RESUMO
Repetitive sequences account for more than 50% of the human genome. Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease associated with reduction in the copy number of the D4Z4 repeat mapping to 4q35. By an unknown mechanism, D4Z4 deletion causes an epigenetic switch leading to de-repression of 4q35 genes. Here we show that the Polycomb group of epigenetic repressors targets D4Z4 in healthy subjects and that D4Z4 deletion is associated with reduced Polycomb silencing in FSHD patients. We identify DBE-T, a chromatin-associated noncoding RNA produced selectively in FSHD patients that coordinates de-repression of 4q35 genes. DBE-T recruits the Trithorax group protein Ash1L to the FSHD locus, driving histone H3 lysine 36 dimethylation, chromatin remodeling, and 4q35 gene transcription. This study provides insights into the biological function of repetitive sequences in regulating gene expression and shows how mutations of such elements can influence the progression of a human genetic disease.
Assuntos
Epigênese Genética , Distrofia Muscular Facioescapuloumeral/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , RNA não Traduzido/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetinae , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/fisiopatologia , Proteínas do Grupo Polycomb , Elementos de Resposta , Fatores de Transcrição/metabolismoRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6-) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.
Assuntos
Calpaína , Proteínas Musculares , Distrofia Muscular Facioescapuloumeral , Proteínas , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Animais , Calpaína/genética , Calpaína/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Proteínas dos Microfilamentos , Desenvolvimento Muscular/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
Overexpression of facioscapulohumeral muscular dystrophy region gene 1 (FRG1) in mice, frogs and worms leads to muscular and vascular abnormalities. Nevertheless, the mechanism that follows FRG1 overexpression and finally leads to muscular defects is currently unknown. Here, we show that the earliest phenotype displayed by mice overexpressing FRG1 is a postnatal muscle-growth defect. Long before the development of muscular dystrophy, FRG1 mice also exhibit a muscle regeneration impairment. Ex vivo and in vivo experiments revealed that FRG1 overexpression causes myogenic stem cell activation and proliferative, clonogenic and differentiation defects. A comparative gene expression profiling of muscles from young pre-dystrophic wild-type and FRG1 mice identified differentially expressed genes in several gene categories and networks that could explain the emerging tissue and myogenic stem cell defects. Overall, our study provides new insights into the pathways regulated by FRG1 and suggests that muscle stem cell defects could contribute to the pathology of FRG1 mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doenças Autoimunes/metabolismo , Proteínas de Transporte/metabolismo , Extensões da Superfície Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Macrófagos/fisiologia , Células-Tronco Multipotentes/fisiologia , Distrofia Muscular Facioescapuloumeral/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Transporte/genética , Linhagem Celular , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a Ácido Graxo , Camundongos , Desenvolvimento Muscular/genética , Multimerização Proteica/genética , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno/genética , Transgenes/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Several reports showed that hematopoietic stem cells (HSCs) participate in muscle regeneration, raising hope for their therapeutic potential for degenerative muscle diseases. However, proof that HSCs are able to reprogram their fate and enter a myogenic pathway, remains elusive. We demonstrate that murine bone marrow (BM)-derived hematopoietic cells, carrying reporter genes controlled by muscle-specific regulatory elements from the Myf5, myosin light chain (MLC3F), or MCK genes, are induced by myoblasts to activate muscle-specific genes. This potential resides in the more undifferentiated progenitors, expressing surface markers typical of HSCs. Comparative gene expression profiling of CD45(+)/Sca1(+) cells isolated from muscle or BM shows that hematopoietic cells participate to muscle regeneration, by undergoing a profound although incomplete myogenic reprogramming on interaction with the muscle microenviroment. These cells undergo specification and differentiation independently from Pax7 and MyoD, and lack Pax7-associated properties, such as self-renewal and proliferation, distinguishing from satellite cells. Our findings indicate that hematopoietic cells, on seeding in the muscle, become a distinct cell population endowed with myogenic potential.
Assuntos
Células da Medula Óssea/fisiologia , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/fisiologia , Desenvolvimento Muscular/genética , Mioblastos/fisiologia , Fator de Transcrição PAX7/fisiologia , Animais , Células da Medula Óssea/citologia , Linhagem da Célula , Células Cultivadas , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Fator de Transcrição PAX7/deficiência , Fator de Transcrição PAX7/genéticaRESUMO
DNA microarrays are powerful tools for the analysis of gene expression on a genomic scale. The importance of individual regulatory events for the process under study can however not be deduced unequivocally without additional experiments. We devised a strategy to identify central regulators of cancer drug responses by combining the results of microarray experiments with efficient methods for phenotypic testing of candidate genes. We exposed murine FL5.12 pro-B cells to cisplatin, camptothecin, methotrexate or paclitaxel, respectively and analysed the patterns of gene expression with cDNA microarrays. Drug-specific regulatory events as well as intersections between different apoptotic pathways, including previously studied responses to staurosporine and interleukin-3 (IL-3) deprivation, were identified. Genes shared by at least three pathways were chosen for further analysis. Ectopic expression of three such genes, TEAP, GP49B, and Lipin1 was found to have an anti-proliferative effect on pro-B cells. Interestingly, we identified hemoglobin alpha as a strong pro-apoptotic regulator. While hemoglobin-expressing cells were growing normally in the presence of IL-3, they displayed accelerated apoptosis with similar kinetics as Bax overexpressing cells upon IL-3 removal. The pro-apoptotic effect of hemoglobin was suppressed by Bcl-2 and was characterized by enhanced stimulation of caspase activity.
Assuntos
Antineoplásicos/farmacologia , Apoptose/genética , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Etiquetas de Sequências Expressas , Citometria de Fluxo , Perfilação da Expressão Gênica , Interleucina-3/farmacologia , Camundongos , Dados de Sequência Molecular , Fenótipo , Estaurosporina/farmacologiaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy with a strong epigenetic component. It is associated with deletion of a macrosatellite repeat leading to over-expression of the nearby genes. Among them, we focused on FSHD region gene 1 (FRG1) since its over-expression in mice, Xenopus laevis and Caenorhabditis elegans, leads to muscular dystrophy-like defects, suggesting that FRG1 plays a relevant role in muscle biology. Here we show that, when over-expressed, FRG1 binds and interferes with the activity of the histone methyltransferase Suv4-20h1 both in mammals and Drosophila. Accordingly, FRG1 over-expression or Suv4-20h1 knockdown inhibits myogenesis. Moreover, Suv4-20h KO mice develop muscular dystrophy signs. Finally, we identify the FRG1/Suv4-20h1 target Eid3 as a novel myogenic inhibitor that contributes to the muscle differentiation defects. Our study suggests a novel role of FRG1 as epigenetic regulator of muscle differentiation and indicates that Suv4-20h1 has a gene-specific function in myogenesis.