RESUMEN
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
The Xenopus oocyte is known to accumulate filamentous or F-actin in the nucleus, but it is currently unknown whether F-actin also accumulates in embryo nuclei. Using fluorescence-labeled actin reporters, we examined the actin distribution in Xenopus embryonic cells and found that F-actin accumulates in nuclei during the blastula stage but not during the gastrula stage. To further investigate nuclear F-actin, we devised a Xenopus egg extract that reproduces the formation of nuclei in which F-actin accumulates. Using this extract, we found that F-actin accumulates primarily at the subnuclear membranous region and is essential to maintain chromatin binding to the nuclear envelope in well-developed nuclei. We also provide evidence that nuclear F-actin increases the structural stability of nuclei and contributes to chromosome alignment on the mitotic spindle at the following M phase. These results suggest the physiological importance of nuclear F-actin accumulation in rapidly dividing large Xenopus blastula cells.
Asunto(s)
Blástula/citología , Cromatina/metabolismo , Membrana Nuclear/metabolismo , Xenopus laevis/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Blástula/metabolismo , Sistema Libre de Células , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Óvulo/citología , Óvulo/metabolismo , Xenopus laevis/embriologíaRESUMEN
Histone chaperones are a group of histone-binding proteins that facilitate the assembly of nucleosomes, the fundamental structural units of chromatin in eukaryotes. In nucleosome assembly, deposition of a histone H3-H4 tetramer onto DNA is the first and critical step, which is mediated by the histone chaperones HIRA and CAF-1. HIRA and CAF-1 are reportedly involved in DNA replication independent (RI) and replication coupled nucleosome assembly, respectively. However, the mechanisms by which they mediate histone deposition remain unclear. In this study, we focused on the mechanism by which HIRA induces RI-nucleosome assembly. We looked for HIRA domains that are required for nucleosome assembly and its localization to chromatin. We used cell-free extracts from Xenopus eggs that carry out RI-nucleosome assembly of plasmid DNA. We confirmed that HIRA formed stable complexes with Asf1, another histone H3-H4 chaperone, and the HIRA-Asf1 complex was solely responsible for RI-nucleosome assembly in egg extracts. We further demonstrated that the HIRA N-terminus containing the WD40 domain, which comprises seven WD40 repeats, and the B domain, to which Asf1 binds, were essential for RI-nucleosome assembly; the three WD40 repeats from the N-terminus were especially critical. Using egg extracts that reproduce nuclear formation accompanying the duplication of chromatin, we also demonstrated that the Hir domain was indispensable for the binding of HIRA to chromatin. Thus, the WD40 and B domains are the core elements for inducing RI-nucleosome assembly. Hir domain regulates the binding to chromatin. Based on these findings, similarities and differences between HIRA and CAF-1 are discussed.
Asunto(s)
Extractos Celulares , Replicación del ADN , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Nucleosomas/metabolismo , Repeticiones WD40 , Animales , Proteínas de Ciclo Celular/química , Humanos , Oocitos , Factores de Transcripción/química , Xenopus laevisRESUMEN
Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.
Asunto(s)
Proteínas F-Box/metabolismo , Oocitos/citología , Oocitos/metabolismo , Proteínas Proto-Oncogénicas c-mos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis , Ciclosoma-Complejo Promotor de la Anafase , Animales , Proteínas F-Box/química , Meiosis , Metafase , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oocitos/enzimología , Fosforilación/efectos de los fármacos , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas de Xenopus/química , Xenopus laevis/embriologíaRESUMEN
At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.
Asunto(s)
Calcineurina/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Cloruro de Calcio/farmacología , Extractos Celulares/química , Ciclina B/metabolismo , Activación Enzimática/efectos de los fármacos , Exocitosis , Femenino , Masculino , Meiosis/efectos de los fármacos , Ácido Ocadaico/farmacología , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/enzimología , Oocitos/metabolismo , Fosforilación/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismoRESUMEN
In the Xenopus laevis intestine during metamorphosis, which is triggered by thyroid hormone (TH), the adult epithelium develops and replaces the larval one undergoing apoptosis. We have previously shown that progenitor/stem cells of the adult epithelium originate from some differentiated larval epithelial cells. To investigate molecular mechanisms underlying larval epithelial dedifferentiation into the adult progenitor/stem cells, we here focused on nuclear lamin A (LA) and lamin LIII (LIII), whose expression is generally known to be correlated with the state of cell differentiation. We analyzed the spatiotemporal expression of LA and LIII during X. laevis intestinal remodeling by reverse transcription PCR, Western blotting, and immunohistochemistry. At the onset of natural metamorphosis, when the adult epithelial progenitor cells appear as small islets, the expression of LA is down-regulated, but that of LIII is up-regulated only in the islets. Then, as the adult progenitor cells differentiate, the expression of LA is up-regulated, whereas that of LIII is down-regulated in the adult cells. As multiple intestinal folds form, adult epithelial cells positive for LIII become restricted only to the troughs of the folds. In addition, we have shown that TH up- or down-regulates the expression of these lamins in the premetamorphic intestine as during natural metamorphosis. These results indicate that TH-regulated expression of LA and LIII closely correlates with dedifferentiation of the epithelial cells in the X. laevis intestine, suggesting the involvement of the lamins in the process of dedifferentiation during amphibian metamorphosis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Mucosa Intestinal/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Hormonas Tiroideas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/crecimiento & desarrollo , Animales , Desdiferenciación Celular , Células Epiteliales/metabolismo , Intestinos/citología , Intestinos/crecimiento & desarrollo , Lamina Tipo A/genética , Lamina Tipo B/genética , Larva/metabolismo , Células Madre/citología , Células Madre/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMEN
The ability to reprogram in vivo a somatic cell after differentiation is quite limited. One of the most impressive examples of such a process is transdifferentiation of pigmented epithelial cells (PECs) to lens cells during lens regeneration in newts. However, very little is known of the molecular events that allow newt cells to transdifferentiate. Histone B4 is an oocyte-type linker histone that replaces the somatic-type linker histone H1 during reprogramming mediated by somatic cell nuclear transfer (SCNT). We found that B4 is expressed and required during transdifferentiation of PECs. Knocking down of B4 decreased proliferation and increased apoptosis, which resulted in considerable smaller lens. Furthermore, B4 knockdown altered gene expression of key genes of lens differentiation and nearly abolished expression of gamma-crystallin. These data are the first to show expression of oocyte-type linker histone in somatic cells and its requirement in newt lens transdifferentiation and suggest that transdifferentiation in newts might share common strategies with reprogramming after SCNT.
Asunto(s)
Histonas/metabolismo , Cristalino/metabolismo , Salamandridae/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Proliferación Celular , Transdiferenciación Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Histonas/genética , Inmunohistoquímica , Cristalino/citología , Datos de Secuencia Molecular , Técnicas de Transferencia Nuclear , Regeneración/genética , Regeneración/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salamandridae/genética , gamma-Cristalinas/genética , gamma-Cristalinas/metabolismoRESUMEN
In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear lamina architecture and nuclear morphology and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development.
Asunto(s)
Blástula/metabolismo , Cromatina/metabolismo , Lamina Tipo B/metabolismo , Membrana Nuclear/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Núcleo Celular/metabolismo , Humanos , Unión Proteica , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Receptor de Lamina BRESUMEN
The nucleosome is the fundamental structural unit of eukaryotic chromatin. During mitosis, duplicated nucleosome fibers are organized into a pair of rod-shaped structures (chromatids) within a mitotic chromosome. However, it remains unclear whether nucleosome assembly is indeed an essential prerequisite for mitotic chromosome assembly. We combined mouse sperm nuclei and Xenopus cell-free egg extracts depleted of the histone chaperone Asf1 and found that chromatid-like structures could be assembled even in the near absence of nucleosomes. The resultant "nucleosome-depleted" chromatids contained discrete central axes positive for condensins, although they were more fragile than normal nucleosome-containing chromatids. Combinatorial depletion experiments underscored the central importance of condensins in mitotic chromosome assembly, which sheds light on their functional cross-talk with nucleosomes in this process.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Proteínas de Unión al ADN/metabolismo , Mitosis , Complejos Multiproteicos/metabolismo , Nucleosomas/metabolismo , Oocitos/metabolismo , Xenopus laevis/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Sistema Libre de Células , Cromátides/metabolismo , Cromatina/metabolismo , Masculino , Ratones , Oocitos/química , Espermatozoides/metabolismoRESUMEN
In meiotic cell cycles, meiosis I (MI) is followed by meiosis II (MII) without an intervening S phase, whereas in mitotic cell cycles, an S phase necessarily alternates with an M phase. For the study of mitotic cell cycles, extracts prepared from unfertilized and parthenogenetically activated Xenopus eggs have been very useful as they can perform the progression of mitotic cycles in vitro. To establish a cell-free system to study the regulatory mechanisms of meiotic transition from MI to MII, extracts have been prepared from maturing Xenopus oocytes isolated from ovaries, stimulated with progesterone to induce the resumption of meiosis, and arrested at meiotic metaphase I by cold treatment. In oocyte extracts, the activity of cyclin B-Cdc2 complexes, the M phase inducer, fluctuates in the same manner as it does in maturing oocytes during the MI to MII transition period. By the use of oocyte extracts, it has been found that incomplete inactivation of Cdc2 at the end of MI is required for meiotic M-M transition. The meiotic extract should provide a useful tool to elucidate molecular mechanisms of meiotic M to M transition, including a role of Mos/mitogen-activated protein kinase cascade in the suppression of S phase entry after MI exit. In this chapter, we describe methods for the preparation and the uses of meiotic extracts. As a comparison, we also include a protocol for the preparation of mitotic extracts.
Asunto(s)
Extractos Celulares , Meiosis/fisiología , Oocitos/citología , Oocitos/fisiología , Animales , Sistema Libre de Células , Femenino , Masculino , Espermatozoides/fisiología , Xenopus laevisRESUMEN
In order to obtain the cytological basis for the periodic flattening and rounding-up of activated amphibian eggs, the surface ultrastructure and the cortical microfilament organization were studied in Xenopus laevis. Scanning electron microscopy (SEM) of the egg surface revealed that the density of microvilli at the animal pole region decreased significantly when the periodic flattening started, but increased again concomitantly with the commencement of the rounding-up. Isolated pieces of the cortices stained with rhodamine-phalloidin exhibited the periodic disorganization and reorganization of a meshwork with bright dots probably corresponding to microvilli, in good synchrony with the decrease and increase of the microvilli density. Study of appropriate batches of eggs in which the moving front of surface contraction waves (SCWs; 1) can be localized revealed that the decrease and increase of the microvilli density correspond to SCW-1 and -2, respectively. SEM and the cytochemical examination of the eggs from which the germinal vesicle (GV) had been removed revealed that none of these changes occurred in the enucleated eggs. These observations suggest that the GV-dependent regulation of the microfilament organization in an egg cortex constitutes the cytological basis for the SCWs and for the periodic flattening and rounding-up of denuded eggs.
RESUMEN
The molecular events associated with decondensation of human sperm nuclei were analyzed by incubating sperm with egg extracts from an amphibian, Bufo japonicus. Acid-urea-Triton polyacrylamide gel electrophoresis (AUT-PAGE) showed that the nuclear basic proteins of human sperm consist mainly of protamines (HPI, HPII) with minor amounts of nucleosomal histones. On incubation of lysolecithin (LC)- and dithiothreitol (DTT)-treated human sperm with the egg extract, the nuclei lost HPI and HPII within 15 min in association with extensive nuclear decondensation, and the acquirement of a whole set of nucleosomal histones. Incubation of LC-DTT-sperm with nucleoplasmin purified from Bufo eggs also induced nuclear decondensation and loss of protamines within 30 min. Native-PAGE and Western blot analyses of incubation medium indicated tight association of the released protamines to nucleoplasmin, strongly suggesting that protamines are removed from sperm nuclei not enzymatically but by their specific binding to nucleoplasmin. On incubation of LC-DTT-sperm with nucleoplasmin and exogenous nucleosomal core histones, micrococcal nuclease-protected DNA fragments were released, although their unit repeat length was slightly less than that of somatic nucleosomes. Thus remodeling of human sperm during fertilization can be mimicked under defined conditions with nucleoplasmin and exogenous histones.
RESUMEN
In Xenopus laevis, nucleoplasmin from fully grown oocytes is not highly phosphorylated, but is more extensively phosphorylated during oocyte maturation to retain this state until mid-blastula transition. Incubation of demembranated sperm with nucleoplasmin from oocytes or mature eggs revealed that egg nucleoplasmin is twice as potent as oocyte nucleoplasmin in removing sperm-specific basic proteins from chromatin (protamine-removing activity: PRA). Dephosphorylation of egg nucleoplasmin by alkaline phosphatase induced a remarkable decline of PRA in nucleoplasmin. Treatment of oocyte nucleoplasmin with cdc2 protein kinase induced an increase of the extent of phosphorylation, but to a level lower than that exhibited by egg nucleoplasmin, suggesting the involvement of other unspecified kinase(s) in phosphorylating nucleoplasmin during oocyte maturation. Incubation of sperm with cdc2 kinase induced selective phosphorylation of sperm-specific basic proteins, accompanied by their enhanced removal from sperm chromatin upon exposure to high-salt solutions. These results suggest that removal of sperm-specific basic proteins from sperm chromatin in fertilized eggs is facilitated by phosphorylation of both nucleoplasmin and sperm-specific basic proteins.
Asunto(s)
Fertilización/fisiología , Meiosis/genética , Óvulo/citología , Óvulo/fisiología , Vertebrados/fisiología , Animales , Calcineurina/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Proteínas de Ciclo Celular/fisiología , Proteínas F-Box/fisiología , Meiosis/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-mos/fisiología , Proteínas de Xenopus/fisiologíaRESUMEN
Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or "intranuclear DNA density." Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans.
Asunto(s)
Núcleo Celular/metabolismo , Empaquetamiento del ADN , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Tamaño del Núcleo Celular , Tamaño de la Célula , Sistema Libre de Células/metabolismo , Cromosomas/genética , Cromosomas/metabolismo , ADN de Helmintos/genética , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Femenino , Conformación de Ácido Nucleico , Ploidias , Xenopus laevisAsunto(s)
Sistema Libre de Células , Ensamble y Desensamble de Cromatina , Óvulo/citología , Xenopus laevis/genética , Animales , Ciclo Celular , Clonación de Organismos , Replicación del ADN , Chaperonas de Histonas/fisiología , Histonas , Técnicas de Transferencia Nuclear , Nucleoplasminas/fisiología , Factores de Transcripción/fisiologíaRESUMEN
The centrosome, consisting of a pair of centrioles surrounded by pericentriolar material, directs the formation of bipolar spindles during mitosis. Aberrant centrosome number can promote chromosome instability, which is implicated in tumorigenesis. Thus, centrosome duplication needs to be tightly regulated to occur only once per cell cycle. Separase, a cysteine protease that triggers sister chromatid separation, is involved in centriole disengagement, which licenses centrosomes for the next round of duplication. However, at least two questions remain unsolved: what is the substrate relevant to the disengagement, and how does separase, activated at anaphase onset, act on the disengagement that occurs during late mitosis. Here, we show that kendrin, also named pericentrin, is cleaved by activated separase at a consensus site in vivo and in vitro, and this leads to the delayed release of kendrin from the centrosome later in mitosis. Furthermore, we demonstrate that expression of a noncleavable kendrin mutant suppresses centriole disengagement and subsequent centriole duplication. Based on these results, we propose that kendrin is a novel and crucial substrate for separase at the centrosome, protecting the engaged centrioles from premature disengagement and thereby blocking reduplication until the cell passes through mitosis.
Asunto(s)
Proteínas de Unión a Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/fisiología , Endopeptidasas/metabolismo , Mitosis/fisiología , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Separasa , XenopusRESUMEN
It is known that differentiated cells can be reprogrammed to an undifferentiated state in oocyte cytoplasm after nuclear transfer. Recently, some reports suggested that Xenopus egg extracts have the ability to reprogram mammalian somatic cells. Reprogramming events of mammalian cells after Xenopus egg extract treatment and after cell culture of extract-treated cells have not been elucidated. In this experiment, we examined reprogramming events in reversibly permeabilized or nonpermeabilized porcine fibroblast cells after Xenopus egg extract treatment. The Xenopus egg-specific histone B4 was assembled on porcine chromatin and nuclear lamin LIII was incorporated into nuclei. Deacetylation of histone H3 at lysine 9 in extract-treated cells was detected in nonpermeabilized cells, suggesting that a part of reprogramming may be induced even in nonpermeabilized cells. Following culture of extract-treated cells, the cells began to express the pluripotent marker genes such as POU5F1 (OCT4) and SOX2 and to form colonies. Reactivation of the OCT4 gene in extract-treated cells was also confirmed in bovine fibroblasts transformed with an OCT4-EGFP construct. These results suggest that nuclei of mammalian cells can be partially reprogrammed to an embryonic state by Xenopus egg extracts and the remodeled cells partly dedifferentiate after cell culture. A system using egg extracts may be useful for understanding the mechanisms and processes of dedifferentiation and reprogramming of mammalian somatic cells after nuclear transfer.
Asunto(s)
Extractos Celulares/farmacología , Reprogramación Celular/efectos de los fármacos , Óvulo/química , Xenopus laevis , Acetilación , Animales , Bovinos , Permeabilidad de la Membrana Celular , Células Cultivadas , Femenino , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Histonas/farmacología , Laminas/farmacología , Masculino , Factor 3 de Transcripción de Unión a Octámeros/genética , PorcinosRESUMEN
Telomeres are regulated by a homeostatic mechanism that includes telomerase and telomeric repeat binding proteins, TRF1 and TRF2. Recently, it has been hypothesized that telomeres assume distinct configurations in a cell-cycle-dependent manner, although direct biochemical evidence is lacking. Here we demonstrated that Xenopus TRF1 (xTRF1) associates with telomere chromatin specifically in mitotic Xenopus egg extracts, and dissociates from it upon mitotic exit. Both the N-terminal TRF-homology (TRFH) domain and the linker region connecting the TRFH domain and the C-terminal Myb domain are required for this cell-cycle-dependent association of xTRF1 with chromatin. In contrast, Xenopus TRF2 (xTRF2) associates with chromatin throughout the cell cycle. We showed that Polo-like kinase (Plx1) phosphorylates xTRF1 in vitro. Moreover, the mitotic xTRF1-chromatin association was significantly impaired when Plx1 was immunodepleted from the extracts. Finally, high telomerase activities were detected in association with replicating interphase chromatin compared with mitotic chromatin. These results indicate that telomere chromatin is actively regulated by cell-cycle-dependent processes, and provide an insight for understanding how telomeres undergo DNA metabolisms during the cell cycle.