RESUMEN
In this issue of Cell, Liu et al. (2018) report the birth of two healthy cloned macaque monkeys using fetal fibroblasts. By artificially enhancing the arsenal of epigenetic modifiers in the oocyte, the authors overcome the earliest roadblocks that take place during somatic cell nuclear transfer (SCNT).
Asunto(s)
Haplorrinos , Macaca , Animales , Clonación de Organismos , Fibroblastos , Técnicas de Transferencia Nuclear , Oocitos , PrimatesRESUMEN
Regeneration-competent vertebrates are considered to suppress inflammation faster than non-regenerating ones. Hence, understanding the cellular mechanisms affected by immune cells and inflammation can help develop strategies to promote tissue repair and regeneration. Here, we took advantage of naturally occurring tail regeneration-competent and -incompetent developmental stages of Xenopus tadpoles. We first establish the essential role of the myeloid lineage for tail regeneration in the regeneration-competent tadpoles. We then reveal that upon tail amputation there is a myeloid lineage-dependent change in amputation-induced apoptosis levels, which in turn promotes tissue remodelling, and ultimately leads to the relocalization of the regeneration-organizing cells responsible for progenitor proliferation. These cellular mechanisms failed to be executed in regeneration-incompetent tadpoles. We demonstrate that regeneration incompetency is characterized by inflammatory myeloid cells whereas regeneration competency is associated with reparative myeloid cells. Moreover, treatment of regeneration-incompetent tadpoles with immune-suppressing drugs restores myeloid lineage-controlled cellular mechanisms. Collectively, our work reveals the effects of differential activation of the myeloid lineage on the creation of a regeneration-permissive environment and could be further exploited to devise strategies for regenerative medicine purposes.
Asunto(s)
Linaje de la Célula/fisiología , Células Mieloides/fisiología , Regeneración/fisiología , Cola (estructura animal)/fisiología , Xenopus laevis/fisiología , Animales , Apoptosis/efectos de los fármacos , Matriz Extracelular/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Inmunosupresores/farmacología , Larva/fisiología , Regeneración/efectos de los fármacos , Medicina Regenerativa/métodosRESUMEN
Cohesin is implicated in establishing and maintaining pluripotency. Whether this is because of essential cohesin functions in the cell cycle or in gene regulation is unknown. Here we tested cohesin's contribution to reprogramming in systems that reactivate the expression of pluripotency genes in the absence of proliferation (embryonic stem [ES] cell heterokaryons) or DNA replication (nuclear transfer). Contrary to expectations, cohesin depletion enhanced the ability of ES cells to initiate somatic cell reprogramming in heterokaryons. This was explained by increased c-Myc (Myc) expression in cohesin-depleted ES cells, which promoted DNA replication-dependent reprogramming of somatic fusion partners. In contrast, cohesin-depleted somatic cells were poorly reprogrammed in heterokaryons, due in part to defective DNA replication. Pluripotency gene induction was rescued by Myc, which restored DNA replication, and by nuclear transfer, where reprogramming does not require DNA replication. These results redefine cohesin's role in pluripotency and reveal a novel function for Myc in promoting the replication-dependent reprogramming of somatic nuclei.
Asunto(s)
Células Madre Embrionarias/fisiología , Regulación de la Expresión Génica , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Reprogramación Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Humanos , Ratones , Datos de Secuencia Molecular , Oocitos/metabolismo , Células Madre Pluripotentes/fisiología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Xenopus , CohesinasRESUMEN
Nuclear transfer to oocytes is an efficient way to transcriptionally reprogram somatic nuclei, but its mechanisms remain unclear. Here, we identify a sequence of molecular events that leads to rapid transcriptional reprogramming of somatic nuclei after transplantation to Xenopus oocytes. RNA-seq analyses reveal that reprogramming by oocytes results in a selective switch in transcription toward an oocyte rather than pluripotent type, without requiring new protein synthesis. Time-course analyses at the single-nucleus level show that transcriptional reprogramming is induced in most transplanted nuclei in a highly hierarchical manner. We demonstrate that an extensive exchange of somatic- for oocyte-specific factors mediates reprogramming and leads to robust oocyte RNA polymerase II binding and phosphorylation on transplanted chromatin. Moreover, genome-wide binding of oocyte-specific linker histone B4 supports its role in transcriptional reprogramming. Thus, our study reveals the rapid, abundant, and stepwise loading of oocyte-specific factors onto somatic chromatin as important determinants for successful reprogramming.
Asunto(s)
Reprogramación Celular/genética , Cromatina/metabolismo , Histonas/fisiología , Oocitos/metabolismo , Xenopus/embriología , Animales , Células Cultivadas , Reprogramación Celular/fisiología , Genoma , Ratones , Técnicas de Transferencia Nuclear , Especificidad de Órganos , ARN/genética , Análisis de Secuencia de ARN , Xenopus/genéticaRESUMEN
Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Citrulina/metabolismo , Histonas/química , Histonas/metabolismo , Células Madre Pluripotentes/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Arginina/química , Arginina/metabolismo , Sitios de Unión , Reprogramación Celular/genética , Cromatina/química , ADN/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica , Hidrolasas/metabolismo , Ratones , Células Madre Pluripotentes/citología , Unión Proteica , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Proteómica , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.
Asunto(s)
Metilación de ADN/genética , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/genética , Espermatozoides/metabolismo , Animales , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/biosíntesis , Histonas , Humanos , Masculino , Ranidae/genética , Ranidae/crecimiento & desarrollo , Espermátides/crecimiento & desarrollo , Espermátides/metabolismo , Espermatozoides/crecimiento & desarrolloRESUMEN
Amphibian oocytes can rapidly and efficiently reprogram the transcription of transplanted somatic nuclei. To explore the factors and mechanisms involved, we focused on nuclear actin, an especially abundant component of the oocyte's nucleus (the germinal vesicle). The existence and significance of nuclear actin has long been debated. Here, we found that nuclear actin polymerization plays an essential part in the transcriptional reactivation of the pluripotency gene Oct4 (also known as Pou5f1). We also found that an actin signaling protein, Toca-1, enhances Oct4 reactivation by regulating nuclear actin polymerization. Toca-1 overexpression has an effect on the chromatin state of transplanted nuclei, including the enhanced binding of nuclear actin to gene regulatory regions. This is the first report showing that naturally stored actin in an oocyte nucleus helps transcriptional reprogramming in a polymerization-dependent manner.
Asunto(s)
Actinas/metabolismo , Núcleo Celular/metabolismo , Reprogramación Celular , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Oocitos/metabolismo , Xenopus , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Ensamble y Desensamble de Cromatina , Proteínas de Unión a Ácidos Grasos , Regulación del Desarrollo de la Expresión Génica , Ratones , Polimerizacion , Transducción de Señal , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression.
Asunto(s)
Acetiltransferasas/fisiología , Núcleo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , ARN Mensajero/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular Tumoral , Humanos , Proteínas de Transporte Nucleocitoplasmático/fisiología , ARN Mensajero/clasificación , Proteínas de Unión al ARN/fisiología , XenopusRESUMEN
How various layers of epigenetic repression restrict somatic cell nuclear reprogramming is poorly understood. The transfer of mammalian somatic cell nuclei into Xenopus oocytes induces transcriptional reprogramming of previously repressed genes. Here, we address the mechanisms that restrict reprogramming following nuclear transfer by assessing the stability of the inactive X chromosome (Xi) in different stages of inactivation. We find that the Xi of mouse post-implantation-derived epiblast stem cells (EpiSCs) can be reversed by nuclear transfer, while the Xi of differentiated or extraembryonic cells is irreversible by nuclear transfer to oocytes. After nuclear transfer, Xist RNA is lost from chromatin of the Xi. Most epigenetic marks such as DNA methylation and Polycomb-deposited H3K27me3 do not explain the differences between reversible and irreversible Xi. Resistance to reprogramming is associated with incorporation of the histone variant macroH2A, which is retained on the Xi of differentiated cells, but absent from the Xi of EpiSCs. Our results uncover the decreased stability of the Xi in EpiSCs, and highlight the importance of combinatorial epigenetic repression involving macroH2A in restricting transcriptional reprogramming by oocytes.
Asunto(s)
Reprogramación Celular/genética , Inestabilidad Cromosómica/genética , Variación Genética , Histonas/genética , Células Madre Pluripotentes/química , ARN no Traducido/química , ARN no Traducido/genética , Inactivación del Cromosoma X/genética , Animales , Diferenciación Celular/genética , Células Cultivadas , Femenino , Fibroblastos/química , Fibroblastos/citología , Fibroblastos/metabolismo , Histonas/química , Masculino , Ratones , Ratones Transgénicos , Oocitos/química , Oocitos/citología , Oocitos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN Largo no Codificante , Transcripción Genética , XenopusRESUMEN
Incompatibilities between the nucleus and the cytoplasm of sufficiently distant species result in developmental arrest of hybrid and nucleocytoplasmic hybrid (cybrid) embryos. Several hypotheses have been proposed to explain their lethality, including problems in embryonic genome activation (EGA) and/or nucleo-mitochondrial interactions. However, conclusive identification of the causes underlying developmental defects of cybrid embryos is still lacking. We show here that while over 80% of both Xenopus laevis and Xenopus (Silurana) tropicalis same-species androgenetic haploids develop to the swimming tadpole stage, the androgenetic cybrids formed by the combination of X. laevis egg cytoplasm and X. tropicalis sperm nucleus invariably fail to gastrulate properly and never reach the swimming tadpole stage. In spite of this arrest, these cybrids show quantitatively normal EGA and energy levels at the stage where their initial gastrulation defects are manifested. The nucleocytoplasmic incompatibility between these two species instead results from a combination of factors, including a reduced emission of induction signal from the vegetal half, a decreased sensitivity of animal cells to induction signals, and differences in a key embryonic protein (Xbra) concentration between the two species, together leading to inefficient induction and defective convergence-extension during gastrulation. Indeed, increased exposure to induction signals and/or Xbra signalling partially rescues the induction response in animal explants and whole cybrid embryos. Altogether, our study demonstrates that the egg cytoplasm of one species may not support the development promoted by the nucleus of another species, even if this nucleus does not interfere with the cytoplasmic/maternal functions of the egg, while the egg cytoplasm is also capable of activating the genome of that nucleus. Instead, our results provide evidence that inefficient signalling and differences in the concentrations of key proteins between species lead to developmental defects in cybrids. Finally, they show that the incompatibilities of cybrids can be corrected by appropriate treatments.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Larva/metabolismo , Transducción de Señal/genética , Xenopus laevis/metabolismo , Xenopus/metabolismo , Animales , Núcleo Celular/genética , Quimera , Citoplasma/genética , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Femenino , Gastrulación/genética , Larva/genética , Masculino , Morfogénesis/genética , Técnicas de Transferencia Nuclear , Óvulo/citología , Óvulo/metabolismo , Xenopus/embriología , Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/genéticaRESUMEN
Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.
Asunto(s)
Cromatina/metabolismo , Proteínas del Huevo/metabolismo , Proteínas Nucleares/metabolismo , Interacciones Espermatozoide-Óvulo , Espermátides/metabolismo , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Electroforesis en Gel de Poliacrilamida , Femenino , Immunoblotting , Masculino , Espectrometría de Masas , Proteínas Nucleares/aislamiento & purificación , Unión Proteica , Mapeo de Interacción de Proteínas , Isoformas de Proteínas , Extractos de Tejidos , Proteínas de Xenopus/aislamiento & purificación , Xenopus laevis/metabolismoRESUMEN
When transplanted into Xenopus oocytes, the nuclei of mammalian somatic cells are reprogrammed to express stem cell genes such as Oct4, Nanog, and Sox2. We now describe an experimental system in which the pluripotency genes Sox2 and Oct4 are repressed in retinoic acid-treated ES cells but are reprogrammed up to 100% within 24 h by injection of nuclei into the germinal vesicle (GV) of growing Xenopus oocytes. The isolation of GVs in nonaqueous medium allows the reprogramming of individual injected nuclei to be seen in real time. Analysis using fluorescence recovery after photobleaching shows that nuclear transfer is associated with an increase in linker histone mobility. A simultaneous loss of somatic H1 linker histone and incorporation of the oocyte-specific linker histone B4 precede transcriptional reprogramming. The loss of H1 is not required for gene reprogramming. We demonstrate both by antibody injection experiments and by dominant negative interference that the incorporation of B4 linker histone is required for pluripotency gene reactivation during nuclear reprogramming. We suggest that the binding of oocyte-specific B4 linker histone to chromatin is a key primary event in the reprogramming of somatic nuclei transplanted to amphibian oocytes.
Asunto(s)
Núcleo Celular/metabolismo , Histonas/metabolismo , Oocitos/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Cromatina/metabolismo , Femenino , Células HeLa , Humanos , Técnicas In Vitro , Técnicas de Transferencia Nuclear , Factor 3 de Transcripción de Unión a Octámeros/genética , Oocitos/citología , Oocitos/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Regiones Promotoras Genéticas , Factores de Transcripción SOXB1/genética , Activación Transcripcional/efectos de los fármacos , Tretinoina/farmacología , Xenopus , Proteínas de Xenopus/genéticaRESUMEN
Xenopus is one of the premier model systems to study cell and developmental biology in vivo in vertebrates. Here we briefly review how this South African frog came to be favored by a large community of scientists after the explosive growth of molecular biology and examine some of the original discoveries arising from this sturdy frog. Experimental embryology started in Rana but developed in newt embryos for historical reasons. A long lineage of mentorship, starting with Theodor Boveri, Hans Spemann, Fritz Baltzer, Ernst Hadorn, and Michail Fischberg, used newt embryos. In Oxford, Fischberg made the transition to Xenopus laevis because it was widely available for human pregnancy tests and laid eggs year-round, and he fortuitously isolated a one-nucleolus mutant. This mutant allowed nuclear transfer experiments showing that genetic information is not lost during cell differentiation and the demonstration that the nucleolus is the locus of transcription of the large ribosomal RNAs. With the advent of DNA cloning, the great equalizer among all fields of biology, microinjected Xenopus oocytes became an indispensable tool, providing the first living-cell mRNA translation, polymerase II and III transcription, and coupled transcription-translation systems in eukaryotes. Xenopus embryos provide abundant material to study the earliest signaling events during vertebrate development and have been subjected to saturating molecular screens in the genomic era. Many novel principles of development and cell biology owe their origins to this remarkably resilient frog.
Asunto(s)
Clonación de Organismos , Técnicas de Transferencia Nuclear , Animales , Biología , Genoma , Masculino , Oocitos , Xenopus laevis/genéticaRESUMEN
Oocytes have a remarkable ability to reactivate silenced genes in somatic cells. However, it is not clear how the chromatin architecture of somatic cells affects this transcriptional reprogramming. Here, we investigated the relationship between the chromatin opening and transcriptional activation. We reveal changes in chromatin accessibility and their relevance to transcriptional reprogramming after transplantation of somatic nuclei into Xenopus oocytes. Genes that are silenced, but have pre-existing open transcription start sites in donor cells, are prone to be activated after nuclear transfer, suggesting that the chromatin signature of somatic nuclei influences transcriptional reprogramming. There are also activated genes associated with new open chromatin sites, and transcription factors in oocytes play an important role in transcriptional reprogramming from such genes. Finally, we show that genes resistant to reprogramming are associated with closed chromatin configurations. We conclude that chromatin accessibility is a central factor for successful transcriptional reprogramming in oocytes.
Asunto(s)
Reprogramación Celular/genética , Cromatina/metabolismo , Oocitos/metabolismo , Transcripción Genética , Animales , Fibroblastos/citología , Fibroblastos/trasplante , Ratones , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Activación Transcripcional/genética , Transposasas/metabolismo , Xenopus laevis/metabolismoRESUMEN
Nuclear reprogramming by the transplantation of somatic cell nuclei to eggs (in second meiotic metaphase) is always followed by a phase of chromosome replication and cell division before new gene expression is seen. To help understand the mechanism of nuclear reprogramming, we have asked whether the nuclei of normal, nontransformed, nondividing, and terminally differentiated mammalian cells can be directly reprogrammed, without DNA replication, by Xenopus oocytes. We find that nuclei of adult mouse thymocytes and of adult human blood lymphocytes, injected into Xenopus oocytes, are induced to extinguish a differentiation marker and to strongly express oct-4, the most diagnostic mammalian stem cell/pluripotency marker. In the course of 2 days at 18 degrees C, the mammalian oct-4 transcripts are spliced to mature mRNA. We conclude that normal mammalian nuclei can be directly reprogrammed by the nucleus (germinal vesicle) of amphibian oocytes to express oct-4 at a rate comparable to that of oct-4 in mouse ES cells. To our knowledge, this is the first demonstration of a stem cell marker being induced in a differentiated adult human cell nucleus. This is an early step toward the long-term aim of developing a procedure for reprogramming readily accessible human adult cells for cell replacement therapy.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Técnicas de Transferencia Nuclear , Oocitos/citología , Células Madre/citología , Factores de Transcripción , Animales , Secuencia de Bases , Núcleo Celular/genética , Mapeo Cromosómico , Clonación de Organismos , Proteínas de Unión al ADN/fisiología , Femenino , Humanos , Linfocitos/citología , Ratones , Factor 3 de Transcripción de Unión a Octámeros , Reacción en Cadena de la Polimerasa , XenopusRESUMEN
Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.
RESUMEN
Nuclear transplantation in amphibia started in 1952. By this is meant, sexually mature cloned frogs can be obtained from the nuclei of embryo cells, differentiating cells, and larval-differentiated cells. Transplanted nuclei are reprogrammed to entirely new patterns of gene expression. In this chapter, the methods used to transplant living nuclei into enucleated eggs of Xenopus are described. A method also is described for transplanting multiple somatic cell nuclei into nonenucleated oocytes, a procedure that achieves reprogramming of gene expression in the absence of cell division.
Asunto(s)
Transporte Activo de Núcleo Celular , Clonación de Organismos/métodos , Técnicas Genéticas , Xenopus/metabolismo , Animales , Blástula/metabolismo , Bovinos , Diferenciación Celular , División Celular , Núcleo Celular/metabolismo , Femenino , Oocitos/metabolismo , Ovario/metabolismo , Rayos UltravioletaRESUMEN
Transcription factors fulfill a key role in the formation and maintenance of different cell-types during development. It is known that transcription factors largely dissociate from chromosomes during mitosis. We found, previously, that mitosis is also a time when somatic nuclei can be far more easily reprogrammed after nuclear transfer than the nuclei of interphase cells. We refer to this as a mitotic advantage. Here, the rate of exchange of a transcription factor on its designated DNA-binding site is discussed. It is proposed that the Xenopus oocyte could serve as an experimental system in which the duration of binding site occupancy could be usefully analyzed. In particular, the Xenopus oocyte has several characteristics which make it possible to determine accurately the concentration and duration of transcription factor binding. It is proposed that the concentration and time are the key variables which govern the action of transcription factors when they activate genes needed for cell lineage determination.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Mitosis/fisiología , Factores de Transcripción/metabolismo , Xenopus laevis/crecimiento & desarrollo , Animales , Transcripción Genética , Xenopus laevis/metabolismoRESUMEN
Methylation of cytosine deoxynucleotides generates 5-methylcytosine (m(5)dC), a well-established epigenetic mark. However, in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N(6)-methyldeoxyadenosine (m(6)dA) in vertebrate DNA, specifically in Xenopus laevis but also in other species including mouse and human. Our methylome analysis reveals that m(6)dA is widely distributed across the eukaryotic genome and is present in different cell types but is commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.