RESUMO
The variant histones TH2A and TH2B are abundant in the testis, but their roles in spermatogenesis remain elusive. Here, we show that male mutant mice lacking both Th2a and Th2b genes were sterile, with few sperm in the epididymis. In the mutant testis, the lack of TH2B was compensated for by overexpression of H2B, whereas overexpression of H2A was not observed, indicating a decrease in the total histone level. Mutant mice exhibited two defects: incomplete release of cohesin at interkinesis after meiosis I and histone replacement during spermiogenesis. In the mutant testis, secondary spermatocytes at interkinesis accumulated and cohesin was not released normally, suggesting that the retained cohesion of sister chromatids delayed the subsequent entry into meiosis II. In addition, impaired chromatin incorporation of TNP2 and degenerated spermatids were observed in the mutant testis. These results suggest that a loss of TH2A and TH2B function in chromatin dynamics or a decrease in the total histone levels causes defects in both cohesin release and histone replacement during spermatogenesis.
Assuntos
Deleção de Genes , Histonas/genética , Espermatogênese/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/metabolismo , Proteínas de Ligação a DNA , Feminino , Histonas/deficiência , Histonas/metabolismo , Masculino , Meiose , Camundongos Endogâmicos BALB C , Mutação/genética , Proteínas Nucleares/metabolismo , Espermátides/citologia , Espermátides/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Testículo/citologia , Testículo/metabolismo , CoesinasRESUMO
Histone variants TH2a and TH2b are highly expressed in testes, oocytes and zygotes. Our recent analysis suggested that these histone variants enhance the induced generation of pluripotent stem cells (iPSCs) when co-expressed along with four transcription factors, Oct3/4, Sox2, Klf4 and c-Myc (OSKM), and are associated with an open chromatin structure [1]. In the present study, we report the crystal structures of nucleosomes (NCPs) with the mouse histone variants, TH2a and TH2b. The structures revealed two significant changes, as compared to the canonical counterparts: fewer histone-DNA contacts and changes in dimer-dimer interactions between TH2a-TH2a' (L1-loop). In vivo studies with domain swapping and point mutants of the variants revealed that the residues in the histone tails and the TH2a-L1 loop are important for reprogramming. Taken together, our work indicates that the NCP variants with structural modifications and flexible tails are most likely important for enhanced reprogramming of functions.
Assuntos
Histonas/química , Histonas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Nucleossomos/química , Sequência de Aminoácidos , Animais , Células Cultivadas , Reprogramação Celular , Cristalografia por Raios X , Histonas/genética , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Mutação Puntual , Conformação Proteica , Estrutura Terciária de Proteína , Testículo/citologiaRESUMO
B-Myb is one member of the vertebrate Myb family of transcription factors and is ubiquitously expressed. B-Myb activates transcription of a group of genes required for the G2/M cell cycle transition by forming the dREAM/Myb-MuvB-like complex, which was originally identified in Drosophila. Mutants of zebrafish B-myb and Drosophila myb exhibit defects in cell cycle progression and genome instability. Although the genome instability caused by a loss of B-Myb has been speculated to be due to abnormal cell cycle progression, the precise mechanism remains unknown. Here, we have purified a B-Myb complex containing clathrin and filamin (Myb-Clafi complex). This complex is required for normal localization of clathrin at the mitotic spindle, which was previously reported to stabilize kinetochore fibres. The Myb-Clafi complex is not tightly associated with the mitotic spindles, suggesting that this complex ferries clathrin to the mitotic spindles. Thus, identification of the Myb-Clafi complex reveals a previously unrecognized function of B-Myb that may contribute to its role in chromosome stability, possibly, tumour suppression.
Assuntos
Clatrina/metabolismo , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Fuso Acromático/metabolismo , Animais , Clatrina/isolamento & purificação , Proteínas Contráteis/isolamento & purificação , Fibroblastos/metabolismo , Filaminas , Instabilidade Genômica , Células HeLa , Humanos , Camundongos , Proteínas dos Microfilamentos/isolamento & purificação , Mitose , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-myb/isolamento & purificaçãoRESUMO
BACKGROUND: The effects of high-intensity immunity on coronavirus disease 2019 (COVID-19) remain unclear. Antibodies against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are preferentially induced in inpatients with COVID-19 compared with outpatients with milder disease, and immunosuppression is the standard therapy for severe cases. This study investigated the relationship between cross-reactive antibody production against seasonal human coronavirus and the clinical course of COVID-19. METHODS: Among the immunogenic epitopes of SARS-CoV-2, conserved peptides in human coronavirus OC43 were searched and synthesized. Enzyme-linked immunosorbent assay was designed to detect antibodies against synthesized peptides. Antibody titres against S2' cleavage site epitopes near fusion peptides of SARS-CoV-2 and OC43 were determined in the sera of 126 inpatients with COVID-19. The correlation between antibody titres and clinical data was analysed. RESULTS: Inpatients with COVID-19 who produced antibodies against OC43 did not develop severe or fatal pneumonia. Antibody titres against the corresponding epitope of SARS-CoV-2 did not differ between inpatients with severe and mild COVID-19. Antibody titres against the OC43 epitope increased more than those against SARS-CoV-2 during the first 2 weeks of COVID-19. CONCLUSIONS: Immunity to seasonal human coronavirus OC43 effectively enhanced recovery from COVID-19. Detecting cross-reactive antibodies to OC43 may help to predict prognosis for patients with COVID-19.
Assuntos
COVID-19 , Coronavirus Humano OC43 , Anticorpos Antivirais , Reações Cruzadas , Humanos , SARS-CoV-2 , Estações do Ano , Glicoproteína da Espícula de CoronavírusRESUMO
Transcription factor ATF-2 is a nuclear target of stress-activated protein kinases, such as p38, which are activated by various extracellular stresses, including UV light. Here, we show that ATF-2 plays a critical role in hypoxia- and high-cell-density-induced apoptosis and the development of mammary tumors. Compared to wild-type cells, Atf-2(-/-) mouse embryonic fibroblasts (MEFs) were more resistant to hypoxia- and anisomycin-induced apoptosis but remained equally susceptible to other stresses, including UV. Atf-2(-/-) and Atf-2(+/-) MEFs could not express a group of genes, such as Gadd45alpha, whose overexpression can induce apoptosis, in response to hypoxia. Atf-2(-/-) MEFs also had a higher saturation density than wild-type cells and expressed lower levels of Maspin, the breast cancer tumor suppressor, which is also known to enhance cellular sensitivity to apoptotic stimuli. Atf-2(-/-) MEFs underwent a lower degree of apoptosis at high cell density than wild-type cells. Atf-2(+/-) mice were highly prone to mammary tumors that expressed reduced levels of Gadd45alpha and Maspin. The ATF-2 mRNA levels in human breast cancers were lower than those in normal breast tissue. Thus, ATF-2 acts as a tumor susceptibility gene of mammary tumors, at least partly, by activating a group of target genes, including Maspin and Gadd45alpha.
Assuntos
Fator 2 Ativador da Transcrição/genética , Predisposição Genética para Doença , Neoplasias Mamárias Animais/genética , Fator 2 Ativador da Transcrição/análise , Fator 2 Ativador da Transcrição/metabolismo , Animais , Anisomicina/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/metabolismo , Hipóxia Celular/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Genes Reporter , Genes Supressores de Tumor , Humanos , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Serpinas/metabolismo , Células Tumorais CultivadasRESUMO
Bacteria use flagella as propellers to move to favorable environments. Escherichia albertii, a growing cause of foodborne illness and diarrhea, is reportedly non-motile and lacks flagella on its surface. Here, we report that 27 out of 59 E. albertii strains, collected mainly from humans and birds, showed swimming motility when cultured at low osmotic pressure. The biosynthesis of flagella in E. albertii cells was induced under ambient temperature and hypoosmotic pressure: conditions which resemble aquatic environments. Flagellar induction increased E. albertii survival in the intestinal epithelial cell culture containing gentamicin. Although genes involved in chemotaxis are not present in the E. albertii genome, the addition of glutamic acid, an amino acid known to regulate the internal cell osmolarity, augmented the proportion of swimming cells by 35-fold. These results suggest that flagellar biosynthesis and motility in E. albertii cells are controlled by their internal and external osmolarity.
Assuntos
Escherichia/fisiologia , Flagelos/metabolismo , Pressão Osmótica/fisiologia , Animais , Aves/microbiologia , Quimiotaxia/genética , Ecossistema , Escherichia/isolamento & purificação , Escherichia/metabolismo , Flagelos/genética , Regulação Bacteriana da Expressão Gênica , Interação Gene-Ambiente , Genótipo , Humanos , Técnicas Microbiológicas , Movimento (Física) , Concentração Osmolar , FilogeniaRESUMO
The transcription factor ATF-2, a member of the ATF/CREB family, is a target of p38 that are involved in stress-induced apoptosis and in Toll-like receptor (TLR)-mediated signaling. Phosphorylation of ATF-2 at Thr-71 was enhanced by treating of RAW264.7 macrophage cells with either LPS, MALP-2, or CpG-ODN. LPS treatment enhanced the trans-activation capacity of ATF-2. Among multiple LPS-induced genes, the LPS-induced expression of Socs-3 was significantly reduced by the treatment of RAW264.7 cells with an Atf-2 siRNA. Transcription from the Socs-3 promoter was synergistically stimulated by ATF-2 and LPS, whereas it was suppressed by Atf-2 siRNA. Histone deacetylase 1 (HDAC1) interacted with ATF-2 after LPS treatment, but not before treatment. Treatment of RAW264.7 cells with trichostatin A, an inhibitor of HDAC, suppressed the LPS-induced Socs-3 expression, suggesting that HDAC1 positively regulates the LPS-induced transcription of Socs-3. Thus, ATF-2 plays an important role in TLR-mediated transcriptional control in macrophage cells.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Regulação da Expressão Gênica , Macrófagos/imunologia , Receptores Toll-Like/genética , Fator 2 Ativador da Transcrição/genética , Animais , Linhagem Celular , Histona Desacetilase 1 , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Lipopolissacarídeos/imunologia , Camundongos , Fosforilação , Regiões Promotoras Genéticas , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Transcrição GênicaRESUMO
The promyelocytic leukemia (PML) tumor suppressor protein accumulates in PML nuclear bodies (PML-NBs), and can induce growth arrest, cellular senescence and apoptosis. PML has also been localized in the cytoplasm, although its function in this localization remains elusive. A general property of primary cancers is their high glycolytic rate which results from increased glucose consumption. However, the mechanism by which cancer cells up-regulate glycolysis is not well understood. Here, we have shown that cytoplasmic PML (cPML) directly interacts with M2-type pyruvate kinase (PKM2), a key regulator of carbon fate. PKM2 determines the proportion of carbons derived from glucose that are used for glycolytic energy production. Over-expression of PML-2KA mutant in the cytoplasm, which was generated by mutagenesis of the nuclear localization signals of PML, in MCF-7 breast cancer cells suppressed PKM2 activity and the accumulation of lactate. PKM2 exists in either an active tetrameric form which has high affinity for its substrate phosphoenolpyruvate (PEP) or a less active dimeric form which has low affinity for its substrate. Over-expression of PML-2KA suppressed the activity of the tetrameric form of PKM2, but not the dimeric form. Our findings suggest that cPML plays a role in tumor metabolism through its interaction with PKM2.
Assuntos
Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Piruvato Quinase/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Metabolismo Energético/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Camundongos , Camundongos SCID , Mutagênese Sítio-Dirigida , Mutação , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , Piruvato Quinase/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Th2a and Th2b are the testis-specific histone variants highly expressed during spermatogenesis. Approximately 4% of the genome is retained in nucleosomes in mature human sperm, which is enriched at loci of developmental importance. Our recent studies revealed that the mouse histone variant homologs TH2a and TH2b are involved in reprogramming. In the present work, we report three nucleosome structures (NCPs) with human testis-specific histone variants hTh2a and hTh2b, [hGcH (hTh2a-hTh2b-H3-H4), hGcHV1 (hTh2a-H2b-H3-H4) and hGcHV2 (H2a-hTh2b-H3-H4)] and a 146-base pair (bp) duplex DNA fragment at ~3.0Å resolutions. These crystal structures revealed two major changes within the nucleosomes, either with hTh2a, hTh2b or both variants, as compared to the canonical counterpart. First, the H-bonding interactions between the L1-L1' interfaces mediated by the hTh2a/hTh2a' L1-loops are lost. Second, the histone dimer-DNA contacts are considerably reduced, and these changes are localized around ±31 to 35-bp from the nucleosome entry/exit sites. Thus, the modified functional residues at the N- and C-terminal ends of histone variants are responsible for the observed structural changes and regulate the gene expression through specific structural alterations in the chromatin by modulating the chromatin-associated binding proteins.
Assuntos
Histonas/genética , Nucleossomos/química , Cromatina/química , Montagem e Desmontagem da Cromatina/genética , Cristalização , DNA/química , Regulação da Expressão Gênica , Variação Genética , Humanos , Ligação de Hidrogênio , MasculinoRESUMO
There are two major methods of reprogramming: generation of induced pluripotent stem cells (iPSCs) by overexpressing embryonic stem cell-specific transcription factors (OCT4, SOX2, KLF4, and c-MYC) and somatic cell nuclear transfer by oocyte-specific factors. Previously, we reported oocyte-enriched histone variants TH2A, TH2B, and the histone chaperone nucleoplasmin (NPM2) enhance the reprogramming by OSKM in mice by inducing open chromatin structure. In this study, we showed that human TH2A, TH2B, and NPM2 enhance the OSKM-induced reprogramming of adult and neonatal human dermal fibroblasts and umbilical vein endothelial cells. Pluripotency of iPSCs generated by coexpressing OSKM, TH2A, TH2B, and NPM2 was shown by in vitro and in vivo differentiation assays. These iPSCs gave rise to highly differentiated teratomas compared to iPSCs induced by OSKM alone. Genome-wide analysis suggests a possibility that TH2A, TH2B, and NPM2 might regulate genes that are involved in naïve stem cell stage. Thus, TH2A, TH2B, and NPM2 enhance reprogramming of human somatic cells and improve the quality of human iPSCs.
Assuntos
Reprogramação Celular , Histonas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Histonas/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Nucleoplasminas/genética , Nucleoplasminas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Expression of Oct3/4, Sox2, Klf4, and c-Myc (OSKM) can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Somatic cell nuclear transfer (SCNT) can also be used for reprogramming, suggesting that factors present in oocytes could potentially augment OSKM-mediated induction of pluripotency. Here, we report that two histone variants, TH2A and TH2B, which are highly expressed in oocytes and contribute to activation of the paternal genome after fertilization, enhance OSKM-dependent generation of iPSCs and can induce reprogramming with Klf4 and Oct3/4 alone. TH2A and TH2B are enriched on the X chromosome during the reprogramming process, and their expression in somatic cells increases the DNase I sensitivity of chromatin. In addition, Xist deficiency, which was reported to enhance SCNT reprogramming efficiency, stimulates iPSC generation using TH2A/TH2B in conjunction with OSKM, but not OSKM alone. Thus, TH2A/TH2B may enhance reprogramming by introducing processes that normally operate in zygotes and during SCNT.
Assuntos
Reprogramação Celular , Histonas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Oócitos/metabolismo , Animais , Reprogramação Celular/genética , Cromatina/química , Cromatina/metabolismo , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Histonas/genética , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cromossomo X/genéticaRESUMO
Tumor necrosis factor α (TNF-α) plays an important role in cell proliferation and apoptosis, and defects in TNF-α-induced apoptosis are associated with various diseases. TRIM27 is a tripartite motif (TRIM) protein containing RING finger, B-box, and coiled-coil domains. We recently reported that TRIM27 positively regulates TNF-α-induced apoptosis through deubiquitination of receptor-interacting protein 1 (RIP1). Multiple studies have suggested a link between TNF-α pathway and various diseases, such as diabetes and colitis. Here, we report that Trim27-deficient mice were susceptible to streptozotocin (STZ)-induced diabetes, a mouse model of diabetes. Infiltration of T cells and cleaved caspase-3 signals were enhanced, and ß-cell mass was decreased in Trim27-deficient islets compared to wild-type islets. On the other hand, Trim27-mutation did not affect the dextran sodium sulphate (DSS)-induced colitis. These data support the idea that the TRIM27 mutation is responsible for the development of certain types of diseases.
RESUMO
Tumor necrosis factor alpha (TNF-α) plays a role in apoptosis and proliferation in multiple types of cells, and defects in TNF-α-induced apoptosis are associated with various autoimmune diseases. Here, we show that TRIM27, a tripartite motif (TRIM) protein containing RING finger, B-box, and coiled-coil domains, positively regulates TNF-α-induced apoptosis. Trim27-deficient mice are resistant to TNF-α-d-galactosamine-induced hepatocyte apoptosis. Trim27-deficient mouse embryonic fibroblasts (MEFs) are also resistant to TNF-α-cycloheximide-induced apoptosis. TRIM27 forms a complex with and ubiquitinates the ubiquitin-specific protease USP7, which deubiquitinates receptor-interacting protein 1 (RIP1), resulting in the positive regulation of TNF-α-induced apoptosis. Our findings indicate that the ubiquitination-deubiquitination cascade mediated by the TRIM27-USP7 complex plays an important role in TNF-α-induced apoptosis.
Assuntos
Apoptose , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Animais , Cicloeximida/farmacologia , Fibroblastos/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Células Hep G2 , Hepatócitos/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Poliubiquitina/metabolismo , Multimerização Proteica , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico , Ubiquitina-Proteína Ligases , Peptidase 7 Específica de UbiquitinaRESUMO
Yeast Sin1 binds to the Sty1 kinase, a member of the stress-activated kinases (SAPKs), and is required for stress-induced phosphorylation and activation of the transcription factor Atf1, a homolog of the vertebrate-activating transcription factor-2 (ATF-2). Here we report that mammalian Sin1 plays an important role in the SAPK signaling pathway by binding to both ATF-2 and p38. In response to stress, ATF-2, a member of the ATF/cAMP response element-binding protein family, is phosphorylated by p38/Jun NH2-terminal protein kinase and activates the transcription of apoptosis-related genes. In contrast, in response to serum stimulation, ATF-2 is phosphorylated via the Ras effector pathway and leads to the induction of growth-related genes. We found that Sin1 binds directly to both ATF-2 and p38. Sin1 over-expression enhanced osmotic stress-induced phosphorylation of ATF-2 and ATF-2-mediated transcription, whereas knockdown of Sin1 expression by siRNA suppressed these responses. Moreover, a reduction in Sin1 expression suppressed osmotic stress-induced apoptosis and the expression of Gadd45beta, one of the ATF-2 target genes that is correlated with apoptosis. Decreased Sin1 expression, however, did not affect the serum stimulation-induced phosphorylation of ATF-2. Sin1 may contribute to ATF-2 signaling specificity by acting as a nuclear scaffold.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 2 Ativador da Transcrição/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Expressão Gênica , Humanos , Masculino , Camundongos , Modelos Biológicos , Pressão Osmótica , Fosforilação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Ativação Transcricional , TransfecçãoRESUMO
We have developed a new vector, named pDECAP, to express long double-strand RNA (ds-RNA) from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5'-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response. Transgenic mice embryos expressing long ds-RNA for the transcriptional corepressor Ski from this vector exhibited phenotypes that were remarkably similar to those of Ski-deficient embryos, including defects of neural tube closure and eye formation. Thus, this vector provides a new tool to efficiently generate tissue-specific knockdown mice for studying gene function in whole animal systems.
Assuntos
Proteínas de Ligação a DNA/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , RNA Polimerase II/genética , RNA de Cadeia Dupla/genética , Regiões 5' não Traduzidas/genética , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/genética , Animais , Sequência de Bases , Células Cultivadas , Cruzamentos Genéticos , Citoplasma/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Vetores Genéticos , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase II/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , TransfecçãoRESUMO
Ski is a transcriptional co-repressor and is involved in the negative regulation of tumor growth factor-beta (TGF-beta) signaling. To understand more fully the role of Ski in TGF-beta signaling, we searched for novel Ski-interacting proteins. The identified C184M protein consists of 189 amino acids and contains the leucine-rich region. An association between Ski and C184M involving the leucine-rich region of C184M and the C-terminal coiled-coil motif of Ski was confirmed by glutathione S-transferase pull-down and immunoprecipitation assays. The C184M protein is located in the cytosol, and the C184M and Ski signals are co-localized in the cytoplasm when C184M was co-expressed with Ski in CV-1 cells. The cytoplasmic C184M-Ski complex inhibited the nuclear translocation of Smad2. Consistent with this, the activity of promoter containing the Smad-binding sites was repressed by C184M, and the TGF-beta-induced growth inhibition of mink lung Mv1Lu cells was attenuated by the ectopic expression of C184M. Thus, C184M inhibits TGF-beta signaling in concert with Ski. In hepatocytes, which express significant levels of C184M, the Ski signals were found only in the cytoplasm, supporting the notion that C184M forms a complex with Ski in the cytosol.
Assuntos
Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/fisiologia , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Coativadores de Receptor Nuclear , Ligação Proteica , Transporte Proteico , Proteínas Smad , Fatores de Transcrição , Técnicas do Sistema de Duplo-HíbridoRESUMO
p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.
Assuntos
Proteínas Proto-Oncogênicas c-myb/fisiologia , Proteínas Repressoras/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Linhagem Celular , Proliferação de Células , DNA/química , DNA/metabolismo , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoprecipitação , Interleucina-6/metabolismo , Luciferases/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myb/metabolismo , RNA/química , Complexo Correpressor Histona Desacetilase e Sin3 , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
Transcription factor Glioblastoma-3 (Gli3) is cleaved in the anterior region of the limb bud to generate its repressor form. In contrast, Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity blocks Gli3 processing and then induces the expression of Gli3 target genes, including Gli1. Here we report that the Ski corepressor binds to Gli3 and recruits the histone deacetylase complex. The Gli3-mediated repression was impaired by anti-Ski antibody and in Ski-deficient fibroblasts, and Shh-induced Gli1 gene transcription mediated by full-length Gli3 was inhibited by Ski. Furthermore, a Ski mutation enhanced the digit abnormalities caused by the Gli3 gene mutation. Thus, Ski plays an important role in pattern formation.