RESUMEN
In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examined the effects of E2f1, E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines. We show that in normal dividing progenitor cells E2f1-3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2f1-3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions. Loss of E2f1-3 completely suppressed these phenotypes caused by Rb deficiency. This work contextualizes the activator versus repressor functions of E2f1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.
Asunto(s)
Diferenciación Celular , Factores de Transcripción E2F/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Proteínas Represoras/metabolismo , Alelos , Animales , Apoptosis , Ciclo Celular/genética , Ciclo Celular/fisiología , Proliferación Celular , Factores de Transcripción E2F/deficiencia , Factores de Transcripción E2F/genética , Factor de Transcripción E2F1/deficiencia , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F2/deficiencia , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F2/metabolismo , Factor de Transcripción E2F3/deficiencia , Factor de Transcripción E2F3/genética , Factor de Transcripción E2F3/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Intestino Delgado/citología , Intestino Delgado/metabolismo , Ratones , Ratones Transgénicos , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Proteína de Retinoblastoma/deficiencia , Proteína de Retinoblastoma/metabolismoRESUMEN
The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Fibroblastos/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Fosfohidrolasa PTEN/metabolismo , Células del Estroma/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Matriz Extracelular/metabolismo , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunidad Innata , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Proteína Proto-Oncogénica c-ets-2/deficiencia , Proteína Proto-Oncogénica c-ets-2/metabolismoRESUMEN
The E2F family is conserved from Caenorhabditis elegans to mammals, with some family members having transcription activation functions and others having repressor functions. Whereas C. elegans and Drosophila melanogaster have a single E2F activator protein and repressor protein, mammals have at least three activator and five repressor proteins. Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a(3bki) or E2f3a(1ki), respectively) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development.
Asunto(s)
Factores de Transcripción E2F/metabolismo , Desarrollo Embrionario , Crecimiento , Animales , Células Cultivadas , Factores de Transcripción E2F/deficiencia , Factores de Transcripción E2F/genética , Factor de Transcripción E2F1/deficiencia , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F2/deficiencia , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F2/metabolismo , Factor de Transcripción E2F3/deficiencia , Factor de Transcripción E2F3/genética , Factor de Transcripción E2F3/metabolismo , Pérdida del Embrión/genética , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Eliminación de Gen , Genotipo , Crecimiento/genética , Ratones , Ratones Noqueados , FenotipoRESUMEN
E2F transcription factors regulate the progression of the cell cycle by repression or transactivation of genes that encode cyclins, cyclin dependent kinases, checkpoint regulators, and replication proteins. Although some E2F functions are independent of the Retinoblastoma tumor suppressor (Rb) and related family members, p107 and p130, much of E2F-mediated repression of S phase entry is dependent upon Rb. We previously showed in cultured mouse embryonic fibroblasts that concomitant loss of three E2F activators with overlapping functions (E2F1, E2F2, and E2F3) triggered the p53-p21(Cip1) response and caused cell cycle arrest. Here we report on a dramatic difference in the requirement for E2F during development and in cultured cells by showing that cell cycle entry occurs normally in E2f1-3 triply-deficient epithelial stem cells and progenitors of the developing lens. Sixteen days after birth, however, massive apoptosis in differentiating epithelium leads to a collapse of the entire eye. Prior to this collapse, we find that expression of cell cycle-regulated genes in E2F-deficient lenses is aberrantly high. In a second set of experiments, we demonstrate that E2F3 ablation alone does not cause abnormalities in lens development but rescues phenotypic defects caused by loss of Rb, a binding partner of E2F known to recruit histone deacetylases, SWI/SNF and CtBP-polycomb complexes, methyltransferases, and other co-repressors to gene promoters. Together, these data implicate E2F1-3 in mediating transcriptional repression by Rb during cell cycle exit and point to a critical role for their repressive functions in cell survival.
Asunto(s)
Proliferación Celular , Factor de Transcripción E2F1/fisiología , Factor de Transcripción E2F2/fisiología , Factor de Transcripción E2F3/fisiología , Proteínas Represoras/fisiología , Animales , Apoptosis , Supervivencia Celular , Roturas del ADN de Doble Cadena , Factor de Transcripción E2F1/deficiencia , Factor de Transcripción E2F2/deficiencia , Factor de Transcripción E2F3/deficiencia , Células Epiteliales/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína de Retinoblastoma/fisiología , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
The urea cycle generates arginine that is one of the major precursors for creatine biosynthesis. Here we evaluate levels of creatine and guanidinoacetate (the precursor in the synthesis of creatine) in plasma samples (ns = 207) of patients (np = 73) with different types of urea cycle disorders (ornithine transcarbamylase deficiency (ns = 22; np = 7), citrullinemia type 1 (ns = 60; np = 22), argininosuccinic aciduria (ns = 81; np = 31), arginase deficiency (ns = 44; np = 13)). The concentration of plasma guanidinoacetate positively correlated (p < 0.001, R2 = 0.64) with levels of arginine, but not with glycine in all patients with urea cycle defects, rising to levels above normal in most samples (34 out of 44) of patients with arginase deficiency. In contrast to patients with guanidinoacetate methyltransferase deficiency (a disorder of creatine synthesis characterized by elevated guanidinoacetate concentrations), creatine levels were normal (32 out of 44) or above normal (12 out of 44) in samples from patients with arginase deficiency. Creatine levels correlated significantly, but poorly (p < 0.01, R2 = 0.1) with guanidinoacetate levels and, despite being overall in the normal range in patients with all other urea cycle disorders, were occasionally below normal in some patients with argininosuccinic acid synthase and lyase deficiency. Creatine levels positively correlated with levels of methionine (p < 0.001, R2 = 0.16), the donor of the methyl group for creatine synthesis. The direct correlation of arginine levels with guanidinoacetate in patients with urea cycle disorders explains the increased concentration of guanidino compounds in arginase deficiency. Low creatine levels in some patients with other urea cycle defects might be explained by low protein intake (creatine is naturally present in meat) and relative or absolute intracellular arginine deficiency.
RESUMEN
Eukaryotic gene expression requires the coordinated activity of many macromolecular machines including transcription factors and RNA polymerase, the spliceosome, mRNA export factors, the nuclear pore, the ribosome and decay machineries. Yeast carrying mutations in genes encoding components of these machineries were examined using microarrays to measure changes in both pre-mRNA and mRNA levels. We used these measurements as a quantitative phenotype to ask how steps in the gene expression pathway are functionally connected. A multiclass support vector machine was trained to recognize the gene expression phenotypes caused by these mutations. In several cases, unexpected phenotype assignments by the computer revealed functional roles for specific factors at multiple steps in the gene expression pathway. The ability to resolve gene expression pathway phenotypes provides insight into how the major machineries of gene expression communicate with each other.
Asunto(s)
Perfilación de la Expresión Génica , Expresión Génica/genética , Expresión Génica/fisiología , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , ARN Helicasas DEAD-box , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma , Vectores Genéticos/genética , Familia de Multigenes/genética , Mutación/genética , Nucleotidiltransferasas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Biosíntesis de Proteínas/genética , ARN Helicasas/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética/genéticaRESUMEN
The simian virus 40 large T antigen contributes to neoplastic transformation, in part, by targeting the Rb family of tumor suppressors. There are three known Rb proteins, pRb, p130, and p107, all of which block the cell cycle by preventing the transcription of genes regulated by the E2F family of transcription factors. T antigen interacts directly with Rb proteins and disrupts Rb-E2F complexes both in vitro and in cultured cells. Consequently, T antigen is thought to inhibit transcriptional repression by the Rb family proteins by disrupting their interaction with E2F proteins, thus allowing E2F-dependent transcription and the expression of cellular genes needed for entry into S phase. This model predicts that active E2F-dependent transcription is required for T-antigen-induced transformation. To test this hypothesis, we have examined the status of Rb-E2F complexes in murine enterocytes. Previous studies have shown that T antigen drives enterocytes into S phase, resulting in intestinal hyperplasia, and that the induction of enterocyte proliferation requires T-antigen binding to Rb proteins. In this paper, we show that normal growth-arrested enterocytes contain p130-E2F4 complexes and that T-antigen expression destroys these complexes, most likely by stimulating p130 degradation. Furthermore, unlike their normal counterparts, enterocytes expressing T antigen contain abundant levels of E2F2 and E2F3a. Concomitantly, T-antigen-induced intestinal proliferation is reduced in mice lacking either E2F2 alone or both E2F2 and E2F3a, but not in mice lacking E2F1. These studies support a model in which T antigen eliminates Rb-E2F repressive complexes so that specific activator E2Fs can drive S-phase entry.
Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Factor de Transcripción E2F2/metabolismo , Enfermedades Gastrointestinales/virología , Hiperplasia/virología , Virus 40 de los Simios/patogenicidad , Animales , Factor de Transcripción E2F2/deficiencia , Factor de Transcripción E2F4/metabolismo , Enterocitos/química , Enterocitos/virología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína p130 Similar a la del Retinoblastoma/metabolismoRESUMEN
Viruses are intracellular parasites that must use the host machinery to multiply. Identification of the host factors that perform essential functions in viral replication is thus of crucial importance to the understanding of virus-host interactions. Here we describe Ded1p, a highly conserved DExD/H-box translation factor, as a possible host factor recruited by the yeast L-A double-stranded RNA (dsRNA) virus. We found that Ded1p interacts specifically and strongly with Gag, the L-A virus coat protein. Further analysis revealed that Ded1p interacts with the L-A virus in an RNA-independent manner and, as a result, L-A particles can be affinity purified via this interaction. The affinity-purified L-A particles are functional, as they are capable of synthesizing RNA in vitro. Critically, using purified L-A particles, we demonstrated that Ded1p specifically promotes L-A dsRNA replication by accelerating the rate of negative-strand RNA synthesis in vitro. In light of these data, we suggest that Ded1p may be a part of the long sought after activity shown to promote yeast viral dsRNA replication. This and the fact that Ded1p is also required for translating brome mosaic virus RNA2 in yeast thus raise the intriguing possibility that Ded1p is one of the key host factors favored by several evolutionarily related RNA viruses, including the human hepatitis C virus.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Fúngicas/fisiología , ARN Helicasas/fisiología , ARN Viral/biosíntesis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/virología , Totivirus/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/farmacología , Secuencia Conservada , ARN Helicasas DEAD-box , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacología , Productos del Gen gag/metabolismo , ARN Helicasas/química , ARN Helicasas/farmacología , Totivirus/metabolismo , Totivirus/ultraestructura , Transcripción Genética , Virión/genética , Virión/metabolismo , Virión/ultraestructura , Replicación ViralRESUMEN
The DHH1 gene in the yeast Saccharomyces cerevisiae encodes a putative RNA helicase of remarkable sequence similarity to several other DExD/H-box proteins, including Xp54 in Xenopus laevis and Ste13p in Schizosaccharomyces pombe. We show here that over-expression of Xp54, an integral component of the stored messenger ribonucleoprotein (mRNP) particles, can rescue the loss of Dhh1p in yeast. Localization and sedimentation studies showed that Dhh1p exists predominantly in the cytoplasm and is present in large complexes whose sizes appear to vary according to the growth stage of the cell culture. In addition, deletion of dhh1, when placed in conjunction with the mutant dbp5 and ded1 alleles, resulted in a synergistically lethal effect, suggesting that Dhh1p may have a role in mRNA export and translation. Finally, similar to Ste13p, Dhh1p is required for sporulation in the budding yeast. Taken together, our data provide evidence that the functions of Dhh1p are conserved through evolution.
Asunto(s)
ARN Helicasas/metabolismo , Proteínas de Unión al ARN , Saccharomyces cerevisiae/enzimología , Proteínas de Schizosaccharomyces pombe , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citoplasma/enzimología , ARN Helicasas DEAD-box , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Expresión Génica , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Sustancias Macromoleculares , Mutación , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Regiones Promotoras Genéticas/genética , ARN Helicasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Proteínas de Xenopus/genéticaRESUMEN
UNLABELLED: Genetic and epigenetic events that alter gene expression and/or protein function or localization are thought to be the primary mechanism that drives tumorigenesis and governs the clinical behavior of cancers. Yet, a number of studies have shown that the effects of oncogene expression or tumor suppressor ablation are highly dependent on cell type. The molecular basis for this cell-type specificity and how it contributes to tumorigenesis are unknown. Here, expression of a truncated SV40 large T antigen in murine intestinal crypts promoted the formation of numerous adenomatous polyps in the colon and small intestine. In contrast, when the same T-antigen construct is expressed in villous enterocytes, the consequences are limited to hyperplasia and dysplasia. The T-antigen-induced polyps show high levels of the proto-oncogene c-Myc protein even though there is no transport of ß-catenin to the nucleus. Targeting the expression of viral oncogenes to intestinal crypts or villi provides a murine model system for studying cell-type specific effects in tumorigenesis, and is particularly relevant to the study of APC/ß-catenin-independent pathways contributing to the generation of intestinal polyps. IMPLICATIONS: This mouse model system describes the formation of colon polyps in the absence of Wnt/ß-catenin signaling.
Asunto(s)
Pólipos Adenomatosos/patología , Compartimento Celular , Intestinos/patología , Proteínas Oncogénicas Virales/metabolismo , Células Madre/metabolismo , Pólipos Adenomatosos/metabolismo , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Apoptosis , Carcinogénesis/patología , Mucosa Intestinal/metabolismo , Ratones Transgénicos , Modelos Biológicos , Mutación/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1(ΔG)) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1(ΔG/ΔG) mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1(ΔG/ΔG) MEFs display a magnitude of E2F target gene derepression similar to that of Rb1(-/-) cells, even though Rb1(ΔG/ΔG) cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1(ΔG/ΔG) MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G1 to arrest proliferation. Some Rb1(ΔG/ΔG) mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.
Asunto(s)
Factores de Transcripción E2F/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Puntos de Control de la Fase S del Ciclo Celular/genética , Adenocarcinoma/genética , Alelos , Animales , Sitios de Unión , Línea Celular , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Fibroblastos/citología , Marcación de Gen , Ratones , Ratones Noqueados , Mutación , Neoplasias Hipofisarias/genética , Fase S/genéticaRESUMEN
The evolutionarily ancient arm of the E2f family of transcription factors consisting of the two atypical members E2f7 and E2f8 is essential for murine embryonic development. However, the critical tissues, cellular processes, and molecular pathways regulated by these two factors remain unknown. Using a series of fetal and placental lineage-specific cre mice, we show that E2F7/E2F8 functions in extraembryonic trophoblast lineages are both necessary and sufficient to carry fetuses to term. Expression profiling and biochemical approaches exposed the canonical E2F3a activator as a key family member that antagonizes E2F7/E2F8 functions. Remarkably, the concomitant loss of E2f3a normalized placental gene expression programs, corrected placental defects, and fostered the survival of E2f7/E2f8-deficient embryos to birth. In summary, we identified a placental transcriptional network tightly coordinated by activation and repression through two distinct arms of the E2F family that is essential for extraembryonic cell proliferation, placental development, and fetal viability.
Asunto(s)
Factor de Transcripción E2F7/fisiología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/fisiología , Placentación/fisiología , Proteínas Represoras/fisiología , Animales , Biomarcadores/metabolismo , Western Blotting , Proliferación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Factor de Transcripción E2F3/genética , Factor de Transcripción E2F3/metabolismo , Embrión de Mamíferos/citología , Femenino , Perfilación de la Expresión Génica , Genes Letales , Integrasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The retinoblastoma tumor suppressor (pRb) regulates cell cycle entry, progression and exit by controlling the activity of the E2F-family of transcription factors. During cell cycle exit pRb acts as a transcriptional repressor by associating with E2F proteins and thereby inhibiting their ability to stimulate the expression of genes required for S phase. Indeed, many tumors harbor mutations in the RB gene and the pRb-E2F pathway is compromised in nearly all types of cancers. In this report we show that both pRb and its interacting partners, the transcriptional factors E2F1-2-3, act as positive modulators of detoxification pathways important for metabolizing and clearing xenobiotics--such as toxins and drugs--from the body. Using a combination of conventional molecular biology techniques and microarray analysis of specific cell populations, we have analyzed the detoxification pathway in murine samples in the presence or absence of pRb and/or E2F1-2-3. In this report, we show that both pRb and E2F1-2-3 act as positive modulators of detoxification pathways in mice, challenging the conventional view of E2F1-2-3 as transcriptional repressors negatively regulated by pRb. These results suggest that mutations altering the pRb-E2F axis may have consequences beyond loss of cell cycle control by altering the ability of tissues to remove toxins and to properly metabolize anticancer drugs, and might help to understand the formation and progression rates of different types of cancer, as well as to better design appropriate therapies based on the particular genetic composition of the tumors.
Asunto(s)
Inactivación Metabólica , Redes y Vías Metabólicas , Proteína de Retinoblastoma/metabolismo , Xenobióticos/metabolismo , Animales , Animales Recién Nacidos , Regulación hacia Abajo/genética , Factores de Transcripción E2F/deficiencia , Factores de Transcripción E2F/metabolismo , Inactivación Metabólica/genética , Hígado/metabolismo , Redes y Vías Metabólicas/genética , Ratones , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/deficiencia , Transcripción GenéticaRESUMEN
The E2f3 locus encodes two Rb-binding gene products, E2F3a and E2F3b, which are differentially regulated during the cell cycle and are thought to be critical for cell cycle progression. We targeted the individual inactivation of E2f3a or E2f3b in mice and examined their contributions to cell proliferation and development. Chromatin immunoprecipitation and gene expression experiments using mouse embryo fibroblasts deficient in each isoform showed that E2F3a and E2F3b contribute to G(1)/S-specific gene expression and cell proliferation. Expression of E2f3a or E2f3b was sufficient to support E2F target gene expression and cell proliferation in the absence of other E2F activators, E2f1 and E2f2, suggesting that these isoforms have redundant functions. Consistent with this notion, E2f3a(-/-) and E2f3b(-/-) embryos developed normally, whereas embryos lacking both isoforms (E2f3(-/-)) died in utero. We also find that E2f3a and E2f3b have redundant and nonredundant roles in the context of Rb mutation. Analysis of double-knockout embryos suggests that the ectopic proliferation and apoptosis in Rb(-/-) embryos is mainly mediated by E2f3a in the placenta and nervous system and by both E2f3a and E2f3b in lens fiber cells. Together, we conclude that the contributions of E2F3a and E2F3b in cell proliferation and development are context dependent.
Asunto(s)
Proliferación Celular , Factor de Transcripción E2F3/genética , Factor de Transcripción E2F3/fisiología , Desarrollo Embrionario , Genes de Retinoblastoma , Animales , Línea Celular , Sistema Nervioso Central/embriología , Inmunoprecipitación de Cromatina , Embrión de Mamíferos/fisiología , Desarrollo Embrionario/genética , Eritropoyesis/genética , Ojo/embriología , Femenino , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistema Nervioso Periférico/embriología , Placenta/metabolismo , Placenta/patología , Embarazo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Activación Transcripcional , Trofoblastos/citología , Trofoblastos/fisiologíaRESUMEN
The inactivation of the retinoblastoma (Rb) tumor suppressor gene in mice results in ectopic proliferation, apoptosis, and impaired differentiation in extraembryonic, neural, and erythroid lineages, culminating in fetal death by embryonic day 15.5 (E15.5). Here we show that the specific loss of Rb in trophoblast stem (TS) cells, but not in trophoblast derivatives, leads to an overexpansion of trophoblasts, a disruption of placental architecture, and fetal death by E15.5. Despite profound placental abnormalities, fetal tissues appeared remarkably normal, suggesting that the full manifestation of fetal phenotypes requires the loss of Rb in both extraembryonic and fetal tissues. Loss of Rb resulted in an increase of E2f3 expression, and the combined ablation of Rb and E2f3 significantly suppressed Rb mutant phenotypes. This rescue appears to be cell autonomous since the inactivation of Rb and E2f3 in TS cells restored placental development and extended the life of embryos to E17.5. Taken together, these results demonstrate that loss of Rb in TS cells is the defining event causing lethality of Rb(-/-) embryos and reveal the convergence of extraembryonic and fetal functions of Rb in neural and erythroid development. We conclude that the Rb pathway plays a critical role in the maintenance of a mammalian stem cell population.