RESUMO
We previously reported that the rat aldolase C 115 bp promoter is sufficient to ensure the brain specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice. We identify in a further reduced 84 bp promoter several putative binding sites for the transcriptional factors Sp1, USF, AP1, and AP2. Deletion or mutation of these partially overlapping binding sites results in inactivation of the cognate transgenes. Moreover, we show that the 115 bp sequence is able to direct bidirectional transcription in vivo but, surprisingly, transcriptional activity in the opposite direction is no more brain specific.
Assuntos
Frutose-Bifosfato Aldolase/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , Transgenes/genética , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Reporter/genética , Células HeLa , Humanos , Pulmão/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação/genética , Miocárdio/metabolismo , Especificidade de Órgãos , Ratos , TATA Box/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
The desmin gene encodes an intermediate filament protein that is present in skeletal, cardiac, and smooth muscle cells. This study shows that the 4-kb upstream region of the murine desmin promoter directs expression of a lacZ reporter gene throughout the heart from E7.5 and in skeletal muscle and vascular smooth muscle cells from E9. 5. The distal fragment (-4005/-2495) is active in arterial smooth muscle cells but not in venous smooth muscle cells or in the heart in vivo. It contains a CArG/octamer overlapping element (designated CArG4) that can bind the serum response factor (SRF) and an Oct-like factor. The desmin distal fragment can replace a SM22alpha regulatory region (-445/-126) that contains two CArG boxes, to cis-activate a minimal (-125/+65) SM22alpha promoter fragment in arterial smooth muscle cells of transgenic embryos. lacZ expression was abolished when mutations were introduced into the desmin CArG4 element that abolished the binding of SRF and/or Oct-like factor. These data suggest that a new type of combined CArG/octamer element plays a prominent role in the regulation of the desmin gene in arterial smooth muscle cells, and SRF and Oct-like factor could cooperate to drive specific expression in these cells.
Assuntos
Desmina/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Musculares/genética , Músculo Liso Vascular/metabolismo , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica/genética , Células 3T3 , Motivos de Aminoácidos , Animais , Sequência de Bases , Sistema Cardiovascular/embriologia , Sistema Cardiovascular/crescimento & desenvolvimento , Sistema Cardiovascular/metabolismo , Células Cultivadas , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Coração Fetal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes , Genes Reporter , Óperon Lac , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Dados de Sequência Molecular , Desenvolvimento Muscular , Proteínas Musculares/deficiência , Proteínas Musculares/fisiologia , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Músculo Liso Vascular/citologia , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Proteínas Nucleares/farmacologia , Fator de Resposta Sérica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transfecção , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
The promoter of the calbindin-D 9k (CaBP9k) gene, previously analyzed in transgenic mice, contains all of the information necessary for expression of a transgene similar to the endogenous gene and also for an appropriate response to vitamin D. In the present study we first investigated the role of a putative vitamin D-responsive element (9k/VDRE), located at nucleotides -489 to -445 on the rat CaBP9k promoter gene, using transgenic mice. As expected, the pattern of transgene expression in mice carrying this putative VDRE mutated in its whole promoter context was similar to that in mice bearing the wild-type sequence. These transgenic mice also responded to 1,25-dihydroxyvitamin D3 in the same way as those bearing the wild-type transgene and as those carrying a transgene with a large deletion (from -2894 to -117) eliminating the putative 9k/VDRE. Thus, the putative 9k/VDRE is not required for the control of rat CaBP9k gene expression by vitamin D in vivo. We also found that responsiveness to 1,25-dihydroxyvitamin D3 depends on the site at which the transgene is integrated into the host genome, in a tissue-specific manner. These data together with the fact that vitamin D-responsive sequences are present in a two-module region (from -3731 to -2894 and/or -117 to +365) and that this region does not contain any classical VDRE show that the CaBP9k gene is submitted to a non-conventional control by vitamin D.
Assuntos
Elementos de Resposta/efeitos dos fármacos , Proteína G de Ligação ao Cálcio S100/genética , Vitamina D/farmacologia , Animais , Ligação Competitiva , Calbindinas , Cloranfenicol O-Acetiltransferase/genética , DNA/genética , Dimerização , Duodeno/fisiologia , Rim/fisiologia , Pulmão/fisiologia , Camundongos , Camundongos Transgênicos/genética , Mutação , Ratos , Receptores de Calcitriol/química , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Elementos de Resposta/fisiologia , Receptores X de Retinoides , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transgenes/efeitos dos fármacosRESUMO
Na-G is a putative sodium (or cationic) channel expressed in neurons and glia of the PNS, in restricted neuronal subpopulations of the brain, and in several tissues outside the nervous system, like lung and adrenal medulla. To analyze the mechanisms underlying tissue-specific expression of this channel, we isolated the 5' region of the corresponding gene and show that Na-G mRNA transcription proceeds from a single promoter with multiple initiation sites. By transgenic mice studies, we demonstrate that 600 bp containing the Na-G proximal promoter region and the first exon are sufficient to drive the expression of a beta-galactosidase reporter gene in neurons of both CNS and PNS, whereas expression in Schwann cells depends on more remote DNA elements lying in the region between -6,500 and -1,050 bp upstream of the main transcription initiation sites. Crucial elements for lung-specific expression seem to be located in the region between -1,050 and -375 bp upstream of the promoter. Using in vivo footprint experiments, we demonstrate that several sites of the Na-G proximal promoter region are bound specifically by nuclear proteins in dorsal root ganglion neurons, as compared with nonexpressing hepatoma cells.
Assuntos
Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Sequências Reguladoras de Ácido Nucleico , Células de Schwann/metabolismo , Canais de Sódio/genética , Animais , Sequência de Bases , Sistema Nervoso Central/metabolismo , Pegada de DNA , DNA Complementar/genética , Éxons/genética , Gânglios Espinais/metabolismo , Genes Reporter , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Músculos/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neurônios Aferentes/metabolismo , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Sistema Nervoso Periférico/metabolismo , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes de Fusão/biossíntese , Canais de Sódio/biossíntese , Transcrição Gênica , Canais de Sódio Disparados por Voltagem , beta-Galactosidase/biossínteseRESUMO
Here, we investigated changes in apoptosis during tumor progression by analyzing the effect of coexpressing various antiapoptotic genes on the multistage process of c-myc-induced hepatocarcinogenesis in transgenic mice. Whereas continuous c-myc gene overexpression in the liver led to cellular hepatocarcinoma, the coexpression of the bcl-2 gene inhibited the emergence of liver tumors, by inhibiting a pretumoral phase characterized by increased proliferation and apoptosis. This antioncogenic effect was specific to Bcl-2 and was not shared by other antiapoptotic genes such as bcl-xL and a dominant negative form of p53. Thus, we have shown that Bcl-2 can have a tumor suppressor effect in vivo on c-myc-induced hepatocarcinogenesis during the emergence of neoplastic foci.
Assuntos
Apoptose , Genes bcl-2 , Genes myc , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Animais , Progressão da Doença , Genes p53 , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piruvato Quinase/genética , Proteína bcl-XRESUMO
Fas ligand (CD95L) and tumor necrosis factor-alpha (TNF-alpha) are pivotal inducers of hepatocyte apoptosis. Uncontrolled activation of these two systems is involved in several forms of liver injury. Although the broad antiapoptotic action of Bcl-2 and Bcl-xL has been clearly established in various apoptotic pathways, their ability to inhibit the Fas/CD95- and TNF-alpha-mediated apoptotic signal has remained controversial. We have demonstrated that the expression of BCL-2 in hepatocytes protects them against Fas-induced fulminant hepatitis in transgenic mice. The present study shows that transgenic mice overexpressing BCL-XL in hepatocytes are also protected from Fas-induced apoptosis in a dose-dependent manner. Bcl-xL and Bcl-2 were protective without any change in the level of endogenous Bcl-xL or Bax and inhibited hepatic caspase-3-like activity. In vivo injection of TNF-alpha caused massive apoptosis and death only when transcription was inhibited. Under these conditions, PK-BCL-XL mice were partially protected from liver injury and death but PK-BCL-2 mice were not. A similar differential protective effect of Bcl-xL and Bcl-2 transgenes was observed when Fas/CD95 was activated and transcription blocked. These results suggest that apoptosis triggered by activation of both Fas/CD95 and TNF-alpha receptors is to some extent counteracted by the transcription-dependent protective effects, which are essential for the antiapoptotic activity of Bcl-2 but not of Bcl-xL. Therefore, Bcl-xL and Bcl-2 appear to have different antiapoptotic effects in the liver whose characterization could facilitate their use to prevent the uncontrolled apoptosis of hepatocytes.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Inibidores de Caspase , Inibidores Enzimáticos/farmacologia , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos/genética , NF-kappa B/metabolismo , Piruvato Quinase/genética , Fator de Necrose Tumoral alfa/farmacologia , Proteína bcl-X , Receptor fas/farmacologia , Receptor fas/fisiologiaRESUMO
Mutations in the adenomatous polyposis coli gene or activating mutations in the beta-catenin gene itself are thought to be responsible for the excessive beta-catenin signaling involved in intestinal carcinogenesis. We generated transgenic mice that expressed large amounts of a NH2-terminally truncated mutant beta-catenin (deltaN131beta-catenin) in the intestine. These mice had multifocal dysplastic lesions in the small intestine, reminiscent of the early lesions observed in the mouse models of familial adenomatous polyposis. The number of apoptotic cells in the villi of these transgenic mice was 3-4-fold higher than in nontransgenic mice. Expression of the truncated beta-catenin mutant in the kidney led to the development of severe polycystic kidney disease. Our findings support the concept that deregulation of the beta-catenin signaling pathway is the major oncogenic consequence of adenomatous polyposis coli mutations in intestinal neoplasia.
Assuntos
Adenoma/genética , Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Intestinais/genética , Mutação , Transativadores , Polipose Adenomatosa do Colo/genética , Animais , Caderinas/genética , Enteropatias/genética , Camundongos , Camundongos Transgênicos , beta CateninaRESUMO
Myogenin, one of the MyoD family of proteins, is expressed early during somitogenesis and is required for myoblast fusion in vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a beta-galactosidase transgene during embryogenesis. We show here that mutation of one of the DNA motifs present in this region, the MEF3 motif, abolished correct expression of this beta-galactosidase transgene. We have found that the proteins that bind to the MEF3 site are homeoproteins of the Six/sine oculis family. Antibodies directed specifically against Six1 or Six4 proteins reveal that each of these proteins is present in the embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and transient transfection experiments confirm that Six1 and Six4 are able to transactivate a reporter gene containing MEF3 sites. Altogether these results establish Six homeoproteins as a family of transcription factors controlling muscle formation through activation of one of its key regulators, myogenin.
Assuntos
Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Músculo Esquelético/metabolismo , Miogenina/genética , Transativadores , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Conservada , Coração Fetal/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/embriologia , Mutagênese Sítio-Dirigida , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligodesoxirribonucleotídeos , Baço/metabolismo , Transfecção , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a 115-base pair (bp) promoter fragment was able to ensure the brain-specific expression of the chloramphenicol acetyltransferase (CAT) reporter gene in transgenic mice, but only at a low level (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). Here we show that in vivo activation of this promoter at a high level requires cooperation between an upstream 0.6-kilobase pair (kb) fragment and far upstream sequences. In the 0.6-kb region, a 28-bp DNA element is shown to include overlapping in vitro binding sites for POU domain regulatory proteins and for the Winged Helix hepatocyte nuclear factor-3beta factor. An hepatocyte nuclear factor-3beta-binding site previously described in the short proximal promoter fragment is also shown to interact in vitro with POU proteins, although with a lower affinity than the 28-bp motif. Additional binding sites for POU factors were detected in the upstream 0.6-kb sequences. Progressive deletion in this region resulted in decreased expression levels of the transgenes in mice, suggesting synergistic interactions between these multiple POU-binding sites. We propose that DNA elements characterized by a dual binding specificity for both POU domain and Winged Helix transcription factors could play an essential role in the brain-specific expression of the aldolase C gene and other neuronal genes.
Assuntos
Encéfalo/enzimologia , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sítios de Ligação , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/genética , Sequência Consenso , Metilação de DNA , Ativação Enzimática , Frutose-Bifosfato Aldolase/biossíntese , Camundongos , Camundongos Transgênicos , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
The calbindin-D9K gene encodes a vitamin D-induced calcium-binding protein that is expressed as a marker of small intestine differentiation. We have shown that 4580 base pairs of its 5' DNA regulatory region can target reporter transgene expression in the intestine and cause this transgene to respond like the endogenous gene to vitamin D active metabolite and that the homeoprotein Cdx2 is bound to the TATA box in the intestine. We now show that the 4580 base pairs construct confers a differentiated pattern of reporter transgene expression in the intestine and that cooperation between the proximal promoter and a distal element located in an opened chromatin structure is responsible for the intestinal expression and vitamin D responsiveness of the transgene. Gel shift and footprinting assays using duodenal nuclear extracts indicate that this distal element contains a Cdx2-binding site. Finally, a mutation in this distal Cdx2-binding site dramatically decreases intestinal expression in transgenic mice. This report, using an in vivo approach, demonstrates the crucial role of Cdx2 for the transcription of an intestinal gene.
Assuntos
Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/metabolismo , Regiões Promotoras Genéticas , Proteína G de Ligação ao Cálcio S100/genética , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Fator de Transcrição CDX2 , Calbindinas , Calcitriol/farmacologia , Cloranfenicol O-Acetiltransferase/genética , Colo , Desoxirribonuclease I , Duodeno , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Camundongos , Camundongos Transgênicos , Microvilosidades/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , Sequências Reguladoras de Ácido Nucleico , Proteína G de Ligação ao Cálcio S100/análise , Proteína G de Ligação ao Cálcio S100/biossíntese , Transativadores , Deficiência de Vitamina D/metabolismoRESUMO
The aldolase B gene is transcribed at a high level in the liver, kidney, and small intestine. This high level of gene expression results from cooperation between a weak but liver-specific promoter and an intronic activator. A deletional study of this activator present in the first intron allowed us to ascribe the maximal enhancer function to a 400-base pair (bp) fragment (+1916 to + 2329). This enhancer is highly liver-specific and enhances the activity of heterologous minimal promoters in a position and distance-independent fashion in transiently transfected Hep G2 hepatoma cells. The aldolase B enhancer is composed of two domains, a 200-bp module (Ba) inactive by itself but which synergizes with another 200-bp module (Bb) that alone retains 25% of the total enhancer activity. The Bb sequence is 76% homologous between human and rat genes and contains several binding sites for liver-enriched nuclear factors. By electrophoretic mobility shift assays, we demonstrated that elements 5 and 7 bind hepatic nuclear factor 1 (HNF1), whereas element 2 binds hepatic nuclear factor 4 (HNF4). A functional analysis of the enhancer whose elements have been mutated demonstrated that mutation of any of the HNF1 sites totally suppressed enhancer activity, whereas mutation of the HNF4-binding site reduced it by 80%.
Assuntos
Elementos Facilitadores Genéticos , Frutose-Bifosfato Aldolase/genética , Íntrons , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Fator 4 Nuclear de Hepatócito , Humanos , Camundongos , Camundongos Transgênicos , Mutagênese , Sondas de Oligonucleotídeos , Fosfoproteínas/metabolismo , Ratos , Fatores de Transcrição/metabolismoRESUMO
In order to identify regulatory elements that direct widespread in vivo expression of a linked gene, we have examined one of the human aldolase A alternative promoters, the ubiquitous pH promoter, which is active in most foetal and adult tissues. We have used the pH promoter region to drive expression of an heterologous CAT reporter gene in transgenic mice. We show that a short 820 bp pH promoter fragment is able to confer a ubiquitous and reproducible activity pattern on the CAT reporter gene in most of the transgenic lines analysed, with a particularly high level of expression in adult skeletal muscle. Activity of this transgene was detected from early embryonic stages. Therefore, this pH promoter region appears to be a powerful tool to direct ubiquitous and early expression of a transgene in vivo. Deletion analysis revealed that: (i) the region between -651 and -369 bp relative to the pH promoter transcription start site includes DNA elements capable of overriding effects of the surrounding chromatin at the integration site, (ii) the region between -285 and -211 bp is involved in pH promoter tissue-specific expression pattern in skeletal muscle and/or nervous tissues, (iii) the region located between -211 and -108 bp is necessary for its ubiquitous and muscle-predominant activity and (iv) the most proximal region downstream from -108 bp is still sufficient to confer an activity in brain and lung.
Assuntos
Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Músculo Esquelético/metabolismo , Regiões Promotoras Genéticas , Animais , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Genes Reporter , Humanos , Camundongos , Camundongos Transgênicos , TransgenesRESUMO
Transgenic mice expressing either human proinsulin cDNA or mutated proinsulin cDNA in the liver were created. The human proinsulin cDNA was mutated to generate a protein cleavable by the ubiquitous prohormone convertase furin, thus leading to mature insulin peptide. All transgenic lines expressed human C-peptide in the blood, whose level varied according to nutritional conditions. High performance liquid chromatography fractionation of mouse serum revealed that mutant proinsulin was effectively processed into mature insulin in vivo. This transgenic mouse model provides a useful tool for further prospects of gene therapy of insulin-dependent diabetes mellitus.
Assuntos
Regulação da Expressão Gênica , Insulina/genética , Fígado/metabolismo , Proinsulina/genética , Animais , Carboidratos/farmacologia , Humanos , Insulina/metabolismo , Camundongos , Camundongos Transgênicos , Mutagênese , Proinsulina/metabolismo , Processamento de Proteína Pós-Traducional , TransgenesRESUMO
Erythropoietin (Epo) is known to control the erythroid developmental program through various biologic activities including maintenance of viability, cell proliferation, and/or cell maturation. In vitro experiments showed massive apoptosis in cultures of Epo-deprived colony-forming unit-erythroid (CFU-E) progenitors, demonstrating the Epo requirement of late-stage erythroid progenitors for survival. Based on these data, a model has been proposed whereby from CFU-E to proerythroblast stages, Epo acts rather as a survival factor than a proliferating factor. To investigate the relationship between Epo dependence and apoptotic mechanisms, we generated transgenic mice expressing the antiapoptotic human bcl-2 gene product in erythroid progenitors. Transgenic animals developed without any evidence of erythropoietic disorders. In vitro studies showed that overexpression of bcl-2 in erythroid progenitors delayed, but did not prevent the loss of CFU-E from Epo-deprivation. By measuring burst-forming unit-erythroid (BFU-E) and CFU-E-derived colonies, an enhanced sensitivity to low levels of Epo was demonstrated in adult bone marrow of transgenic mice with respect to nontransgenic animals. No spontaneous erythroid colonies were, however, observed in vitro in the absence of the cytokine, indicating that overexpression of bcl-2 is not sufficient to induce by itself a complete erythroid differentiation. Taken together, our data indicate that targeted erythroid overexpression of bcl-2 fails to alter the normal erythropoietic development in vivo and that erythroid progenitors remain strictly dependent on Epo for their survival.
Assuntos
Apoptose , Células Precursoras Eritroides/citologia , Eritropoetina/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Animais , Células da Medula Óssea/efeitos dos fármacos , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Células Precursoras Eritroides/metabolismo , Eritropoetina/administração & dosagem , Eritropoetina/farmacologia , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Transgenes , Células Tumorais CultivadasRESUMO
Anti-Müllerian hormone (AMH) induces the regression of Müllerian ducts in the male foetus; it is secreted by prepubertal testicular Sertoli cells and repressed at puberty. Using an AMH promoter/Simian virus 40 (SV40) oncogene fusion gene, we generated transgenic mouse lines exhibiting heritable Sertoli cell tumorigenesis. One cell line, derived from an adult male, expressed mRNAs characteristic of mature Sertoli cells, but no AMH. Two other cell lines were obtained from pretumoral testes at 6.5 days. One was cloned to yield SMAT1, whose expression pattern was characteristic of prepubertal Sertoli cells, namely no transferrin and high SF-I and AMH expression. SMAT1 also secretes AMH protein into the culture medium and expresses the AMH receptor. To the best of our knowledge, this is the first Sertoli cell line stably expressing AMH and its receptor. Our results show that, in targeted oncogenesis, the timing of cell line derivation plays a critical role even when using a developmentally regulated promoter.
Assuntos
Glicoproteínas , Inibidores do Crescimento/metabolismo , Receptores de Peptídeos/metabolismo , Células de Sertoli/metabolismo , Hormônios Testiculares/metabolismo , Animais , Hormônio Antimülleriano , Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular , DNA Recombinante/genética , Feminino , Expressão Gênica , Inibidores do Crescimento/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Oncogenes/genética , Neoplasias Ovarianas/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , RNA Viral/análise , Receptores de Fatores de Crescimento Transformadores beta , Tumor de Células de Sertoli/genética , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/imunologia , Hormônios Testiculares/genética , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologia , Testículo/química , Células Tumorais CultivadasRESUMO
The rat Calbindin-D9K (CaBP9K) gene is mainly expressed in intestine, uterus, and lung and is regulated in a complex tissue-specific manner. To analyze the role of potential regulatory elements, previously defined by DNaseI hypersensivity, we made transgenic mice containing truncated rat CaBP9K fusion gene with simian virus 40 large T antigen and the chloramphenicol acetyltransferase as reporter genes. The transgenes contained CaBP9K promoter fragments with 5' end points at -4400, -1011, and -117 base pairs (bp), whereas the 3' end points was at +365 bp. Northern blot analysis of T antigen expression and chloramphenicol acetyltransferase enzyme-linked immunosorbent assay indicated that a positive element, probably the distal intestine-specific DNaseI HS, necessary to target the expression of the transgene in the intestine, is present between -4400 and -1011 bp. The cephalo-caudal gradient of expression of the transgene along the small intestine was similar to those of the endogenous gene, but an ectopic expression of the transgene was observed in the colon. The -1011 transgene was expressed in epithelial alveolar cells of the lung, in renal proximal tubule cells, and in uterine myometrium, as judged from immunocytochemical, histological, and Northern blot analyses. The shortest, -117 construct was only expressed in uterine myometrium, and it was under a strict estrogen dependence like the endogenous gene. Finally, responsiveness to vitamin D in the duodenum was observed with the largest, -4400 construct. Thus, different tissues utilize distinct cis-acting elements to direct and regulate the expression of the rat CaBP9K gene.
Assuntos
Dietilestilbestrol/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteína G de Ligação ao Cálcio S100/genética , Vitamina D/farmacologia , Animais , Sequência de Bases , Calbindinas , Cloranfenicol O-Acetiltransferase/genética , Primers do DNA , Feminino , Mucosa Intestinal/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Ratos , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Transgenes , Útero/metabolismoRESUMO
Expression in mice of transgenes directed by regulatory regions of the rat aldolase B gene requires the presence of a B element located in the first intron, while constructs devoid of this intronic enhancer are silent. Histo- and immunochemical staining of transgenic tissue sections showed that the longer transgene was expressed in the proximal tubular cells of the kidney, enterocytes located in small intestine villi and liver parenchymal cells. In the liver, a maximal expression was observed in perivenous hepatocytes, while the transgene was weakly active in periportal hepatocytes, which reproduced the pattern of functional zonation already reported for other glycolytic and gluconeogenic genes in the liver. We also established that the transgene retained the necessary elements for a correct chronological expression during development but was lacking elements necessary for activation by high carbohydrate diet. Instead, transgene expression was paradoxically stimulated in fasted animals, suggesting that the endogenous gene, which must be active under both glycolytic and gluconeogenic conditions, could possess distinct elements activating it in fasted as well as in carbohydrate-fed animals; the former element might be conserved in the transgene and the latter one might be lost.
Assuntos
Elementos Facilitadores Genéticos , Frutose-Bifosfato Aldolase/biossíntese , Regulação Enzimológica da Expressão Gênica , Íntrons , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Northern Blotting , Encéfalo/enzimologia , Cloranfenicol O-Acetiltransferase/biossíntese , Desenvolvimento Embrionário e Fetal , Feminino , Frutose-Bifosfato Aldolase/genética , Idade Gestacional , Gluconeogênese , Glicólise , Imuno-Histoquímica , Intestinos/enzimologia , Túbulos Renais Proximais/enzimologia , Fígado/citologia , Fígado/embriologia , Fígado/enzimologia , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Gravidez , Ratos , Proteínas Recombinantes/biossíntese , Baço/enzimologiaRESUMO
The L-type pyruvate kinase (L-PK) gene is regulated by diet and hormones and expressed at high levels in the hepatocytes, enterocytes, and proximal tubular cells of the kidney and at low levels in the endocrine pancreatic cells. Two regulatory regions have been shown to be important in transgenic mice to confer on a reporter gene a similar tissue-specific and diet-responsive expression: a proximal promoter fragment, with binding sites for the tissue-specific hepatocyte nuclear factors 1 and 4, and presence of the glucose-response element (GIRE) and a distal activator corresponding to a liver-specific hypersensitive site at -3000 bp with respect to the cap site. Although the proximal promoter is able to confer by itself tissue-specific expression on a reporter gene, its activity in vivo is strongly stimulated by the distal activator. To determine the possible role of the distal region on diet responsiveness and tissue specificity of the L-PK gene expression, we have created lines of transgenic mice in which the gene for SV40 T antigen (Tag) was directed by composite regulatory sequences consisting of the L-PK promoter and different enhancers: either the SV40 early enhancer (SV) or the H enhancer of the aldolase A gene (H). The induction of the composite H-PK/Tag and SV-PK/Tag transgenes by a carbohydrate-rich diet in the liver was similar to that of the endogenous L-PK gene. This suggests that in fasted mice the L-PK promoter, and especially the GIRE, is able to silence the activating influence of a strong viral enhancer such as the SV40 enhancer. The H-PK/Tag mice expressed the transgene similarly to the endogenous gene, except in the pancreas, where expression was practically undetectable. Consistently, whereas L-PK/Tag mice develop insulinomas, H-PK/Tag mice develop only hepatomas. In contrast, the transgene expression was partly aberrant in SV-PK/Tag mice. In addition to a normal activation of the transgene in the liver, a strong expression was also detected in the kidney medulla, whereas the transgene was practically silent in enterocytes. Finally, the effect of the distal region (-2070 to -3200) on an ubiquitous promoter was tested by ligating the distal L-PK gene fragment in front of a thymidine kinase/CAT transgene. Such a transgene was constantly expressed in the pancreas and, strikingly, in the brain. It appears, therefore, that the L-PK distal activator exhibits, by itself, a certain neuropancreatic specificity required in combination with the proximal promoter for L-PK gene expression in pancreas endocrine cells.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/genética , Piruvato Quinase/metabolismo , Ativação Transcricional/genética , Transgenes/genética , Animais , Antígenos Transformantes de Poliomavirus/análise , Antígenos Transformantes de Poliomavirus/genética , Núcleo Celular/química , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/genética , Dieta , Elementos Facilitadores Genéticos/genética , Jejum , Frutose-Bifosfato Aldolase/genética , Insulinoma , Neoplasias Hepáticas Experimentais , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Pâncreas/química , Neoplasias Pancreáticas , Piruvato Quinase/genética , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão , Vírus 40 dos Símios/imunologia , Timidina Quinase/genéticaRESUMO
Fas is an apoptosis-signalling cell surface antigen that has been shown to trigger cell death upon specific ligand or antibody binding. Treatment of mice with an anti-Fas antibody causes fulminant hepatic failure due to massive apoptosis. To test a putative protective effect of the anti-apoptotic Bcl-2 protein, transgenic mice were generated to express the human bcl-2 gene product in hepatocytes. Early onset of massive hepatic apoptosis leading to death was observed in all nontransgenic mice treated with an anti-Fas antibody. By contrast, hepatic apoptosis was delayed and dramatically reduced in transgenic animals, yielding a 93% survival rate. These results demonstrate that Bcl-2 is able to protect from in vivo Fas-mediated cytotoxicity, and could be of significance for preventing fulminant hepatic failure due to viral hepatitis in humans.
Assuntos
Anticorpos/toxicidade , Apoptose/fisiologia , Encefalopatia Hepática/prevenção & controle , Fígado/patologia , Proteínas Proto-Oncogênicas/biossíntese , Proto-Oncogenes , Receptor fas/fisiologia , Animais , Northern Blotting , Western Blotting , Proteínas de Ligação ao GTP/biossíntese , Encefalopatia Hepática/patologia , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Receptor fas/imunologiaRESUMO
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.