RESUMO
Rhabdoid tumors (RT) are rare and highly aggressive pediatric neoplasms. Their epigenetically-driven intertumoral heterogeneity is well described; however, the cellular origin of RT remains an enigma. Here, we establish and characterize different genetically engineered mouse models driven under the control of distinct promoters and being active in early progenitor cell types with diverse embryonic onsets. From all models only Sox2-positive progenitor cells give rise to murine RT. Using single-cell analyses, we identify distinct cells of origin for the SHH and MYC subgroups of RT, rooting in early stages of embryogenesis. Intra- and extracranial MYC tumors harbor common genetic programs and potentially originate from fetal primordial germ cells (PGCs). Using PGC specific Smarcb1 knockout mouse models we validate that MYC RT originate from these progenitor cells. We uncover an epigenetic imbalance in MYC tumors compared to PGCs being sustained by epigenetically-driven subpopulations. Importantly, treatments with the DNA demethylating agent decitabine successfully impair tumor growth in vitro and in vivo. In summary, our work sheds light on the origin of RT and supports the clinical relevance of DNA methyltransferase inhibitors against this disease.
Assuntos
Tumor Rabdoide , Animais , Células Germinativas/patologia , Humanos , Camundongos , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Proteína SMARCB1/genética , Análise de Célula Única , TranscriptomaRESUMO
Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancreatic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability upon extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed striking differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines.
Assuntos
Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , NF-kappa B/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and therapy resistance. Here, we show that low expression of κB-Ras GTPases is frequently detected in PDAC and correlates with higher histologic grade. In a model of KRasG12D-driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival. κB-Ras deficiency promotes acinar-to-ductal metaplasia (ADM) during tumour initiation as well as tumour progression through intrinsic effects on proliferation and invasion. κB-Ras proteins are also required for acinar regeneration after pancreatitis, demonstrating a general role in control of plasticity. Molecularly, upregulation of Ral GTPase activity and Sox9 expression underlies the observed phenotypes, identifying a previously unrecognized function of Ral signalling in ADM. Our results provide evidence for a tumour suppressive role of κB-Ras proteins and highlight low κB-Ras levels and consequent loss of Ral control as risk factors, thus emphasizing the necessity for therapeutic options that allow interference with Ral-driven signalling.
Assuntos
Células Acinares/metabolismo , Carcinoma Ductal Pancreático/genética , GTP Fosfo-Hidrolases/genética , Neoplasias Pancreáticas/genética , Pancreatite/genética , Proteínas/genética , Células Acinares/patologia , Idoso , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Estimativa de Kaplan-Meier , Masculino , Metaplasia/genética , Metaplasia/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/metabolismo , Proteínas/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
The TATA-box Binding Protein (TBP) plays a central role in regulating gene expression and is the first step in the process of pre-initiation complex (PIC) formation on promoter DNA. The lifetime of TBP at the promoter site is controlled by several cofactors including the Modifier of transcription 1 (Mot1), an essential TBP-associated ATPase. Based on ensemble measurements, Mot1 can use adenosine triphosphate (ATP) hydrolysis to displace TBP from DNA and various models for how this activity is coupled to transcriptional regulation have been proposed. However, the underlying molecular mechanism of Mot1 action is not well understood. In this work, the interaction of Mot1 with the DNA/TBP complex was investigated by single-pair Förster resonance energy transfer (spFRET). Upon Mot1 binding to the DNA/TBP complex, a transition in the DNA/TBP conformation was observed. Hydrolysis of ATP by Mot1 led to a conformational change but was not sufficient to efficiently disrupt the complex. SpFRET measurements of dual-labeled DNA suggest that Mot1's ATPase activity primes incorrectly oriented TBP for dissociation from DNA and additional Mot1 in solution is necessary for TBP unbinding. These findings provide a framework for understanding how the efficiency of Mot1's catalytic activity is tuned to establish a dynamic pool of TBP without interfering with stable and functional TBP-containing complexes.
Assuntos
Adenosina Trifosfatases/fisiologia , DNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores Associados à Proteína de Ligação a TATA/fisiologia , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Catálise , DNA Fúngico/química , Escherichia coli , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismoRESUMO
Single-pair Förster resonance energy transfer (spFRET) has become an important tool for investigating conformational dynamics in biological systems. To extract dynamic information from the spFRET traces measured with total internal reflection fluorescence microscopy, we extended the hidden Markov model (HMM) approach. In our extended HMM analysis, we incorporated the photon-shot noise from camera-based systems into the HMM. Thus, the variance in Förster resonance energy transfer (FRET) efficiency of the various states, which is typically a fitted parameter, is explicitly included in the analysis estimated from the number of detected photons. It is also possible to include an additional broadening of the FRET state, which would then only reflect the inherent flexibility of the dynamic biological systems. This approach is useful when comparing the dynamics of individual molecules for which the total intensities vary significantly. We used spFRET with the extended HMM analysis to investigate the dynamics of TATA-box-binding protein (TBP) on promoter DNA in the presence of negative cofactor 2 (NC2). We compared the dynamics of two promoters as well as DNAs of different length and labeling location. For the adenovirus major late promoter, four FRET states were observed; three states correspond to different conformations of the DNA in the TBP-DNA-NC2 complex and a four-state model in which the complex has shifted along the DNA. The HMM analysis revealed that the states are connected via a linear, four-well model. For the H2B promoter, more complex dynamics were observed. By clustering the FRET states detected with the HMM analysis, we could compare the general dynamics observed for the two promoter sequences. We observed that the dynamics from a stretched DNA conformation to a bent conformation for the two promoters were similar, whereas the bent conformation of the TBP-DNA-NC2 complex for the H2B promoter is approximately three times more stable than for the adenovirus major late promoter.
Assuntos
DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cadeias de Markov , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Transcrição/metabolismo , DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Proteína de Ligação a TATA-Box/química , Fatores de Transcrição/químicaRESUMO
Rhabdoid tumors are caused by the deletion of SMARCB1, whose protein encodes the SMARCB1 subunit of the chromatin remodeling complex SWI/SNF that is involved in global chromatin organization and gene expression control. Simultaneously inhibiting the main players involved in the deregulated transcription machinery is a promising option for preventing exaggerated tumor cell proliferation and survival as it may bypass compensatory mechanisms. In support of this hypothesis, we report efficient impairment of cellular proliferation and strong induction of cell death elicited by inhibition of bromodomain protein BRD4 and transcription kinase CDK9 using small molecular compounds. Combination of both compounds efficiently represses antiapoptotic genes and the oncogene MYC. Our results provide a novel approach for the treatment of RT.
RESUMO
CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed-with significant delay-by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Fatores de Transcrição , Ativação Transcricional , Proteína Supressora de Tumor p53/genéticaRESUMO
Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Triazinas/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Neoplasias/genética , Fosforilação , Roscovitina , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Rhabdoid tumors are highly aggressive tumors occurring in infants and very young children. Despite multimodal and intensive therapy prognosis remains poor. Molecular analyses have uncovered several deregulated pathways, among them the CDK4/6-Rb-, the WNT- and the Sonic hedgehog (SHH) pathways. The SHH pathway is activated in rhabdoid tumors by GLI1 overexpression. Here, we demonstrate that arsenic trioxide (ATO) inhibits tumor cell growth of malignant rhabdoid tumors in vitro and in a mouse xenograft model by suppressing Gli1. Our data uncover ATO as a promising therapeutic approach to improve prognosis for rhabdoid tumor patients.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Óxidos/farmacologia , Tumor Rabdoide/tratamento farmacológico , Fatores de Transcrição/metabolismo , Animais , Apoptose , Trióxido de Arsênio , Ciclo Celular , Proliferação de Células , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Prognóstico , Transdução de Sinais , Proteína GLI1 em Dedos de ZincoRESUMO
BACKGROUND: Rhabdoid tumors are highly aggressive malignancies affecting infants and very young children. In many instances these tumors are resistant to conventional type chemotherapy necessitating alternative approaches. METHODS: Proliferation assays (MTT), apoptosis (propidium iodide/annexin V) and cell cycle analysis (DAPI), RNA expression microarrays and western blots were used to identify synergism of the HDAC (histone deacetylase) inhibitor SAHA with fenretinide, tamoxifen and doxorubicin in rhabdoidtumor cell lines. RESULTS: HDAC1 and HDAC2 are overexpressed in primary rhabdoid tumors and rhabdoid tumor cell lines. Targeting HDACs in rhabdoid tumors induces cell cycle arrest and apoptosis. On the other hand HDAC inhibition induces deregulated gene programs (MYCC-, RB program and the stem cell program) in rhabdoid tumors. These programs are in general associated with cell cycle progression. Targeting these activated pro-proliferative genes by combined approaches of HDAC-inhibitors plus fenretinide, which inhibits cyclinD1, exhibit strong synergistic effects on induction of apoptosis. Furthermore, HDAC inhibition sensitizes rhabdoid tumor cell lines to cell death induced by chemotherapy. CONCLUSION: Our data demonstrate that HDAC inhibitor treatment in combination with fenretinide or conventional chemotherapy is a promising tool for the treatment of chemoresistant rhabdoid tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Histona Desacetilases/administração & dosagem , Histona Desacetilases/metabolismo , Tumor Rabdoide/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Sinergismo Farmacológico , Fenretinida/administração & dosagem , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Tumor Rabdoide/patologia , VorinostatRESUMO
In cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV), the activation of mitogen-activated protein kinase (MAPK) pathways plays a crucial role early after virus infection as well as during reactivation. In order to systematically identify viral proteins activating MAPK pathways in KSHV-infected cells, a clone collection of KSHV open reading frames (ORFs) was screened for induction of the serum response element (SRE), as SRE is induced by MAPKs. The strongest induction of the SRE was found with ORF73 (latency-associated nuclear antigen 1, or Lana-1), although weaker activation was also found with the kaposin B isoform, ORF54 (dUTPase) and ORF74 (G-protein-coupled receptor). The bipartite SRE is bound by a ternary complex consisting of serum response factor (SRF) and ternary complex factor. Lana-1 bound directly to SRF, but also to the MED25 (ARC92/ACID-1), MED15 (PCQAP) and MED23 (Sur-2) subunits of the Mediator complex, a multi-subunit transcriptional co-activator complex for RNA polymerase II. Lana-1-induced SRE activation was inhibited by the dominant-negative N-terminal domain of the MED25 mediator subunit, suggesting that this subunit mediates Lana-1-induced SRE activation. In summary, these data suggest a model in which Lana-1 acts as an adaptor between the transcription factor SRF and the basal transcriptional machinery.
Assuntos
Antígenos Virais/fisiologia , Herpesvirus Humano 8/fisiologia , Complexo Mediador/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Nucleares/fisiologia , Elemento de Resposta Sérica , Linhagem Celular , Genes Reporter , Humanos , Luciferases/metabolismo , Modelos Biológicos , Ligação Proteica , Ativação TranscricionalRESUMO
RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5' and 3' regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.
Assuntos
Regulação da Expressão Gênica , RNA Polimerase II/metabolismo , Serina/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Anticorpos Monoclonais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Epitopos , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Humanos , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Fosforilação , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Subunidades Proteicas/metabolismo , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/imunologiaRESUMO
Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters in vitro. Its association with TBP prevents the binding of TFIIB and leads to inhibition of preinitiation complex formation. Here, we investigate the association of NC2 subunit-alpha with human RNA polymerase II promoter regions by using gene-specific ChIP and genome-wide promoter ChIPchip analyses. We find NC2alpha associated with a large number of human promoters, where it peaks close to the core regions. NC2 occupancy in vivo positively correlates with mRNA levels, which perhaps reflects its capacity to stabilize TBP on promoter regions. In single gene analyses, we confirm core promoter binding and in addition map the NC2 complex to enhancer proximal regions. High-occupancy histone genes display a stable NC2/TFIIB ratio during the cell cycle, which otherwise varies markedly from one gene to another. The latter is at least in part explained by an observed negative correlation of NC2 occupancy with the presence of the TFIIB recognition element in core promoter regions. Our data establish the genome-wide basis for general and gene-specific functions of NC2 in mammalian cells.
Assuntos
Genoma Humano , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Linfócitos B/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transformação Celular Viral , Imunoprecipitação da Cromatina , Expressão Gênica , Humanos , Células Jurkat , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/análise , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIB/antagonistas & inibidores , Fator de Transcrição TFIIB/genéticaRESUMO
Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2.TBP with DNA. We show that NC2.TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP.TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2.TBP displays a K(D) for DNA of approximately 2 x 10(-9) m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP.DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence.
Assuntos
DNA/genética , DNA/metabolismo , Fosfoproteínas/metabolismo , TATA Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Sequência de Bases , Cromatina/genética , Cromatina/metabolismo , Genes Virais/genética , Humanos , Cinética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , Especificidade por Substrato , Termodinâmica , Fator de Transcrição TFIIA/metabolismoRESUMO
Transcriptional activity of the TATA-binding protein (TBP) is controlled by a variety of proteins. The BTAF1 protein (formerly known as TAF(II)170/TAF-172 and the human ortholog of Saccharomyces cerevisiae Mot1p) and the NC2 complex composed of NC2alpha (DRAP1) and NC2beta (Dr1) are able to bind to TBP directly and regulate RNA polymerase II transcription both positively and negatively. Here, we present evidence that the NC2alpha subunit interacts with BTAF1. In contrast, the NC2beta subunit is not able to associate with BTAF1 and seems to interfere with the BTAF1-TBP interaction. Addition of NC2alpha or the NC2 complex can stimulate the ability of BTAF1 to interact with TBP. This function is dependent on the presence of ATP in cell extracts but does not involve the ATPase activity of BTAF1 nor phosphorylation of NC2alpha. Together, our results constitute the first evidence of the physical cooperation between BTAF1 and NC2alpha in TBP regulation and provide a framework to understand transcription functions of NC2alpha and NC2beta in vivo.
Assuntos
Proteínas Repressoras/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIID/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/genética , DNA/metabolismo , Humanos , Técnicas In Vitro , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIID/genética , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Tirosina/químicaRESUMO
An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. The RTA transcription activator of Kaposi's sarcoma-associated herpesvirus (KSHV) acts as a molecular switch for lytic reactivation. Here we demonstrate that KSHV RTA recruits CBP, the SWI/SNF chromatin remodeling complex, and the TRAP/Mediator coactivator into viral promoters through interactions with a short acidic sequence in the carboxyl region and that this recruitment is essential for RTA-dependent viral gene expression. The Brg1 subunit of SWI/SNF and the TRAP230 subunit of TRAP/Mediator were shown to interact directly with RTA. Consequently, genetic ablation of these interactions abolished KSHV lytic replication. These results demonstrate that the recruitment of CBP, SWI/SNF, and TRAP/Mediator complexes by RTA is the principal mechanism to direct well-controlled viral gene expression and thereby viral lytic reactivation.