RESUMO
The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated and left ventricular noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, we have heart specifically interfered scox expression, the single Drosophila Sco orthologue. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reduction in COX activity and they undergo a metabolic switch from OXPHOS to glycolysis, mimicking the clinical features found in patients harbouring Sco mutations. The major cardiac defects observed are produced by a significant increase in apoptosis, which is dp53-dependent. Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans.
Assuntos
Apoptose , Cardiomiopatias/enzimologia , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Miocárdio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Drosophila/enzimologia , Drosophila/genética , Proteínas de Drosophila/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Proteína Supressora de Tumor p53/genéticaRESUMO
Fast and slow TnI are co-expressed in E11.5 embryos, and fast TnI is present from the very beginning of myogenesis. A novel green fluorescent protein (GFP) reporter mouse lines (FastTnI/GFP lines) that carry the primary and secondary enhancer elements of the mouse fast troponin I (fast TnI), in which reporter expression correlates precisely with distribution of the endogenous fTnI protein was generated. Using the FastTnI/GFP mouse model, we characterized the early myogenic events in mice, analyzing the migration of GFP+ myoblasts, and the formation of primary and secondary myotubes in transgenic embryos. Interestingly, we found that the two contractile fast and slow isoforms of TnI are expressed during the migration of myoblasts from the somites to the limbs and body wall, suggesting that both participate in these events. Since no sarcomeres are present in myoblasts, we speculate that the function of fast TnI in early myogenesis is, like Myosin and Tropomyosin, to participate in cell movement during the initial myogenic stages. genesis
Assuntos
Rastreamento de Células/métodos , Regulação da Expressão Gênica no Desenvolvimento , Mioblastos/metabolismo , Troponina I/genética , Animais , Extremidades/embriologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Modelos Animais , Desenvolvimento Muscular/genética , Miosinas/genética , Miosinas/metabolismo , Isoformas de Proteínas , Tropomiosina/genética , Tropomiosina/metabolismo , Troponina I/metabolismoRESUMO
Although tight quantitative control of gene expression is required to ensure that organs and tissues function correctly, the transcriptional mechanisms underlying this process still remain poorly understood. Here, we describe novel and evolutionary conserved secondary enhancers that are needed for the regulation of the expression of Troponin I genes. Secondary enhancers are silent when tested individually in electroporated muscles but interact with the primary enhancers and are required to precisely control the appropriate timing, the tissue and fibre specificity, and the quantitative expression of these genes during muscle differentiation. Synergism is completely dependent of the fully conserved MEF2 site present on the primary enhancers core of skeletal muscle Troponin I genes. Thus, while each of these paired enhancers has a different function, the concerted action of both is crucial to recapitulate endogenous gene expression. Through comparative genomics, we predict that this mechanism has also arisen in other mammalian muscle genes. Our results reveal the existence of a novel mechanism, conserved from flies to mammals, to fine-tune gene expression in each muscle and probably other tissues.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo , Troponina I/genética , Animais , Sítios de Ligação , Células Cultivadas , Sequência Conservada , Óperon Lac , Fatores de Transcrição MEF2 , Masculino , Camundongos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Fatores de Regulação Miogênica/metabolismo , Ratos , Ratos WistarRESUMO
CF2 and Mef2 influence a variety of developmental muscle processes at distinct stages of development. Nevertheless, the exact nature of the CF2-Mef2 relationship and its effects on muscle building remain yet to be resolved. Here, we explored the regulatory role of CF2 in the Drosophila embryo muscle formation. To address this question and not having proper null CF2 mutants we exploited loss or gain of function strategies to study the contribution of CF2 to Mef2 transcription regulation and to muscle formation. Our data point to CF2 as a factor involved in the regulation of muscle final size and/or the number of nuclei present in each muscle. This function is independent of its role as a Mef2 collaborative factor in the transcriptional regulation of muscle-structural genes. Although Mef2 expression patterns do not change, reductions or increases in parallel in CF2 and Mef2 transcript abundance were observed in interfered and overexpressed CF2 embryos. Since CF2 expression variations yield altered Mef2 expression levels but with correct spatio-temporal Mef2 expression patterns, it can be concluded that only the mechanism controlling expression levels is de-regulated. Here, it is proposed that CF2 regulates Mef2 expression through a Feedforward Loop circuit.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/embriologia , Desenvolvimento Muscular/fisiologia , Músculos/embriologia , Fatores de Regulação Miogênica/biossíntese , RNA Mensageiro/biossíntese , Fatores de Transcrição/metabolismo , Animais , Padronização Corporal/fisiologia , Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Regulação Miogênica/genética , RNA Mensageiro/genética , Fatores de Transcrição/genéticaRESUMO
The distinct muscles of an organism accumulate different quantities of structural proteins, but always maintaining their stoichiometry. However, the mechanisms that control the levels of these proteins and that co-ordinate muscle gene expression remain to be defined. The paramyosin/miniparamyosin gene encodes two thick filament proteins transcribed from two different promoters. We have analysed the regulatory regions that control expression of this gene and that are situated in the two promoters, the 5' and the internal promoters, both in vivo and in silico. A distal muscle enhancer containing three conserved MEF2 motifs is essential to drive high levels of paramyosin expression in all the major embryonic, larval and adult muscles. This enhancer shares sequence motifs, as well as its structure and organisation, with at least four co-regulated muscle enhancers that direct similar patterns of expression. However, other elements located downstream of the enhancer are also required for correct gene expression. Other muscle genes with different patterns of expression, such as miniparamyosin, are regulated by other basic mechanisms. The expression of miniparamyosin is controlled by two enhancers, AB and TX, but a BF modulator is required to ensure the correct levels of expression in each particular muscle. We propose a mechanism of transcriptional regulation in which similar enhancers are responsible for the spatio-temporal expression of co-regulated genes. However, it is the interaction between enhancers which ensures that the correct amounts of protein are expressed at any particular time in a cell, adapting these levels to their specific needs. These mechanisms may not be exclusive to neural or muscle tissue and might represent a general mechanism for genes that are spatially and temporally co-regulated.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Músculos/embriologia , Músculos/metabolismo , Regiões Promotoras Genéticas , Tropomiosina/genética , Motivos de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Drosophila melanogaster , Genes Reporter , Genoma , Modelos Genéticos , Dados de Sequência Molecular , Neurônios/metabolismo , Nucleotídeos/química , Filogenia , Plasmídeos/metabolismo , Biossíntese de Proteínas , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Transgenes , Tropomiosina/biossíntese , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/metabolismoRESUMO
Formation of the contractile apparatus in muscle cells requires co-ordinated activation of several genes and the proper assembly of their products. To investigate the role of TnT (troponin T) in the mechanisms that control and co-ordinate thin-filament formation, we generated transgenic Drosophila lines that overexpress TnT in their indirect flight muscles. All flies that overexpress TnT were unable to fly, and the loss of thin filaments themselves was coupled with ultrastructural perturbations of the sarcomere. In contrast, thick filaments remained largely unaffected. Biochemical analysis of these lines revealed that the increase in TnT levels could be detected only during the early stages of adult muscle formation and was followed by a profound decrease in the amount of this protein as well as that of other thin-filament proteins such as tropomyosin, troponin I and actin. The decrease in thin-filament proteins is not only due to degradation but also due to a decrease in their synthesis, since accumulation of their mRNA transcripts was also severely diminished. This decrease in expression levels of the distinct thin-filament components led us to postulate that any change in the amount of TnT transcripts might trigger the down-regulation of other co-regulated thin-filament components. Taken together, these results suggest the existence of a mechanism that tightly co-ordinates the expression of thin-filament genes and controls the correct stoichiometry of these proteins. We propose that the high levels of unassembled protein might act as a sensor in this process.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Musculares/biossíntese , Músculos/metabolismo , Troponina T/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/ultraestrutura , Retroalimentação Fisiológica , Voo Animal , Músculos/ultraestrutura , Doenças Musculares/etiologia , Doenças Musculares/patologia , Pupa , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Sarcômeros/ultraestrutura , Troponina T/biossíntese , Troponina T/genéticaRESUMO
Hematopoiesis is a complex process regulated by both cell intrinsic and cell extrinsic factors. Alterations in the expression of critical genes during hematopoiesis can modify the balance between stem cell differentiation and proliferation, and may ultimately give rise to leukemia and other diseases. AF10 is a transcription factor that has been implicated in the development of leukemia following chromosomal rearrangements between the AF10 gene and one of at least two other genes, MLL and CALM. The link between AF10 and leukemia, together with the known interactions between AF10 and hematopoietic regulators, suggests that AF10 may be important in hematopoiesis and in leukemic transformation. Here we show that AF10 is important for proper hematopoietic differentiation. The induction of hematopoietic differentiation in both human hematopoietic cell lines and murine total bone marrow cells triggers a decrease of AF10 mRNA and protein levels, particularly in stem cells and multipotent progenitors. Gain- and loss-of-function studies demonstrate that over- or under-expression of AF10 leads to apoptotic cell death in stem cells and multipotent progenitors. We conclude that AF10 plays a key role in the maintenance of multipotent hematopoietic cells.
Assuntos
Apoptose , Diferenciação Celular , Proliferação de Células , Hematopoese , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Sobrevivência Celular , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Humanos , Leucemia/metabolismo , Leucemia/patologia , Camundongos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
The aim of this study was to identify the genetic defect in two patients having cardiac dysfunction accompanied by neurological symptoms, and in one case MRI evidence of cortical and cerebellar atrophy with hyperintensities in the basal ganglia. Muscle biopsies from each patient revealed single and combined mitochondrial respiratory chain deficiency. The complete mtDNA sequencing of both patients revealed two transitions in the mitochondrial tRNA(Val) gene (MT-TV) (m.1628C>T in Patient 1, and m.1644G>A in Patient 2). The functional and molecular analyses reported here suggest that the MT-TV gene should be routinely considered in the diagnosis of mitochondrial cardiomyopathies.
Assuntos
Cardiomiopatias/genética , Doenças Mitocondriais/genética , Mutação , RNA de Transferência de Valina/genética , RNA/genética , Adolescente , Adulto , Análise Mutacional de DNA , DNA Mitocondrial/química , DNA Mitocondrial/genética , Humanos , Masculino , RNA Mitocondrial , Análise de Sequência de DNARESUMO
The creation of the contractile apparatus in muscle involves the co-activation of a group of genes encoding muscle-specific proteins and the production of high levels of protein in a short period of time. We have studied the transcriptional control of six Drosophila muscle genes that have similar expression profiles and we have compared these mechanisms with those employed to control the distinct expression profiles of other Drosophila genes. The regulatory elements controlling the transcription of co-expressed muscle genes share an Upstream Regulatory Element and an Intronic Regulatory Element. Moreover, similar clusters of MEF2 and CF2 binding sites are present in these elements. Here, we demonstrate that CF2 depletion alters the relative expression of thin and thick filament components. We propose that the appropriate rapid gene expression responses during muscle formation and the maintenance of each muscle type is guaranteed in Drosophila by equivalent duplicate enhancer-like elements. This mechanism may be exceptional and restricted to muscle genes, reflecting the specific requirement to mediate rapid muscle responses. However, it may also be a more general mechanism to control the correct levels of gene expression during development in each cell type.