RESUMEN
Exceptional genomic stability is one of the hallmarks of mouse embryonic stem (ES) cells. However, the genes contributing to this stability remain obscure. We previously identified Zscan4 as a specific marker for two-cell embryo and ES cells. Here we show that Zscan4 is involved in telomere maintenance and long-term genomic stability in ES cells. Only 5% of ES cells express Zscan4 at a given time, but nearly all ES cells activate Zscan4 at least once during nine passages. The transient Zscan4-positive state is associated with rapid telomere extension by telomere recombination and upregulation of meiosis-specific homologous recombination genes, which encode proteins that are colocalized with ZSCAN4 on telomeres. Furthermore, Zscan4 knockdown shortens telomeres, increases karyotype abnormalities and spontaneous sister chromatid exchange, and slows down cell proliferation until reaching crisis by passage eight. Together, our data show a unique mode of genome maintenance in ES cells.
Asunto(s)
Células Madre Embrionarias/metabolismo , Inestabilidad Genómica , Telómero/genética , Telómero/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proliferación Celular , Aberraciones Cromosómicas , Células Madre Embrionarias/citología , Células Madre Embrionarias/patología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Cariotipificación , Meiosis/genética , Meiosis/fisiología , Ratones , Transporte de Proteínas , Recombinación Genética/genética , Intercambio de Cromátides Hermanas/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
INTRODUCTION: X-linked PLAC1 is highly expressed in placenta during embryogenesis, and when ablated in mice, causes aberrant placental cell layer organization. It is also highly expressed in many types of cancer cell-lines. Although it has been shown that it promotes AKT phosphorylation in cancer cells, the exact mechanism by which it influences placental layer differentiation is unclear. METHODS: To investigate the mechanism of action of PLAC1 we did cell fractionation and immunoprecipitation of the protein and Mass Spectrometry analysis to identify its interaction partners. The associated proteins were directly tested for interactions by co-transfection with PLAC1 and immunoprecipitation. Mutations in the ZP-N domain of PLAC1 were introduced to assess its involvement in the interactions. RESULTS: We provide evidence that Desmoglein-2 (DSG2), a component of the membrane-associated desmosomal complex, directly interacts with PLAC1. Mutations of cysteines in ZP-N domain disrupt the interaction between PLAC1 and DSG-2. DISCUSSION: Because desmosomes are responsible for establishing lateral cell-cell junctions, we suggest that direct interaction with the lateral junction protein complex may be implicated in the PLAC1 effects on cell-cell interactions, and thereby on the layer structure of the placenta.
Asunto(s)
Comunicación Celular , Desmosomas/metabolismo , Proteínas Gestacionales/fisiología , Animales , Células COS , Comunicación Celular/genética , Diferenciación Celular , Células Cultivadas , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Placenta/citología , Placenta/metabolismo , Placenta/fisiología , Embarazo , Proteínas Gestacionales/metabolismo , Unión Proteica , Trofoblastos/citología , Trofoblastos/metabolismo , Trofoblastos/fisiologíaRESUMEN
We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.
Asunto(s)
Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Hibridación in Situ , Animales , Línea Celular , Ratones , Ratones Endogámicos , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.
Asunto(s)
Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre/citología , Transcripción Genética , Animales , Animales Recién Nacidos , Blastocisto/citología , Blastocisto/metabolismo , Biología Computacional , ADN Complementario/metabolismo , Bases de Datos Genéticas , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Oocitos/citología , Oocitos/metabolismo , Análisis de Componente Principal , ARN Mensajero/metabolismo , Análisis de Secuencia de ADNRESUMEN
Retinoic acid (RA) is one of the most potent inducers of differentiation of mouse embryonic stem cells (ESCs). However, previous studies show that RA treatment of cells cultured in the presence of a leukemia inhibitory factor (LIF) also result in the upregulation of a gene called Zscan4, whose transient expression is a marker for undifferentiated ESCs. We explored the balance between these two seemingly antagonistic effects of RA. ESCs indeed differentiated in the presence of LIF after RA treatment, but colonies of undifferentiated ESCs eventually emerged from these differentiated cells - even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies - after transfer to the standard ESC medium - retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Tretinoina/farmacología , Animales , Técnicas de Cultivo de Célula , Linaje de la Célula , Células Madre Embrionarias/citología , Factor Inhibidor de Leucemia/farmacología , Ratones , Regulación hacia ArribaRESUMEN
Microarray expression profiling of a collection of 15,000 mouse genes with placental and embryonic RNAs revealed candidates for placental-enriched genes, three of which we have confirmed and further characterized. One, Plac1, strongly expressed in all trophoblast-derived cells in the placenta, has been described earlier (Genomics 68 (2000) 305). Here we report that of the other two, Plac8 expression is restricted to the spongiotrophoblast layer during development, whereas Plac9 is weakly expressed though highly enriched in placenta. For both, cDNAs with complete open reading frames were recovered and exon-intron structures inferred from comparisons of mouse cDNA and genomic sequence. The predicted proteins (112 and 108 amino acids) both contain putative signal peptides, with a coiled-coil segment of mPLAC9 as the only other detected motif. Genomic sequence comparisons reveal that in addition to an apparent pseudogene on chromosome 1, Plac8 is expressed at mouse cytoband 5e3. It is tightly conserved in human in a syntenically equivalent ortholog at 4q21.23. Plac9 is present in a single copy on chromosome 14, with a syntenically equivalent human ortholog at 10q22.3. Putative promoter regions up to 10 kb 5' of the transcription units for Plac1, Plac8, and Plac9 contain sites for widely-expressed transcription factors which, by analogy to other instances, may be sufficient to explain placental enrichment.
Asunto(s)
Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Placenta/metabolismo , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , ADN Complementario/química , ADN Complementario/genética , Embrión de Mamíferos/metabolismo , Exones , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes/genética , Hibridación in Situ , Intrones , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
A catalog of mouse genes expressed in early embryos, embryonic and adult stem cells was assembled, including 250000 ESTs, representing approximately 39000 unique transcripts. The cDNA libraries, enriched in full-length clones, were condensed into the NIA 15 and 7.4K clone sets, freely distributed to the research community, providing a standard platform for expression studies using microarrays. They are essential tools for studying mammalian development and stem cell biology, and to provide hints about the differential nature of embryonic and adult stem cells.
Asunto(s)
Embrión de Mamíferos , Biblioteca de Genes , Ratones/genética , Células Madre , Animales , Clonación Molecular , Genómica , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The developmental potency of mouse embryonic stem (ES) cells, which is the ability to contribute to a whole embryo, is known to deteriorate during long-term cell culture. Previously, we have shown that ES cells oscillate between Zscan4(-) and Zscan4(+) states, and the transient activation of Zscan4 is required for the maintenance of telomeres and genome stability of ES cells. Here we show that increasing the frequency of Zscan4 activation in mouse ES cells restores and maintains their developmental potency in long-term cell culture. Injection of a single ES cell with such increased potency into a tetraploid blastocyst gives rise to an entire embryo with a higher success rate. These results not only provide a means to rejuvenate ES cells by manipulating Zscan4 expression, but also indicate the active roles of Zscan4 in the long-term maintenance of ES cell potency.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Poliploidía , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Telómero/metabolismoRESUMEN
Networks of transcription factors (TFs) are thought to determine and maintain the identity of cells. Here we systematically repressed each of 100 TFs with shRNA and carried out global gene expression profiling in mouse embryonic stem (ES) cells. Unexpectedly, only the repression of a handful of TFs significantly affected transcriptomes, which changed in two directions/trajectories: one trajectory by the repression of either Pou5f1 or Sox2; the other trajectory by the repression of either Esrrb, Sall4, Nanog, or Tcfap4. The data suggest that the trajectories of gene expression change are already preconfigured by the gene regulatory network and roughly correspond to extraembryonic and embryonic fates of cell differentiation, respectively. These data also indicate the robustness of the pluripotency gene network, as the transient repression of most TFs did not alter the transcriptomes.
Asunto(s)
Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/genética , Animales , Análisis por Conglomerados , Perfilación de la Expresión Génica , Silenciador del Gen , Ratones , Modelos Biológicos , Interferencia de ARN , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
To examine transcription factor (TF) network(s), we created mouse ESC lines, in each of which 1 of 50 TFs tagged with a FLAG moiety is inserted into a ubiquitously controllable tetracycline-repressible locus. Of the 50 TFs, Cdx2 provoked the most extensive transcriptome perturbation in ESCs, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. ChIP-Seq revealed that CDX2 binds to promoters of upregulated target genes. By contrast, genes downregulated by CDX2 did not show CDX2 binding but were enriched with binding sites for POU5F1, SOX2, and NANOG. Genes with binding sites for these core TFs were also downregulated by the induction of at least 15 other TFs, suggesting a common initial step for ESC differentiation mediated by interference with the binding of core TFs to their target genes. These ESC lines provide a fundamental resource to study biological networks in ESCs and mice.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Redes Reguladoras de Genes/fisiología , Factores de Transcripción/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Factor de Transcripción CDX2 , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Inmunoprecipitación de Cromatina , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Factor de Transcripción GATA3/fisiología , Redes Reguladoras de Genes/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Inmunoprecipitación , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Decreasing oocyte competence with maternal aging is a major factor in human infertility. To investigate the age-dependent molecular changes in a mouse model, we compared the expression profiles of metaphase II oocytes collected from 5- to 6-week-old mice with those collected from 42- to 45-week-old mice using the NIA 22K 60-mer oligo microarray. Among approximately 11,000 genes whose transcripts were detected in oocytes, about 5% (530) showed statistically significant expression changes, excluding the possibility of global decline in transcript abundance. Consistent with the generally accepted view of aging, the differentially expressed genes included ones involved in mitochondrial function and oxidative stress. However, the expression of other genes involved in chromatin structure, DNA methylation, genome stability and RNA helicases was also altered, suggesting the existence of additional mechanisms for aging. Among the transcripts decreased with aging, we identified and characterized a group of new oocyte-specific genes, members of the human NACHT, leucine-rich repeat and PYD-containing (NALP) gene family. These results have implications for aging research as well as for clinical ooplasmic donation to rejuvenate aging oocytes.
Asunto(s)
Envejecimiento/fisiología , Biomarcadores/metabolismo , Perfilación de la Expresión Génica , Oocitos/fisiología , Animales , Femenino , Humanos , Metafase , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , FilogeniaRESUMEN
Large-scale gene expression profiling was performed on embryo-derived stem cell lines to identify molecular signatures of pluripotency and lineage specificity. Analysis of pluripotent embryonic stem (ES) cells, extraembryonic-restricted trophoblast stem (TS) cells, and terminally-differentiated mouse embryo fibroblast (MEF) cells identified expression profiles unique to each cell type, as well as genes common only to ES and TS cells. Whereas most of the MEF-specific genes had been characterized previously, the majority (67%) of the ES-specific genes were novel and did not include known differentiated cell markers. Comparison with microarray data from embryonic material demonstrated that ES-specific genes were underrepresented in all stages sampled, whereas TS-specific genes included known placental markers. Investigation of four novel TS-specific genes showed trophoblast-restricted expression in cell lines and in vivo, whereas one uncharacterized ES-specific gene, Esg-1, was found to be exclusively associated with pluripotency. We suggest that pluripotency requires a set of genes not expressed in other cell types, whereas lineage-restricted stem cells, like TS cells, express genes predictive of their differentiated lineage.
Asunto(s)
Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Células Madre Pluripotentes/fisiología , Proteínas de Xenopus , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , Linaje de la Célula/genética , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Genes Esenciales/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Familia de Multigenes/genética , Factor 3 de Transcripción de Unión a Octámeros , Especificidad de Órganos/genética , Células Madre Pluripotentes/química , Células Madre Pluripotentes/citología , Proteínas/genética , Células Madre Totipotentes/química , Células Madre Totipotentes/fisiología , Factores de Transcripción/genética , Trofoblastos/química , Trofoblastos/citología , Trofoblastos/fisiologíaRESUMEN
A set of 7407 cDNA clones (NIA mouse 7.4K) was assembled from >20 cDNA libraries constructed mainly from early mouse embryos, including several stem cell libraries. The clone set was assembled from embryonic and newborn organ libraries consisting of ~120,000 cDNA clones, which were initially re-arrayed into a set of ~11,000 unique cDNA clones. A set of tubes was constructed from the racks in this set to prevent contamination and potential mishandling errors in all further re-arrays. Sequences from this set (11K) were analyzed further for quality and clone identity, and high-quality clones with verified identity were re-arrayed into the final set (7.4K). The set is freely available, and a corresponding database was built to provide comprehensive annotation for those clones with known identity or homology, and has been made available through an extensive Web site that includes many link-outs to external databases and analysis servers.