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Our recent studies revealed the role of mouse Aprataxin PNK-like Factor (APLF) in development. Nevertheless, the comprehensive characterization of mouse APLF remains entirely unexplored. Based on domain deletion studies, here we report that mouse APLF's Acidic Domain and Fork Head Associated (FHA) domain can chaperone histones and repair DNA like the respective human orthologs. Immunofluorescence studies in mouse embryonic stem cells showed APLF co-localized with γ-tubulin within and around the centrosomes and govern the number and integrity of centrosomes via PLK4 phosphorylation. Enzymatic analysis established mouse APLF as a kinase. Docking studies identified three putative ATP binding sites within the FHA domain. Site-directed mutagenesis showed that R37 residue within the FHA domain is indispensable for the kinase activity of APLF thereby regulating the centrosome number. These findings might assist us comprehend APLF in different pathological and developmental conditions and reveal non-canonical kinase activity of proteins harbouring FHA domains that might impact multiple cellular processes.
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Centrosoma , Células Madre Embrionarias de Ratones , Proteínas Serina-Treonina Quinasas , Animales , Centrosoma/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , FosforilaciónRESUMEN
Visualizing and quantifying the numerous factors that regulate murine pre-implantation embryonic development is technically challenging. Here, we present a protocol for the isolation of pre-implantation embryos at multiple stages of embryonic development to study gene expression. We describe steps for isolating RNA and cDNA synthesis from a small number of embryos. We then detail an immunofluorescence assay for the detection and localization of protein of interest by confocal microscopy in the pre-implantation embryos. For complete details on the use and execution of this protocol, please refer to Varghese et al.1.
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Desarrollo Embrionario , Perfilación de la Expresión Génica , Femenino , Embarazo , Animales , Ratones , ADN Complementario , Desarrollo Embrionario/genética , Microscopía Confocal , ARNRESUMEN
BACKGROUND: Endometrial cancer (EC) arises from uterine endometrium tissue and is the most prevalent cancer of the female reproductive tract in developed countries. It has been predicted that the global prevalence of EC will increase in part because of its positive association with economic growth and lifestyle. The majority of EC presented with endometrioid histology and mutations in the tumor suppressor gene PTEN, resulting in its loss of function. PTEN negatively regulates the PI3K/Akt/mTOR axis of cell proliferation and thus serves as a tumorigenesis gatekeeper. Through its chromatin functions, PTEN is also implicated in genome maintenance procedures. However, our comprehension of how DNA repair occurs in the absence of PTEN function in EC is inadequate. METHODS: We utilized The Cancer Genome Atlas (TCGA) data analysis to establish a correlation between PTEN and DNA damage response genes in EC, followed by a series of cellular and biochemical assays to elucidate a molecular mechanism utilizing the AN3CA cell line model for EC. RESULTS: The TCGA analyses demonstrated an inverse correlation between the expression of the damage sensor protein of nucleotide excision repair (NER), DDB2, and PTEN in EC. The transcriptional activation of DDB2 is mediated by the recruitment of active RNA polymerase II to the DDB2 promoter in the PTEN-null EC cells, revealing a correlation between increased DDB2 expression and augmented NER activity in the absence of PTEN. CONCLUSION: Our study indicated a causal relationship between NER and EC that may be exploited in disease management.
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Neoplasias Endometriales , Fosfatidilinositol 3-Quinasas , Femenino , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Reparación del ADN/genética , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Endometrio/patología , Daño del ADN , Rayos Ultravioleta , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismoRESUMEN
Inhibition of PKC (PKCi) signaling maintains pluripotency of embryonic stem cells (ESCs) across different mammalian species. However, the position of PKCi maintained ESCs in the pluripotency continuum is largely unknown. Here we demonstrate that mouse ESCs when cultured continuously, with PKCi, for 75 days are retained in naïve state of pluripotency. Gene expression analysis and proteomics studies demonstrated enhanced naïve character of PKCi maintained ESCs in comparison to classical serum/LIF (S/L) supported ESCs. Molecular analysis revealed that activation of PKCζ isoform associate with primed state of pluripotency, present in epiblast-like stem cells generated in vitro while inhibition of PKCζ phosphorylation associated with naïve state of pluripotency in vitro and in vivo. Phosphoproteomics and chromatin modification enzyme array based studies showed loss in DNA methyl transferase 3B (DNMT3B) and its phosphorylation level upon functional inhibition of PKCζ as one of the crucial components of this regulatory pathway. Unlike ground state of pluripotency maintained by MEK/GSK3 inhibitor in addition to LIF (2i/LIF), loss in DNMT3B is a reversible phenomenon in PKCi maintained ESCs. Absence of phosphorylation of c-MYC, RAF1, SPRY4 while presence of ERF, DUSP6, CIC and YAP1 phosphorylation underlined the phosphoproteomics signature of PKCi mediated maintenance of naïve pluripotency. States of pluripotency represent the developmental continuum and the existence of PKCi mediated mouse ESCs in a distinct state in the continuum of pluripotency (DiSCo) might contribute to the establishment of stages of murine embryonic development that were non-permissible till date.
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Glucógeno Sintasa Quinasa 3 , Células Madre Embrionarias de Ratones , Animales , Ratones , Glucógeno Sintasa Quinasa 3/metabolismo , Células Madre Embrionarias , Transducción de Señal , Fosforilación , Mamíferos/metabolismoRESUMEN
Pluripotent character is described as the potency of cells to differentiate into all three germ layers. The best example to reinstate the term lies in the context of embryonic stem cells (ESCs). Pluripotent ESC describes the in vitro status of those cells that originate during the complex process of embryogenesis. Pre-implantation to post-implantation development of embryo embrace cells with different levels of stemness. Currently, four states of pluripotency have been recognized, in the progressing order of "naïve," "poised," "formative," and "primed." Epigenetics act as the "conductor" in this "orchestra" of transition in pluripotent states. With a distinguishable gene expression profile, these four states associate with different epigenetic signatures, sometimes distinct while otherwise overlapping. The present review focuses on how epigenetic factors, including DNA methylation, bivalent chromatin, chromatin remodelers, chromatin/nuclear architecture, and microRNA, could dictate pluripotent states and their transition among themselves.
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Células Madre Pluripotentes , Células Madre Pluripotentes/metabolismo , Epigénesis Genética , Estratos Germinativos/metabolismo , Células Madre Embrionarias/metabolismo , Cromatina/genética , Cromatina/metabolismo , Diferenciación Celular/genéticaRESUMEN
Dynamicity and flexibility of the chromatin landscape are critical for most of the DNA-dependent processes to occur. This higher-order packaging of the eukaryotic genome into the chromatin is mediated by histones and associated non-histone proteins that determine the states of chromatin. Histone chaperones- "the guardian of genome stability and epigenetic information" controls the chromatin accessibility by escorting the nucleosomal and non-nucleosomal histones as well as their variants. This distinct group of molecules is involved in all facets of histone metabolism. The selectivity and specificity of histone chaperones to the histones determine the maintenance of the chromatin in an open or closed state. This review highlights the functional implication of the network of histone chaperones in shaping the chromatin function in the development of an organism. Seminal studies have reported embryonic lethality at different stages of embryogenesis upon perturbation of some of the chaperones, suggesting their essentiality in development. We hereby epitomize facts and functions that emphasize the relevance of histone chaperones in orchestrating different embryonic developmental stages starting from gametogenesis to organogenesis in multicellular organisms.
RESUMEN
Our recent findings demonstrated that the histone chaperone and DNA repair factor aprataxin and PNK-like factor (APLF) could regulate epithelial to mesenchymal transition (EMT) during the reprogramming of murine fibroblasts and in breast cancer metastasis. Therefore, we investigated the function of APLF in EMT associated with mouse development. Here, we show that APLF is predominantly enhanced in trophectoderm (TE) and lineages derived from TE in pre- and post-implantation embryos. Downregulation of APLF induced the hatching of embryos in vitro, with a significant increase in Cdh1 and Cdx2 expression. Aplf short hairpin RNA-microinjected embryos failed to implant in vivo Rescue experiments neutralized the knockdown effects of APLF both in vitro and in vivo Reduced expression of Snai2 and Tead4, and the gain in Cdh1 and sFlt1 (also known as Flt1) level, marked the differentiation of APLF-knocked down trophoblast stem cells that might contribute towards the impaired implantation of embryos. Hence, our findings suggest a novel role for APLF during implantation and post-implantation development of mouse embryos. We anticipate that APLF might contribute to the establishment of maternal-fetal connection, as its fine balance is required to achieve implantation and thereby attain proper pregnancy.
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Chaperonas de Histonas , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Animales , Factor de Transcripción CDX2 , Cadherinas , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Implantación del Embrión , Transición Epitelial-Mesenquimal , Femenino , Ratones , Proteínas de Unión a Poli-ADP-Ribosa , Embarazo , TrofoblastosRESUMEN
Thanks to the advancement in science and technology and a significant number of cancer research programs being carried out throughout the world, the prevention, prognosis and treatment of breast cancer are improving with a positive and steady pace. However, a stern thoughtful attention is required for the metastatic breast cancer cases-the deadliest of all types of breast cancer, with a character of relapse even when treated. In an effort to explore the less travelled avenues, we summarize here studies underlying the aspects of histone epigenetics in breast cancer metastasis. Authoritative reviews on breast cancer epigenetics are already available; however, there is an urgent need to focus on the epigenetics involved in metastatic character of this cancer. Here we put forward a comprehensive review on how different layers of histone epigenetics comprising of histone chaperones, histone variants and histone modifications interplay to create breast cancer metastasis landscape. Finally, we propose a hypothesis of integrating histone-epigenetic factors as biomarkers that encompass different breast cancer subtypes and hence could be exploited as a target of larger population.
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Abnormal proliferation and disrupted differentiation of hematopoietic progenitors mark leukemia. Histone cell cycle regulator A (HIRA), a histone chaperone, regulates hemogenic to hematopoietic transition involved in normal hematopoiesis. But, its role remains unexplored in leukemia, a case of dysregulated hematopoiesis. Here, the Cancer Cell Line Encyclopedia database analysis showed enhanced HIRA mRNA expression in cells of hematopoietic and lymphoid origin with maximal expression in the chronic myeloid leukemia (CML) cell line, K562. This observation was further endorsed by the induced expression of HIRA in CML patient samples compared to healthy individuals and Acute Myeloid Leukemia patients. Downregulation of HIRA in K562 cells displayed cell cycle arrest, loss in proliferation, presence of polyploidy with significant increase in CD41+ population thereby limiting proliferation but inducing differentiation of leukemia cells to megakaryocyte fate. Induced megakaryocyte differentiation of mouse Hira-knockout hematopoietic progenitors in vivo further confirmed the in vitro findings in leukemia cells. Molecular analysis showed the involvement of MKL1/GATA2/H3.3 axis in dictating differentiation of CML cells to megakaryocytes. Thus, HIRA could be exploited for differentiation induction therapy in CML and in chronic pathological conditions involving low platelet counts.
RESUMEN
DNA damage-specific histone chaperone Aprataxin PNK-like factor (APLF) regulates mesenchymal-to-epithelial transition (MET) during cellular reprogramming. We investigated the role of APLF in epithelial-to-mesenchymal transition (EMT) linked to breast cancer invasiveness and metastasis. Here, we show that a significant manifestation of APLF is present in tumor sections of patients with invasive ductal carcinoma when compared to their normal adjacent tissues. APLF was significantly induced in triple negative breast cancer (TNBC) cells, MDAMB-231, in comparison to invasive MCF7 or normal MCF10A breast cells and supported by studies on invasive breast carcinoma in The Cancer Genome Atlas (TCGA). Functionally, APLF downregulation inhibited proliferative capacity, altered cell cycle behavior, induced apoptosis and impaired DNA repair ability of MDAMB-231 cells. Reduction in APLF level impeded invasive, migratory, tumorigenic and metastatic potential of TNBC cells with loss in expression of genes associated with EMT while upregulation of MET-specific gene E-cadherin (CDH1). So, here we provided novel evidence for enrichment of APLF in breast tumors, which could regulate metastasis-associated EMT in invasive breast cancer. We anticipate that APLF could be exploited as a biomarker for breast tumors and additionally could be targeted in sensitizing cancer cells towards DNA damaging agents.
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Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Regulación hacia Arriba , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Trasplante de Neoplasias , Análisis de Matrices Tisulares , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Induction of pluripotency in differentiated cells through the exogenous expression of the transcription factors Oct4, Sox2, Klf4 and cellular Myc involves reprogramming at the epigenetic level. Histones and their metabolism governed by histone chaperones constitute an important regulator of epigenetic control. We hypothesized that histone chaperones facilitate or inhibit the course of reprogramming. For the first time, we report here that the downregulation of histone chaperone Aprataxin PNK-like factor (APLF) promotes reprogramming by augmenting the expression of E-cadherin (Cdh1), which is implicated in the mesenchymal-to-epithelial transition (MET) involved in the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Downregulation of APLF in MEFs expedites the loss of the repressive MacroH2A.1 (encoded by H2afy) histone variant from the Cdh1 promoter and enhances the incorporation of active histone H3me2K4 marks at the promoters of the pluripotency genes Nanog and Klf4, thereby accelerating the process of cellular reprogramming and increasing the efficiency of iPSC generation. We demonstrate a new histone chaperone (APLF)-MET-histone modification cohort that functions in the induction of pluripotency in fibroblasts. This regulatory axis might provide new mechanistic insights into perspectives of epigenetic regulation involved in cancer metastasis.
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Proteínas Portadoras/metabolismo , Fibroblastos/metabolismo , Chaperonas de Histonas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Puntos de Control del Ciclo Celular/genética , Diferenciación Celular/genética , Reprogramación Celular/genética , Ensayo de Unidades Formadoras de Colonias , Reparación del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Regulación hacia Abajo/genética , Embrión de Mamíferos/citología , Células Epiteliales/citología , Femenino , Fibroblastos/citología , Técnicas de Silenciamiento del Gen , Células HEK293 , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Lisina/metabolismo , Masculino , Mesodermo/citología , Metilación , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión a Poli-ADP-Ribosa , Regiones Promotoras Genéticas/genética , Regulación hacia Arriba/genéticaRESUMEN
Flow cytometry is a reliable method for identification and purification of live cells from a heterogeneous population. Since permeabilized cells cannot be sorted live in a FACS sorter, its application in isolation of functional cells largely depends on antibodies for surface markers. In various fields of biology we find intracellular markers that reveal subpopulations of biological significance. Cell cycle stage specific molecules, metastatic signature molecules, stemness associated proteins etc. are examples of potential markers that could improve the research and therapy enormously. Currently their use is restricted by lack of techniques that allow live detection. Even though a few methods like aptamers, droplet-based microfluidics and smartflares are reported, their application is limited. Here, for the first time we report a simple, cost-effective and efficient method of live sorting of cells based on the expression of an intracellular marker using a fluorophore-tagged binding peptide. The target molecule selected was a histone chaperone, HIRA, the expression of which can predict the fate of differentiating myoblast. Our results confirm that the peptide shows specific interaction with its target; and it can be used to separate cells with differential expression of HIRA. Further, this method offers high purity and viability for the isolated cells.
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Proteínas de Ciclo Celular/metabolismo , Separación Celular/métodos , Chaperonas de Histonas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Permeabilidad de la Membrana Celular , Células Cultivadas , Citometría de Flujo/métodos , Fluoresceína-5-Isotiocianato/metabolismo , Ratones , Datos de Secuencia Molecular , Células Madre Embrionarias de Ratones/metabolismo , Fragmentos de Péptidos/metabolismo , Coloración y EtiquetadoRESUMEN
RUNX1 (Runt-related transcription factor 1) is indispensable for the generation of hemogenic endothelium. However, the regulation of RUNX1 during this developmental process is poorly understood. We investigated the role of the histone chaperone HIRA (histone cell cycle regulation-defective homolog A) from this perspective and report that HIRA significantly contributes toward the regulation of RUNX1 in the transition of differentiating mouse embryonic stem cells from hemogenic to hematopoietic stage. Direct interaction of HIRA and RUNX1 activates the downstream targets of RUNX1 implicated in generation of hematopoietic stem cells. At the molecular level, HIRA-mediated incorporation of histone H3.3 variant within the Runx1 +24 mouse conserved noncoding element is essential for the expression of Runx1 during endothelial to hematopoietic transition. An inactive chromatin at the intronic enhancer of Runx1 in absence of HIRA significantly repressed the transition of cells from hemogenic to hematopoietic fate. We expect that the HIRA-RUNX1 axis might open up a novel approach in understanding leukemogenesis in future.
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Proteínas de Ciclo Celular/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Endotelio Vascular/citología , Regulación de la Expresión Génica , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Chaperonas de Histonas/fisiología , Factores de Transcripción/fisiología , Animales , Western Blotting , Proteínas de Ciclo Celular/antagonistas & inhibidores , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Endotelio Vascular/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células Madre Hematopoyéticas/metabolismo , Chaperonas de Histonas/antagonistas & inhibidores , Humanos , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/antagonistas & inhibidores , Saco Vitelino/citología , Saco Vitelino/metabolismoRESUMEN
Embryonic Stem Cells (ESCs) are derived from the inner cell mass of blastocysts. They have the unique potency to differentiate into diverse lineages. Hence, they are bestowed with the term pluripotency. Several mechanisms have been implicated in maintaining the pluripotency of ESCs. This review will focus on the role of signaling pathways in regulating ESC pluripotency among diverse mammalian species. A novel phylogenetic approach has been designed to understand the structural basis of divergence in the signaling pathways which modulate pluripotency among different species. Detailed insight into different signaling mechanisms indicates inhibition of Extracellular Related Kinase 1/2 (ERK 1/2) signaling as the key component regulating the pluripotency of ESCs. On the basis of recent advances made in this field, it can be hypothesized that expression of the transcription factor KLF4 and inhibition of ERK signaling may promote the establishment and maintenance of true ESCs from different mammalian species.
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Blastocisto/metabolismo , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/biosíntesis , Transducción de SeñalRESUMEN
Embryonic stem cell (ESC) pluripotency is orchestrated by distinct signaling pathways that are often targeted to maintain ESC self-renewal or their differentiation to other lineages. We showed earlier that inhibition of PKC signaling maintains pluripotency in mouse ESCs. Therefore, in this study, we investigated the importance of protein kinase C signaling in the context of rat ESC (rESC) pluripotency. Here we show that inhibition of PKC signaling is an efficient strategy to establish and maintain pluripotent rESCs and to facilitate reprogramming of rat embryonic fibroblasts to rat induced pluripotent stem cells. The complete developmental potential of rESCs was confirmed with viable chimeras and germ line transmission. Our molecular analyses indicated that inhibition of a PKCζ-NF-κB-microRNA-21/microRNA-29 regulatory axis contributes to the maintenance of rESC self-renewal. In addition, PKC inhibition maintains ESC-specific epigenetic modifications at the chromatin domains of pluripotency genes and, thereby, maintains their expression. Our results indicate a conserved function of PKC signaling in balancing self-renewal versus differentiation of both mouse and rat ESCs and indicate that targeting PKC signaling might be an efficient strategy to establish ESCs from other mammalian species.
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Células Madre Embrionarias/enzimología , Células Madre Pluripotentes/enzimología , Proteína Quinasa C-epsilon/metabolismo , Transducción de Señal/fisiología , Animales , Células Madre Embrionarias/citología , Indoles/farmacología , Maleimidas/farmacología , MicroARNs/metabolismo , FN-kappa B/metabolismo , Células Madre Pluripotentes/citología , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future.
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Células Madre Embrionarias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcriptoma , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular , Células Nutrientes , Fibroblastos/metabolismo , Genes Esenciales , Estratos Germinativos/citología , Ratones , Ratas , Ratas Endogámicas F344 , Ratas Long-Evans , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/citologíaRESUMEN
In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.
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Linaje de la Célula , Proteínas de Unión al ADN/metabolismo , Proteínas Musculares/metabolismo , Factores de Transcripción/metabolismo , Animales , Blastocisto/citología , Blastocisto/metabolismo , Masa Celular Interna del Blastocisto/citología , Masa Celular Interna del Blastocisto/metabolismo , Blastómeros/citología , Blastómeros/metabolismo , Western Blotting , Factor de Transcripción CDX2 , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Macaca mulatta , Ratones , Ratones Transgénicos , Proteínas Musculares/genética , Interferencia de ARN , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genéticaRESUMEN
The purification and characterization of an extracellular levansucrase enzyme produced by novel nitrogen-fixer Acetobacter nitrogenifigens strain RG1(T) is described. Culture conditions were optimized for maximum levansucrase production. Levansucrase purified to homogeneity by tenfold purification has a molecular weight of 65 kDa, contained four cysteine residues, polymerized raffinose and was stable for 21 days at pH 6.0 when stored at 4 °C or -20 °C but was vulnerable to DTT and ß-mercaptoethanol. Interestingly, this enzyme showed enhanced hydrolytic and polymerization activity in the presence of mercuric ion which, to our knowledge, is the first report for any levansucrase enzyme characterized so far. Evidences obtained from Native PAGE, tryptophan fluorescence study and activity measurements at different temperatures and in the presence of thiol modifying agents, show that mercuric ion stabilizes the enzyme. Levan, synthesized by the enzyme, has a molecular weight of 7,080 kDa and was shown to be a homopolymer of fructose.