RESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe disease with substantial economic consequences for the swine industry. The DEAD-box helicase 3 (DDX3X) is an RNA helicase that plays a crucial role in regulating RNA metabolism, immunological response, and even RNA virus infection. However, it is unclear whether it contributes to PRRSV infection. Recent studies have found that the expression of DDX3X considerably increases in Marc-145 cells when infected with live PRRSV strains Ch-1R and SD16; however, it was observed that inactivated viruses did not lead to any changes. By using the RK-33 inhibitor or DDX3X-specific siRNAs to reduce DDX3X expression, there was a significant decrease in the production of PRRSV progenies. In contrast, the overexpression of DDX3X in host cells substantially increased the proliferation of PRRSV. A combination of transcriptomics and metabolomics investigations revealed that in PRRSV-infected cells, DDX3X gene silencing severely affected biological processes such as ferroptosis, the FoxO signalling pathway, and glutathione metabolism. The subsequent transmission electron microscopy (TEM) imaging displayed the typical ferroptosis features in PRRSV-infected cells, such as mitochondrial shrinkage, reduction or disappearance of mitochondrial cristae, and cytoplasmic membrane rupture. Conversely, the mitochondrial morphology was unchanged in DDX3X-inhibited cells. Furthermore, silencing of the DDX3X gene changed the expression of ferroptosis-related genes and inhibited the virus proliferation, while the drug-induced ferroptosis inversely promoted PRRSV replication. In summary, these results present an updated perspective of how PRRSV infection uses DDX3X for self-replication, potentially leading to ferroptosis via various mechanisms that promote PRRSV replication.
Assuntos
RNA Helicases DEAD-box , Ferroptose , Vírus da Síndrome Respiratória e Reprodutiva Suína , Replicação Viral , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Ferroptose/fisiologia , Suínos , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Linhagem CelularRESUMO
Primordial germ cell 7 (PGC7)(Dppa3 or Stella) is a small inherently disordered protein that is mainly expressed in oocytes and plays a vital role in the regulation of DNA methylation reprogramming in imprinted loci through interaction with other proteins. Most of PGC7-deficient zygotes are blocked at two-cell stage with an increased tri-methylation at lysine 27 of histone H3 (H3K27me3) level in the nucleus. Our previous work has indicated that PGC7 interacts with yin-yang1 (YY1) that is essential for the recruitment of enhancer of zeste homolog 2 (EZH2)-containing Polycomb repressive complex 2 (PRC2) to H3K27me3 modification sites. Here, we found that the presence of PGC7 weakened the interaction between YY1 and PRC2 without disrupting the assembly of core subunits of the PRC2 complex. In addition, PGC7 promoted AKT to phosphorylate serine 21 of EZH2, resulting in inhibition of EZH2 activity and the dissociation of EZH2 from YY1, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 both promoted EZH2 to enter the pronuclei but without disturbing the subcellular localization of YY1 and caused an increase in the level of H3K27me3 in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos. In summary, PGC7 could affect zygotic genome activation during early embryonic development by regulating the level of H3K27me3 through regulation of PRC2 recruitment, EZH2 activity, and subcellular localization.NEW & NOTEWORTHY PGC7 and YY1 interaction inhibits recruitment of PRC2 by YY1. PGC7 promotes AKT and EZH2 interaction to increase pEZH2-S21 level, which weakens YY1 and EZH2 interaction, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 promote EZH2 to enter the pronuclei, and increase H3K27me3 level in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos, which ultimately affects early embryo development.
Assuntos
Histonas , Complexo Repressor Polycomb 2 , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Metilação de DNA , Células Germinativas/metabolismoRESUMO
DNA methylation is an epigenetic modification that plays a vital role in a variety of biological processes, including the regulation of gene expression, cell differentiation, early embryonic development, genomic imprinting, and X chromosome inactivation. PGC7 is a maternal factor that maintains DNA methylation during early embryonic development. One mechanism of action has been identified by analyzing the interactions between PGC7 and UHRF1, H3K9 me2, or TET2/TET3, which reveals how PGC7 regulates DNA methylation in oocytes or fertilized embryos. However, the mechanism by which PGC7 regulates the post-translational modification of methylation-related enzymes remains to be elucidated. This study focused on F9 cells (embryonic cancer cells), which display high levels of PGC7 expression. We found that both knockdown of Pgc7 and inhibition of ERK activity resulted in increased genome-wide DNA methylation levels. Mechanistic experiments confirmed that inhibition of ERK activity led to the accumulation of DNMT1 in the nucleus, ERK phosphorylated DNMT1 at ser717, and DNMT1 Ser717-Ala mutation promoted the nuclear localization of DNMT1. Moreover, knockdown of Pgc7 also caused downregulation of ERK phosphorylation and promoted the accumulation of DNMT1 in the nucleus. In conclusion, we reveal a new mechanism by which PGC7 regulates genome-wide DNA methylation via phosphorylation of DNMT1 at ser717 by ERK. These findings may provide new insights into treatments for DNA methylation-related diseases.
Assuntos
Metilação de DNA , Epigênese Genética , Núcleo Celular/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Impressão Genômica , Processamento de Proteína Pós-Traducional , Proteínas Cromossômicas não HistonaRESUMO
The gene dosage at the imprinted Dlk1-Dio3 locus is critical for cell growth and development. A relatively high gene expression within the Dlk1-Dio3 region, especially the active expression of Gtl2, has been identified as the only reliable marker for cell pluripotency. The DNA methylation state of the IG-DNA methylated regions (DMR), which is located upstream of the Gtl2 gene, dominantly contributes to the control of gene expression in the Dlk1-Dio3 locus. However, the precise mechanism underlying the regulation of DNA methylation in the IG-DMR remains largely unknown. Here, we use the F9 embryonal carcinoma cell line, a low pluripotent cell model, to identify the mechanism responsible for DNA methylation in the IG-DMR, and find that the interaction of PGC7 with UHRF1 is involved in maintaining DNA methylation and inducing DNA hypermethylation in the IG-DMR region. PGC7 and UHRF1 cooperatively bind in the IG-DMR to regulate the methylation of DNA and histones in this imprinted region. PGC7 promotes the recruitment of DNMT1 by UHRF1 to maintain DNA methylation in the IG-DMR locus. The interaction between PGC7 and UHRF1 strengthens their binding to H3K9me3 and leads to further enrichment of H3K9me3 in the IG-DMR by recruiting the specific histone methyltransferase SETDB1. Consequently, the abundance of H3K9me3 promotes DNMT3A to bind to the IG-DMR and increases DNA methylation level in this region. In summary, we propose a new mechanism of DNA methylation regulation in the IG-DMR locus and provide further insight into the understanding of the difference in Gtl2 expression levels between high and low pluripotent cells.
Assuntos
Metilação de DNA , RNA Longo não Codificante , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , DNA/metabolismo , Impressão Genômica , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
FOXC1, a transcription factor involved in cell differentiation and embryogenesis, is demonstrated to be a negative regulator of Nanog in this study. FOXC1 is up-regulated in retinoic acid-induced differentiation of F9 Embryonal Carcinoma (EC) cells; furthermore, FOXC1 specifically inhibits the core pluripotency factor Nanog by binding to the proximal promoter. Overexpression of FOXC1 in F9 or knockdown in 3T3 results in the down-regulation or up-regulation of Nanog mRNA and proteins, respectively. In order to explain the mechanism by which FOXC1 inhibits Nanog expression, we identified the co-repressor HDAC2 from the FOXC1 interactome. FOXC1 recruits HDAC2 to Nanog promoter to decrease H3K27ac enrichment, resulting in transcription inhibition of Nanog. To the best of our knowledge, this is the first report that FOXC1 is involved in the epigenetic regulation of gene expression.
Assuntos
Células-Tronco de Carcinoma Embrionário/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 2/metabolismo , Proteína Homeobox Nanog/genética , Regiões Promotoras Genéticas , Tretinoína/farmacologia , Animais , Antineoplásicos/farmacologia , Células-Tronco de Carcinoma Embrionário/efeitos dos fármacos , Células-Tronco de Carcinoma Embrionário/patologia , Epigênese Genética , Fatores de Transcrição Forkhead/genética , Células HEK293 , Histona Desacetilase 2/genética , Humanos , Camundongos , Células NIH 3T3 , Proteína Homeobox Nanog/metabolismoRESUMO
Retinoic acid (RA) plays a key role in pluripotent cell differentiation. In F9 embryonic carcinoma cells, RA can induce differentiation towards somatic lineages via the Ras-extracellular signal-regulated kinase (Ras/Erk) pathway, but the mechanism through which it induces the Erk1/2 phosphorylation is unclear. Here, we show that miR-485 is a positive regulator that targets α/ß-hydrolase domain-containing protein 2 (Abhd2), which can result in Erk1/2 phosphorylation and triggers differentiation. RA up-regulates miR-485 and concurrently down-regulates Abhd2. We verified that Abhd2 is targeted by miR-485 and they both can influence the phosphorylation of Erk1/2. In summary, RA can mediate cell differentiation by phosphorylating Erk1/2 via miR-485 and Abhd2.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hidrolases/genética , MicroRNAs/genética , Interferência de RNA , Tretinoína/farmacologia , Animais , Biomarcadores , Células-Tronco de Carcinoma Embrionário , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , FosforilaçãoRESUMO
Somatic cell nuclear transfer (SCNT) is a very powerful technique used to produce genetically identical or modified animals. However, the cloning efficiency in mammals remains low. In this study, we aimed to explore the effects of vitamin C (Vc)-treated donor cells on cloned embryos. As a result, Vc treatment relaxed the chromatin of donor cells and improved cloned embryo development. RNA sequencing was adopted to investigate the changes in the transcriptional profiles in early embryos. We found that Vc treatment increased the expression of genes involved in the cell-substrate adherens junction. Gene ontology (GO) analysis revealed that Vc treatment facilitated the activation of autophagy, which was deficient in cloned two-cell embryos. Rapamycin, an effective autophagy activator, increased the formation of cloned blastocysts (36.0% vs. 25.6%, p < 0.05). Abnormal expression of some coding genes and long non-coding RNAs in cloned embryos was restored by Vc treatment, including the zinc-finger protein 641 (ZNF641). ZNF641 compensation by means of mRNA microinjection improved the developmental potential of cloned embryos. Moreover, Vc treatment rescued some deficient RNA-editing sites in cloned two-cell embryos. Collectively, Vc-treated donor cells improved the development of the cloned embryo by affecting embryonic transcription. This study provided useful resources for future work to promote the reprogramming process in SCNT embryos.
Assuntos
Ácido Ascórbico/farmacologia , Blastocisto/efeitos dos fármacos , Clonagem de Organismos/métodos , Oócitos/efeitos dos fármacos , Transcriptoma , Vitaminas/farmacologia , Animais , Autofagia , Blastocisto/metabolismo , Bovinos/genética , Células Cultivadas , Feminino , Masculino , Técnicas de Transferência Nuclear , Oócitos/metabolismoRESUMO
The ability to self-renew is one of the most important properties of embryonic stem (ES) cells. Pluripotin (SC1), a small molecule with high activity and low toxicity, promotes self-renewal in mouse ES cells. SC1 can noticeably change the morphology of retinoic acid (RA)-induced F9 embryonic carcinoma cells (F9 cells). However, in the long term, RA and SC1 together cause cell apoptosis. When being added after 18-24 h of RA-induced F9 cell differentiation, SC1 transitorily activated Nanog and Oct4. Both Nanog and Oct4 were downregulated when SC1 and RA were added simultaneously. On the other hand, Klf4 was continually activated when SC1 was added between 6 and 24 h. Phosphorylated Erk1/2 protein levels were reduced from 6 to 24 h, whereas unphosphorylated Erk1 protein levels remained unchanged. A higher concentration of SC1 promoted cell self-renewal by strengthening the inhibition of Erk1/2 protein phosphorylation in F9 cells. Furthermore, SC1 and RA affect global DNA methylation by influencing the expressions of methylation-associated proteins, including Dnmt3b, Dnmt3l, Tet1, Tet2, and Tet3. In conclusion, SC1 inhibits the differentiation of RA-induced F9 cells mainly by reducing the levels of phosphorylated Erk1/2 and enhancing the expression of Klf4, although it also reduces DNA methylation, which may have an additional effect on ES cell differentiation.
Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Tretinoína/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Carcinoma Embrionário/genética , Carcinoma Embrionário/metabolismo , Carcinoma Embrionário/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 4 Semelhante a Kruppel , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Primordial germ cell 7 (PGC7), a maternal factor essential for early development, plays a critical role in the regulation of DNA methylation, transcriptional repression, chromatin condensation, and cell division and the maintenance of cell pluripotentiality. Despite the fundamental roles of PGC7 in these cellular processes, only a few molecular and functional interactions of PGC7 have been reported. Here, a streptavidin-biotin affinity purification technique combined with LC-MS/MS was used to analyze potential proteins that interact with PGC7. In total, 291 potential PGC7-interacting proteins were identified. Through an in-depth bioinformatic analysis of potential interactors, we linked PGC7 to critical cellular processes including translation, RNA processing, cell cycle, and regulation of heterochromatin structure. To better understand the functional interactions of PGC7 with its potential interactors, we constructed a protein-protein interaction network using the STRING database. In addition, we discussed in detail the interactions between PGC7 and some of its newly validated partners. The identification of these potential interactors of PGC7 expands our knowledge on the PGC7 interactome and provides a valuable resource for understanding the diverse functions of this protein.
Assuntos
Redes Reguladoras de Genes , Heterocromatina/metabolismo , Mapeamento de Interação de Proteínas , Proteômica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Animais , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Ciclo Celular/genética , Cromatografia Líquida , Proteínas Cromossômicas não Histona , Clonagem Molecular , Metilação de DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Heterocromatina/química , Humanos , Camundongos , Anotação de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética , Espectrometria de Massas em TandemRESUMO
BACKGROUND/AIMS: Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. METHODS: In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. RESULTS: SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. CONCLUSION: SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1.
Assuntos
Autorrenovação Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/antagonistas & inibidores , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Adaptadora GRB2/antagonistas & inibidores , Proteína Adaptadora GRB2/metabolismo , Fator Inibidor de Leucemia/química , MAP Quinase Quinase Quinases/metabolismo , Camundongos , MicroRNAs/química , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/metabolismo , Análise de Sequência de RNA , Proteínas Son Of Sevenless/antagonistas & inibidores , Proteínas Son Of Sevenless/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
DNA methylation is an important epigenetic modification that needs to be carefully controlled as a prerequisite for normal early embryogenesis. Compelling evidence now suggests that four maternal-effect proteins, primordial germ cell 7 (PGC7), zinc finger protein 57 (ZFP57), tripartite motif-containing 28 (TRIM28) and DNA methyltransferase (cytosine-5) 1 (DNMT1) are involved in the maintenance of DNA methylation. However, it is still not fully understood how these maternal-effect proteins maintain the DNA methylation imprint. We noticed that a feature common to these proteins is the presence of significant levels of intrinsic disorder so in this study we started from an intrinsic disorder perspective to try to understand these maternal-effect proteins. To do this, we firstly analysed the intrinsic disorder predispositions of PGC7, ZFP57, TRIM28 and DNMT1 by using a set of currently available computational tools and secondly conducted an intensive literature search to collect information on their interacting partners and structural characterization. Finally, we discuss the potential effect of intrinsic disorder on the function of these proteins in maintaining DNA methylation.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de ProteínasRESUMO
Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression.
Assuntos
Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/metabolismo , Animais , Linhagem Celular , Fator de Transcrição GATA1/genética , Proteínas de Homeodomínio/genética , Camundongos , Células-Tronco Embrionárias Murinas , Proteína Homeobox Nanog , Regiões Promotoras Genéticas/genéticaRESUMO
Hesx1, a homeobox gene expressed in embryonic stem cells (ESCs), has been implicated in the core transcription factors governing the pluripotent state. However, data about the underlying mechanism of how Hesx1 is involved in maintaining pluripotency is still scarce. In this study, we find Hesx1 responds to multiple pluripotency-related pathway inhibitors as well as LIF stimulation. Particularly, the expression of Hesx1 can be readily induced by dual inhibition (2i) of glycogen synthase kinase 3 and mitogen-activated protein kinase. Forced expression of Hesx1 can partially compensate for the withdrawal of either LIF or each component of 2i. We also demonstrate that LIF and each inhibitor of 2i can induce Hesx1 independent of one another. We tentatively put forward that Hesx1 is a common downstream target of LIF- and 2i-mediated self-renewal signaling pathways and plays an important role in maintaining ESC identity. Our study extends the methods of identifying the missing crucial factors in establishing ESC pluripotency.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes/fisiologia , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Homeodomínio/genética , Fator Inibidor de Leucemia/farmacologia , Camundongos Transgênicos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais , Tretinoína/farmacologiaRESUMO
Vitamin C (Vc), also known as ascorbic acid, is involved in many important metabolic and physiological reactions in the body. Here, we report that Vc enhances the expression of Nanog and inhibits retinoic acid-induced differentiation of embryonic stem cells. We investigated Vc regulation of Nanog through Janus kinase/signal transducer and activator of transcription pathway using cell signaling pathway profiling systems, and further confirmed by specific pathway inhibition. Using overexpression and knockdown strategies, we demonstrated that STAT2 is a new positive regulator of Nanog and is activated by phosphorylation following Vc treatment. In addition, site mutation analysis identified that STAT2 physically occupies the Nanog promoter, which was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Taken together, our data suggest a role for Vc in Nanog regulation networks and reveal a novel role for STAT2 in regulating Nanog expression.
Assuntos
Ácido Ascórbico/farmacologia , Células-Tronco de Carcinoma Embrionário/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Proteínas de Homeodomínio/biossíntese , Janus Quinases/metabolismo , Fator de Transcrição STAT2/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco de Carcinoma Embrionário/citologia , Células-Tronco de Carcinoma Embrionário/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/genética , Camundongos , Proteína Homeobox Nanog , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Vitamin C (Vc) has been widely studied in cell and embryo culture, and has recently been demonstrated to promote cellular reprogramming. The objective of this study was to identify a suitable Vc concentration that, when used to treat adult bovine fibroblasts serving as donor cells for nuclear transfer, improved donor-cell physiology and the developmental potential of the cloned embryos that the donor nuclei were used to create. A Vc concentration of 0.15 mM promoted cell proliferation and increased donor-cell 5-hydroxy methyl cytosine levels 2.73-fold (P < 0.05). The blastocyst rate was also significantly improved after nuclear transfer (39.6% treated vs. 26.0% control, P < 0.05); the average number of apoptotic cells in cloned blastocysts was significantly reduced (2.2 vs. 4.4, P < 0.05); and the inner cell mass-to-trophectoderm ratio (38.25% vs. 30.75%, P < 0.05) and expression of SOX2 (3.71-fold, P < 0.05) and POU5F1 (3.15-fold, P < 0.05) were significantly increased. These results suggested that Vc promotes cell proliferation, decreases DNA methylation levels in donor cells, and improves the developmental competence of bovine somatic-cell nuclear transfer embryos.
Assuntos
Ácido Ascórbico/farmacologia , Clonagem de Organismos , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Técnicas de Transferência Nuclear , Animais , Bovinos , Embrião de Mamíferos/citologia , FemininoRESUMO
BACKGROUND: Colony morphology of embryonic stem (ES) cells contributes to the maintenance of undifferentiated ES cells. Small molecule 3,4-dihydropy-rimido[4,5-d]pyrimidine (SC1), an inhibitor of ERK1- and RasGAP-dependent signaling pathways, can maintain the compact colony morphology of ES cells. However, information on the influence of SC1 on cell morphological change remains lacking. METHODS: In this study, mouse ES cells J1 and embryonic carcinoma (EC) cells F9 were cultured in SC1-containing medium to determine the effect of SC1 on cell morphology. RESULTS: SC1 promotes a more compact morphology of J1 mouse ES cells and induces colony growth of F9 EC cells. Furthermore, the cell adhesion protein E-cadherin is a downstream target of SC1, and E-cadherin is critical for SC1-mediated colony growth of F9 EC cells. CONCLUSIONS: SC1 maintains and induces compact colony morphology of pluripotent cells, and its downstream target, E-cadherin, is involved in the colony phenotype of F9 EC cells. These results explored the potential role of SC1 in morphological change and gene expression in pluripotent cells.
Assuntos
Caderinas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Embrionárias de Células Germinativas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos , Neoplasias Embrionárias de Células Germinativas/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Pluripotentes/patologia , Ensaio Tumoral de Célula-TroncoRESUMO
Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine ß-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in ß-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.
Assuntos
Caseínas/genética , Resistência à Doença/genética , Marcação de Genes/veterinária , Mastite Bovina/genética , Muramidase/genética , Dedos de Zinco/genética , Animais , Sequência de Bases , Bovinos , Clonagem de Organismos/veterinária , Feminino , Fibroblastos/enzimologia , Genes Reporter , Genômica , Humanos , Mastite Bovina/prevenção & controle , Dados de Sequência Molecular , Técnicas de Transferência Nuclear/veterinária , Organismos Geneticamente Modificados , Alinhamento de SequênciaRESUMO
Successful in vitro maturation (IVM) and oocyte quality both affect the subsequent development of cloned embryos derived from somatic-cell nuclear transfer (SCNT). Developmental competence is usually lower in oocytes matured in vitro compared with those that matured in vivo, possibly due to insufficient levels of oocyte-secreted factors (OSFs) and disrupted oocyte-cumulus communication. This study investigated the effects of OSFs secreted by denuded oocytes (DOs) during IVM on the subsequent developmental competence of cloned bovine embryos. Cumulus-oocyte complexes (COCs) from antral follicles of slaughtered-cow ovaries collected from an abattoir were divided into four groups: COCs co-cultured with and without DOs in maturation media used for SCNT, as well as COCs co-cultured with and without DOs in maturation media used for in vitro fertilization (IVF). Based on the developmental competence and embryo quality of bovine embryos generated from these four groups, we found that co-culturing the COCs with DOs enhanced the in vitro development of IVF and cloned bovine embryos, and potentially generated more high-quality cloned blastocysts that possessed locus-specific histone modifications at levels similar to in vitro-fertilized embryos. These results strongly suggest that co-culturing COCs with DOs enhances subsequent developmental competence of cloned bovine embryo.
Assuntos
Clonagem de Organismos/métodos , Meios de Cultivo Condicionados/farmacologia , Células do Cúmulo/metabolismo , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Oogênese/fisiologia , Acetilação , Animais , Apoptose , Blastocisto/citologia , Bovinos , Técnicas de Cocultura , Desenvolvimento Embrionário , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Cultura Primária de Células , Processamento de Proteína Pós-TraducionalRESUMO
Embryonic stem cells (ESCs) can proliferate indefinitely in vitro and differentiate into cells of all three germ layers. These unique properties make them exceptionally valuable for drug discovery and regenerative medicine. However, the practical application of ESCs is limited because it is difficult to derive and culture ESCs. It has been demonstrated that CHIR99021 (CHIR) promotes self-renewal and enhances the derivation efficiency of mouse (m)ESCs. However, the downstream targets of CHIR are not fully understood. In this study, we identified CHIR-regulated genes in mESCs using microarray analysis. Our microarray data demonstrated that CHIR not only influenced the Wnt/ß-catenin pathway by stabilizing ß-catenin, but also modulated several other pluripotency-related signaling pathways such as TGF-ß, Notch and MAPK signaling pathways. More detailed analysis demonstrated that CHIR inhibited Nodal signaling, while activating bone morphogenetic protein signaling in mESCs. In addition, we found that pluripotency-maintaining transcription factors were up-regulated by CHIR, while several developmental-related genes were down-regulated. Furthermore, we found that CHIR altered the expression of epigenetic regulatory genes and long intergenic non-coding RNAs. Quantitative real-time PCR results were consistent with microarray data, suggesting that CHIR alters the expression pattern of protein-encoding genes (especially transcription factors), epigenetic regulatory genes and non-coding RNAs to establish a relatively stable pluripotency-maintaining network.
Assuntos
Células-Tronco Embrionárias/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Longo não Codificante/genética , Transcrição Gênica/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteína Nodal/genética , Proteína Nodal/metabolismo , RNA Longo não Codificante/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND/AIMS: [corrected] Embryonic stem cells (ES cells) have the capacity to propagate indefinitely, maintain pluripotency, and differentiate into any cell type under defined conditions. As a result, they are considered to be the best model system for research into early embryonic development. AICA ribonucleotide (AICAR) is an activator of AMP-activated protein kinase (AMPK) that is thought to affect ES cell function, but its role in ES cell fate decision is unclear. METHODS: In this study, we performed microarray analysis to investigate AICAR downstream targets and further understand its effect on ES cells. RESULTS: Our microarray data demonstrated that AICAR can significantly up-regulate pluripotency-associated genes and down-regulate differentiation-associated transcription factors. Although AICAR cannot maintain ES cell identity without LIF, it can antagonize the action of RA-induced differentiation. Using those differentially expressed genes identified, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the Database for Annotation, Visualization and Integrated Discovery (DAVID) online system. AICAR was not only shown to influence the AMPK pathway, but also act on other signaling pathways such as BMP, MAPK and TGF-ß, to maintain the stemness of J1 ES cells. Furthermore, AICAR modulated ES cell epigenetic modification by altering the expression of epigenetic-associated proteins, including Dnmt3a, Dnmt3b, Smarca2, Mbd3, and Arid1a, or through regulating the transcription of long intervening non-coding RNA (lincRNA). CONCLUSION: Taken together, our work suggests that AICAR is capable of maintaining ES cell self-renewal and pluripotency, which could be useful in future medical treatment.