RESUMEN
Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.
Asunto(s)
Pérdida del Embrión/genética , Pérdida del Embrión/patología , Mutación , Placenta/patología , Placentación/genética , Animales , Femenino , Ratones , Ratones Noqueados , Embarazo , Células Madre/metabolismo , Células Madre/patología , Trofoblastos/metabolismo , Trofoblastos/patologíaRESUMEN
An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.
Asunto(s)
Heterocromatina/genética , Heterocromatina/metabolismo , Células Madre Embrionarias de Ratones/fisiología , Proteína Homeótica Nanog/metabolismo , Animales , Línea Celular , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Regulación hacia Abajo , Eliminación de Gen , Ratones , Proteína Homeótica Nanog/genética , Dominios Proteicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trofoblastos/citología , Animales , Diferenciación Celular/genética , Línea Celular , Autorrenovación de las Células/genética , Proteínas de Unión al ADN/química , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/química , Trofoblastos/metabolismoRESUMEN
Embryonic stem (ES) cells are in a dynamic equilibrium of distinct functional states, characterized by the heterogeneous expression of critical pluripotency factors and regulated by a spectrum of reversible histone modifications. Maintenance of this equilibrium is a hallmark of pluripotency. Here we find that the ADP-ribosyltransferases Parp1 and Parp7 play a critical role in safeguarding this state by occupying key pluripotency genes, notably Nanog, Pou5f1, Sox2, Stella, Tet1 and Zfp42, thereby protecting them from progressive epigenetic repression. In the absence of either Parp1 or Parp7, or upon inhibition of the ADP-ribosylating activity, ES cells exhibit a decrease in ground state pluripotency as they cannot maintain the typical heterogeneity characteristic of the metastable state. As a consequence, they display a higher propensity to differentiate. These findings place Parp1 and Parp7 at the genetic-epigenetic interface of pluripotency networks, fine-tuning the transcriptional heterogeneity and thereby determining the developmental plasticity of ES cells.
Asunto(s)
ADP Ribosa Transferasas/fisiología , Células Madre Embrionarias/enzimología , Células Madre Pluripotentes/enzimología , Poli(ADP-Ribosa) Polimerasas/fisiología , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , Animales , Diferenciación Celular , Línea Celular , Cromatina/química , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Metilación , Ratones , Mutación , Células Madre Pluripotentes/citología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de Transcripción/metabolismo , Trofoblastos/citología , Trofoblastos/enzimologíaRESUMEN
Macrophages exert a wide variety of functions, which necessitate a high level of plasticity on the chromatin level. In the work presented here, we analyzed the role of the polycomb group protein Bmi1 during the acute response of bone marrow derived macrophages (BMDM) to lipopolysaccharide (LPS). Unexpectedly, we observed that Bmi1 was rapidly induced at the protein level and transiently phosphorylated upon LPS treatment. The induction of Bmi1 was dependent on MAP-kinase signaling. LPS treatment of BMDM in the absence of Bmi1 resulted in a pronounced increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Our results identify Bmi1 as a repressor of IL-10 expression during macrophage activation.
Asunto(s)
Interleucina-10/biosíntesis , Activación de Macrófagos , Macrófagos/inmunología , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Línea Celular , Interleucina-10/metabolismo , Lipopolisacáridos/inmunología , Sistema de Señalización de MAP Quinasas , Activación de Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Complejo Represivo Polycomb 1 , Interferencia de ARN , ARN Interferente Pequeño , Receptor Toll-Like 4/metabolismoRESUMEN
BAD, a member of the BCL2 family, exhibits an original mode of regulation by phosphorylation. In the present report, we examine the role of the kinase C-RAF in this process. We show that the inducible activation of C-RAF promotes the rapid phosphorylation of BAD on Serine-112 (Ser-75 in the human protein), through a cascade involving the kinases MEK and RSK. Our findings reveal a new aspect of the regulation of BAD protein and its control by the RAF pathway: we find that C-RAF activation promotes BAD poly-ubiquitylation in a phosphorylation-dependent fashion, and increases the turn-over of this protein through proteasomal degradation.
Asunto(s)
Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ubiquitinación , Proteína Letal Asociada a bcl/metabolismo , Animales , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Serina/metabolismo , Proteína Letal Asociada a bcl/genéticaRESUMEN
Gene loci that are hypermethylated and repressed in embryonic (ESCs) but hypomethylated and expressed in trophoblast (TSCs) stem cells are very rare and may have particularly important roles in early developmental cell fate decisions, as previously shown for Elf5. Here, we assessed another member of this small group of genes, Placenta Expressed Transcript 1 (Plet1), for its function in establishing trophoblast lineage identity and modulating trophoblast differentiation. We find that Plet1 is tightly repressed by DNA methylation in ESCs but expressed on the cell surface of TSCs and trophoblast giant cells. In hypomethylated ESCs that are prone to acquire some trophoblast characteristics, Plet1 is required to confer a trophoblast-specific gene expression pattern, including up-regulation of Elf5. Plet1 displays an unusual biphasic expression profile during TSC differentiation and thus may be pivotal in balancing trophoblast self-renewal and differentiation. Furthermore, overexpression and CRISPR/Cas9-mediated knockout in TSCs showed that high Plet1 levels favour differentiation towards the trophoblast giant cell lineage, whereas lack of Plet1 preferentially induces syncytiotrophoblast formation. Thus, the endogenous dynamics of Plet1 expression establish important patterning cues within the trophoblast compartment by promoting differentiation towards the syncytiotrophoblast or giant cell pathway in Plet1-low and Plet1-high cells, respectively.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Proteínas Gestacionales/genética , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Trofoblastos/metabolismo , Animales , Metilación de ADN , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Gestacionales/metabolismo , Células Madre/fisiología , Trofoblastos/fisiologíaRESUMEN
Through the histone methyltransferase EZH2, the Polycomb complex PRC2 mediates H3K27me3 and is associated with transcriptional repression. PRC2 regulates cell-fate decisions in model organisms; however, its role in regulating cell differentiation during human embryogenesis is unknown. Here, we report the characterization of EZH2-deficient human embryonic stem cells (hESCs). H3K27me3 was lost upon EZH2 deletion, identifying an essential requirement for EZH2 in methylating H3K27 in hESCs, in contrast to its non-essential role in mouse ESCs. Developmental regulators were derepressed in EZH2-deficient hESCs, and single-cell analysis revealed an unexpected acquisition of lineage-restricted transcriptional programs. EZH2-deficient hESCs show strongly reduced self-renewal and proliferation, thereby identifying a more severe phenotype compared to mouse ESCs. EZH2-deficient hESCs can initiate differentiation toward developmental lineages; however, they cannot fully differentiate into mature specialized tissues. Thus, EZH2 is required for stable ESC self-renewal, regulation of transcriptional programs, and for late-stage differentiation in this model of early human development.
Asunto(s)
Diferenciación Celular/genética , Autorrenovación de las Células/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Células Madre Embrionarias Humanas/metabolismo , Animales , Proliferación Celular/genética , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Ratones , Complejo Represivo Polycomb 2/genética , Proteínas del Grupo Polycomb/genética , Eliminación de Secuencia , Análisis de la Célula IndividualRESUMEN
BACKGROUND: We have previously described a RAF oncogene driven transgenic mouse model for non small cell lung cancer (NSCLC). Here we examine whether tumor initiation and growth requires the stem cell self-renewal factor Bmi1. PRINCIPAL FINDINGS: In order to evaluate Bmi1 function in NSCLC two founder lines that differ in incidence and latency of tumor formation were compared. Ablation of Bmi1 expression in both lines had a dramatically decreased tumor growth. As the line with shorter latency matched the life span of Bmi1 knock out mice, these mice were chosen for further study. The absence of Bmi1 did not decrease the number of tumor initiation in these mice as only the size and not the number of tumors decreased. Reduction in tumor growth resulted from an increase in cell death and decrease in cell cycle progression that corresponded with up-regulation of the p16(INK4a) and p19(ARF). SIGNIFICANCE: The data identifies Bmi1 as an important factor for expansion but not initiation of RAF driven NSCLC.