RESUMO
We previously reported that the activation of the M promoter of the human choline acetyltransferase (ChAT) gene by butyrate and trapoxin in transfected CHP126 cells is blocked by PD98059, a specific mitogen-activated protein kinase kinase (MEK) inhibitor (E. Espinos and M. J. Weber, Mol. Brain Res. 56:118-124, 1998). We now report that the transcriptional effects of histone deacetylase inhibitors are mediated by an H7-sensitive serine/threonine protein kinase. Activation of the ChAT promoter by butyrate and trapoxin was blocked by 50 microM H7 in both transient- and stable-transfection assays. Overexpression of p300, a coactivator protein endowed with histone acetyltransferase activity, stimulated the ChAT promoter and had a synergistic effect on butyrate treatment. These effects were blocked by H7 and by overexpressed adenovirus E1A 12S protein. Moreover, both H7 and PD98059 suppressed the activation of the Rous sarcoma virus (RSV) and simian virus 40 promoters by butyrate in transfection experiments. Similarly, the induction of the cellular histone H1(0) gene by butyrate in CHP126 cells was blocked by H7 and by PD98059. Previous data (L. Cuisset, L. Tichonicky, P. Jaffray, and M. Delpech, J. Biol. Chem. 272:24148-24153, 1997) showed that the induction of the H1(0) gene by butyrate is blocked by okadaic acid, an inhibitor of protein phosphatases. We now show that the activation of the ChAT and RSV promoters by butyrate in transfected CHP126 cells is also blocked by 200 nM okadaic acid. Western blotting and in vivo metabolic labeling experiments showed that butyrate has a biphasic effect on histone H3 phosphorylation, i.e., depression for up to 16 h followed by stimulation. The data thus strongly suggest that the transcriptional effects of histone deacetylase inhibitors are mediated through the activation of MEK1 and of an H7-sensitive protein kinase in addition to protein phosphatases.
Assuntos
Regulação da Expressão Gênica/genética , Peptídeos , Proteínas Serina-Treonina Quinases/genética , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Acetilação , Acetiltransferases/genética , Proteínas E1A de Adenovirus/genética , Antibacterianos/farmacologia , Butiratos/farmacologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Colina O-Acetiltransferase/genética , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Genes Reporter/genética , Histona Acetiltransferases , Inibidores de Histona Desacetilases , Histonas/genética , Humanos , Ácido Okadáico/farmacologia , Fosforilação , Regiões Promotoras Genéticas/genética , Fatores de Transcrição , Ativação Transcricional/efeitos dos fármacos , Transfecção , Fatores de Transcrição de p300-CBPRESUMO
We have cloned the 5' region of human aromatic L-amino acid decarboxylase (AADC) gene in a cosmid and an overlapping lambda clone, and sequenced the first five exons. A 61 base pair (bp) non-coding, first exon containing for the 5' end of a human pheochromocytoma AADC cDNA was localized 16 kb upstream of exon 2, in which translation is initiated. The transcription start site was localized by RNAse mapping, primer extension and reverse transcription-PCR. The non-conventional cap site was preceded by a modified TATA box at position -29. A strong promoter was characterized in the 560 bp region upstream of the cap site by linkage to the reporter gene LacZ, and transfection in human neuroblastoma SK-N-BE and SK-N-BE-K2 cells. Using a series of constructs bearing a varying length of 5' flanking region, three positive regulatory elements have been localized in the -560 to -394, -244 to -200 and -147 to -1 regions. Negative regulatory elements were localized in the -9000 to -560 and -394 to -316 regions. Surprisingly, constructs comprising all or the major part of intron 1 were inactive, suggesting the presence of a silencer in the first intron, or incorrect splicing events. The construct containing 560 bp of 5' flanking sequence did not express in human cholinergic neuroepithelioma cells MC-I-XC, and in three non-neuronal cell lines which displayed high AADC activities: human pancreatic carcinoma cells AsPC-1, rat insulinoma cells RINm5F and mouse anterior pituitary cells AtT20. These data suggest that we have identified a neuron-specific AADC promoter. An extensive search for a second promoter responsible for AADC gene expression in non-neuronal cells only gave negative results.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Neurônios/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
The neuronal promoter of human aromatic l-amino acid decarboxylase gene has been analysed to elucidate the mechanisms of neuron type-specific expression. The (-560/+92) promoter segment was sufficient to direct luciferase expression at a higher level in SK-N-BE neuroblastoma cells, than in CHP126 neuroepithelia, HepG2 hepatoma or SK-Hep1 epithelioma cells. Deletions experiments showed that this segment contained a neuronal-specific (element T1) and a SK-N-BE-specific (element N1) cis-activating sequences. Element T1 (-72/-36) bound Sp1 and NF-Y proteins, and unidentified neuronal-specific factors. Element N1 (-102/-72) bound cell-specific factors, identified as HNF-3, N-Oct-3/Brn-2 and N-Oct-2. HNF-3 proteins recognized the sequence TCAGTAAATA that matches the consensus motif. Oct-1, N-Oct-2 and N-Oct-3 bound the AAATAATGC sequence that overlaps the HNF-3 binding site. In addition, we show that the HNF-3 binding sites from aldolase C and HNF-3beta gene promoters also bind N-Oct-2 and N-Oct-3 proteins. These data suggest a functional interplay of winged helix/forkhead and POU-domain transcription factors on a variety of neuronal gene promoters.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/genética , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Alça-Hélice , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Células COS , Proteínas de Ligação a DNA/fisiologia , Fator 3-alfa Nuclear de Hepatócito , Fator 3-beta Nuclear de Hepatócito , Fator 3-gama Nuclear de Hepatócito , Proteínas de Homeodomínio , Humanos , Neurônios/citologia , Proteínas Nucleares/fisiologia , Fatores do Domínio POU , Fatores de Transcrição/fisiologia , Células Tumorais CultivadasRESUMO
The promoter regions of human choline acetyltransferase (ChAT) and aromatic L-amino acid decarboxylase (AADC) genes have been analyzed by transient transfection assays. AADC gene is transcribed from two alternative noncoding first exons, 1N and 1NN, expressed in pheochomocytoma and hepatoma cells, respectively. 5' flanking sequences of exon 1 N (from 9000 to 147 bp) display promoter activity in SK-N-BE neuroblastoma cells, but not in MC-I-XC cholinergic neuroepithelioma cells, and in AADC-rich non-neuronal cells. On the contrary, 5' flanking sequences of exon 1 NN (from 1117 to 119 bp) display high promoter activity in human hepatoma cells HepG2, but not in SK-N-BE cells, suggesting high degrees of specificity of promoters N and NN for AADC-expressing neuronal and non-neuronal cells, respectively. Preliminary evidence suggests that leukemia inhibitory factor suppresses the activity of the neuronal promoter in cultured sympathetic neurons. Two alternative first exons, R and M, have been localized in human ChAT gene, and the corresponding promoters characterized in cholinergic PC12 and NG-108-15 cells, and in non-cholinergic neuro2A cells. Several positively or negatively acting cis elements have been localized in the two promoters, as well as a cAMP-inducible, enhancer-like element in the second intron. Among the various cell lines studied, there was no correlation between promoter activities and the expression of the endogenous ChAT gene, suggesting that the fine-tuning of ChAT gene expression is controlled by silencer elements which remain to be localized.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/genética , Colina O-Acetiltransferase/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Neurotransmissores/biossíntese , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Células Cultivadas , Éxons , Humanos , Dados de Sequência Molecular , Neurônios/metabolismo , RatosRESUMO
We have studied the effects of transforming growth factor beta 1 (TGF beta 1) on the survival of embryonic motoneurons in culture. For this purpose, E14 rat embryo motoneurons were purified to more than 90% homogeneity by cell sorting and cultured at low density on monolayers of cortex astrocytes. Subnanomolar concentrations of TGF beta 1 (40-500 pM) increased the survival of motoneurons 2-fold after 9-11 days in culture. The increase in choline acetyltransferase (ChAT) activity per culture caused by TGF beta 1 was attributable to its effects on survival. Comparable results were found with motoneurons cultured on lysed astrocytes, suggesting that the effects of the factor are not mediated by non-neuronal cells, but that motoneurons are a target for TGF beta 1.
Assuntos
Neurônios Motores/citologia , Medula Espinal/citologia , Fatores de Crescimento Transformadores/farmacologia , Fibras Adrenérgicas/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Neurônios Motores/efeitos dos fármacos , Ratos , Medula Espinal/efeitos dos fármacosRESUMO
Motoneurons from E14 rat embryos have been retrogradely labelled with a carbocyanine derivative and purified approximately 20-fold by density gradient centrifugation. For certain experiments, motoneurons have been purified to near homogeneity by cell sorting. These neurons were then cultured at low density on various substrata. Laminin stimulated neurite outgrowth of the identified motoneurons and stimulated choline acetyltransferase (ChAT) development by 2-fold; these cultures could not be maintained for more than 4 days. Motoneurons could be cultured for at least 7 days on monolayers of both living and lysed astrocytes from newborn cortex. The cells survived equally well on both substrata but neurite outgrowth was less elaborated on lysed astrocytes on which motoneurons displayed a mono- or bipolar, rather than a multipolar, morphology. Furthermore, ChAT developed at a 2-fold higher level on lysed, rather than living, newborn astrocytes. Such a difference was not observed using astrocytes from E14 rat embryos. In the latter case, both living and lysed astrocytes supported ChAT development at the same rate as lysed newborn astrocytes. Consequently, living astrocytes from newborn rat cortex are relatively less permissive for ChAT expression. On the other hand, both living and lysed astrocytes from newborn cortex induced ChAT expression by sympathetic neurons to the same extent. We propose that, in addition to retrograde factors from muscle origin, cell surface molecules expressed by embryonic astrocytes stimulate ChAT expression by motoneurons. Such molecules would be present in newborn astrocytes in an intracellular form and could be unmasked by cell lysis. Different cell surface molecules may induce ChAT activity in sympathetic neurons.
Assuntos
Astrócitos/citologia , Colina O-Acetiltransferase/metabolismo , Técnicas de Cultura/métodos , Regulação da Expressão Gênica , Neurônios Motores/enzimologia , Animais , Separação Celular , Córtex Cerebral/citologia , Embrião de Mamíferos , Neurônios Motores/citologia , Plásticos , RatosRESUMO
The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1 alpha) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the A1 gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 bp DNA fragment extending from -982 to -634 bp upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 alpha F1 gene and of several highly expressed plant genes.
Assuntos
Genes de Plantas , Família Multigênica , Fatores de Alongamento de Peptídeos/genética , Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Quimera , Clonagem Molecular , Éxons , Íntrons , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos , Regiões Promotoras Genéticas , Protoplastos/metabolismo , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Motoneurons from E14 rat embryos have been retrogradely labeled with the carbocyanine derivative dil and purified 12-fold by centrifugation on a density gradient made of Nycodenz, as assessed by the increase in CAT activity per cell and in the percentage of dil-labeled cells. A 20- to 36-fold purification was achieved by the microdissection of the labeled lumbar motor columns followed by density-gradient centrifugation. Motoneurons were then purified to near homogeneity by fluorescence-activated cell sorting. In certain experiments, motoneurons were purified by a novel cell-sorting procedure that only uses light-scatter parameters and does not necessitate retrograde labeling. In cultures of motoneurons enriched by centrifugation, muscle-conditioned media (CM) had only a marginal effect on the overall neuronal survival but increased up to 10-fold the number of labeled motoneurons surviving after 7 d in culture. CAT activity per culture was quantitatively stimulated to the same extent, suggesting that the increase in CAT activity caused by CM mostly reflected better survival of motoneurons. In cultures of motoneurons purified by cell sorting, CM increased both survival and CAT expression per motoneuron. We have partially purified 2 factors from CM that stimulate CAT activity per motoneuron 2- to 3-fold without affecting their survival. One of these factors is probably identical to the factor involved in the cholinergic/noradrenergic choice of sympathetic neurons (Fukada, 1985).
Assuntos
Colina O-Acetiltransferase/metabolismo , Neurônios Motores/enzimologia , Animais , Carbocianinas , Diferenciação Celular/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Centrifugação com Gradiente de Concentração , Meios de Cultura/análise , Meios de Cultura/farmacologia , Embrião de Mamíferos , Citometria de Fluxo , Corantes Fluorescentes , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Músculos/citologia , Ratos , Medula Espinal/citologia , Frações Subcelulares/enzimologiaRESUMO
Two overlapping cosmids containing the 5' end of human choline acetyltransferase (ChAT) gene have been cloned. Using heterologous probes, we localized two alternative first exons homologous to rodent ChAT exons R and M (Misawa et al.: J Biol Chem 267:20392-20399, 1992). The sequence of rodent exon N was not conserved in the human gene. Northern blot analysis of mRNA purified from the human neuroepithelioma cell lines LA-N2 and MC-I-XC revealed that both exons R and M were transcribed in mRNA species of 6.0 and 2.5 kb. Only the 6-kb species was detected with both R- and M-specific probes in the neuroepithelioma cell line CHP126. Reverse transcription-polymerase chain reaction (RT-PCR) analysis suggested that the major mRNA species in MC-I-XC and CHP126 cells contained the proximal part of exon M spliced to exon 1, which contains the alternative ACG initiation codon. RT-PCR also allowed the characterization of a mRNA species containing exon R spliced to exon 1, but no species containing both exon R and the distal part of exon M could be detected. RT-PCR was also used to evidence an alternative exon (tentatively numbered exon 8) in the coding sequence.
Assuntos
Colina O-Acetiltransferase/genética , Éxons/genética , Processamento Alternativo , Animais , Sequência de Bases , Southern Blotting , Células Cultivadas , Clonagem Molecular , Genes , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo GenéticoRESUMO
The molecular forms of acetylcholinesterase (AcChE) have been studied in primary cultures of newborn rat sympathetic neurons grown either in the absence (CM- cultures) or in the presence (CM+ cultures) of muscle conditioned medium. The cultures were treated with a mitotic poison to eliminate non-neuronal cells. CAT activity increased with time in culture 4- to 20-fold faster in CM+ than in CM- cultures. In agreement with previous experiments (J.P. Swerts, A. Le Van Thaï, A. Vigny, and M.J. Weber, 1983, Develop. Biol. 100, 1-11), AcChE activity developed at a 3-fold lower rate in CM+ than in CM- cultures. This deficit in AcChE activity in CM+ cultures resulted from a deficit in the number of enzyme molecules immunoprecipitable with an antiserum raised against rat brain AcChE. In both types of cultures, AcChE forms were separated by sucrose gradient sedimentation into three main peaks corresponding to the 16 S and 10 S forms and a mixture of the 6.5 and 4 S forms. In 3-day-old CM+ and CM- cultures, the 16 S form represented 2% of the total activity. After 12-26 days, the percentage of 16 S form raised to 15-30% in CM- cultures, but remained lower than 5% in CM+ cultures. This difference was also observed when AcChE molecular forms were analyzed in the presence of protease inhibitors. A similar result was obtained by comparing cultures grown with and without a macromolecular factor partially purified from conditioned medium. These results suggest that an inverse relationship exists between the presence of 16 S AcChE and the presence of cholinergic synapses in these cultures.
Assuntos
Acetilcolinesterase/metabolismo , Isoenzimas/metabolismo , Neurônios/enzimologia , Sistema Nervoso Simpático/enzimologia , Acetilcolinesterase/isolamento & purificação , Animais , Células Cultivadas , Colina O-Acetiltransferase/metabolismo , Isoenzimas/isolamento & purificação , Cinética , Músculos/enzimologia , Ratos , Fatores de TempoRESUMO
The enzymatic machinery for neurotransmitter synthesis and breakdown have been compared in sister cultures of newborn rat sympathetic neurons grown for 12-28 days either in the presence (CM+ cultures) or in the absence (CM- cultures) of a culture medium conditioned by rat skeletal muscle cells. Neuron numbers, total protein, and lactate dehydrogenase activities were identical in CM+ and CM- cultures. Choline acetyltransferase activity was 27- to 100-fold higher in homogenates of CM+ than CM- cultures, whereas acetylcholinesterase activity was 2.5-fold lower. The activities of tyrosine hydroxylase (TOH), DOPA decarboxylase, and dopamine beta-hydroxylase were all about twofold lower in homogenates from CM+ cultures. All these effects were also observed in homogenates of sympathetic neuron cultures grown with and without a macromolecular factor partially purified from CM (Weber, J. (1981). Biol. Chem. 256, 3447-3453.). Experiments of mixing homogenates from CM+ and CM- cultures suggested that the differences in each of the enzyme activities did not result from differences in the concentrations of hypothetical reversible enzyme activators and/or inhibitors. In addition, the deficit in TOH activity in CM+ cultures resulted from a decrease in the enzymatic Vmax with no significant variation in the apparent Km's for the substrate and the cofactor. An identical decrease in the Vmax was observed if TOH was assayed under phosphorylating or nonphosphorylating conditions, suggesting that this decrease did not result from differences in the state of enzyme phosphorylation. Immunoprecipitation curves of TOH activity by an anti-TOH antiserum were parallel when performed on homogenates from CM+ and CM- cultures, suggesting a difference in the number of enzyme molecules without detectable alteration of their kinetic properties.