RESUMO
Arsenic, an established carcinogen and toxicant, occurs in drinking water and food and affects millions of people worldwide. Arsenic appears to interfere with gene expression through epigenetic processes, such as DNA methylation and post-translational histone modifications. We investigated the effects of arsenic on histone residues in vivo as well as in vitro. Analysis of H3K9Ac and H3K9me3 in CD4+ and CD8+ sorted blood cells from individuals exposed to arsenic through drinking water in the Argentinean Andes showed a significant decrease in global H3K9me3 in CD4+ cells, but not CD8+ cells, with increasing arsenic exposure. In vitro studies of inorganic arsenic-treated T lymphocytes (Jurkat and CCRF-CEM, 0.1, 1, and 100 µg/L) showed arsenic-related modifications of H3K9Ac and changes in the levels of the histone deacetylating enzyme HDAC2 at very low arsenic concentrations. Further, in vitro exposure of kidney HEK293 cells to arsenic (1 and 5 µM) altered the protein levels of PCNA and DNMT1, parts of a gene expression repressor complex, as well as MAML1. MAML1 co-localized and interacted with components of this complex in HEK293 cells, and in silico studies indicated that MAML1 expression correlate with HDAC2 and DNMT1 expression in kidney cells. In conclusion, our data suggest that arsenic exposure may lead to changes in the global levels of H3K9me3 and H3K9Ac in lymphocytes. Also, we show that arsenic exposure affects the expression of PCNA and DNMT1-proteins that are part of a gene expression silencing complex.
Assuntos
Arsênio/efeitos adversos , Histonas/metabolismo , Linfócitos/efeitos dos fármacos , Adulto , Arsênio/sangue , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Água Potável , Epigênese Genética , Feminino , Inativação Gênica/efeitos dos fármacos , Células HEK293 , Código das Histonas/efeitos dos fármacos , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histonas/genética , Humanos , Células Jurkat , Linfócitos/metabolismo , Pessoa de Meia-Idade , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
Mastermind-like 1 (MAML1) is a transcriptional coregulator that has been associated with early development of many systems such as neuronal, muscular and urogenital. The present study aimed to explore the genome wide effects of MAML1 on DNA methylation and RNA expression in human embryonic kidney cells. Infinium HumanMethylation450 BeadChip Illumina array, methylation-sensitive high-resolution melt technique, Chip Analysis Methylation Pipeline and RNA profiling approaches were used to study MAML1 effects on the epigenome. We found that 11802 CpG sites were differentially methylated in MAML1-expressing cells while only 225 genes were differentially expressed. MAML1 overexpression induced more global differential hypermethylation than hypomethylation changes. In addition, the differentially methylated regions were mapped predominantly to 3'untranslated regions, intragenic regions and gene bodies and to a lesser extent to gene regulatory sequences. Gene ontology analysis revealed that the differentially changed genes (including HOXC11, HTATIP2, SLFN12 and SOX11) are involved in the regulation of urogenital system development, cell adhesion and embryogenesis. This study is the first report that shows the global effect of a single coregulator on DNA methylation and gene expression. Our results stress and support the effects of transcriptional coregulators on the cell methylome.
Assuntos
Ilhas de CpG , Metilação de DNA , Proteínas de Ligação a DNA/genética , Rim/citologia , Fatores de Transcrição/genética , Transcriptoma , Acetiltransferases/genética , Acetiltransferases/metabolismo , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Rim/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Earlier studies demonstrated the involvement of the p300 histone acetyltransferase in Notch signaling but the precise mechanisms by which p300 might modulate Notch function remains to be investigated. In this study, we show that p300 acetylates Notch1 ICD in cell culture assay and in vitro, and conserved lysines located within the Notch C-terminal nuclear localization signal are essential for Notch acetylation. MAML1 and CSL, which are components of the Notch transcription complex, enhance Notch acetylation and we suggest that MAML1 increases Notch acetylation by potentiating p300 autoacetylation. Furthermore, MAML1-dependent acetylation of Notch1 ICD by p300 decreases the ubiquitination of Notch1 ICD in cellular assays. CDK8 has been shown to target Notch1 for ubiquitination and proteosomal degradation. We show that CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300. Therefore, we speculate that acetylation of Notch1 might be a mechanism to regulate Notch activity by interfering with ubiquitin-dependent pathways.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptor Notch1/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinação , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Quinase 8 Dependente de Ciclina/metabolismo , Células HEK293 , Humanos , Lisina/química , Lisina/metabolismo , Camundongos , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Transcrição GênicaRESUMO
The Mastermindlike (MAML) family, comprising human MAML1, MAML2, and MAML3, are transcriptional regulators in Notch signaling. MAML proteins contain two consensus sites for SUMOylation at Lysine217 and Lysine299 that are conserved in humans, mice, and Xenopus. In this report, we show that MAML1 is SUMOylated at both sites. The E2-conjugating enzyme UBC9 is essential for MAML1 SUMOylation, and the E3 ligase PIAS1 stimulates this activity. Mutation of the lysines abolishes SUMOylation of MAML1 and strongly increases MAML1-activated transcription in cell culture assays. The protease SENP1 reverses SUMOylation of MAML1 and potentiates the transcription factor activity of MAML1. Furthermore, SUMOylation enhances MAML1 interaction with HDAC7, which decreases MAML1 transcriptional activity. Taken together, our data indicate that SUMOylation of MAML1 is a mechanism for repressing MAML1 activity by influencing its interaction with HDAC7.
Assuntos
Proteínas de Ligação a DNA/genética , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Proteínas Nucleares , Transativadores , Fatores de Transcrição/metabolismoRESUMO
Glycogen synthase kinase 3beta (GSK3beta) is involved in several cellular signaling systems through regulation of the activity of diverse transcription factors such as Notch, p53 and beta-catenin. Mastermind-like 1 (MAML1) was originally identified as a Notch coactivator, but has also been reported to function as a transcriptional coregulator of p53, beta-catenin and MEF2C. In this report, we show that active GSK3beta directly interacts with the MAML1 N-terminus and decreases MAML1 transcriptional activity, suggesting that GSK3beta might target a coactivator in its regulation of gene expression. We have previously shown that MAML1 increases global acetylation of histones, and here we show that the GSK3 inhibitor SB41, further enhances MAML1-dependent histone acetylation in cells. Finally, MAML1 translocates GSK3beta to nuclear bodies; this function requires full-length MAML1 protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Glicogênio Sintase Quinase 3 beta , Histonas/metabolismo , Humanos , Estrutura Terciária de Proteína , Receptor Notch1/metabolismo , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
MAML1 is a transcriptional coregulator originally identified as a Notch coactivator. MAML1 is also reported to interact with other coregulator proteins, such as CDK8 and p300, to modulate the activity of Notch. We, and others, previously showed that MAML1 recruits p300 to Notch-regulated genes through direct interactions with the DNA-CSL-Notch complex and p300. MAML1 interacts with the C/H3 domain of p300, and the p300-MAML1 complex specifically acetylates lysines of histone H3 and H4 tails in chromatin in vitro. In this report, we show that MAML1 potentiates p300 autoacetylation and p300 transcriptional activation. MAML1 directly enhances p300 HAT activity, and this coincides with the translocation of MAML1, p300 and acetylated histones to nuclear bodies.
Assuntos
Transativadores/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Linhagem Celular , Histonas/metabolismo , Estrutura Terciária de Proteína , Deleção de Sequência , Transativadores/química , Transativadores/genética , Fatores de Transcrição de p300-CBP/químicaRESUMO
The nuclear receptor steroidogenic factor-1 (SF1) is critical for development and function of steroidogenic tissues. Posttranslational modifications are known to influence the transcriptional capacity of SF1, and it was previously demonstrated that serine 203 is phosphorylated. In this paper we report that serine 203 is phosphorylated by a cyclin-dependent kinase 7 (CDK7)-mediated process. As part of the CDK-activating kinase complex, CDK7 is a component of the basal transcription factor TFIIH, and phosphorylation of SF1 as well as SF1-dependent transcription was clearly reduced in cells carrying a mutation that renders the CDK-activating kinase complex unable to interact with the TFIIH core. Coimmunoprecipitation analyses revealed that SF1 and CDK7 reside in the same complex, and kinase assays demonstrated that immunoprecipitated CDK7 and purified TFIIH phosphorylate SF1 in vitro. The CDK inhibitor roscovitine blocked phosphorylation of SF1, and an inactive form of CDK7 repressed the phosphorylation level and the transactivation capacity of SF1. Structural studies have identified phosphoinositides as potential ligands for SF1. Interestingly, we found that mutations designed to block phospholipid binding dramatically decreased the level of SF1 phosphorylation. Together our results suggest a connection between ligand occupation and phosphorylation and association with the basic transcriptional machinery, indicating an intricate regulation of SF1 transactivation.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Fator Esteroidogênico 1/metabolismo , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Chlorocebus aethiops , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Células HeLa , Humanos , Imunoprecipitação , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Fosfolipídeos/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Purinas/farmacologia , Roscovitina , Serina/metabolismo , Fator Esteroidogênico 1/genética , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição , TransfecçãoRESUMO
Ligand activation of Notch leads to the release of Notch IC (the intracellular receptor domain), which translocates to the nucleus and interacts with the DNA-binding protein CSL to control expression of specific target genes. In addition to ligand-mediated activation, Notch signalling can be further modulated by interactions of Notch IC with a number of other proteins. MAML1 has previously been shown to act co-operatively with the histone acetyltransferase p300 in Notch IC-mediated transcription. In the present study we show that the N-terminal domain of MAML1 directly interacts with both p300 and histones, and the p300-MAML1 complex specifically acetylates histone H3 and H4 tails in chromatin. Furthermore, p300 acetylates MAML1 and evolutionarily conserved lysine residues in the MAML1 N-terminus are direct substrates for p300-mediated acetylation. The N-terminal domain of MAML1 contains a proline repeat motif (PXPAAPAP) that was previously shown to be present in p53 and important for the p300-p53 interaction. We show that the MAML1 proline repeat motif interacts with p300 and enhances the activity of the MAML1 N-terminus in vivo. These findings suggest that the N-terminal domain of MAML1 plays an important role in Notch-regulated transcription, by direct interactions with Notch, p300 and histones.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Prolina/química , Receptores Notch/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Linhagem Celular , Cromatina/metabolismo , DNA Complementar , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Spodoptera , Transativadores , Fatores de TranscriçãoRESUMO
The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liver-specific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regulatory module of the transcription apparatus. Using a system that has been reconstituted from purified transcription factors, as well as one consisting of unfractionated nuclear extract from which TRAP/SMCC/Mediator has been depleted by specific antibodies, we demonstrate a strong dependence of HNF-4 function on this coactivator. Importantly, we further show a TRAP/SMCC/Mediator-dependence for HNF-4 transcriptional activation from chromatin templates. The latter involves cooperation with the histone acetyltransferase-containing coactivator p300, in accord with a synergistic mode of action of the two divergent coactivators. We also show that HNF-4 and TRAP/SMCC/Mediator can interact physically. This interaction likely involves primary HNF-4 activation function 2 (AF-2)-dependent interactions with the TRAP220 subunit of TRAP/SMCC/Mediator and secondary (AF-2-independent) interactions with TRAP170/RGR1. Finally, recruitment experiments using immobilized templates strongly suggest that the functional consequences of the physical interaction probably are manifested at a postrecruitment step in the activation pathway.
Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA , DNA/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Acetiltransferases/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proteínas de Transporte/química , Proteínas de Ciclo Celular/metabolismo , Extratos Celulares/química , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Cromatina/química , DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Fator 4 Nuclear de Hepatócito , Histona Acetiltransferases , Humanos , Proteínas Imediatamente Precoces , Substâncias Macromoleculares , Complexo Mediador , Subunidade 1 do Complexo Mediador , Proteínas de Membrana , Iniciação Traducional da Cadeia Peptídica/fisiologia , Fosfoproteínas/química , Ligação Proteica/fisiologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transativadores/química , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição de p300-CBPRESUMO
Ligand activation of Notch receptors leads to release of the intracellular receptor domain (Notch IC), which translocates to the nucleus and interacts with the DNA-binding protein RBP-Jkappa to control expression of specific target genes. A number of proteins have been shown to interact with Notch ICs and to modulate target gene activation, but the precise function of and interplay between these factors is not known. This report investigates the Notch IC-interacting proteins, p300, PCAF, and Mastermind-like 1 (MAML1), in an in vitro transcription system with purified factors and naked DNA or chromatin templates. MAML1, RBP-Jkappa, and Notch IC are all required for optimal transcription from DNA, whereas transcription from chromatin requires, in addition, p300, which interacts with MAML1. The transcriptional activity of p300 requires acetyl coenzyme A, indicating that it functions as a histone acetyltransferase when mediating Notch IC function. PCAF is unable to promote transcription on its own but enhances Notch IC-mediated transcription from chromatin in conjunction with p300. These data define a critical role for p300 in the potentiation of Notch IC function by MAML1 and PCAF, provide the first evidence for cooperativity between PCAF and p300 in Notch IC function, and also indicate direct effects of RBP-Jkappa, Notch IC, and MAML1 on the general transcription machinery.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Histona Acetiltransferases , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Proteínas de Membrana/genética , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch1 , Receptor Notch4 , Receptores Notch , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Moldes Genéticos , Transativadores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Transcrição de p300-CBPRESUMO
Immortalized cell lines are widely used in genetic and epigenetic studies, from exploration of basic molecular pathways to evaluation of disease-specific cellular properties. They are also used in biotechnology, e.g., in drug toxicity tests and vaccine production. Cellular and genetic uniformity is the main feature of immortalized cell lines and it has been particularly advantageous in functional genomic research, which has in recent years been expanded to include epigenetic mechanisms of gene expression regulation. Using the MS-HRM technique, we demonstrated heterogeneity in locus-specific methylation patterns in different cell cultures of four human cell lines: HEK293, HEK293T, LCL and DU145. Our results show that some human immortalized cell lines consist of cells that differ in the methylation status of specific loci, i.e., that they are epigenetically heterogeneous. We show that even two cultures of the same cell line obtained from different laboratories can differ in the methylation status of the specific loci. The results indicated that epigenetic uniformity of the cell lines cannot be assumed in experiments which utilize cell cultures and that the methylation status of the specific loci in the immortalized cell lines should be re-characterized and carefully profiled before epigenetic studies are performed.
Assuntos
Epigênese Genética , Análise de Sequência de DNA , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Bases , Metilação de DNA , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Expressão Gênica , Loci Gênicos , Variação Genética , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Dados de Sequência Molecular , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Temperatura de Transição , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1). MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ubiquitinação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/química , Genes Reporter , Células HeLa , Proteínas de Homeodomínio/genética , Humanos , Lisina/metabolismo , Fatores de Transcrição HES-1 , Fatores de Transcrição/química , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Mastermind-like 1 (MAML1) is a transcriptional coregulator of activators in various signaling pathways, such as Notch, p53, myocyte enhancer factor 2C (MEF2C) and beta-catenin. In earlier studies, we demonstrated that MAML1 enhanced p300 acetyltransferase activity, which increased the acetylation of Notch by p300. In this study, we show that MAML1 strongly induced acetylation of the transcription factor early growth response-1 (EGR1) by p300, and increased EGR1 protein expression in embryonic kidney cells. EGR1 mRNA transcripts were also upregulated in the presence of MAML1. We show that MAML1 physically interacted with, and acted cooperatively with EGR1 to increase transcriptional activity of the EGR1 and p300 promoters, which both contain EGR1 binding sites. Bioinformatics assessment revealed a correlation between p300, EGR1 and MAML1 copy number and mRNA alterations in renal clear cell carcinoma and p300, EGR1 and MAML1 gene alterations were associated with increased overall survival. Our findings suggest MAML1 may be a component of the transcriptional networks which regulate EGR1 target genes during nephrogenesis and could also have implications for the development of renal cell carcinoma.
Assuntos
Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Fatores de Transcrição/genética , Acetilação , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Dosagem de Genes , Genômica , Células HCT116 , Células HEK293 , Humanos , Neoplasias Renais/metabolismo , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , RNA Mensageiro/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Transcriptional coactivators showing physical and functional interactions with PPARgamma include the protein acetyl transferase p300, the TRAP/Mediator complex that interacts with the general transcription machinery, and the highly regulated PGC-1alpha. We show that PGC-1alpha directly interacts with TRAP/Mediator, through the PPARgamma-interacting subunit TRAP220, and stimulates TRAP/Mediator-dependent function on DNA templates. Further, while ineffective by itself, PGC-1alpha stimulates p300-dependent histone acetylation and transcription on chromatin templates in response to PPARgamma. These functions are mediated by largely independent PPARgamma, p300, and TRAP220 interaction domains in PGC-1alpha, whereas p300 and TRAP220 show ligand-dependent interactions with a common region of PPARgamma. Apart from showing PGC-1alpha functions both in chromatin remodeling and in preinitiation complex formation or function (transcription), these results suggest a key role for PGC-1alpha, through concerted but dynamic interactions, in coordinating these steps.
Assuntos
Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteína p300 Associada a E1A , Genes Reporter , Células HeLa , Humanos , Complexo Mediador , Subunidade 1 do Complexo Mediador , Camundongos , Subunidades Proteicas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição GênicaRESUMO
The TRAP (thyroid hormone receptor-associated proteins) transcription coactivator complex (also known as Mediator) was first isolated as a group of proteins that facilitate the function of the thyroid hormone receptor. This complex interacts physically with several nuclear receptors through the TRAP220 subunit, and with diverse activators through other subunits. TRAP220 has been reported to show ligand-enhanced interaction with peroxisome proliferator-activated receptor gamma(2) (PPAR gamma(2)), a nuclear receptor essential for adipogenesis. Here we show that Trap220(-/-) fibroblasts are refractory to PPAR gamma(2)-stimulated adipogenesis, but not to MyoD-stimulated myogenesis, and do not express adipogenesis markers or PPAR gamma(2) target genes. These defects can be restored by expression of exogenous TRAP220. Further indicative of a direct role for TRAP220 in PPAR gamma(2) function via the TRAP complex, TRAP functions directly as a transcriptional coactivator for PPAR gamma(2) in a purified in vitro system and interacts with PPAR gamma(2) in a ligand- and TRAP220-dependent manner. These data indicate that TRAP220 acts, via the TRAP complex, as a PPAR gamma(2)-selective coactivator and, accordingly, that it is specific for one fibroblast differentiation pathway (adipogenesis) relative to another (myogenesis).
Assuntos
Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Proteínas de Transporte/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Biomarcadores/análise , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular , Fibroblastos , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Subunidade 1 do Complexo Mediador , Camundongos , Desenvolvimento Muscular , Músculos/citologia , Músculos/metabolismo , Proteína MyoD/metabolismo , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores dos Hormônios Tireóideos/química , Receptores dos Hormônios Tireóideos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The N-terminal domain of c-Myc plays a key role in cellular transformation and is involved in both activation and repression of target genes as well as in modulated proteolysis of c-Myc via the proteasome. Given this functional complexity, it has been difficult to clarify the structures within the N terminus that contribute to these different processes as well as the mechanisms by which they function. We have used a simplified yeast model system to identify the primary determinants within the N terminus for (i) chromatin remodeling of a promoter, (ii) gene activation from a chromatin template in vivo, and (iii) interaction with highly purified Gcn5 complexes as well as other chromatin-remodeling complexes in vitro. The results identify two regions that contain autonomous chromatin opening and gene activation activity, but both regions are required for efficient interaction with chromatin-remodeling complexes in vitro. The conserved Myc boxes do not play a direct role in gene activation, and Myc box II is not generally required for in vitro interactions with remodeling complexes. The yeast SAGA complex, which is orthologous to the human GCN5-TRRAP complex that interacts with Myc in human cells, plays a role in Myc-mediated chromatin opening at the promoter but may also be involved in later steps of gene activation.