RESUMO
Macrophages play a pivotal role in atherosclerosis through a variety of events related to cellular oxidative stress. This process is mainly due to an excessive production of reactive oxygen species whose elimination occurs through antioxidant systems including the thioredoxin (Trx) system. In this paper, we investigated whether the Trx system would exhibit circadian rhythmicity in dexamethasone synchronized cultured macrophages and monitored the impact of the rhythmicity of Trx-1 on markers of atherosclerosis. We found that the clock-related genes BMAL-1, PER-2, CRY-1 and REV ERB α exhibited a robust circadian expression. However, the Trx genes family (Trx-1, Trx-2, TrxR1 and TXNIP) did not exhibit a circadian expression at the mRNA level in spite of the presence of E-box elements within the promoter regions of TrxR1 and TXNIP genes. Nevertheless, both Trx-1 and TXNIP exhibited a circadian expression at the protein level and proteasome inhibition abolished the rhythmicity of Trx-1. Moreover, we found a link between low Trx-1 level and elevated atherogenic markers such as 4-HNE, TNF-α and cholesterol accumulation in macrophages. Our results indicate that the Trx gene family does not exhibit the same circadian regulation and that the presence of E-box elements in the TXNIP promoter is not sufficient to ensure a circadian rhythmicity at the transcriptional level. In addition, since a link was found between a low level of Trx-1 protein during circadian rhythm and high levels of atherogenic markers, administration of Trx-1 at certain time points could be an interesting approach to protect against atherosclerosis development.
Assuntos
Aterosclerose , Macrófagos Peritoneais , Animais , Ritmo Circadiano , Macrófagos Peritoneais/metabolismo , Camundongos , Estresse Oxidativo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.
Assuntos
Angiotensina II/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces , Fatores de Transcrição/metabolismo , Ativação Transcricional , Androstadienos/farmacologia , Animais , Sítios de Ligação , Butadienos/farmacologia , Células CHO , Linhagem Celular , Núcleo Celular/metabolismo , Cricetinae , Análise Mutacional de DNA , Proteína 1 de Resposta de Crescimento Precoce , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Sistema de Sinalização das MAP Quinases , Mutagênese Sítio-Dirigida , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Transfecção , Tirosina/metabolismo , Regulação para Cima , WortmaninaRESUMO
In hepatocytes, the amount of the Spot 14 (S14) protein is closely related to the full expression of enzymes involved in the glycolytic and lipogenic pathways. In the present study we address the role played by this protein in the control of transcription of the L-type pyruvate kinase (L-PK) gene in primary hepatocytes. We show that human S14, which by itself does not bind to the L-PK promoter, physically interacts with the human chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) and induces the switch of this factor from a repressor to an activator. However, the enhancing activity of S14 and COUP-TF1 depends on the presence of a proximal GC-rich box (the L0 element) that specifically binds nuclear proteins from the livers of rats fed a glucose-rich diet. Moreover, the L0 element, which strongly binds dephosphorylated specificity protein 1 (Sp1), loses all affinity when this factor is phosphorylated by cAMP-dependent protein kinase. Mutations that affect binding of Sp1 and nuclear proteins to the L0 box also decrease basal transcription and impair glucose responsiveness of the promoter. These results therefore shed light on the mechanism by which the S14 protein, whose concentration rapidly rises after glucose intake, contributes to the full activity of the L-PK promoter.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas/química , Proteínas/metabolismo , Piruvato Quinase/genética , Fator de Transcrição Sp1/química , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Fator I de Transcrição COUP , Núcleo Celular/metabolismo , Células Cultivadas , Galinhas , AMP Cíclico/metabolismo , Glucose/metabolismo , Glutationa Transferase/metabolismo , Hepatócitos/metabolismo , Humanos , Masculino , Mutação , Proteínas Nucleares , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Type II secreted phospholipase A(2) (sPLA(2)) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA(2), which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA(2) was not detected in control vascular smooth muscle cells (VSMC). Treatment of VSMC with agents that increase intracellular cAMP (eg, forskolin, dibutyryl [db]-cAMP) resulted in a time- and concentration-dependent increase in sPLA(2) gene expression. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent inhibition of forskolin-induced mRNA by protein kinase A inhibitor. Electrophoretic mobility shift analysis of nuclear proteins from forskolin-treated and db-cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oligonucleotides from the rat promoter revealed greater binding than in control VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, also blocked the binding of nuclear C/EBP to the C/EBP site of the rat promoter induced by db-cAMP and forskolin. Binding was unchanged with the use of CRE consensus oligonucleotides. Antibodies revealed the specific formation of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP-ss and -delta antibodies. Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat copies of the C/EBP element from the rat sPLA(2) promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression vector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP. H89 inhibited this activations. We therefore conclude that the increases in sPLA(2) mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-ss and -delta acting through a protein kinase A signaling pathway.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Fosfolipases A/genética , Sulfonamidas , Fatores de Transcrição , Animais , Bucladesina/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/farmacologia , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/farmacologia , Células Cultivadas , Colforsina/farmacologia , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Isoquinolinas/farmacologia , Luciferases/genética , Masculino , Músculo Liso Vascular/enzimologia , Oligonucleotídeos/farmacologia , Fosfolipases A/biossíntese , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
Secreted phospholipases A(2) is a family of small molecular weight and calcium-dependent enzymes of which the members list is presently growing. Among these enzymes, the synovial type IIA and the type V phospholipases A(2) are involved in inflammation. Although their actual mechanism is still a subject of debate, new therapeutic strategies can result from the knowledge of the regulations of their gene expression. The human genes of the type IIA and type V phospholipases A(2) are located on the chromosome 1 at close positions and transcribed in reverse orientations. These genes can therefore be regulated by common elements but only the regulation of the type IIA phospholipase A(2) gene expression has been extensively studied. Pro-inflammatory cytokines upregulate while the growth factors downregulate the type IIA phospholipase A(2) gene expression. Interleukin-6 and interleukin-1beta exert their effects at least partially at the transcriptional level. The transcriptional regulation of the type IIA phospholipase A(2) gene is cell- and species-specific. The activity of the human promoter is controlled by the CAAT-enhancer binding protein (C/EBP) factors while that of the rat promoter is regulated by nuclear factor kappaB (NF-kappaB) and C/EBPs. Furthermore, the human promoter is constitutively repressed in hepatocytes by single strand DNA binding proteins whose effects are relieved by C/EBP factors while the glucocorticoid receptor interacts with C/EBPs in chondrocytes to achieve full basal and interleukin-1beta-stimulated transcription activity. Other factors like CTF/NF1 and Sp1 might be involved in the regulation of both the rat and human promoter. Peroxisome proliferator-activated receptors could contribute to the stimulation of the rat promoter by NF-kappaB in vascular smooth muscle cells. The study of the coactivators and coinhibitors associated to these transcription factors will give a better understanding of the diversity and complexity of the transcriptional regulations of the type IIA phospholipase A(2) gene.
Assuntos
Regulação Enzimológica da Expressão Gênica , Inflamação/enzimologia , Fosfolipases A/genética , Animais , Sequência de Bases , Sítios de Ligação , Citocinas/metabolismo , Substâncias de Crescimento/metabolismo , Humanos , Dados de Sequência Molecular , Fosfolipases A/metabolismo , Regiões Promotoras Genéticas , Alinhamento de Sequência , Transdução de Sinais , TATA Box , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
L-type pyruvate kinase gene expression is modulated by hormonal and nutritional conditions. Here, we show by transient transfections in hepatocytes in primary culture that both the glucose response element and the contiguous hepatocyte nuclear factor 4 (HNF4) binding site (L3) of the promoter were negative cyclic AMP (cAMP) response elements and that cAMP-dependent inhibition through L3 requires HNF4 binding. Another HNF4 binding site-dependent construct was also inhibited by cAMP. However, HNF4 mutants whose putative PKA-dependent phosphorylation sites have been mutated still conferred cAMP-sensitive transactivation of a L3-dependent reporter gene. Overexpression of the CREB binding protein (CBP) increased the HNF4-dependent transactivation but this effect remained sensitive to cAMP inhibition.
Assuntos
AMP Cíclico/fisiologia , Proteínas de Ligação a DNA , Regiões Promotoras Genéticas , Piruvato Quinase/genética , Animais , Sítios de Ligação , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Fator 4 Nuclear de Hepatócito , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Elementos de Resposta , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Glucose-regulated transcription of the L-type pyruvate kinase (L-PK) gene is mediated through its glucose response element (GlRE/L4 box) composed of two degenerated E-boxes. Upstream stimulatory factor (USF) is a component of the transcriptional glucose response complex built up on the GlRE. Cooperation of the GlRE with the contiguous binding site (L3 box) for the orphan nuclear receptor hepatocyte nuclear factor 4 (HNF4) has also been suggested. We compared by transient transfection assays the effects of USF2a and other basic helix-loop-helix leucine zipper (bHLH-LZ) factors (TFE3, c-Myc, SREBP/ADD1) on the activity and glucose responsiveness of a minimal L-PK promoter directed by oligomerized glucose response units (L4L3 boxes). We found that: (i) although USF2a is intrinsically a moderate transcriptional activator, it has a strong stimulatory effect on the activity of the L4L3-based reporter construct in hepatocyte-derived cells and interferes with the glucose responsiveness; (ii) despite its potent ability as a transactivator, TFE3 alone is barely active on the GlRE in hepatocyte-derived cells; (iii) TFE3 as USF2a acts synergistically with HNF4 and abolishes glucose responsiveness of the promoter when overexpressed; (iv) in contrast, overexpression of HNF4 alone stimulates activity of the promoter without interfering with glucose responsiveness; (v) SREBP/ADD1 has a very weak activity on the L4L3 elements, only detectable in the presence of HNF4, and c-Myc does not interact with the GIRE of the L-PK promoter. Our studies indicate that different bHLH-LZ transcription factors known to recognize CACGTG-type E-boxes are not equivalent in acting through the L-PK glucose response element, with USF proteins being especially efficient in hepatocyte-derived cells.
Assuntos
Proteínas de Ligação a DNA , Glucose/metabolismo , Sequências Hélice-Alça-Hélice , Zíper de Leucina , Piruvato Quinase/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Ligação Competitiva , Células COS , Linhagem Celular , Cloranfenicol O-Acetiltransferase , Sequência Consenso , Eletroforese em Gel de Poliacrilamida , Fator 4 Nuclear de Hepatócito , Fígado , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , TransfecçãoRESUMO
USF1 and USF2 are ubiquitous transcription factors of the basic helix-loop-helix leucine zipper family. They form homo- and heterodimers and recognize a CACGTG motif termed E box. In the liver, USF binding activity is mainly accounted for by the USF1/USF2 heterodimer, which binds in vitro the glucose/carbohydrate response elements (GlRE/ChoRE) of glucose-responsive genes. To assign a physiological role of USFs in vivo, we have undertaken the disruption of USF1 and USF2 genes in mice. We present here the generation of USF1-deficient mice. In the liver of these mice, we demonstrate that USF2 remaining dimers can compensate for glucose responsiveness, even though the level of total USF binding activity is reduced by half as compared with wild type mice. The residual USF1 binding activity was similarly reduced in the previously reported USF2 -/- mice in which an impaired glucose responsiveness was observed (Vallet, V. S., Henrion, A. A., Bucchini, D., Casado, M. , Raymondjean, M., Kahn, A., and Vaulont, S. (1997) J. Biol. Chem. 272, 21944-21949). Taken together, these results clearly suggest differential transactivating efficiencies of USF1 and USF2 in promoting the glucose response. Furthermore, they support the view that USF2 is the functional transactivator of the glucose-responsive complex.
Assuntos
Regulação da Expressão Gênica/genética , Glucose/farmacologia , Fígado/fisiologia , Fatores de Transcrição/genética , Animais , Proteínas de Ligação a DNA/análise , Dimerização , Camundongos , Camundongos Knockout , Proteínas Nucleares , Conformação Proteica , Proteínas/genética , Piruvato Quinase/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genética , Fatores Estimuladores UpstreamRESUMO
Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNA-binding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein-when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Células COS , Carcinoma Hepatocelular , Extratos Celulares , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Privação de Alimentos/fisiologia , Fator 4 Nuclear de Hepatócito , Humanos , Fígado/enzimologia , Fígado/metabolismo , Neoplasias Hepáticas , Dados de Sequência Molecular , Mutação , Fosfoproteínas/genética , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Ratos , Ratos Wistar , Serina/metabolismo , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Upstream stimulatory factors (USF) 1 and 2 belong to the Myc family of transcription factors characterized by a basic/helix loop helix/leucine zipper domain responsible for dimerization and DNA binding. These ubiquitous factors form homo- and heterodimers and recognize in vitro a CACGTG core sequence termed E box. Through binding to E boxes of target genes, USF factors have been demonstrated to activate gene transcription and to enhance expression of some genes in response to various stimuli. In particular, in the liver USF1 and USF2 have been shown to bind in vitro glucose/carbohydrate response elements of glycolytic and lipogenic genes and have been proposed, from ex vivo experiments, to be involved in their transcriptional activation by glucose. However, the direct involvement of these factors in gene expression and nutrient gene regulation in vivo has not yet been demonstrated. Therefore, to gain insight into the specific role of USF1 and USF2 in vivo, and in particular to determine whether the USF products are required for the response of genes to glucose, we have created, by homologous recombination, USF2 -/- mice. In this paper, we provide the first evidence that USF2 proteins are required in vivo for a normal transcriptional response of L-type pyruvate kinase and Spot 14 genes to glucose in the liver.
Assuntos
Expressão Gênica , Glucose/metabolismo , Sequências Hélice-Alça-Hélice , Fígado/metabolismo , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ligação a DNA/fisiologia , Carboidratos da Dieta/farmacologia , Dimerização , Marcação de Genes , Fígado/embriologia , Camundongos , Camundongos Knockout , Proteínas Nucleares , Regiões Promotoras Genéticas , Proteínas/genética , Piruvato Quinase/genética , RNA Mensageiro/metabolismo , Fatores Estimuladores UpstreamRESUMO
Ubiquitous upstream stimulatory factors (USF1, USF2a and USF2b) are members of the basic-helix-loop-helix-leucine-zipper family of transcription factors that have been shown to be involved in the transcriptional response of the L-type pyruvate kinase (L-PK) gene to glucose. To understand the mechanisms of action of the USF2 isoforms, we initiated a series of co-transfection assays with deletion mutants and Ga14-USF2 fusions. The transactivating efficiency of the different native and mutant factors was determined at similar DNA binding activity. We found that: (i) exons 3- and 5-encoded regions are activation domains, (ii) a modulator domain encoded by exon 4 could be necessary to their additive action, (iii) a hexapeptide encoded by the first 5' codons of exon 6 is indispensable for transmitting activation due to both exon 3- and exon 5-encoded domains to the transcriptional machinery. Therefore, USF2 presents a modular structure and mediates transcriptional activation thanks to two non-autonomous activation domains dependent on an auxiliary peptide for expressing their activating potential.
Assuntos
Proteínas de Ligação a DNA , Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sequência de Bases , Primers do DNA , Células HeLa , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/química , Fatores Estimuladores UpstreamRESUMO
Upstream stimulatory factors (USF/MLTF) belong to the c-myc family of transcription factors. Through binding to target DNA as dimers, the ubiquitous USF proteins regulate a variety of genes. USF proteins are encoded by two genes, USF1 and USF2. Protein sequences of USF1 and 2 are highly homologous across species, suggesting functional conservation. To determine whether the genomic organization was conserved between USF1 and USF2, we isolated the murine USF1 gene and characterized its genomic structure. Both genes are similarly organized in 10 exons spanning over 10 kbp. By the 5'-rapid amplification of cDNA ends and S1 nuclease mapping methods, exon 1 was defined and the transcription initiation sites were mapped. The sequence of 8 kb of the gene, including 1.75 kb of 5'-flanking DNA, was determined. The promoter region is GC rich and lacks a typical TATA or CCAAT element. Strikingly, a comparison of the murine and human untranslated sequences reveals regions that exhibit greater than 73% sequence identity. A genomic alignment of the dimerization and DNA binding domains is presented for five genes of the c-myc family, suggesting a hypothetical common ancestor gene.
Assuntos
Proteínas de Ligação a DNA , Genes myc , Família Multigênica , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Éxons , Regulação da Expressão Gênica , Sequências Hélice-Alça-Hélice , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-myc/biossíntese , Técnica de Amplificação ao Acaso de DNA Polimórfico , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Fatores Estimuladores UpstreamRESUMO
The calbindin-D9K (CaBP9k) gene is mainly expressed in differentiated duodenal epithelial cells and is used as a model for studying the molecular mechanisms of intestine-specific transcription. The gene has been cloned, two major DNase-I-hypersensitive sites in the duodenum have been described, and a vitamin-D-response element has been identified. We have now analysed the transcription factors and regulatory sequences involved in the transcription of the CaBP9k gene in the intestine in ex vivo and in vitro experiments. Transfection experiments in intestinal (CaCo-2) and non-intestinal (HeLa) cell lines defined two regions in the 5'-flanking sequences of the rat CaBP9k gene. A minimal proximal region (-117 to +20) promoted transcription in both intestinal expressing and non-expressing cell lines. Tissue specificity was conferred by the sequences situated further upstream, which are responsible for complete repression in the non-intestinal cells. Intestinal transcription was specified by the proximal region, containing a specialized TATA box, and a distal region, which contains a previously described intestinal DNase-I-hypersensitive site. In vitro DNase I footprinting, electrophoretic mobility shift assays and antibody supershift assays were used to examine the factors bound to the proximal promoter region (-800 to +80 bp). Rat duodenal nuclear extracts protected 12 sites. Some of them appear to be binding sites for ubiquitous (nuclear factor 1) or hepatic-enriched sites (hepatocyte nuclear factors 1 and 4, enhancer binding protein alpha and beta factors. DNA binding studies and transfection experiments indicated that an intestine-specific transcription factor, caudal homeobox-2, binds to the TATA box of the rat CaBP9k gene. These data contribute to our understanding of the control of the intestinal transcription of the CaBP9k gene and demonstrate that several trans-acting factors, other than the vitamin D receptor, may be factors for intestine-specific CaBP9k gene expression.
Assuntos
Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/metabolismo , Sequências Reguladoras de Ácido Nucleico , Proteína G de Ligação ao Cálcio S100/biossíntese , TATA Box , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Fator de Transcrição CDX2 , Calbindinas , Núcleo Celular/metabolismo , Neoplasias do Colo , Sequência Consenso , Pegada de DNA , Desoxirribonuclease I , Duodeno/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas , Ratos , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Proteína G de Ligação ao Cálcio S100/genética , Homologia de Sequência do Ácido Nucleico , Transativadores , Transfecção , Células Tumorais CultivadasRESUMO
The ubiquitous upstream stimulatory factor (USF) transcription factors encoded by two distinct genes (USF1 and USF2) exist under the form of various dimers able to bind E-boxes. We report the molecular cloning and functional characterization of USF2 isoforms, corresponding to a 44-kDa subunit, USF2a, and a new 38-kDa subunit, USF2b, generated by differential splicing. Using specific anti-USF antibodies, we define the different binding complexes in various nuclear extracts. In vivo, the USF1/USF2a heterodimer represents over 66% of the USF binding activity whereas the USF1 and USF2a homodimers represent less than 10%, which strongly suggests an in vivo preferential association in heterodimers. In particular, an USF1/USF2b heterodimer accounted for almost 15% of the USF species in some cells. The preferential heterodimerization of USF subunits was reproduced ex vivo, while the in vitro association of cotranslated subunits, or recombinant USF proteins, appeared to be random. In transiently transfected HeLa or hepatoma cells, USF2a and USF1 homodimers transactivated a minimal promoter with similar efficiency, whereas USF2b, which lacks an internal 67-amino acid domain, was a poor transactivator. Additionally, USF2b was an efficient as USF1 and USF2a homodimers in transactivating the liver-specific pyruvate kinase gene promoter.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma Hepatocelular , Linhagem Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , Primers do DNA , DNA Complementar , Biblioteca Gênica , Variação Genética , Células HeLa , Sequências Hélice-Alça-Hélice , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Neoplasias Hepáticas , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/química , Transfecção , Células Tumorais Cultivadas , Fatores Estimuladores UpstreamRESUMO
More than 400 sequence alterations have been identified in the whole coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene corresponding to the 27 exons and their exon-intron boundaries. However, in some CF chromosomes, no mutation has yet been detected. In such cases, we have explored the promoter and the sequence up to position -1000 from the cap site, by using denaturing gradient gel electrophoresis. This study concerning 35 CF chromosomes has allowed us to identify three novel sequence variations located in the 5' upstream region of the gene. The T to G substitution located at position -895 from the cap site could be considered as a polymorphic variation. The second substitution (C to T at position -816) has been detected on only one CF chromosome, but does not concern a regulatory DNA element previously described. Conversely, the third substitution (a T to G substitution at position -741 from the cap site) is located at a potential AP-1 binding site. We have investigated, by electrophoretic mobility shift assay, the ability of this region to bind nuclear factors. We have found that the normal sequence between -740/-745 does not bind either the AP-1 transcription factor or AP-1 related proteins, and that the T to G-741 mutated sequence exhibits an abnormal binding pattern suggesting the possible deleterious effect of still unknown negative trans-acting factors.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Sequências Reguladoras de Ácido Nucleico/genética , Sequência de Bases , Regulador de Condutância Transmembrana em Fibrose Cística , Humanos , Dados de Sequência Molecular , Mutação Puntual , Ligação ProteicaRESUMO
We have shown previously that fatty acid synthase (FAS) gene expression is positively regulated by glucose in rat adipose tissue and liver. In the present study, we have identified in the first intron of the gene a sequence closely related to known glucose-responsive elements such as in the L-pyruvate kinase and S14 genes, including a putative upstream stimulatory factor/major late transcription factor (USF/MLTF) binding site (E-box) (+ 292 nt to + 297 nt). Location of this sequence corresponds to a site of hypersensitivity to DNase I which is present in the liver but not in the spleen. Moreover, using this information from a preliminary report of the present work, others have shown that a + 283 nt to + 303 nt sequence of the FAS gene can confer glucose responsiveness to a heterologous promoter. The protein binding to this region has been investigated in vitro by a combination of DNase I footprinting and gel-retardation experiments with synthetic oligonucleotides and known nuclear proteins. DNase I footprinting experiments using a + 161 nt to + 405 nt fragment of the FAS gene demonstrate that a region from + 290 nt to + 316 nt is protected by nuclear extracts from liver and spleen. This region binds two ubiquitous nuclear factors, USF/MLTF and the CAAT-binding transcription factor/nuclear factor 1 (CTF/NF1). Binding of these factors is similar in nuclear extracts from liver which does or does not express the FAS gene as observed for glucose-responsive elements in the L-pyruvate kinase and S14 genes. This suggests a posttranslational modification of a factor of the complex after glucose stimulation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/farmacologia , Ácido Graxo Sintases/genética , Regulação Enzimológica da Expressão Gênica , Proteínas Nucleares/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Genes , Dados de Sequência Molecular , RNA Mensageiro/genética , RatosRESUMO
The rat L-type pyruvate kinase gene is transcribed either from promoter L in the liver or promoter L' in erythroid cells. We have now cloned and functionally characterized an erythroid-specific enhancer, mapped in the fetal liver as hypersensitive site B (HSSB) at 3.7 kilobases upstream from the promoter L'. Protein-DNA interactions were examined in the 200-base pair core of the site by in vivo footprinting experiments. In the fetal liver, footprints were revealed at multiple GATA and CACC/GT motifs, whose association is the hallmark of erythroid-specific regulatory sequences. Functional analysis of the HSSB element in transgenic mice revealed properties of a cell-restricted enhancer. Indeed, this element was able to activate the linked ubiquitous herpes simplex virus thymidine kinase promoter in erythroid tissues. The activation was also observed in a variety of nonerythroid tissues known to synthesize GATA-binding factors. In the context of L'-PK transgenes, HSSB was not needed for an erythroid-specific activation of the L' promoter, while it was required to stimulate the L' promoter activity to a proper level. Finally, HSSB cannot be replaced by strong ubiquitous viral or cellular enhancers, suggesting a preferential interaction of the HSSB region with the L' promoter.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Fígado/enzimologia , Regiões Promotoras Genéticas , Piruvato Quinase/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Desoxirribonuclease I , Fatores de Ligação de DNA Eritroide Específicos , Feto , Biblioteca Genômica , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Piruvato Quinase/biossíntese , Ratos , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Transcrição Gênica , Dedos de ZincoRESUMO
L-type pyruvate kinase (L-PK) gene transcription is induced by glucose through its glucose response element (GlRE) composed of two degenerated E boxes able to bind in vitro ubiquitous upstream stimulator factor (USF) proteins. Here we demonstrate in vivo, by transient transfections in hepatoma cells, that (i) native USF proteins synthesized from expression vectors can act as transactivators of the L-PK promoter via the GlRE, stimulating transcription without glucose and, therefore, decreasing the glucose responsiveness of the promoter; (ii) expression of the truncated USF proteins, able to bind the GlRE but devoid of the NH2-terminal activation domain, represses the activation of the L-PK promoter by glucose; and (iii) a similar repression of the glucose effect is observed upon expression of mutant USF proteins devoid of the basic DNA binding domain, able to dimerize with endogenous USF but not to bind the GlRE. We conclude that USF proteins are components of the transcriptional glucose response complex assembled on the L-PK gene promoter.
Assuntos
Proteínas de Ligação a DNA , Glucose/metabolismo , Regiões Promotoras Genéticas , Piruvato Quinase/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Mutação , Oligodesoxirribonucleotídeos , Piruvato Quinase/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Células Tumorais Cultivadas , Fatores Estimuladores UpstreamRESUMO
The ubiquitously expressed upstream stimulatory factor (USF) involved in the transcription of a wide variety of cellular genes is defined as dimers of c-myc-related proteins, composed of a basic helix-loop-helix/leucine zipper region. The USF family consists of different members that split into two groups: MLTF or USF1 and USF2 or FIP. We present here evidence that USF1 and USF2 are distinct closely related genes in human, rat, and mouse. Based on the recent cloning of rat and human new cDNAs, we have isolated genomic clones encompassing the murine USF2 gene, which consists of at least 10 exons spanning a minimum of 10 kb of genomic DNA. Unexpectedly, the organization of USF2 appears very split up by introns (0.08 to over 6 kb in size), compared to the myc gene structure. The entire gene (but the larger intron) and 1.6 kb of the 5' flanking region were sequenced. This 5' flanking region is GC-rich, contains several putative transcription binding sites, and has no apparent TATA box. Gene mapping of murine USF2 and USF1 has been determined by in situ hybridization, indicating the localization of USF2 on chromosome 7 and of USF1 on chromosomes 1 and 11.
Assuntos
Mapeamento Cromossômico , Proteínas de Ligação a DNA , Camundongos/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Éxons , Biblioteca Genômica , Sequências Hélice-Alça-Hélice , Humanos , Íntrons , Zíper de Leucina , Dados de Sequência Molecular , Ratos , Mapeamento por Restrição , Fatores Estimuladores UpstreamRESUMO
The L-pyruvate kinase (L-PK) gene is slightly active in normal and tumoral endocrine pancreatic tissues while, in vivo, this gene is not transcribed in the exocrine pancreas. Nevertheless, the L-PK gene is re-expressed at a very low level in cultured 266.6 cells derived from an exocrine pancreas carcinoma. The L-PK gene is early activated in endodermal tissues, e.g. yolk sac and primitive intestine; it remains transcribed in fetal pancreas. In adult, L-PK gene expression is restricted to some endocrine cells. Hepatocyte nuclear factor (HNF) 1 and HNF4 are the main tissue-restricted transcription factors involved in tissue-specific expression of the L-PK gene. HNF1 concentration is similar in liver and all pancreatic cells. HNF4 concentration is high in liver, much lower in islets of Langerhans, endocrine pancreatic tumors, and cultured insulinoma cells, and is scarcely detectable in adult exocrine pancreas. This distribution of HNF4 parallels the expression of the L-PK gene. In vivo footprinting experiments show that the HNF1 binding site is similarly occupied in both adult liver and adult pancreas, in which this gene is practically inactive. In this latter tissue, however, the HNF4 binding site is differently occupied with respect to the liver. Since the chromatin structure remains open around the L-PK promoter in pancreas, the L-PK gene can probably be re-expressed under certain circumstances, for instance in cancerous pancreatic cells.