RESUMEN
Flying insects oscillate their wings at high frequencies of up to 1,000 Hz and produce large mechanical forces of 80 W per kilogram of muscle. They utilize a pair of perpendicularly oriented indirect flight muscles that contain fibrillar, stretch-activated myofibres. In contrast, all other, more slowly contracting, insect body muscles have a tubular muscle morphology. Here we identify the transcription factor Spalt major (Salm) as a master regulator of fibrillar flight muscle fate in Drosophila. salm is necessary and sufficient to induce fibrillar muscle fate. salm switches the entire transcriptional program from tubular to fibrillar fate by regulating the expression and splicing of key sarcomeric components specific to each muscle type. Spalt function is conserved in insects evolutionarily separated by 280 million years. We propose that Spalt proteins switch myofibres from tubular to fibrillar fate during development, a function potentially conserved in the vertebrate heart--a stretch-activated muscle sharing features with insect flight muscle.
Asunto(s)
Evolución Biológica , Secuencia Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Músculos/anatomía & histología , Músculos/fisiología , Factores de Transcripción/metabolismo , Empalme Alternativo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Systematic genetic approaches have provided deep insight into the molecular and cellular mechanisms that operate in simple unicellular organisms. For multicellular organisms, however, the pleiotropy of gene function has largely restricted such approaches to the study of early embryogenesis. With the availability of genome-wide transgenic RNA interference (RNAi) libraries in Drosophila, it is now possible to perform a systematic genetic dissection of any cell or tissue type at any stage of the lifespan. Here we apply these methods to define the genetic basis for formation and function of the Drosophila muscle. We identify a role in muscle for 2,785 genes, many of which we assign to specific functions in the organization of muscles, myofibrils or sarcomeres. Many of these genes are phylogenetically conserved, including genes implicated in mammalian sarcomere organization and human muscle diseases.
Asunto(s)
Drosophila melanogaster/embriología , Genes de Insecto/genética , Animales , Biología Computacional , Estudio de Asociación del Genoma Completo , Biblioteca Genómica , Larva , Masculino , Músculos/embriología , Interferencia de ARNRESUMEN
In yeast, protein kinase A (PKA) adjusts transcriptional profiles, metabolic rates, and cell growth in accord with carbon source availability. PKA affects gene expression mostly via the transcription factors Msn2 and Msn4, two key regulators of the environmental stress response. Here we analyze the role of the PKA-Msn2 signaling module using an Msn2 allele that harbors serine-to-alanine substitutions at six functionally important PKA motifs (Msn2A6) . Expression of Msn2A6 mimics low PKA activity, entails a transcription profile similar to that of respiring cells, and prevents formation of colonies on glucose-containing medium. Furthermore, Msn2A6 leads to high oxygen consumption and hence high respiratory activity. Substantially increased intracellular concentrations of several carbon metabolites, such as trehalose, point to a metabolic adjustment similar to diauxic shift. This partial metabolic switch is the likely cause for the slow-growth phenotype in the presence of glucose. Consistently, Msn2A6 expression does not interfere with growth on ethanol and tolerated is to a limited degree in deletion mutant strains with a gene expression signature corresponding to nonfermentative growth. We propose that the lethality observed in mutants with hampered PKA activity resides in metabolic reprogramming that is initiated by Msn2 hyperactivity.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas de Unión al ADN/fisiología , Frecuencia de los Genes , Glucosa/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Elementos de Respuesta , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Transducción de Señal , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
Bone resorbing osteoclasts are specialized macrophages that cannot differentiate in the absence of c-Fos, a member of the dimeric transcription factor AP-1 (activator protein-1). However, osteoclast differentiation in the absence of c-Fos can be rescued in vitro and in vivo by Fra1, a Fos-like protein and transcriptional target of c-Fos. To enable AP-1 proteins binding to DNA, c-Fos or Fra1 must heterodimerize with a partner such as c-Jun, JunB and JunD. In this study, we investigated the dimerization partners of c-Fos and Fra1 required for osteoclast differentiation using synthetic "single-chain" AP-1 dimers in which c-Fos or Fra1 is tethered via a linker to Jun proteins. When c-Fos was analyzed in combination with any Jun protein, including a c-Jun mutant lacking major phosphorylation sites for c-Jun amino-terminal kinase (JNK), osteoclasts were efficiently formed from c-Fos-deficient hematopoietic precursors. However, Fra1 in combination with any Jun protein could not rescue osteoclastogenesis. The ability to rescue was compared to transcriptional activity measured in transient transfection assays using promoters driven by consensus AP-1 sites or a composite AP-1/NFAT binding site. These data show that a single Jun/c-Fos dimer is sufficient for osteoclast differentiation, likely due to its transactivation ability for a broader range of promoters, in particular consensus AP-1 sites. We propose that Fra1 together with a dimerization partner different from Jun proteins can rescue osteoclast differentiation in c-Fos-deficient precursors.
Asunto(s)
Osteoclastos/citología , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-fos/química , Animales , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Diferenciación Celular , Células Cultivadas , Dimerización , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos/química , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Fosforilación , Proteínas Proto-Oncogénicas c-fos/deficiencia , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factor de Transcripción AP-1/química , Factor de Transcripción AP-1/metabolismoRESUMEN
Transactivation/transformation-domain associated protein (TRRAP) is a component of several multi-protein HAT complexes implicated in both transcriptional regulation and DNA repair. We recently identified Trrap, the murine ortholog of TRRAP, as an essential protein implicated in mitotic progression control, although its target genes are not known. In the present study, we analyzed the expression profiles of Trrap-responsive genes, using cDNA microarray in mitotic cells. From a panel of 17 664 transcript elements, we found that loss of Trrap leads to expression alteration of a large fraction of genes at mitotic stage. Functional classification of these genes indicates that Trrap influences a variety of cellular processes including cell cycle progression, cytoskeleton and cell adhesion, protein turnover, metabolism and signal transduction. The majority (71%) of differentially expressed genes was down-regulated in Trrap- deficient cells, whereas the rest were up-regulated, suggesting that Trrap may also play a role in transcriptional silencing. ChIP analysis revealed that Trrap might regulate gene expression by participating in acetylation of histone H4 and/or H3 depending on target genes and cell cycle stage. Our study indicates that Trrap regulates the expression of a wide range of genes in both quiescence and mitotic stages. Removal of the Trrap protein is associated with both increased and decreased gene expression.
Asunto(s)
Ciclo Celular/genética , Eliminación de Gen , Regulación de la Expresión Génica , Proteínas Nucleares/deficiencia , Proteínas Nucleares/metabolismo , Acetilación , Proteínas Adaptadoras Transductoras de Señales , Animales , Cromatina/genética , Cromatina/metabolismo , Fibroblastos , Perfilación de la Expresión Génica , Genómica , Histonas/metabolismo , Ratones , Ratones Noqueados , Mitosis/genética , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Polyomavirus (Py) large and small tumorantigens together are competent to induce S phase in growth-arrested mouse fibroblasts. The capacity of the large tumorantigen to bind the pocket proteins, pRB, p130 and p107, is important for the transactivation of DNA synthesis enzymes and the cyclins E and A, while the interference of small tumorantigen with protein phosphatase PP2A causes a destabilization of the cdk2 inhibitor p27, and thus leads to strong cyclin E- and cyclin A-dependent cdk2 activity. Py small tumorantigen, in addition, is able to transactivate cyclin A. Hence, this protein might have a much wider effect on gene expression in arrested mouse fibroblasts than hitherto suspected. This may have a profound part in the known capacity of Py to form tumors in mice. Therefore, it was interesting to gain an insight into the spectrum of transcriptional deregulation by Py tumorantigens. Accordingly, we performed microarray analysis of quiescent mouse fibroblasts in the absence and presence of small or large tumorantigen. We found that the viral proteins can induce or repress a great variety of genes beyond those involved in the S phase induction and DNA synthesis. The results of the microarray analysis were confirmed for selected genes by several methods, including real-time PCR. Interestingly, a mutation of the binding site for pocket proteins in case of LT and for PP2A in case of ST has a variable effect on the deregulation of genes by the viral proteins depending on the gene in question. In fact, some genes are transactivated by LT as well as ST completely independent of an interaction with their major cellular targets, pocket proteins and PP2A, respectively.
Asunto(s)
Antígenos Transformadores de Poliomavirus/metabolismo , Fibroblastos/metabolismo , Expresión Génica , Poliomavirus/metabolismo , Células 3T3 , Animales , Antígenos Transformadores de Poliomavirus/genética , Western Blotting , Cadherinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Decorina , Proteínas de la Matriz Extracelular , Fibroblastos/citología , Ratones , Componente 6 del Complejo de Mantenimiento de Minicromosoma , Miosinas/metabolismo , Neurofibromatosis 2/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Poliomavirus/inmunología , Proteoglicanos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidoresRESUMEN
Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in C. glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologs CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG) pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species and it required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.
RESUMEN
The Igf2r imprinted cluster is an epigenetic silencing model in which expression of a ncRNA silences multiple genes in cis. Here, we map a 250 kb region in mouse embryonic fibroblast cells to show that histone modifications associated with expressed and silent genes are mutually exclusive and localized to discrete regions. Expressed genes were modified at promoter regions by H3K4me3 + H3K4me2 + H3K9Ac and on putative regulatory elements flanking active promoters by H3K4me2 + H3K9Ac. Silent genes showed two types of nonoverlapping profile. One type spread over large domains of tissue-specific silent genes and contained H3K27me3 alone. A second type formed localized foci on silent imprinted gene promoters and a nonexpressed pseudogene and contained H3K9me3 + H4K20me3 +/- HP1. Thus, mammalian chromosome arms contain active chromatin interspersed with repressive chromatin resembling the type of heterochromatin previously considered a feature of centromeres, telomeres, and the inactive X chromosome.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Genoma , Impresión Genómica , Histonas/metabolismo , Animales , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , Cromosomas , Embrión de Mamíferos , Fibroblastos/metabolismo , Silenciador del Gen , Metilación , Ratones , Mapeo Físico de CromosomaRESUMEN
Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints.