RESUMO
Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis, SphK1, dephosphorylation, and SGPP1 were differentially correlated with GFI1 levels in MM cells. We detected GFI1 occupancy on the SGGP1 gene in MM cells in a predicted enhancer region at the 5' end of intron 1, which correlated with decreased SGGP1 expression and increased S1P levels in GFI1 overexpressing cells, regardless of their p53 status. The high S1P:Ceramide intracellular ratio in MM cells protected c-Myc protein stability in a PP2A-dependent manner. The decreased MM viability by SphK1 inhibition was dependent on the induction of autophagy in both p53WT and p53mut MM. An autophagic blockade prevented GFI1 support for viability only in p53mut MM, demonstrating that GFI1 increases MM cell survival via both p53WT inhibition and upregulation of S1P independently. Therefore, GFI1 may be a key therapeutic target for all types of MM that may significantly benefit patients that are highly resistant to current therapies.
RESUMO
Key osteoclast (OCL) regulatory gene promoters in bone marrow-derived monocytes harbor bivalent histone modifications that combine activating Histone 3 lysine 4 tri-methyl (H3K4me3) and repressive H3K27me3 marks, which upon RANKL stimulation resolve into repressive or activating architecture. Enhancer of zeste homologue 2 (EZH2) is the histone methyltransferase component of the polycomb repressive complex 2, which catalyzes H3K27me3 modifications. Immunofluorescence microscopy reveals that EZH2 localization during murine osteoclastogenesis is dynamically regulated. Using EZH2 knockdown and small molecule EZH2 inhibitor GSK126, we show that EZH2 plays a critical epigenetic role in OCL precursors (OCLp) during the first 24 hours of RANKL activation. RANKL triggers EZH2 translocation into the nucleus where it represses OCL-negative regulators MafB, Irf8, and Arg1. Consistent with its cytoplasmic localization in OCLp, EZH2 methyltransferase activity is required during early RANKL signaling for phosphorylation of AKT, resulting in downstream activation of the mTOR complex, which is essential for induction of OCL differentiation. Inhibition of RANKL-induced pmTOR-pS6RP signaling by GSK126 altered the translation ratio of the C/EBPß-LAP and C/EBPß-LIP isoforms and reduced nuclear translocation of the inhibitory C/EBPß-LIP, which is necessary for transcriptional repression of the OCL negative-regulatory transcription factor MafB. EZH2 in multinucleated OCL is primarily cytoplasmic and mature OCL cultured on bone segments in the presence of GSK126 exhibit defective cytoskeletal architecture and reduced resorptive activity. Here we present new evidence that EZH2 plays epigenetic and cytoplasmic roles during OCL differentiation by suppressing MafB transcription and regulating early phases of PI3K-AKT-mTOR-mediated RANKL signaling, respectively. Consistent with its cytoplasmic localization, EZH2 is required for cytoskeletal dynamics during resorption by mature OCL. Thus, EZH2 exhibits complex roles in supporting osteoclast differentiation and function. © 2019 American Society for Bone and Mineral Research.
Assuntos
Reabsorção Óssea , Osteoclastos , Animais , Reabsorção Óssea/genética , Diferenciação Celular , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética , Camundongos , Fosfatidilinositol 3-QuinasesRESUMO
We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1ß, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBPß) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBPß leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBPß DBDs, along with the C/EBPß C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding l-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBPß-Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with l-arginine had significantly decreased IL1B transcription and reduced C/EBPß's association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBPß to cognate DNA and in transcription of the C/EBPß-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein-protein interactions and can serve as druggable targets for regulating gene promoter activity.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Interleucina-1beta/genética , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/química , Linhagem Celular , Cristalografia por Raios X , Humanos , Camundongos , Simulação de Acoplamento Molecular , Regiões Promotoras Genéticas , Conformação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/química , Transativadores/químicaRESUMO
Interleukin 1ß is a pro-inflammatory cytokine important for both normal immune responses and chronic inflammatory diseases. The regulation of the 31â¯kDa proIL-1ß precursor coded by the IL1B gene has been extensively studied in myeloid cells, but not in lymphoid-derived CD4 T cells. Surprisingly, we found that some CD4 T cell subsets express higher levels of proIL-1ß than unstimulated monocytes, despite relatively low IL1B mRNA levels. We observed a significant increase in IL1B transcription and translation in CD4 T cells upon ex vivo CD3/CD28 activation, and a similar elevation in the CCR5+ effector memory population compared to CCR5- T cells in vivo. The rapid and vigorous increase in IL1B gene transcription for stimulated monocytes has previously been associated with the presence of Spi-1/PU.1 (Spi1), a myeloid-lineage transcription factor, pre-bound to the promoter. In the case of CD4 T cells, this increase occurred despite the lack of detectable Spi1 at the IL1B promoter. Additionally, we found altered epigenetic regulation of the IL1B locus in CD3/CD28-activated CD4 T cells. Unlike monocytes, activated CD4 T cells possess bivalent H3K4me3+/H3K27me3+ nucleosome marks at the IL1B promoter, reflecting low transcriptional activity. These results support a model in which the IL1B gene in CD4 T cells is transcribed from a low-activity bivalent promoter independent of Spi1. Accumulated cytoplasmic proIL-1ß may ultimately be cleaved to mature 17â¯kDa bioactive IL-1ß, regulating T cell polarization and pathogenic chronic inflammation.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Interleucina-1beta/genética , Monócitos/fisiologia , Transcrição Gênica/genética , Biomarcadores/metabolismo , Antígenos CD28/genética , Complexo CD3/genética , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Humanos , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Receptores CCR5/genética , Ativação Transcricional/genéticaRESUMO
Aberrant activation of EGFR in human cancers promotes tumorigenesis through stimulation of AKT signaling. Here, we determined that the discoidina neuropilin-like membrane protein DCBLD2 is upregulated in clinical specimens of glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. In multiple cancer cell lines, EGFR activated phosphorylation of tyrosine 750 (Y750) of DCBLD2, which is located within a recently identified binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, phosphorylation of DCBLD2 Y750 recruited TRAF6, leading to increased TRAF6 E3 ubiquitin ligase activity and subsequent activation of AKT, thereby enhancing EGFR-driven tumorigenesis. Moreover, evaluation of patient samples of gliomas and HNCs revealed an association among EGFR activation, DCBLD2 phosphorylation, and poor prognoses. Together, our findings uncover a pathway in which DCBLD2 functions as a signal relay for oncogenic EGFR signaling to promote tumorigenesis and suggest DCBLD2 and TRAF6 as potential therapeutic targets for human cancers that are associated with EGFR activation.
Assuntos
Carcinogênese , Receptores ErbB/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Neoplasias Encefálicas/etiologia , Células Cultivadas , Glioma/etiologia , Neoplasias de Cabeça e Pescoço/etiologia , Humanos , Proteínas de Membrana/genética , Fosforilação , Transdução de SinaisRESUMO
Interleukin-1ß and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPß and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators.
Assuntos
Genes Precoces/genética , Interleucina-1beta/genética , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética , Animais , Transporte Biológico/efeitos dos fármacos , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ontologia Genética , Loci Gênicos/genética , Glucose/metabolismo , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Monócitos/metabolismo , Nucleossomos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Prostaglandin E2 (PGE2), IL-23 and IL-1ß are implicated in inflammatory bowel disease susceptibility, likely in part by modulating IL-17 producing CD4(+) T helper (Th17) cells. To better understand how these three mediators affect Th17 cell memory responses, we characterized the gene expression profiles of activated human peripheral CD4(+) effector memory T cells and sorted Th17 memory cells from healthy donors concurrent with IL17A mRNA induction mediated by PGE2 and/or IL-23 plus IL-1ß. We discovered that PGE2 and IL-23 plus IL-1ß differentially regulate Th17 cytokine expression and synergize to induce IL-17A, but not IL-17F. IL-23 plus IL-1ß preferentially induce IL-17F expression. The addition of PGE2 to IL-23 plus IL-1ß only enhances IL-17A expression as mediated by the PGE2 EP4 receptor, and promotes a switch from an IL-17F to an IL-17A predominant immune response. The human Th17 HuT-102 cell line was also found to constitutively express IL-17A, but not IL-17F. We went on to show that the IL17A and IL17F loci have divergent epigenetic architectures in unstimulated HuT-102 and primary Th17 cells and are poised for preferential expression of IL17A. We conclude that the chromatin for IL17A and IL17F are distinctly regulated, which may play an important role in mucosal health and disease.
Assuntos
Histonas/metabolismo , Interleucina-17/genética , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Células Th17/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , AMP Cíclico/metabolismo , Histonas/genética , Humanos , Memória Imunológica/imunologia , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Prostaglandinas E/metabolismo , RNA Mensageiro/biossíntese , Transdução de Sinais/imunologia , Células Th17/imunologiaRESUMO
Gram-negative bacteria release lipopolysaccharide (LPS) into the bloodstream. Here, it engages Toll-like receptor (TLR) 4 expressed in human lung microvascular endothelia (HMVEC-Ls) to open the paracellular pathway through Src family kinase (SFK) activation. The signaling molecules that couple TLR4 to the SFK-driven barrier disruption are unknown. In HMVEC-Ls, siRNA-induced silencing of TIRAP/Mal and overexpression of dominant-negative TIRAP/Mal each blocked LPS-induced SFK activation and increases in transendothelial [(14)C]albumin flux, implicating the MyD88-dependent pathway. LPS increased TRAF6 autoubiquitination and binding to IRAK1. Silencing of TRAF6, TRAF6-dominant-negative overexpression, or preincubation of HMVEC-Ls with a cell-permeable TRAF6 decoy peptide decreased both LPS-induced SFK activation and barrier disruption. LPS increased binding of both c-Src and Fyn to GST-TRAF6 but not to a GST-TRAF6 mutant in which the three prolines in the putative Src homology 3 domain-binding motif (amino acids 461-469) were substituted with alanines. A cell-permeable decoy peptide corresponding to the same proline-rich motif reduced SFK binding to WT GST-TRAF6 compared with the Pro â Ala-substituted peptide. Finally, LPS increased binding of activated Tyr(P)(416)-SFK to GST-TRAF6, and preincubation of HMVEC-Ls with SFK-selective tyrosine kinase inhibitors, PP2 and SU6656, diminished TRAF6 binding to c-Src and Fyn. During the TRAF6-SFK association, TRAF6 catalyzed Lys(63)-linked ubiquitination of c-Src and Fyn, whereas SFK activation increased tyrosine phosphorylation of TRAF6. The TRAF6 decoy peptide blocked both LPS-induced SFK ubiquitination and TRAF6 phosphorylation. Together, these data indicate that the proline-rich Src homology 3 domain-binding motif in TRAF6 interacts directly with activated SFKs to couple LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls.
Assuntos
Permeabilidade Capilar/fisiologia , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/metabolismo , Quinases da Família src/metabolismo , Motivos de Aminoácidos , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Células Endoteliais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inativação Gênica , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/agonistas , Fator 6 Associado a Receptor de TNF/genética , Receptor 4 Toll-Like/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética , Quinases da Família src/genéticaRESUMO
The C/EBP family of proteins represents an important group of bZIP transcription factors that are key to the regulation of essential functions such as cell cycle, hematopoiesis, skeletal development, and host immune responses. They are also intimately associated with tumorigenesis and viral disease. These proteins are regulated at multiple levels, including gene induction, alternative translational initiation, post-translational modification, and protein-protein interaction. This review attempts to integrate recent reports with more than 20 years of previous effort focused on this fascinating collection of regulators.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Diferenciação Celular , Dimerização , Células-Tronco Hematopoéticas/citologia , Humanos , Fosforilação , Ligação Proteica , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Transativadores/metabolismoRESUMO
The tumor necrosis factor (TNF) receptor associated factor (TRAF) class of intracellular signal transducers is responsible for mediating many of the activation events initiated by TNF receptor (TNFR) and Toll-like/Interleukin-1, -17, and -18 receptor (TIR) families. Investigation of the mechanism by which TRAF6 is activated has demonstrated that two critical domains of the molecule required for activation and downstream signaling are involved in an interaction which renders the molecule inactive and structurally closed, as well as incapable of auto-ubiquitination. Contrary to its assumed role as a direct mediator of protein-protein interaction, TRAF auto-ubiquitination is a means of sustaining an open conformation active in downstream signaling. Furthermore, the inferred cis-function of TRAF auto-ubiquitination is now demonstrated to act in trans and requires both the RING-Zinc (RZ) fingers region and coiled-coil domain. We also observed that both the RZ fingers region and the MATH domain are targets for ubiquitination. Although TRAF6 ubiquitination has emerged as a hallmark of activation, trans-ubiquitination induced by two TRAF6 muteins is insufficient for NF-kappaB activation.
Assuntos
Fator 6 Associado a Receptor de TNF/química , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação/fisiologia , Western Blotting , Linhagem Celular , Humanos , Imunoprecipitação , NF-kappa B/metabolismo , Conformação Proteica , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
The immediate early (IE) proteins of human cytomegalovirus (hCMV) have diverse roles in directing viral and host cell transcription. Among these is the ability of IE2 to induce transcription of the IL1B gene that codes for IL-1beta in monocytes. This function is partially explained by interaction between IE2 and the host cell transcription factor Spi-1/PU.1 (Spi-1). We now show that maximal IE2 function also depends on productive interactions localizing to two C/EBP sites on the IL1B promoter suggesting either bi- or tri-molecular interactions between IE2, Spi-1 and C/EBPbeta at two different locations on the promoter. The IE2 interaction region on Spi-1 was previously mapped to the DNA-binding ETS domain and overlaps the region of Spi-1 that interacts with the transcription factor C/EBPbeta, a factor known to be critical for the induction of IL1B in response to Toll/IL-1 receptor (TIR) family signal transduction. The Spi-1 interacting region of IE2 maps to amino acids 315-328, a sequence that also interacts with the bZIP domain of C/EBPbeta. An expression vector coding for amino acids 291-364 of IE2 can suppress LPS induction of a co-transfected IL1B enhancer-promoter fragment in a monocyte cell line. This inhibition is likely the result of competition between Spi-1 and C/EBPbeta, thus blunting gene induction.
Assuntos
Citomegalovirus/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Interleucina-1beta/genética , Transativadores/metabolismo , Transcrição Gênica , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Citomegalovirus/imunologia , Células HeLa , Humanos , Proteínas Imediatamente Precoces/química , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/química , TransfecçãoRESUMO
MOTIVATION: Mutual information can be used to explore covarying positions in biological sequences. In the past, it has been successfully used to infer RNA secondary structure conformations from multiple sequence alignments. In this study, we show that the same principles allow the discovery of transcription factor amino acids that are coevolving with nucleotides in their DNA-binding targets. RESULTS: Given an alignment of transcription factor binding domains, and a separate alignment of their DNA target motifs, we demonstrate that mutually covarying base-amino acid positions may indicate possible protein-DNA contacts. Examples explored in this study include C2H2 zinc finger, homeodomain and bHLH DNA-binding motif families, where a number of known base-amino acid contacting positions are identified. Mutual information analyses may aid the prediction of base-amino acid contacting pairs for particular transcription factor families, thereby yielding structural insights from sequence information alone. Such inference of protein-DNA contacting positions may guide future experimental studies of DNA recognition.
Assuntos
Algoritmos , DNA/química , DNA/genética , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Biologia Computacional/métodos , Dados de Sequência Molecular , Ligação ProteicaRESUMO
gp130-linked cytokines such as interleukin-6 (IL-6) stimulate the formation of tyrosine-phosphorylated signal transducer and activator of transcription 3 (P-STAT3), which activates many genes, including the STAT3 gene itself. The resulting increase in the concentration of unphosphorylated STAT3 (U-STAT3) drives a second wave of expression of genes such as RANTES, IL6, IL8, MET, and MRAS that do not respond directly to P-STAT3. Thus, U-STAT3 sustains cytokine-dependent signaling at late times through a mechanism completely distinct from that used by P-STAT3. Many U-STAT3-responsive genes have kappaB elements that are activated by a novel transcription factor complex formed when U-STAT3 binds to unphosphorylated NFkappaB (U-NFkappaB), in competition with IkappaB. The U-STAT3/U-NFkappaB complex accumulates in the nucleus with help from the nuclear localization signal of STAT3, activating a subset of kappaB-dependent genes. Additional genes respond to U-STAT3 through an NFkappaB-independent mechanism. The role of signal-dependent increases in U-STAT3 expression in regulating gene expression is likely to be important in physiological responses to gp130-linked cytokines and growth factors that activate STAT3, and in cancers that have constitutively active P-STAT3.
Assuntos
Regulação da Expressão Gênica , Interleucina-6/farmacologia , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica , Sítios de Ligação , Biomarcadores/metabolismo , Mama/citologia , Mama/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Imunoprecipitação da Cromatina , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Imunoprecipitação , NF-kappa B/genética , Sinais de Localização Nuclear , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Transdução de SinaisRESUMO
Transcription factor (TF) proteins recognize a small number of DNA sequences with high specificity and control the expression of neighbouring genes. The evolution of TF binding preference has been the subject of a number of recent studies, in which generalized binding profiles have been introduced and used to improve the prediction of new target sites. Generalized profiles are generated by aligning and merging the individual profiles of related TFs. However, the distance metrics and alignment algorithms used to compare the binding profiles have not yet been fully explored or optimized. As a result, binding profiles depend on TF structural information and sometimes may ignore important distinctions between subfamilies. Prediction of the identity or the structural class of a protein that binds to a given DNA pattern will enhance the analysis of microarray and ChIP-chip data where frequently multiple putative targets of usually unknown TFs are predicted. Various comparison metrics and alignment algorithms are evaluated (a total of 105 combinations). We find that local alignments are generally better than global alignments at detecting eukaryotic DNA motif similarities, especially when combined with the sum of squared distances or Pearson's correlation coefficient comparison metrics. In addition, multiple-alignment strategies for binding profiles and tree-building methods are tested for their efficiency in constructing generalized binding models. A new method for automatic determination of the optimal number of clusters is developed and applied in the construction of a new set of familial binding profiles which improves upon TF classification accuracy. A software tool, STAMP, is developed to host all tested methods and make them publicly available. This work provides a high quality reference set of familial binding profiles and the first comprehensive platform for analysis of DNA profiles. Detecting similarities between DNA motifs is a key step in the comparative study of transcriptional regulation, and the work presented here will form the basis for tool and method development for future transcriptional modeling studies.
Assuntos
DNA/química , DNA/genética , Família Multigênica/genética , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Análise por Conglomerados , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Rapid induction of transcription is known to be mediated by factors which bind DNA following post-translational modification. We report here that non-tyrosine phosphorylated (NTP)-Stat1 is involved in a cooperative interaction with Spi-1/PU.1 and IRF8 to form a pre-associated, poised complex for IL1B gene induction. A double point mutation at a putative STAT binding site, which overlaps this composite Spi-1 x IRF8 site located in the LPS and IL-1 response element (LILRE), inhibited human IL1B LPS-dependent reporter activity to about 10 percent of the control wild type vector. Chromatin immunoprecipitation revealed stimulation-independent constitutive binding of IRF8, Spi-1 and NTP-Stat1 at the LILRE, while binding of C/EBP beta was activated at an adjacent C/EBP beta site after LPS stimulation. In contrast to Stat1, IRF8 was tyrosine phosphorylated following LPS treatment. Supporting the involvement of NTP-Stat1, LPS-induced IL1B reporter activity in monocytes was enhanced by ectopic expression of NTP-Stat1 Y701F. In contrast, co-expression of a Y211F IRF8 mutein functioned as a dominant-negative inhibitor of LPS-induced IL1B reporter activity. In vitro DNA binding using extracts from LPS-treated monocytes confirmed that the LILRE enhancer constitutively binds a trimolecular complex containing IRF8, Spi-1 and NTP-Stat1. Binding studies using in vitro-expressed proteins revealed that NTP-Stat1 enhanced the binding of Spi-1 and IRF8 to the LILRE. Co-expression of TRAF6, an LPS surrogate, with Spi-1 and IRF8 enhanced IL1B reporter activity in HEK293R cells, which was dramatically reduced when Y211F IRF8 was co-expressed. These results suggest that the rapid transcriptional induction of an important inflammatory gene is dependent upon constitutive cooperative binding of a Spi-1 x IRF8 x NTP-Stat1 complex to the LILRE, which primes the gene for immediate induction following IRF8 phosphorylation. Phosphorylation of chromatin pre-associated factors like IRF8 may be an important strategy for the rapid transcriptional activation of genes involved in innate immunity.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Lipopolissacarídeos/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT1/fisiologia , Transativadores/metabolismo , Ativação Transcricional/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Fatores Reguladores de Interferon/fisiologia , Camundongos , Fosforilação , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas/fisiologia , Elementos de Resposta , Fator de Transcrição STAT1/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional/genética , Tirosina/metabolismoRESUMO
Interleukin 1 (IL-1) has been implicated in the reorganization of the actin cytoskeleton. An expression vector encoding a PKB/Akt pleckstrin-homology domain fused to a fluorescent protein was used to detect phosphoinositide 3-kinase (PI 3-kinase) products. It was observed that PI 3-kinase was activated either by treatment with IL-1 or by expression of either TRAF6, Src, MyD88 or dominant-positive PI 3-kinase, and resulted in the formation of long filopodia-like cellular protrusions that appeared to branch at membrane sites consisting of clusters of phosphoinositide. This depended upon a TRAF6 polyproline motif and Src catalytic activity, and was blocked by inhibitors of PI 3-kinase, Src and Ras. Using both conventional and split fluorescent protein probes fused to expressed TRAF6 and Src in living cells, the polyproline sequence of TRAF6 and the Src-homology 3 (SH3) domain of Src were shown to be required for interaction between these two proteins. Interaction occurred within the cytoplasm, and not at either the cell membrane or cytoplasmic sequestosomes. In addition, co-transfection of vectors expressing fluorescent-protein-fused TRAF6 and non-fluorescent MyD88, IRAK1 and IRAK2 revealed an inverse correlation between increased sequestosome formation and activation of both PI 3-kinase and NF-kappaB. Although a key factor in TRAF6-dependent activation of PI 3-kinase, ectopic expression of Src was insufficient for NF-kappaB activation and, in contrast to NF-kappaB, was not inhibited by IRAK2.
Assuntos
Citoesqueleto/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Actinas/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Humanos , Interleucina-1/farmacologia , Modelos Biológicos , NF-kappa B/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Pseudópodes/fisiologia , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/fisiologia , TransfecçãoRESUMO
Elucidating the role of glucocorticoid in regulating gene expression is crucial to developing effective strategies against inflammatory diseases such as arthritis. In this report we demonstrate that glucocorticoid inhibits transcription directed by the IL-lbeta gene (IL1B) upstream induction sequence (UIS) enhancer, and to a much lesser extent by the tissue-specific basal promoter. Within the enhancer, three transcription factor binding sites, previously demonstrated by us to be important for the induction of IL1B by lipopolysaccharide, are now shown to be directly inhibited by the synthetic glucocorticoid, dexamethasone. We also previously showed that one of these sites could bind a novel STAT-like factor, while the other two bound heterodimers containing NF-IL6(C/EBPbeta). Although it has been reported by others that NF-IL6 homodimers can interact with glucocorticoid receptor (GR) to enhance transcription of the alpha1-acid glycoprotein gene, it now appears that glucocorticoid represses DNA binding of NF-IL6 heterodimers as well as the novel STAT-like factor to the critical sites within the IL1B UIS. Thus, GR likely disrupts the DNA binding capability of critical IL1B factors via transrepression.
Assuntos
Dexametasona/farmacologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1/genética , Precursores de Proteínas/genética , Transativadores/antagonistas & inibidores , Sítios de Ligação , Proteína delta de Ligação ao Facilitador CCAAT/antagonistas & inibidores , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Células Cultivadas , Cloranfenicol O-Acetiltransferase/análise , Cloranfenicol O-Acetiltransferase/genética , DNA/metabolismo , Glucocorticoides/farmacologia , Humanos , Lipopolissacarídeos/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Transdução de Sinais , Transativadores/metabolismoRESUMO
The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1beta. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBPbeta carboxyl-terminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/química , DNA/química , Escherichia coli/metabolismo , Arginina/química , Western Blotting , Proteína beta Intensificadora de Ligação a CCAAT/química , Cátions , Núcleo Celular/metabolismo , Relação Dose-Resposta a Droga , Vetores Genéticos , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Interleucina-1/metabolismo , Luciferases/metabolismo , Modelos Biológicos , Modelos Genéticos , Modelos Moleculares , Monócitos/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/química , Proteínas Proto-Oncogênicas/química , Transativadores/química , Ativação TranscricionalRESUMO
Interleukins (IL) 1 and 6 are important cytokines that function via the activation, respectively, of the transcription factors NF-kappaB and STAT3. We have observed that a specific type of kappa B DNA sequence motif supports both NF-kappaB p65 homodimer binding and cooperativity with non-tyrosine-phosphorylated STAT3. This activity, in contrast to that mediated by kappaB DNA motifs that do not efficiently bind p65 homodimers, is shown to be uniquely dependent upon signal transduction through the carboxyl terminus of TRAF6. Furthermore, STAT3 and p65 are shown to physically interact, in vivo, and this interaction appears to inhibit the function of "classical" STAT3 GAS-like binding sites. The distinct p50 form of NF-kappaB is also shown to interact with STAT3. However, in contrast to p65, p50 cooperates with STAT3 bound to GAS sites. These data argue for a novel transcription factor cross-talk mechanism that may help resolve inconsistencies previously reported regarding the mechanism of IL-1 inhibition of IL-6 activity during the acute-phase response.