RESUMEN
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.
Asunto(s)
Apoptosis , Cardiomiopatías/enzimología , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Miocardio/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Drosophila/enzimología , Drosophila/genética , Proteínas de Drosophila/genética , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Proteína p53 Supresora de Tumor/genéticaRESUMEN
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
Asunto(s)
Rastreo Celular/métodos , Regulación del Desarrollo de la Expresión Génica , Mioblastos/metabolismo , Troponina I/genética , Animales , Extremidades/embriología , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Transgénicos , Modelos Animales , Desarrollo de Músculos/genética , Miosinas/genética , Miosinas/metabolismo , Isoformas de Proteínas , Tropomiosina/genética , Tropomiosina/metabolismo , Troponina I/metabolismoRESUMEN
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.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica , Músculo Esquelético/metabolismo , Miocitos Cardíacos/metabolismo , Troponina I/genética , Animales , Sitios de Unión , Células Cultivadas , Secuencia Conservada , Operón Lac , Factores de Transcripción MEF2 , Masculino , Ratones , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Factores Reguladores Miogénicos/metabolismo , Ratas , Ratas WistarRESUMEN
The control of muscle-specific expression is one of the principal mechanisms by which diversity is generated among muscle types. In an attempt to elucidate the regulatory mechanisms that control fiber diversity in any given muscle, we have focused our attention on the transcriptional regulation of the Drosophila Troponin T gene. Two, nonredundant, functionally identical, enhancer-like elements activate Troponin T transcription independently in all major muscles of the embryo and larvae as well as in adult somatic and visceral muscles. Here, we propose that the differential but concerted interaction of these two elements underlies the mechanism by which a particular muscle-type establish the correct levels of Troponin T expression, adapting these levels to their specific needs. This mechanism is not exclusive to the Troponin T gene, but is also relevant to the muscle-specific Troponin I gene. In conjunction with in vivo transgenic studies, an in silico analysis of the Troponin T enhancer-like sequences revealed that both these elements are organized in a modular manner. Extending this analysis to the Troponin I and Tropomyosin regulatory elements, the two other components of the muscle-regulatory complex, we have discovered a similar modular organization of phylogenetically conserved domains.
Asunto(s)
Regulación de la Expresión Génica , Músculos/metabolismo , Transcripción Genética , Tropomiosina/genética , Troponina T/genética , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Línea Celular Transformada , Clonación Molecular , Drosophila , Drosophila melanogaster , Electroforesis en Gel de Poliacrilamida , Elementos de Facilitación Genéticos , Genes Reporteros , Immunoblotting , Modelos Genéticos , Datos de Secuencia Molecular , Filogenia , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Tórax/metabolismo , Factores de Tiempo , Transgenes , beta-Galactosidasa/metabolismoRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/embriología , Desarrollo de Músculos/fisiología , Músculos/embriología , Factores Reguladores Miogénicos/biosíntesis , ARN Mensajero/biosíntesis , Factores de Transcripción/metabolismo , Animales , Tipificación del Cuerpo/fisiología , Núcleo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores Reguladores Miogénicos/genética , ARN Mensajero/genética , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Músculos/embriología , Músculos/metabolismo , Regiones Promotoras Genéticas , Tropomiosina/genética , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Drosophila melanogaster , Genes Reporteros , Genoma , Modelos Genéticos , Datos de Secuencia Molecular , Neuronas/metabolismo , Nucleótidos/química , Filogenia , Plásmidos/metabolismo , Biosíntesis de Proteínas , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Transgenes , Tropomiosina/biosíntesis , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/metabolismoRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Musculares/biosíntesis , Músculos/metabolismo , Troponina T/fisiología , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/ultraestructura , Retroalimentación Fisiológica , Vuelo Animal , Músculos/ultraestructura , Enfermedades Musculares/etiología , Enfermedades Musculares/patología , Pupa , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Sarcómeros/ultraestructura , Troponina T/biosíntesis , Troponina T/genéticaRESUMEN
Catalepsy tests performed in rodents treated with drugs that interfere with dopaminergic transmission have been widely used for the screening of drugs with therapeutic potential in the treatment of Parkinson's disease. The basic method for measuring catalepsy intensity is the "standard" bar test. We present here an easy to use microcontroller-based automatic system for recording bar test experiments. The design is simple, compact, and has a low cost. Recording intervals and total experimental time can be programmed within a wide range of values. The resulting catalepsy times are stored, and up to five simultaneous experiments can be recorded. A standard personal computer interface is included. The automated system also permits the elimination of human error associated with factors such as fatigue, distraction, and data transcription, occurring during manual recording. Furthermore, a uniform criterion for timing the cataleptic condition can be achieved. Correlation values between the results obtained with the automated system and those reported by two independent observers ranged between 0.88 and 0.99 (P<0.0001; three treatments, nine animals, 144 catalepsy time measurements).
Asunto(s)
Automatización/métodos , Ciencias de la Conducta/métodos , Catalepsia/diagnóstico , Electrónica/métodos , Neurofisiología/métodos , Procesamiento de Señales Asistido por Computador/instrumentación , Animales , Artefactos , Automatización/instrumentación , Ciencias de la Conducta/instrumentación , Catalepsia/inducido químicamente , Catalepsia/fisiopatología , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Electrónica/instrumentación , Masculino , Neurofisiología/instrumentación , Variaciones Dependientes del Observador , Ratas , Ratas Wistar , Programas InformáticosRESUMEN
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.
Asunto(s)
Apoptosis , Diferenciación Celular , Proliferación Celular , Hematopoyesis , Células Madre/citología , Factores de Transcripción/metabolismo , Animales , Western Blotting , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Supervivencia Celular , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Leucemia/metabolismo , Leucemia/patología , Ratones , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Cardiomiopatías/genética , Enfermedades Mitocondriales/genética , Mutación , ARN de Transferencia de Valina/genética , ARN/genética , Adolescente , Adulto , Análisis Mutacional de ADN , ADN Mitocondrial/química , ADN Mitocondrial/genética , Humanos , Masculino , ARN Mitocondrial , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Elementos de Facilitación Genéticos/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Desarrollo de Músculos/fisiología , Miosinas/biosíntesis , Miosinas/genética , Factores de Transcripción/fisiología , Tropomiosina/biosíntesis , Tropomiosina/genéticaRESUMEN
In bilaterians, the main regulator of muscle contraction is the troponin (Tpn) complex, comprising three closely interacting subunits (C, T, and I). To understand how evolutionary forces drive molecular change in protein complexes, we have compared the gene structures and expression patterns of Tpn genes in insects. In this class, while TpnC is encoded by multiple genes, TpnT and TpnI are encoded by single genes. Their isoform expression pattern is highly conserved within the Drosophilidae, and single orthologous genes were identified in the sequenced genomes of Drosophila pseudoobscura, Anopheles gambiae, and Apis mellifera. Apis expression patterns also support the equivalence of their exon organization throughout holometabolous insects. All TpnT genes include a previously unidentified indirect flight muscle (IFM)-specific exon (10A) that has evolved an expression pattern similar to that of exon 9 in TpnI. Thus, expression patterns, sequence evolution trends, and structural data indicate that Tpn genes and their isoforms have coevolved, building species- and muscle-specific troponin complexes. Furthermore, a clear case can be made for independent evolution of the IFM-specific isoforms containing alanine/proline-rich sequences. Dipteran genomes contain one tropomyosin gene that encodes one or two high-molecular weight isoforms (TmH) incorporating APPAEGA-rich sequences, specifically expressed in IFM. Corresponding exons do not exist in the Apis tropomyosin gene, but equivalent sequences occur in a high-molecular weight Apis IFM-specific TpnI isoform (TnH). Overall, our approach to comparatively analyze supramolecular complexes reveals coevolutionary trends not only in gene families but in isoforms generated by alternative splicing.