstrated an essential role for ERF controlling the exit from naïve pluripotency in a MAPK-dependent manner during the progression to primed pluripotency.
RESUMEN
Totipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.
Asunto(s)
Factor de Unión a CCCTC/metabolismo , Reprogramación Celular , Células Madre Totipotentes/citología , Animales , Sitios de Unión , Factor de Unión a CCCTC/genética , Muerte Celular , Daño del ADN , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Células Madre Totipotentes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The recent availability of somatic haploid cell lines has provided a unique tool for genetic studies in mammals. However, the percentage of haploid cells rapidly decreases in these cell lines, which we recently showed is due to their overgrowth by diploid cells present in the cultures. Based on this property, we have now performed a phenotypic chemical screen in human haploid HAP1 cells aiming to identify compounds that facilitate the maintenance of haploid cells. Our top hit was 10-Deacetyl-baccatin-III (DAB), a chemical precursor in the synthesis of Taxol, which selects for haploid cells in HAP1 and mouse haploid embryonic stem cultures. Interestingly, DAB also enriches for diploid cells in mixed cultures of diploid and tetraploid cells, including in the colon cancer cell line DLD-1, revealing a general strategy for selecting cells with lower ploidy in mixed populations of mammalian cells.
Asunto(s)
Células Madre Embrionarias/citología , Haploidia , Ensayos Analíticos de Alto Rendimiento/métodos , Ploidias , Taxoides/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Separación Celular , Diploidia , Células Madre Embrionarias/metabolismo , Humanos , Ratones , Mitosis/efectos de los fármacos , Mitosis/genética , Taxoides/químicaRESUMEN
MEK inhibition in combination with a glycogen synthase kinase-3ß (GSK3ß) inhibitor, referred as the 2i condition, favors pluripotency in embryonic stem cells (ESCs). However, the mechanisms by which the 2i condition limits ESC differentiation and whether RAS proteins are involved in this phenomenon remain poorly understood. Here we show that RAS nullyzygosity reduces the growth of mouse ESCs (mESCs) and prohibits their differentiation. Upon RAS deficiency or MEK inhibition, ERF (E twenty-six 2 [Ets2]-repressive factor), a transcriptional repressor from the ETS domain family, translocates to the nucleus, where it binds to the enhancers of pluripotency factors and key RAS targets. Remarkably, deletion of Erf rescues the proliferative defects of RAS-devoid mESCs and restores their capacity to differentiate. Furthermore, we show that Erf loss enables the development of RAS nullyzygous teratomas. In summary, this work reveals an essential role for RAS proteins in pluripotency and identifies ERF as a key mediator of the response to RAS/MEK/ERK inhibition in mESCs.
Asunto(s)
Células Madre Embrionarias/citología , Genes ras , Proteínas Represoras/fisiología , Animales , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Eliminación de Gen , Ratones , Ratones Desnudos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Teratoma/genéticaRESUMEN
The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include near-haploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell-sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells.
Asunto(s)
Supervivencia Celular/fisiología , Haploidia , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Proliferación Celular/fisiología , Segregación Cromosómica , Humanos , Ratones , Células Madre Embrionarias de Ratones/fisiologíaRESUMEN
One recurring theme in drug development is to exploit synthetic lethal properties as means to preferentially damage the DNA of cancer cells. We and others have previously developed inhibitors of the ATR kinase, shown to be particularly genotoxic for cells expressing certain oncogenes. In contrast, the mechanisms of resistance to ATR inhibitors remain unexplored. We report here on a genome-wide CRISPR-Cas9 screen that identified CDC25A as a major determinant of sensitivity to ATR inhibition. CDC25A-deficient cells resist high doses of ATR inhibitors, which we show is due to their failure to prematurely enter mitosis in response to the drugs. Forcing mitotic entry with WEE1 inhibitors restores the toxicity of ATR inhibitors in CDC25A-deficient cells. With ATR inhibitors now entering the clinic, our work provides a better understanding of the mechanisms by which these compounds kill cells and reveals genetic interactions that could be used for their rational use.
Asunto(s)
Antineoplásicos/farmacología , Sistemas CRISPR-Cas , Resistencia a Antineoplásicos/genética , Células Madre Embrionarias/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Fosfatasas cdc25/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/enzimología , Células Madre Embrionarias/patología , Estudio de Asociación del Genoma Completo , Humanos , Mitosis/efectos de los fármacos , Terapia Molecular Dirigida , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Transfección , Fosfatasas cdc25/genéticaRESUMEN
Gene expression regulation in Leishmania has been related to post-transcriptional events involving mainly sequences present in the 5' and 3' untranslated regions. PABPs are high-affinity poly(A)-binding proteins that are implicated in the regulation of translation initiation, RNA stability and other important biological processes. We describe a PABP from Leishmania infantum (LiPABP) that shows a very high homology with PABPs from other eukaryotic organisms, including mammals and other parasites. LiPABP conserves the main domains present in other PABPs, maintains poly(A)-binding properties and is phosphorylated by p38 mitogen-activated protein kinase. Using the sera from dogs infected with L. infantum, we demonstrate that LiPABP is expressed in L. infantum promastigotes.