RESUMO
The general transcription factor TFIID is a multiprotein complex that is essential for specific transcription initiation by RNA polymerase II. It is composed of the TATA box-binding protein (TBP) and ~13 different TBP-associated factors (TAFs). Purification of TFIID free of other general transcription factors and coactivators is essential to analyze the transcription regulatory mechanisms in reconstituted systems in vitro. A breakthrough in TFIID purification was the generation of HeLa cell lines that express a FLAG epitope-tagged TBP subunit and immunopurification protocols with monoclonal anti-FLAG antibodies. Purification of TFIID from HeLa nuclear extracts generally required a two-step purification procedure involving phosphocellulose P11 chromatography followed by anti-flag M2 affinity purification (Chiang et al., 1993; Ge et al., 1996) [1,2]. Here we show first that the MED26 (CRSP70) coactivator subunit of Mediator co-purifies with TFIID in the above two-step protocol and interacts strongly with TFIID under high salt conditions. We further show that a MED26-free TFIID complex can be obtained by including a simple additional DE52 chromatography step following P11 fractionation. Thus, we demonstrate that MED26 strongly interacts with TFIID and recommend the use of a P11-DE52-M2 resin affinity three-step purification procedure to obtain MED26-free TFIID for analyzing Mediator-dependent transcription regulatory mechanisms in purified transcription systems in vitro.
Assuntos
Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Células HeLa , Humanos , Complexo Mediador/química , Complexo Mediador/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/isolamento & purificação , Fator de Transcrição TFIID/biossíntese , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/isolamento & purificaçãoRESUMO
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder characterized pathologically by motor neuron degeneration and associated with aggregation of RNA-binding proteins. TATA-binding protein-associated factor 15 (TAF15) accumulates as cytoplasmic aggregates in neuronal cells, and clearance of these aggregates is considered a potential therapeutic strategy for ALS. However, the exact pathogenic mechanism of TAF15-induced neurotoxicity remains to be elucidated. Glycogen synthase kinase-3 (GSK-3) plays a critical role in the protection of ALS pathology. In the present study, we use a transgenic fly model over-expressing human TAF15 to study the protective effects of Shaggy/GSK3ß on TAF15-induced neuronal toxicity in Drosophila brain. Transgenic flies were examined for locomotor activity and lithium treatment. The expression level and solubility of TAF15 were assessed with western blotting, whereas immunohistochemistry was used to assess TAF15 aggregation in Drosophila brain. We have revealed that Shaggy/GSK3ß was abnormally activated in neurons of TAF15-expressing flies and its inhibition can suppress the defective phenotypes, thereby preventing retinal degeneration and locomotive activity caused by TAF15. We have also found that Shaggy/GSK3ß inhibition in neuronal cells leads to a reduction in TAF15 levels. Indeed, the F-box proteins Slimb and archipelago genetically interact with TAF15 and control TAF15 protein level in Drosophila. Importantly, SCFslimb is a critical regulator for Shaggy/GSK3ß-mediated suppression of TAF15-induced toxicity in Drosophila. The present study has provided an in vivo evidence supporting the molecular mechanism of GSK3ß inhibition for protection against TAF15-linked proteinopathies.
Assuntos
Encéfalo/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteínas de Drosophila/biossíntese , Glicogênio Sintase Quinase 3 beta/biossíntese , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fatores Associados à Proteína de Ligação a TATA/toxicidade , Ubiquitina-Proteína Ligases/biossíntese , Animais , Animais Geneticamente Modificados , Encéfalo/patologia , Proteínas de Ciclo Celular/genética , Drosophila , Proteínas de Drosophila/genética , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Locomoção/fisiologia , Masculino , Fatores Associados à Proteína de Ligação a TATA/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Breast cancer is the most common malignant disease of females. Overall, one woman in every nine will get breast cancer at some time in her life. Epidemiological studies have indicated that alcohol consumption has most consistently been associated with breast cancer risk. However, the mechanism of alcohol-associated breast cancer remains to be addressed. Little is known about the effects of alcohol consumption on Brf1 (TFIIIB-related factor 1) expression and RNA Pol III gene (RNA polymerase III-dependent gene) transcription, which are responsible for protein synthesis and tightly linked to cell proliferation, cell transformation, and tumor development. Emerging evidences have indicated that alcohol induces deregulation of Brf1 and Pol III genes to cause the alterations of cell phenotypes and tumor formation. In this paper, we summarize the progresses regarding alcohol-caused increase in the expression of Brf1 and Pol III genes and analysis of its molecular mechanism of breast cancer. As the earlier and accurate diagnosis approach of breast cancer is not available yet, exploring the molecular mechanism and identifying the biomarker of alcohol-associated breast cancer are especially important. Recent studies have demonstrated that Brf1 is overexpressed in most ER+ (estrogen receptor positive) cases of breast cancer and the change in cellular levels of Brf1 reflects the therapeutic efficacy and prognosis of this disease. It suggests that Brf1 may be a potential diagnosis biomarker and a therapeutic target of alcohol-associated breast cancer.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Biomarcadores Tumorais/biossíntese , Neoplasias da Mama , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Consumo de Bebidas Alcoólicas/patologia , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , HumanosRESUMO
OBJECTIVE: X-linked dystonia parkinsonism (XDP) is a neurodegenerative movement disorder caused by a single mutation: SINE-VNTR-Alu (SVA) retrotransposon insertion in TAF1. Recently, a (CCCTCT)n repeat within the SVA insertion has been reported as an age-at-onset (AAO) modifier in XDP. Here we investigate the role of this hexanucleotide repeat in modifying expressivity of XDP. METHODS: We genotyped the hexanucleotide repeat in 355 XDP patients and correlated the repeat number (RN) with AAO (n = 295), initial clinical manifestation (n = 294), site of dystonia onset (n = 238), disease severity (n = 28), and cognitive function (n = 15). Furthermore, we investigated i) repeat instability by segregation analysis and Southern blotting using postmortem brain samples from two affected individuals and ii) relative TAF1 expression in blood RNA from 31 XDP patients. RESULTS: RN showed significant inverse correlations with AAO and with TAF1 expression and a positive correlation with disease severity and cognitive dysfunction. Importantly, AAO (and not RN) was directly associated with whether dystonia or parkinsonism will manifest at onset. RN was lower in patients affected by mouth/tongue dystonia compared with blepharospasm. RN was unstable across germline transmissions with an overall tendency to increase in length and exhibited somatic mosaicism in brain. INTERPRETATION: The hexanucleotide repeat within the SVA insertion acts as a genetic modifier of disease expressivity in XDP. RN-dependent TAF1 repression and subsequent differences in TAF1 mRNA levels in patients may be potentiated in the brain through somatic variability leading to the neurological phenotype. ANN NEUROL 2019;85:812-822.
Assuntos
Expansão das Repetições de DNA/genética , Distúrbios Distônicos/diagnóstico , Distúrbios Distônicos/genética , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Histona Acetiltransferases/genética , Sequências Repetitivas de Ácido Nucleico/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Adulto , Distúrbios Distônicos/metabolismo , Feminino , Expressão Gênica , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Histona Acetiltransferases/biossíntese , Humanos , Masculino , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fator de Transcrição TFIID/biossíntese , Adulto JovemRESUMO
RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.
Assuntos
Fator 1 de Resposta a Butirato/deficiência , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Pâncreas/metabolismo , Animais , Fator 1 de Resposta a Butirato/biossíntese , Fator 1 de Resposta a Butirato/genética , Homeostase , Humanos , Camundongos , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fatores Associados à Proteína de Ligação a TATA/genéticaRESUMO
Brain development involves proliferation, migration and specification of neural progenitor cells, culminating in neuronal circuit formation. Mounting evidence indicates that improper regulation of RNA binding proteins (RBPs), including members of the FET (FUS, EWS, TAF15) family, results in defective cortical development and/or neurodegenerative disorders. However, in spite of their physiological relevance, the precise pattern of FET protein expression in developing neurons is largely unknown. Herein, we found that FUS, EWS and TAF15 expression is differentially regulated during brain development, both in time and in space. In particular, our study identifies a fine-tuned regulation of FUS and EWS during neuronal differentiation, whereas TAF15 appears to be more constitutively expressed. Mechanistically FUS and EWS protein expression is regulated at the post-transcriptional level during neuron differentiation and brain development. Moreover, we identified miR-141 as a key regulator of these FET proteins that modulate their expression levels in differentiating neuronal cells. Thus, our studies uncover a novel link between post-transcriptional regulation of FET proteins expression and neurogenesis.
Assuntos
MicroRNAs/metabolismo , Neurônios/fisiologia , Processamento Pós-Transcricional do RNA , Proteína EWS de Ligação a RNA/biossíntese , Proteína FUS de Ligação a RNA/biossíntese , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismoRESUMO
Brf1 (TFIIB-related factor 1) plays a crucial role in cell transformation and tumorigenesis. However, the significance of Brf1 expression in human HCC (hepatocellular carcinoma) cases remains to be addressed. In this study, biopsies of human HCC, liver tumor samples of mice and cell lines of normal and tumor liver were utilized to determine the alteration of Brf1 expression using cytological and molecular biological approaches. Brf1 expression is increased in human HCC cases, which is correlated with shorter survival times. Levels of Brf1 and Pol III (RNA polymerase III-dependent) gene transcription in HCC patients with alcohol consumption are higher than the cases of non-HCC with or without alcohol intake. Induction of Brf1 and Pol III genes by ethanol in hepatoma cells is higher than in non-tumor cells. Ethanol increases the rate of cell transformation. Repression of Brf1 inhibits alcohol-promoted cell transformation. Alcohol consumption enhances Brf1 expression to promote cell transformation. These studies demonstrate that Brf1 is a new biomarker of HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , TransfecçãoRESUMO
Long-term mammalian spermatogenesis requires proper development of spermatogonial stem cells (SSCs) that replenish the testis with germ cell progenitors during adult life. TAF4b is a gonadal-enriched component of the general transcription factor complex, TFIID, which is required for the maintenance of spermatogenesis in the mouse. Successful germ cell transplantation assays into adult TAF4b-deficient host testes suggested that TAF4b performs an essential germ cell autonomous function in SSC establishment and/or maintenance. To elucidate the SSC function of TAF4b, we characterized the initial gonocyte pool and rounds of spermatogenic differentiation in the context of the Taf4b-deficient mouse testis. Here, we demonstrate a significant reduction in the late embryonic gonocyte pool and a deficient expansion of this pool soon after birth. Resulting from this reduction of germ cell progenitors is a developmental delay in meiosis initiation, as compared to age-matched controls. While GFRα1+ spermatogonia are appropriately present as Asingle and Apaired in wild-type testes, TAF4b-deficient testes display an increased proportion of long and clustered chains of GFRα1+ cells. In the absence of TAF4b, seminiferous tubules in the adult testis either lack germ cells altogether or are found to have missing generations of spermatogenic progenitor cells. Together these data indicate that TAF4b-deficient spermatogenic progenitor cells display a tendency for differentiation at the expense of self-renewal and a renewing pool of SSCs fail to establish during the critical window of SSC development.
Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular/fisiologia , Espermatogênese/fisiologia , Espermatogônias/crescimento & desenvolvimento , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fator de Transcrição TFIID/biossíntese , Animais , Animais Recém-Nascidos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Alcohol consumption in women has been associated with an increased risk of breast cancer, particular in estrogen receptor positive (ER+) cases. Deregulation of RNA polymerase III-dependent (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, leading to an increase in translational capacity to promote cell transformation and tumor formation. Our recent studies demonstrated that alcohol induces Brf1 expression and Pol III gene transcription via ER. Here, we report that Tamoxifen (Tam) inhibits the induction of Brf1 and Pol III genes in ER+ breast cancer cells. Further analysis indicates that alcohol increases c-Jun expression to upregulate the transcription of Brf1 and Pol III genes, whereas Tam reduces c-Jun expression to repress the transcription of Brf1. Repression of cJun decreases cellular levels of ERα and Brf1. Alcohol-dependent increased occupancy of Brf1 in Pol III gene promoters is reduced by Tam. The repression of Brf1 and Pol III genes by Tam reduces alcohol-induced cell proliferation and colony formation. Together, these results indicate that Tam inhibits alcohol-induced Brf1 expression through c-Jun and ERα to downregulate Pol III gene transcription. Our studies uncover a new mechanism of Tam-treated ER+ breast cancer, by which Tam inhibits tumor growth through repressing Pol III gene transcription.
Assuntos
Neoplasias da Mama/metabolismo , Etanol/toxicidade , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , RNA Polimerase III/biossíntese , Tamoxifeno/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Estrogênio/biossíntese , Transdução de Sinais/efeitos dos fármacos , Fatores Associados à Proteína de Ligação a TATA/biossíntese , TransfecçãoRESUMO
Cancer testis antigens (CTAs) have normal expression restricted in the testis and also inappropriate expression in various tumor types. Special and favorable characteristics of these genes, as being immunogenic and therefore having the potential to be used as tumor vaccine, have made them as one of the star attractions of the cancer research. Acute myeloid leukemia (AML) is a highly heterogeneous hematological disorder with various reported changes in gene expression. To find new CTA genes in AML, we analyzed the expression pattern of four testis-specific genes AURKC, OIP5, PIWIL2 and TAF7L using real-time quantitative PCR in 51 AMLs and 6 myelodysplastic syndrome cases in comparison with 33 healthy controls. The expression of the studied genes, noticeably OIP5 and TAF7L, differed between studied groups in a gender-dependent manner. Upregulation of OIP5 was observed only in ~41 % of the female AML patients as compared to the healthy females. The remaining ~59 % of the male AML patients, when compared to the healthy males, displayed downregulation of TAF7L. This finding may affect many aspects of AML such as diagnosis, prognosis assessment and treatment choice.
Assuntos
Proteínas Argonautas/biossíntese , Proteínas Cromossômicas não Histona/biossíntese , Leucemia Mieloide Aguda/genética , Proteínas Serina-Treonina Quinases/biossíntese , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fator de Transcrição TFIID/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Argonautas/análise , Proteínas Argonautas/genética , Aurora Quinase C , Aurora Quinases , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/análise , Proteínas Cromossômicas não Histona/genética , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Caracteres Sexuais , Fatores Associados à Proteína de Ligação a TATA/análise , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/análise , Fator de Transcrição TFIID/genética , Transcriptoma , Adulto JovemRESUMO
We isolated two TATA-binding protein-associated factor (TAF) genes, TAF10 and TAF13, from pepper (Capsicum annuum). The complete coding sequences were amplified using reverse transcriptase-PCR on the basis of conserved sequence information of eggplant and several other plant species. Nucleotide sequence analysis of these two genes revealed that the pepper TAF10 gene encodes a protein of 103 amino acids that belongs to the TAF10 superfamily. The pepper TAF10 gene was highly expressed in the pericarp and placenta, moderately expressed in the stems, flowers, seeds and leaves, and weakly expressed in roots. The TAF13 gene was found to encode a protein of 130 amino acids that belongs to the TAF13 superfamily. The TAF13 gene was highly expressed in the stems, flowers and pericarp, moderately expressed in the leaves, placenta and seeds, and weakly expressed in roots.
Assuntos
Capsicum/genética , Regulação da Expressão Gênica de Plantas , Fatores Associados à Proteína de Ligação a TATA/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Flores/metabolismo , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Folhas de Planta/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/metabolismo , Análise de Sequência de DNA , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fatores de Transcrição TFII/genéticaRESUMO
Translocations or mutations of FUS, EWSR1, and TAF15 (FET) result in distinct genetic diseases. N-terminal translocations of any FET protein to a series of transcription factors yields chimeric proteins that contribute to sarcomagenesis, whereas mutations in the conserved COOH-terminal domain of wild-type FUS were recently shown to cause familial amyotrophic lateral sclerosis. We thus investigated whether the loss of one FUS allele by translocation in liposarcoma may be followed by mutations in either the remaining FUS allele or the paralogous EWSR1. Furthermore, we investigated the strength of the FET promoters and their contributions to sarcomagenesis given the proteins' frequent involvement in oncogenic translocations. We sequenced the respective genomic regions of both FUS and EWSR1 in 96 liposarcoma samples. Additionally, we determined FET transcript and protein levels in several liposarcoma cell lines. We did not observe sequence variations in either FUS or EWSR1. However, protein copy numbers reached an impressive 0.9 and 5.5 Mio of FUS and EWSR1 per tumor cell, respectively. Compared with adipose-derived stem cells, FUS and EWSR1 protein expression levels were elevated on average 28.6-fold and 7.3-fold, respectively. TAF15 mRNA levels were elevated on average 3.9-fold, although with a larger variation between samples. Interestingly, elevated TAF15 mRNA levels did not translate to strongly elevated protein levels, consistent with its infrequent occurrence as translocation partner in tumors. These results suggest that the powerful promoters of FET genes are predominantly responsible for the oncogenic effect of transcription factor translocations in sarcomas.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Lipossarcoma/genética , RNA Mensageiro/genética , Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Alelos , Animais , Sequência de Bases , Proteínas de Ligação a Calmodulina/biossíntese , Linhagem Celular Transformada , Linhagem Celular Tumoral , Éxons , Células HEK293 , Humanos , Lipossarcoma/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Proteína EWS de Ligação a RNA , Proteína FUS de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/biossíntese , Spodoptera/genética , Spodoptera/metabolismo , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Transfecção , Translocação Genética , Regulação para CimaRESUMO
Paget's disease (PD) is characterized by abnormal osteoclasts (OCL) that secrete high IL-6 levels and induce exuberant bone formation. Because measles virus nucleocapsid gene (MVNP) and the p62(P392L) mutation are implicated in PD, marrows from 12 PD patients harboring p62(P392L) and eight normals were tested for MVNP expression and pagetic OCL formation. Eight out of twelve patients expressed MVNP and formed pagetic OCL in vitro, which were inhibited by antisense-MVNP. Four out of twelve patients lacked MVNP and formed normal OCL that were hyperresponsive to RANKL but unaffected by antisense-MVNP. Similarly, mice expressing only p62(P394L) formed normal OCL, while mice expressing MVNP in OCL, with or without p62(P394L), developed pagetic OCL and expressed high IL-6 levels dependent on p38MAPK activation. IL-6 deficiency in MVNP mice abrogated pagetic OCL development in vitro. Mice coexpressing MVNP and p62(P394L) developed dramatic Paget's-like bone lesions. These results suggest that p62(P394L) and IL-6 induction by MVNP play key roles in PD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Vírus do Sarampo/genética , Osteíte Deformante/patologia , Osteíte Deformante/virologia , Osteoclastos/patologia , Animais , Células da Medula Óssea/patologia , Osso e Ossos/patologia , Calcitriol/farmacologia , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Interleucina-6/biossíntese , Vírus do Sarampo/patogenicidade , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/biossíntese , Proteínas do Nucleocapsídeo/genética , Osteíte Deformante/genética , Osteoclastos/efeitos dos fármacos , Ligante RANK/farmacologia , Proteína Sequestossoma-1 , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Nucleares/biossíntese , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Polimerase III/metabolismo , Proteínas de Ligação a RNA/biossíntese , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Proteína de Ligação a TATA-Box/metabolismo , Animais , Fator 1 de Resposta a Butirato , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Depressores do Sistema Nervoso Central/efeitos adversos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Etanol/efeitos adversos , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Regulação da Expressão Gênica/genética , Células Hep G2 , Humanos , Cirrose Hepática Alcoólica/genética , Cirrose Hepática Alcoólica/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-jun/genética , RNA Polimerase III/genética , RNA Ribossômico 5S/biossíntese , RNA Ribossômico 5S/genética , RNA de Transferência/biossíntese , RNA de Transferência/genética , Proteínas de Ligação a RNA/genética , Elementos de Resposta/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Proteína de Ligação a TATA-Box/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genéticaRESUMO
Colon cancer is one of the leading causes of cancer deaths in developed countries due to the absence of tumor specific markers for early diagnosis of the disease, providing adequate sensitivity. Search for diagnostic markers of various types of cancer by proteomic approaches has been limited by large differences in protein centration. We used preliminary extraction of major cellular proteins by 0.2 M sodium chloride in presence of nonionic detergent NP-40 in order to raise the sensitivity of the 2D PAGE detection of low-abundant soluble proteins, some of which may penetrate in blood circulation during carcinogenesis. Application of this procedure prior to 2D comparative analysis of proteomes of normal tissues and matched colon cancer specimens led to selection of ten proteins, which are frequently overexpressed in colon adenocarcinomas. Mass-spectrometric identification of selected proteins led to discovery of two novel protein markers of colon tumors--TAF9 and CISH. Low level of CISH expression in various tissues suggests that it is a novel prospective marker for diagnosis of colon cancer.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/biossíntese , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Proteoma/biossíntese , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Feminino , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteoma/genética , Solubilidade , Proteínas Supressoras da Sinalização de Citocina/genética , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/biossíntese , Fator de Transcrição TFIID/genéticaRESUMO
A method is presented that allows efficient production of antimicrobial peptides in bacteria by means of fusion to the histone fold domain of the human transcription factor TAF12. This small fusion partner drives high-level expression of peptides and leads to their accumulation in an entirely insoluble form, thereby eliminating toxicity to the host. Using the antimicrobial peptide LAH4 as an example, we demonstrate that neither affinity purification of the TAF12 fusion protein nor initial solubilization of inclusion bodies in denaturing buffers is required. Instead, crude insoluble material from bacteria is directly dissolved in formic acid for immediate release of the peptide through chemical cleavage at a unique Asp-Pro site. This is followed by purification to homogeneity in a single chromatographic step. Because of the elevated expression levels of the histone fold domain and its small size (8 kDa), this straightforward purification scheme produces yields in excess of 10 mg active peptide per liter of culture. We demonstrate that TAF12 fusion allows expression of a wide range of antimicrobial peptides as well as efficient isotope labeling for NMR studies.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Sequência de Bases , DNA Recombinante/genética , Vetores Genéticos , Humanos , Isótopos , Dados de Sequência Molecular , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/isolamento & purificaçãoRESUMO
Activating mutations in the RAS proto-oncogene result in constant stimulation of its downstream pathways, further leading to tumorigenesis. Transcription factor IID (TFIID) can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. To investigate potential links between the regulation of TFIID function and the RAS-induced carcinogenesis, we monitored the expression of the TATA box-binding protein and its associated factors (TAF) in human colon carcinoma cells. We primarily identified TAF12 levels as being up-regulated in cell lines bearing natural RAS mutations or stably overexpressing a mutated RAS isoform via a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-dependent pathway. We further showed by electrophoretic mobility shift assays and chromatin immunoprecipitation that the ETS1 protein was interacting with an ETS-binding site on the TAF12 promoter and was regulating TAF12 expression. The binding was enhanced in extracts from oncogenic RAS-transformed cells, pointing to a role in the RAS-mediated regulation of TAF12 expression. Reduction of TAF12 levels by small interfering RNA treatment induced a destabilization of the TFIID complex, enhanced E-cadherin mRNA and protein levels, and reduced migration and adhesion properties of RAS-transformed cells with epithelial to mesenchymal transition. Overall, our study indicates the importance of TAF12 in the process of RAS-induced transformation properties of human colon cells and epithelial to mesenchymal transition, most notably those related to increased motility, by regulating specifically expression of genes such as E-cadherin.
Assuntos
Transformação Celular Neoplásica/genética , Neoplasias Colorretais/genética , Proteína Oncogênica p21(ras)/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Sequência de Bases , Sítios de Ligação , Células CACO-2 , Caderinas/biossíntese , Caderinas/genética , Linhagem Celular , Movimento Celular , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Sequência Consenso , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Invasividade Neoplásica , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Proto-Oncogene Mas , Proteína Proto-Oncogênica c-ets-1/metabolismo , RNA Mensageiro/metabolismo , Fatores Associados à Proteína de Ligação a TATA/antagonistas & inibidores , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fator de Transcrição TFIID/metabolismoRESUMO
Cisplatin has been used effectively to treat various human cancer types; yet, the precise mechanism underlying its cytotoxicity remains unknown. In eukaryotes, progression through G1 is monitored by a checkpoint, which executes G1 arrest in the event of DNA damage to allow time for repair before initiating DNA replication. The retinoblastoma tumor suppressor gene is an integral component of the mammalian G1 checkpoint. The utility of the retinoblastoma gene as a therapeutic for human cancers has been investigated. Intriguingly, the cytotoxicity profile of the retinoblastoma gene therapy closely parallels the clinical targets of cisplatin. It prompted an investigation into the potential role of the checkpoint-induced G1 arrest in cisplatin cytotoxicity. Here, the evidence that G1 arrest induction represents a critical step in cisplatin-induced lytic path is presented. First, cisplatin-treated human cancer cells undergo a prolonged G1 arrest before dying. Second, triggering G1 arrest via infection with a recombinant adenovirus expressing the human retinoblastoma gene is sufficient to potentiate lethality in the absence of cisplatin. Third, the extent of the lethality induced correlates with the G1-arresting potential of the ectopically expressed human retinoblastoma polypeptide. Fourth, human cancer cells resistant to cisplatin do not undergo G1 arrest despite cisplatin treatment. The above mechanism may be exploited to develop therapeutics that preserve the efficacy of cisplatin yet bypass its mutagenicity associated with the formation of secondary tumors.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Fase G1/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Adenoviridae/genética , Western Blotting , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Citometria de Fluxo , Vetores Genéticos , Humanos , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fatores Associados à Proteína de Ligação a TATA/genéticaRESUMO
TATA binding protein (TBP) associated factor 1 (TAF1) is a member of the general transcription machinery. Interference in the function of TAF1 causes a broad transcriptional defect in early development. To explore possible roles of TAF1 in embryonic transcriptional silence and zygotic genome activation, we examined the expression of TAF1 in 1-cell mouse embryos. Using an immunofluorescence assay, TAF1 was not detected in embryos in the first few hours after fertilization. TAF1 appeared in pronuclei 6 h post-fertilization and reached a relatively high level before zygotic genome activation. These data show that besides TBP, another critical member of the general transcription machinery such as TAF1 is also absent or at an extremely low level at the outset of development. Combined deficiency in critical members of the general transcription machinery may account for embryonic transcriptional silence.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Zigoto/metabolismo , Animais , Feminino , Histona Acetiltransferases , Masculino , Camundongos , Fatores Associados à Proteína de Ligação a TATA/biossíntese , Fator de Transcrição TFIID/biossínteseRESUMO
The ATF7 proteins, which are members of the cyclic AMP responsive binding protein (CREB)/activating transcription factor (ATF) family of transcription factors, display quite versatile properties: they can interact with the adenovirus E1a oncoprotein, mediating part of its transcriptional activity; they heterodimerize with the Jun, Fos or related transcription factors, likely modulating their DNA-binding specificity; they also recruit to the promoter a stress-induced protein kinase (JNK2). In the present study, we investigate the functional relationships of ATF7 with hsTAF12 (formerly hsTAF(II)20/15), which has originally been identified as a component of the general transcription factor TFIID. We show that overexpression of hsTAF12 potentiates ATF7-induced transcriptional activation through direct interaction with ATF7, suggesting that TAF12 is a functional partner of ATF7. In support of this conclusion, chromatin immunoprecipitation experiments confirm the interaction of ATF7 with TAF12 on an ATF7-responsive promoter, in the absence of any artificial overexpression of both proteins. We also show that the TAF12-dependent transcriptional activation is competitively inhibited by TAF4. Although both TAF12 isoforms (TAF12-1 and -2, formerly TAF(II)20 and TAF(II)15) interact with the ATF7 activation region through their histone-fold domain, only the largest, hsTAF12-1, mediates transcriptional activation through its N-terminal region.