RESUMO
Stress-induced activation of p53 is an essential cellular response to prevent aberrant cell proliferation and cancer development. The ubiquitin ligase MDM2 promotes p53 degradation and limits the duration of p53 activation. It remains unclear, however, how p53 persistently escapes MDM2-mediated negative control for making appropriate cell fate decisions. Here we report that TBP-like protein (TLP), a member of the TBP family, is a new regulatory factor for the p53-MDM2 interplay and thus for p53 activation. We found that TLP acts to stabilize p53 protein to ensure long-lasting p53 activation, leading to potentiation of p53-induced apoptosis and senescence after genotoxic stress. Mechanistically, TLP interferes with MDM2 binding and ubiquitination of p53. Moreover, single cell imaging analysis shows that TLP depletion accelerates MDM2-mediated nuclear export of p53. We further show that a cervical cancer-derived TLP mutant has less p53 binding ability and lacks a proliferation-repressive function. Our findings uncover a role of TLP as a competitive MDM2 blocker, proposing a novel mechanism by which p53 escapes the p53-MDM2 negative feedback loop to modulate cell fate decisions.
Assuntos
Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Células HCT116 , Células HeLa , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Estabilidade Proteica , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/análise , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/análise , Proteína Supressora de Tumor p53/análise , UbiquitinaçãoRESUMO
Although the majority of gene expression is driven by TATA-binding protein (TBP)-based transcription machinery, it has been reported that TBP-related factors (TRFs) are also involved in the regulation of gene expression. TBP-like protein (TLP), which is one of the TRFs and exhibits the highest affinity to TFIIA among known proteins, has recently been showed to have significant roles in gene regulation. However, how the level of TLP is maintained in vivo has remained unknown. In this study, we explored the mechanism by which TLP protein is turned over in vivo and the factor that maintains the amount of TLP. We showed that TLP is rapidly degraded by the ubiquitin-proteasome system and that tight interaction with TFIIA results in protection of TLP from ubiquitin-proteasome-dependent degradation. The half-life of TLP was shown to be less than a few hours, and the proteasome inhibitor MG132 specifically suppressed TLP degradation. Moreover, knockdown and over-expression experiments showed that TFIIA is engaged in stabilization of TLPin vivo. Thus, we showed a novel characteristic of TLP, that is, interaction with TFIIA is essential to suppress proteasome-dependent turnover of TLP, providing a further insight into TLP-governed gene regulation.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIA/metabolismo , Ubiquitina/metabolismo , Animais , Ligação Competitiva , Regulação da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Camundongos , Ligação Proteica , Estabilidade Proteica , Proteólise , Proteína de Ligação a TATA-Box/metabolismoRESUMO
TBP-TFIIA interaction is involved in the potentiation of TATA box-driven promoters. TFIIA activates transcription through stabilization of TATA box-bound TBP. The precursor of TFIIA is subjected to Taspase1-directed processing to generate α and ß subunits. Although this processing has been assumed to be required for the promoter activation function of TFIIA, little is known about how the processing is regulated. In this study, we found that TBP-like protein (TLP), which has the highest affinity to TFIIA among known proteins, affects Taspase1-driven processing of TFIIA. TLP interfered with TFIIA processing in vivo and in vitro, and direct binding of TLP to TFIIA was essential for inhibition of the processing. We also showed that TATA box promoters are specifically potentiated by processed TFIIA. Processed TFIIA, but not unprocessed TFIIA, associated with the TATA box. In a TLP-knocked-down condition, not only the amounts of TATA box-bound TFIIA but also those of chromatin-bound TBP were significantly increased, resulting in the stimulation of TATA box-mediated gene expression. Consequently, we suggest that TLP works as a negative regulator of the TFIIA processing and represses TFIIA-governed and TATA-dependent gene expression through preventing TFIIA maturation.
Assuntos
Endopeptidases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , TATA Box , Fator de Transcrição TFIIA/metabolismo , Ativação Transcricional , Linhagem Celular , Cromatina/metabolismo , Células HeLa , Humanos , Proteína de Ligação a TATA-Box/metabolismoRESUMO
TBP-like protein (TLP) is one of the metazoan-restricted transcription factors participating in development and differentiation, though the molecular mechanism by which TLP regulates these processes remains unclear. In this study, we investigated the relationship between TLP and myogenesis of mouse C2C12 myoblasts. We found that TLP gene expression decreases during myogenic differentiation. Overexpression and knockdown of TLP revealed that the levels of muscle-specific myosin heavy chain and the myogenic transcription factor myogenin are downregulated by TLP. TLP inhibits the progression of morphological change from myoblasts to myotubes, thereby suppressing myogenesis. We further show that TLP represses the promoter activity of myogenin. The proximal AT-rich sequence of the myogenin promoter is responsible for TLP-mediated transcriptional repression. The results of this study suggest that TLP inhibits myogenesis through downregulation of the myogenin gene.
Assuntos
Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Miogenina/genética , Regiões Promotoras Genéticas , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/genética , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Regulação para Baixo , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , RNA Interferente Pequeno/genética , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/antagonistas & inibidoresRESUMO
TATA-binding protein-like protein (TLP) is involved in development, checkpoint, and apoptosis through potentiation of gene expression. TLP-overexpressing human cells, especially p53-containing cells, exhibited a decreased growth rate and increased proportion of G(1) phase cells. TLP stimulated expression of several growth-related genes including p21 (p21(Waf1/Cip1)). TLP-mediated activation of the p21 upstream promoter in cells was shown by a promoter-luciferase reporter assay. The p53-binding sequence located in the p21 upstream promoter and p53 itself are required for TLP-mediated transcriptional activation. TLP and p53 bound to each other and synergistically enhanced activity of the upstream promoter. TLP specifically activated transcription from the endogenous upstream promoter, and p53 was required for this activation. Etoposide treatment also resulted in activation of the upstream promoter as well as nuclear accumulation of TLP and p53. Moreover, the upstream promoter was associated with endogenous p53 and TLP, and the p53 recruitment was enhanced by TLP. The results of the present study suggest that TLP mediates p53-governed transcriptional activation of the p21 upstream promoter.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Elementos de Resposta/fisiologia , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Transcrição Gênica/fisiologia , Ativação Transcricional/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Células COS , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p21/genética , Etoposídeo/farmacologia , Fase G1/efeitos dos fármacos , Fase G1/fisiologia , Células HeLa , Células Hep G2 , Humanos , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
Accumulating evidence indicates that TBP (TATA-binding protein)-like protein (TLP) contributes to the regulation of stress-mediated cell cycle checkpoint and apoptotic pathways, although its physiological target genes have remained elusive. In the present study, we have demonstrated that human TAp63 is one of the direct transcriptional target genes of TLP. Enforced expression of TLP results in the transcriptional induction of the endogenous TAp63, but not of the other p53 family members such as TAp73 and p53. Consistent with these results, small interference RNA-mediated knockdown led to a significant down-regulation of the endogenous TAp63. Luciferase reporter assay and chromatin immunoprecipitation analysis revealed that the genomic region located at positions -487 to -29, where +1 represents the transcriptional initiation site of TAp63, is required for TLP-dependent transcriptional activation of TAp63 and also TLP is efficiently recruited onto this region. Additionally, cells treated with anti-cancer drug etoposide underwent apoptosis in association with the transcriptional enhancement of TAp63 in a p53-independent manner, and the knockdown of the endogenous TLP reduced etoposide-induced apoptosis through repression of TAp63 expression. Taken together, our present study identifies a TLP-TAp63 pathway that is further implicated in stress-induced apoptosis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Transativadores/biossíntese , Transcrição Gênica/efeitos dos fármacos , Proteínas Supressoras de Tumor/biossíntese , Animais , Apoptose/genética , Galinhas , Técnicas de Silenciamento de Genes , Células HeLa , Células Hep G2 , Humanos , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/genética , Transativadores/genética , Fatores de Transcrição , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Transcription and DNA damage repair act in a coordinated manner. Recent studies have shown that double-strand DNA breaks (DSBs) are repaired in a transcription-coupled manner. Active transcription results in a faster recruitment of DSB repair factors and expedites DNA repair. On the other hand, transcription is repressed by DNA damage through multiple mechanisms. We previously reported that TLP, a TATA box-binding protein (TBP) family member that functions as a transcriptional regulator, is also involved in DNA damage-induced apoptosis. However, the mechanism by which TLP affects DNA damage response was largely unknown. Here we show that TLP-mediated global transcriptional repression after DSBs is crucial for apoptosis induction by DNA-damaging agents such as etoposide and doxorubicin. Compared to control cells, TLP-knockdown cells were resistant to etoposide-induced apoptosis and exhibited an elevated level of global transcription after etoposide exposure. DSBs were efficiently removed in transcriptionally hyperactive TLP-knockdown cells. However, forced transcriptional shutdown using transcriptional inhibitors α-amanitin and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) slowed down DSB repair and resensitized TLP-knockdown cells to etoposide. Taken together, these results indicate that TLP is a critical determinant as to how cells respond to DSBs and triggers apoptosis to cells that have sustained DNA damage.
Assuntos
Apoptose/genética , Proteínas Relacionadas à Autofagia/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Proteínas de Transporte Vesicular/genética , Alfa-Amanitina/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Diclororribofuranosilbenzimidazol/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Transcrição Gênica/genética , Proteínas de Transporte Vesicular/antagonistas & inibidoresRESUMO
Transcription factor IIA (TFIIA) is one of the general transcription factors for RNA polymerase II and composed of three subunits, TFIIAalpha, TFIIAbeta and TFIIAgamma. TFIIAalpha and TFIIAbeta are encoded by a single gene (TFIIAalphabeta) and mature through internal cleavage of TFIIAalphabeta. In this study, we found that structures of TFIIAalphabeta and TFIIAgamma are highly homologous with each mammalian counterpart. Exon-intron organizations of the human and chicken TFIIA genes were also homologous. The sequence of the cleavage region of the chicken TFIIAalphabeta precursor protein was fitted to the consensus cleavage recognition site. It was thus demonstrated that TFIIA is conserved in vertebrates. TFIIA proteins are present ubiquitously in chicken tissues. Fluorescent in situ hybridization revealed that TFIIAalphabeta and TFIIAgamma genes are located in chromosome 5 and a mini-chromosome, respectively. We generated semi-knockout chicken DT40 cells for TFIIAalphabeta and TFIIAgamma genes with high homologous recombination efficiencies, whereas we failed to establish double-knockout cells for each gene. It is thought that both genes for TFIIA are required in vertebrates. TFIIA siRNA resulted in deceleration of cell growth rate, suggesting that, consistent with those of knockout assays, TFIIA is associated with cell growth regulation.
Assuntos
Galinhas/genética , Fator de Transcrição TFIIA/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas/metabolismo , Mapeamento Cromossômico , Éxons , Feminino , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Íntrons , Masculino , Dados de Sequência Molecular , Interferência de RNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Distribuição Tecidual , Fator de Transcrição TFIIA/antagonistas & inibidores , Fator de Transcrição TFIIA/metabolismoRESUMO
The TATA-binding protein (TBP) is a universal transcription factor required for all of the eukaryotic RNA polymerases. In addition to TBP, metazoans commonly express a distantly TBP-related protein referred to as TBP-like protein (TLP/TRF2/TLF). Although the function of TLP in transcriptional regulation is not clear, it is known that TLP is required for embryogenesis and spermiogenesis. In the present study, we investigated the cellular functions of TLP by using TLP knockout chicken DT40 cells. TLP was found to be dispensable for cell growth. Unexpectedly, TLP-null cells exhibited a 20% elevated cell cycle progression rate that was attributed to shortening of the G(2) phase. This indicates that TLP functions as a negative regulator of cell growth. Moreover, we found that TLP mainly existed in the cytoplasm and was translocated to the nucleus restrictedly at the G(2) phase. Ectopic expression of nuclear localization signal-carrying TLP resulted in an increase (1.5-fold) in the proportion of cells remaining in the G(2)/M phase and apoptotic state. Notably, TLP-null cells showed an insufficient G(2) checkpoint when the cells were exposed to stresses such as UV light and methyl methanesulfonate, and the population of apoptotic cells after stresses decreased to 40%. These phenomena in G(2) checkpoint regulation are suggested to be p53 independent because p53 does not function in DT40 cells. Moreover, TLP was transiently translocated to the nucleus shortly (15 min) after stress treatment. The expression of several stress response and cell cycle regulatory genes drifted in a both TLP- and stress-dependent manner. Nucleus-translocating TLP is therefore thought to work by checking cell integrity through its transcription regulatory ability. TLP is considered to be a signal-transducing transcription factor in cell cycle regulation and stress response.
Assuntos
Ciclo Celular , Fase G2 , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Apoptose , Northern Blotting , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Separação Celular , Galinhas , Citoplasma/metabolismo , Citometria de Fluxo , Imuno-Histoquímica , Sinais de Localização Nuclear , Plasmídeos/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Raios UltravioletaRESUMO
TBP (TATA-binding protein)-related factor 2 (TRF2) regulates transcription during a nuber of cellular processes. We previously demonstrated that it is localized in the cytoplasm and is translocated to the nucleus by DNA-damaging agents. However, the cytoplasmic localization of TRF2 is controversial. In this study, we reconfirmed its cytoplasmic localization in various ways and examined its nuclear migration. Stresses such as heat shock, redox agents, heavy metals, and osmotic shock did not affect localization whereas genotoxins such as methyl methanesulfonate (MMS), cisplatin, etoposide, and hydroxyurea caused it to migrate to the nucleus. Adriamycin, mitomycin C and gamma-rays had no obvious effect. We determined optimal conditions for the nuclear migration. The proportions of cells with nuclei enriched for TRF2 were 25-60% and 5-10% for stressed cells and control cells, respectively. Nuclear translocation was observed after 1 h, 4 h and 12 h for cisplatin, etoposide and MMS and hydroxyurea, respectively. The association of TRF2 with the chromatin and promoter region of the proliferating cell nuclear antigen (PCNA) gene, a putative target of TRF2, was increased by MMS treatment. Thus TRF2 may be involved in genotoxin-induced transcriptional regulation.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/química , Dano ao DNA , Mutagênicos/farmacologia , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/análise , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Células COS , Chlorocebus aethiops , Cromatina/química , Cisplatino/farmacologia , Células HeLa , Histonas/análise , Humanos , Metanossulfonato de Metila/farmacologia , Camundongos , Células NIH 3T3 , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transcrição GênicaRESUMO
Microarrays have been extensively used to investigate genome-wide expression patterns. Although this technology has been tremendously successful, several practical issues would benefit from improvements in design. Here we describe a novel, efficient labeling methodology that uses carbodiimide-linked cyanine dyes to directly chemically label cDNA derived from mouse total RNA. Using this protocol, it takes only 10 min at 70 degrees C to complete the cDNA labeling reaction. The directly labeled cDNAs can then be hybridized to 70-mer mouse oligonucleotide arrays for expression profiling studies. Microarray analyses indicate that these cDNAs are uniformly labeled and produce higher signal intensities than conventional enzymatic direct labeling methods and comparable signal intensities to those obtained by conventional indirect labeling methods. Furthermore, verification of our microarray data using a reverse transcription PCR (RT-PCR) method indicates good agreement between the two methods. Thus, we conclude that our simplified cyanine-carbodiimide labeling method, which does not rely on the incorporation of modified nucleotides, will provide a reliable, quicker, and potentially cheaper alternative to established labeling techniques for gene expression analyses.
Assuntos
Carbocianinas , Carbodi-Imidas , DNA Complementar/química , DNA Complementar/genética , Corantes Fluorescentes , Hibridização in Situ Fluorescente/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Coloração e Rotulagem/métodosRESUMO
The cullin-containing E3 ubiquitin ligases play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. We recently identified TIP120A as a cullin-interacting protein and found that TIP120A functions as a negative regulator of a ubiquitin ligase by interfering with the binding of Skp1 and an F box protein to CUL1. Here we show that TIP120A binds to the unneddylated CUL1 but not the neddylated one. The association of TIP120A with CUL1 requires both the N-terminal stalk and the C-terminal globular domain of CUL1. TIP120A efficiently inhibits neddylation of CUL1 but does not affect substrate-independent ubiquitination by CUL1/Rbx1, implying that it blocks the access of Nedd8 to the conjugation site but does not interfere with the interaction of the ubiquitin-conjugating enzyme with Rbx1. Our data suggest that the association/dissociation of TIP120A coupled to neddylation/deneddylation of CUL1 may play an important role in assembly and disassembly of Skp1-Cdc53/cullin-F box ubiquitin ligases.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina , Fatores de Transcrição , Ubiquitinas/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Células HeLa , Humanos , Ligases/metabolismo , Substâncias Macromoleculares , Proteína NEDD8 , Ligação Proteica , Estrutura Terciária de Proteína , Ubiquitina-Proteína LigasesRESUMO
Various transcriptional activators are induced in neurons concomitantly with long-lasting neural activity, whereas only a few transcription factors are known to act as neural activity-inducible transcription repressors. In this study, mRNA of DREAM (DRE-antagonizing modulator), a Ca(2+)-modulated transcriptional repressor, was demonstrated to accumulate in the mouse brain after pentylenetetrazol (PTZ)-induced seizures. Accumulation in the mouse hippocampus reached maximal level in the late phase (at 7-8 h) after PTZ injection. Kainic acid induced the same response. Interestingly, the late induction of DREAM expression required new protein synthesis and was blocked by MK801 suggesting that Ca(2+)-influx via NMDA receptors is necessary for the PTZ-mediated DREAM expression. In situ hybridization revealed that PTZ-induced DREAM mRNA accumulation was observed particularly in the dentate gyrus, cerebral cortex, and piriform cortex. The results of the present study demonstrate that DREAM is a neural activity-stimulated late gene and suggest its involvement in adaptation to long-lasting neuronal activity.
Assuntos
Proteínas de Ligação ao Cálcio , Regulação da Expressão Gênica , Hipocampo/fisiologia , Neurônios/metabolismo , Proteínas Repressoras/genética , Convulsões/metabolismo , Animais , Convulsivantes/farmacologia , Cicloeximida/farmacologia , Maleato de Dizocilpina/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hibridização In Situ , Ácido Caínico/farmacologia , Proteínas Interatuantes com Canais de Kv , Masculino , Camundongos , Fármacos Neuroprotetores/farmacologia , Pentilenotetrazol/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Convulsões/induzido quimicamente , Fatores de TempoRESUMO
We investigated the cis-element for cell-specific expression in the mouse myelin basic protein (MBP) promoter by transfection assay in comparison with that of the glial fibrillary acidic protein (GFAP) promoter. The MBP promoter region including the 1.3 kb upstream region from the transcription start point was found to be highly expressed in NG108-15 cells, a nervous tissue-derived hybrid cell line, but not in fibroblasts. In contrast, the GFAP promoter region including the region 2.6 kb upstream of the transcription start point was found to function dominantly in C6 glioma cells, but not in NG108-15 cells. Deletion analysis revealed that the NFl motif in the MBP promoter (named MBTE) resulted in the loss of preferential expression in NG108-15 cells. The GFAP promoter also possessed another NFI motif (named GFII) and its distance from the transcription start point was very similar to that of the MBP promoter. Base-substitution mutation of GFII to MBTE in the GFAP promoter caused remarkable increase in GFAP promoter activity in NG108-15 cells but only slight increase in it activity in C6 cells. These results suggest that the NFI motif in the MBP promoter (MBTE) functions as a cis-acting promoter element that governs cell-specific transcription.
RESUMO
A new member of the Y-box protein family of the silkworm Bombyx mori (BYB) was co-purified with the fibroin gene enhancer-binding protein FMBP-1, and stimulated the binding of FMBP-1 to its cognate DNA element. However, the stimulatory effect was not specific to FMBP-1, BYB also enhancing the binding of mammalian transcription factors OTF2, SP1 and AP2 to their specific binding elements. Besides the above transcription regulatory factors, BYB facilitated the binding of basal transcription factor TBP, and enhanced transcription from the adenovirus 2 major late promoter in a reconstituted transcription system. Moreover, BYB stimulated the reactions of some restriction endonucleases under cold conditions. The C-terminal region of BYB was sufficient for these stimulatory effects, and the highly conserved cold shock domain (CSD) in the N-terminal region was dispensable. GST-pull down experiments showed that the C-terminal region could interact with DNA independently of the CSD. The above results suggest that the C-terminal region of BYB causes the active interaction of various DNA binding proteins with their targets. Such a function of the C-terminal region of BYB may partly explain the functional diversity of Y-box proteins.
Assuntos
Bombyx/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Insetos/metabolismo , Sequência de Aminoácidos , Animais , Bombyx/anatomia & histologia , Temperatura Baixa , Proteínas de Ligação a DNA/genética , Humanos , Proteínas de Insetos/genética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/metabolismoRESUMO
We identified a 45-kDa protein by 2D electrophoresis that was enhanced following pentylenetetrazol (PTZ)-mediated seizures. Mass-spectrography of this protein revealed the beta subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIbeta), although no evidence for increase in bulk CaMKIIbeta transcripts was obtained. Physicochemical parameters of the 45-kDa species coincided with those of the type 7 isoform of CaMKIIbeta, CaMKIIbeta7. Reverse transcription-polymerase chain reaction revealed the existence of the CaMKIIbeta7 transcript in the mouse brain, but its RNA content was small and was not elevated by PTZ injection. CaMKIIbeta7 protein is thought to be accumulated in the nuclei of brain cells by PTZ-mediated seizure via some cellular mechanisms other than transcriptional and post-transcriptional regulation.
Assuntos
Química Encefálica/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Núcleo Celular/metabolismo , Convulsões/enzimologia , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Convulsivantes/administração & dosagem , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Pentilenotetrazol/administração & dosagem , Proteoma/análise , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/induzido quimicamenteRESUMO
Gene expression changes in accordance with cell growth, differentiation and carcinogenesis. To elucidate the molecular mechanisms for hepatocarcinogenesis as well as maintenance of normal hepatocytes, it is important to identify the genes that have altered expression with carcinogenesis. We established a new and efficient cDNA subtraction method via two cDNA populations. By using this method along with rat hepatomas made by the Soh-Farber protocol, we identified a number of genes, some of which are activated in hepatocellular carcinoma (HCC). These genes include ones which code for a transcription factor and a metabolic enzyme. One particular gene can be used as a tumour marker. Our method is beneficial for the isolation of a wide range of HCC-related genes in rats which, in turn, enables easy identification of their human counterparts. In this review, we describe details of our method and the isolated genes. We also briefly describe transcription factors in the liver.
RESUMO
TATA-binding protein-like protein (TLP) binds to transcription factor IIA (TFIIA) with high affinity, although the significance of this binding is poorly understood. In this study, we investigated the role of TFIIA in transcriptional regulation of the p21(Waf1/Cip1) (p21) gene. It has been shown that TLP is indispensable for p53-activated transcription from an upstream TATA-less promoter of the p21 gene. We found that mutant TLPs having decreased TFIIA-binding ability exhibited weakened transcriptional activation function for the upstream promoter. Activity of the upstream promoter was enhanced considerably by an increased amount of TFIIA in a p53-dependent manner, whereas activity of the TATA-containing downstream promoter was enhanced only slightly. TFIIA potentiated the upstream promoter additively with TLP. Although TFIIA is recruited to both promoters, activity of the upstream promoter was much more dependent on TFIIA. Recruitment of TFIIA and TLP to the upstream promoter was augmented in etoposide-treated cells, in which the amount of TFIIA-TLP complex is increased, and TFIIA-reactive TLP was required for the recruitment of both factors. It was confirmed that etoposide-stimulated transcription depends on TLP. We also found that TFIIA-reactive TLP acts to decrease cell growth rate, which can be explained by interaction of the p21 promoter with the transcription factors that we examined. The results of the present study suggest that the upstream TATA-less promoter of p21 needs TFIIA and TFIIA-reactive TLP for p53-dependent transcriptional enhancement.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Fator de Transcrição TFIIA/metabolismo , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Etoposídeo/farmacologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Regiões Promotoras Genéticas/fisiologia , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIA/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Ativação TranscricionalRESUMO
TBP-like protein (TLP) is involved in transcriptional activation of an upstream promoter of the human p21 gene. TLP binds to p53 and facilitates p53-activated transcription from the upstream promoter. In this study, we clarified that in vitro affinity between TLP and p53 is about one-third of that between TBP and p53. Extensive mutation analyses revealed that the TLP-stimulated function resides in transcription activating domain 1 (TAD1) in the N-terminus of p53. Among the mutants, #22.23, which has two amino acid substitutions in TAD1, exhibited a typical mutant phenotype. Moreover, #22.23 exhibited the strongest mutant phenotype for TLP-binding ability. It is thus thought that TLP-stimulated and p53-dependent transcriptional activation is involved in TAD1 binding of TLP. #22.23 had a decreased transcriptional activation function, especially for the upstream promoter of the endogenous p21 gene, compared with wild-type p53. This mutant did not facilitate p53-dependent growth repression and etoposide-mediated cell-death as wild-type p53 does. Moreover, mutation analysis revealed that middle part of TLP, which is requited for p53 binding, is involved in TLP-stimulated and p53-dependent promoter activation and cell growth repression. These results suggest that activation of the p21 upstream promoter is mediated by interaction between specific regions of TLP and p53.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regiões Promotoras Genéticas , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Transcrição Gênica , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Mutação , Ligação Proteica , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/genética , Proteína Supressora de Tumor p53/químicaRESUMO
Wee1 protein kinase represses CDK1 required for G(2)/M transition. The mouse wee1 promoter contains multiple CACCC-boxes between -306 and +1 that can bind to Krüppel-like factor 3 (KLF3) transcriptional repressor. We found that increasing amounts of intracellular KLF3 decreased the amount of wee1 mRNA. A promoter reporter assay demonstrated that wee1 promoter activity was repressed by KLF3 overexpression. Elimination of the first and fourth CACCC-boxes suppressed KLF3-governed transcriptional repression. A gel-shift assay demonstrated that KLF3 binds to the first, third, and fourth CACCC-boxes with various strengths. Moreover, KLF3 was suggested to interact with the wee1 regulatory region in a physiological condition. Therefore, we concluded that KLF3 is a transcriptional repressor for wee1 gene. In a previous study, we demonstrated that TBP-like protein (TLP) inhibits wee1 promoter function. In this study, we found that the chromosomal wee1 gene is also down-regulated by KLF3. Since KLF3-repressed wee1 promoter function was further inhibited by TLP overexpression regardless of the inhibition degree of KLF3, we propose that TLP and KLF3 repress wee1 promoter independently.