RESUMEN
A single missense variant of the TMPO/LAP2α gene, encoding LAP2 proteins, has been associated with cardiomyopathy in two brothers. To further evaluate its role in cardiac muscle, we included TMPO in our cardiomyopathy diagnostic gene panel. A screening of ~5000 patients revealed three novel rare TMPO heterozygous variants in six males diagnosed with hypertrophic or dilated cardiomypathy. We identified in different cellular models that (1) the frameshift variant LAP2α p.(Gly395Glufs*11) induced haploinsufficiency, impeding cell proliferation and/or producing a truncated protein mislocalized in the cytoplasm; (2) the C-ter missense variant LAP2α p.(Ala240Thr) led to a reduced proximity events between LAP2α and the nucleosome binding protein HMGN5; and (3) the LEM-domain missense variant p.(Leu124Phe) decreased both associations of LAP2α/ß with the chromatin-associated protein BAF and inhibition of the E2F1 transcription factor activity which is known to be dependent on Rb, partner of LAP2α. Additionally, the LAP2α expression was lower in the left ventricles of male mice compared to females. In conclusion, our study reveals distinct altered properties of LAP2 induced by these TMPO/LAP2 variants, leading to altered cell proliferation, chromatin structure or gene expression-regulation pathways, and suggests a potential sex-dependent role of LAP2 in myocardial function and disease.
Asunto(s)
Cardiomiopatías , Cromosomas , Femenino , Masculino , Ratones , Animales , Cardiomiopatías/genética , Cromatina , FenotipoRESUMEN
Lamins are the main components of the nucleoskeleton. Whereas their 3D organization was recently described using cryoelectron tomography, no structural data highlights how they interact with their partners at the interface between the inner nuclear envelope and chromatin. A large number of mutations causing rare genetic disorders called laminopathies were identified in the C-terminal globular Igfold domain of lamins A and C. We here present a first structural description of the interaction between the lamin A/C immunoglobulin-like domain and emerin, a nuclear envelope protein. We reveal that this lamin A/C domain both directly binds self-assembled emerin and interacts with monomeric emerin LEM domain through the dimeric chromatin-associated Barrier-to-Autointegration Factor (BAF) protein. Mutations causing autosomal recessive progeroid syndromes specifically impair proper binding of lamin A/C domain to BAF, thus destabilizing the link between lamin A/C and BAF in cells. Recent data revealed that, during nuclear assembly, BAF's ability to bridge distant DNA sites is essential for guiding membranes to form a single nucleus around the mitotic chromosome ensemble. Our results suggest that BAF interaction with lamin A/C also plays an essential role, and that mutations associated with progeroid syndromes leads to a dysregulation of BAF-mediated chromatin organization and gene expression.
Asunto(s)
Proteínas de Unión al ADN/química , Lamina Tipo A/química , Proteínas de la Membrana/química , Proteínas Nucleares/química , Progeria/metabolismo , Dominios Proteicos , Cristalografía por Rayos X , Proteínas de Unión al ADN/metabolismo , Genes Recesivos , Humanos , Lamina Tipo A/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mutación , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Progeria/genética , Unión Proteica , Multimerización de ProteínaRESUMEN
Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD) and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A.
RESUMEN
Current three-dimensional (3D) genome modeling platforms are limited by their inability to account for radial placement of loci in the nucleus. We present Chrom3D, a user-friendly whole-genome 3D computational modeling framework that simulates positions of topologically-associated domains (TADs) relative to each other and to the nuclear periphery. Chrom3D integrates chromosome conformation capture (Hi-C) and lamin-associated domain (LAD) datasets to generate structure ensembles that recapitulate radial distributions of TADs detected in single cells. Chrom3D reveals unexpected spatial features of LAD regulation in cells from patients with a laminopathy-causing lamin mutation. Chrom3D is freely available on github.
Asunto(s)
Cromatina/genética , Biología Computacional/métodos , Lámina Nuclear/genética , Adulto , Femenino , Genoma , Células HeLa , Humanos , Masculino , Modelos Genéticos , Adulto JovenRESUMEN
A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by LMNA. LMNA mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous LMNA p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient's skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient's mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other LMNA mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype.
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Núcleo Celular/metabolismo , Lamina Tipo A/genética , Lipodistrofia/genética , Distrofias Musculares/genética , Mutación , Acetilación , Adolescente , Animales , Núcleo Celular/patología , Senescencia Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , Lamina Tipo A/metabolismo , Lipodistrofia/complicaciones , Lipodistrofia/metabolismo , Lipodistrofia/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Distrofias Musculares/complicaciones , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Mioblastos/metabolismo , Mioblastos/patología , Cultivo Primario de Células , Piel/metabolismo , Piel/patologíaRESUMEN
Proximity ligation assay (PLA) appears as a quick and easy technique to visualize within fixed cells the occurrence and in situ distribution of protein complexes. PLA has been validated to detect protein-protein interactions within the nuclear compartment. Here, we describe a protocol which allows the detection of interactions between A-type nuclear lamins and either LEM-domain proteins (such as emerin, integrated within the inner nuclear membrane, and LAP2α which accumulates within the nucleoplasm) or gene regulatory factors (e.g., the transcription factor SREBP1). The distinct amounts and patterns of PLA signals obtained for various complexes highlight the pertinence of using PLA to reveal in situ where and to which extent nuclear envelope proteins bind specific partners.
Asunto(s)
Proteínas Portadoras/metabolismo , Fluoroinmunoensayo , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas de Unión al ADN/metabolismo , Laminas/metabolismo , Microscopía Fluorescente , Unión ProteicaRESUMEN
Despite recent progress in the genetic characterization of congenital muscle diseases, the genes responsible for a significant proportion of cases remain unknown. We analysed two branches of a large consanguineous family in which four patients presented with a severe new phenotype, clinically marked by neonatal-onset muscle weakness predominantly involving axial muscles, life-threatening respiratory failure, skin abnormalities and joint hyperlaxity without contractures. Muscle biopsies showed the unreported association of multi-minicores, caps and dystrophic lesions. Genome-wide linkage analysis followed by gene and exome sequencing in patients identified a homozygous nonsense mutation in TRIP4 encoding Activating Signal Cointegrator-1 (ASC-1), a poorly characterized transcription coactivator never associated with muscle or with human inherited disease. This mutation resulted in TRIP4 mRNA decay to around 10% of control levels and absence of detectable protein in patient cells. ASC-1 levels were higher in axial than in limb muscles in mouse, and increased during differentiation in C2C12 myogenic cells. Depletion of ASC-1 in cultured muscle cells from a patient and in Trip4 knocked-down C2C12 led to a significant reduction in myotube diameter ex vivo and in vitro, without changes in fusion index or markers of initial myogenic differentiation. This work reports the first TRIP4 mutation and defines a novel form of congenital muscle disease, expanding their histological, clinical and molecular spectrum. We establish the importance of ASC-1 in human skeletal muscle, identify transcriptional co-regulation as novel pathophysiological pathway, define ASC-1 as a regulator of late myogenic differentiation and suggest defects in myotube growth as a novel myopathic mechanism.
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Codón sin Sentido , Desarrollo de Músculos , Enfermedades Musculares/congénito , Enfermedades Musculares/patología , Factores de Transcripción/genética , Adolescente , Animales , Diferenciación Celular , Línea Celular , Niño , Femenino , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Lactante , Masculino , Ratones , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Linaje , Estabilidad del ARN , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
LAP2-emerin-MAN1 (LEM)-domain proteins are modular proteins characterized by the presence of a conserved motif of about 50 residues. Most LEM-domain proteins localize at the inner nuclear membrane, but some are also found in the endoplasmic reticulum or nuclear interior. Their architecture has been analyzed by predicting the limits of their globular domains, determining the 3D structure of these domains and in a few cases calculating the 3D structure of specific domains bound to biological targets. The LEM domain adopts an α-helical fold also found in SAP and HeH domains of prokaryotes and unicellular eukaryotes. The LEM domain binds to BAF (barrier-to-autointegration factor; BANF1), which interacts with DNA and tethers chromatin to the nuclear envelope. LAP2 isoforms also share an N-terminal LEM-like domain, which binds DNA. The structure and function of other globular domains that distinguish LEM-domain proteins from each other have been characterized, including the C-terminal dimerization domain of LAP2α and C-terminal WH and UHM domains of MAN1. LEM-domain proteins also have large intrinsically disordered regions that are involved in intra- and intermolecular interactions and are highly regulated by posttranslational modifications in vivo.
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Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Cromatografía Liquida , Proteínas de Unión al ADN/química , Escherichia coli , Humanos , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de ProteínaRESUMEN
More than 100 genetic mutations causing X-linked Emery-Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding emerin variants' loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of ß-structures. Analysis of the assembly properties of five EmN variants reveals that del95-99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of emerin-emerin and emerin-lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to emerin aggregation and mislocalization in cells. Our results suggest that emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Distrofias Musculares/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Variación Genética , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/química , Membrana Nuclear/química , Proteínas Nucleares/química , Deficiencias en la Proteostasis/genética , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The nuclear lamina has been shown to interact with the genome through lamina-associated domains (LADs). LADs have been identified by DamID, a proximity labeling assay, and more recently by chromatin immunoprecipitation-sequencing (ChIP-seq) of A- and B-type lamins. LADs form megabase-size domains at the nuclear periphery, they are gene-poor and mostly heterochromatic. Here, we show that the mode of chromatin fragmentation for ChIP, namely bath sonication or digestion with micrococcal nuclease (MNase), leads to the discovery of common but also distinct sets of lamin-interacting domains, or LiDs. Using ChIP-seq, we show the existence of lamin A/C (LMNA) LiDs with distinct gene contents, histone composition enrichment and relationships to lamin B1-interacting domains. The extent of genome coverage of lamin A/C (LMNA) LiDs in sonicated or MNase-digested chromatin is similar (â¼730 megabases); however over half of these domains are uniquely detected in sonicated or MNase-digested chromatin. Sonication-specific LMNA LiDs are gene-poor and devoid of a broad panel of histone modifications, while MNase-specific LMNA LiDs are of higher gene density and are enriched in H3K9me3, H3K27me3 and in histone variant H2A.Z. LMNB1 LiDs are gene-poor and show no or little enrichment in these marks. Comparison of published LMNB1 DamID LADs with LMNB1 and LMNA LiDs identified here by ChIP-seq further shows that LMNA can associate with 'open' chromatin domains displaying euchromatin characteristics, and which are not associated with LMNB1. The differential genomic and epigenetic properties of lamin-interacting domains reflect the existence of distinct LiD populations identifiable in different chromatin contexts, including nuclease-accessible regions presumably localized in the nuclear interior.
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Inmunoprecipitación de Cromatina , Cromatina/genética , Cromatina/metabolismo , Lamina Tipo A/metabolismo , Nucleasa Microcócica/metabolismo , Análisis de Secuencia de ADN , Sonicación , Transporte Activo de Núcleo Celular , Cromatina/química , Eucromatina/química , Eucromatina/genética , Eucromatina/metabolismo , Genómica , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/metabolismo , Humanos , Lámina Nuclear/genética , Lámina Nuclear/metabolismoRESUMEN
Nuclear lamins are involved in many cellular functions due to their ability to bind numerous partners including chromatin and transcription factors, and affect their properties. Dunnigan type familial partial lipodystrophy (FPLD2; OMIM#151660) is caused in most cases by the A-type lamin R482W mutation. We report here that the R482W mutation affects the regulatory activity of sterol response element binding protein 1 (SREBP1), a transcription factor that regulates hundreds of genes involved in lipid metabolism and adipocyte differentiation. Using in situ proximity ligation assays (PLA), reporter assays and biochemical and transcriptomic approaches, we show that interactions of SREBP1 with lamin A and lamin C occur at the nuclear periphery and in the nucleoplasm. These interactions involve the Ig-fold of A-type lamins and are favored upon SREBP1 binding to its DNA target sequences. We show that SREBP1, LMNA and sterol response DNA elements form ternary complexes in vitro. In addition, overexpression of A-type lamins reduces transcriptional activity of SREBP1. In contrast, both overexpression of LMNA R482W in primary human preadipocytes and endogenous expression of A-type lamins R482W in FPLD2 patient fibroblasts, reduce A-type lamins-SREBP1 in situ interactions and upregulate a large number of SREBP1 target genes. As this LMNA mutant was previously shown to inhibit adipogenic differentiation, we propose that deregulation of SREBP1 by mutated A-type lamins constitutes one underlying mechanism of the physiopathology of FPLD2. Our data suggest that SREBP1 targeting molecules could be considered in a therapeutic context.
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Sustitución de Aminoácidos , Lamina Tipo A/genética , Lipodistrofia Parcial Familiar/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Adulto , Femenino , Humanos , Lamina Tipo A/metabolismo , Lipodistrofia Parcial Familiar/genética , Masculino , Persona de Mediana Edad , Mutación Missense , Unión Proteica , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Adulto JovenRESUMEN
OBJECTIVE: The objective of the study is to determine the frequency and the clinical significance of autoantibodies to the pericentromeric heterochromatin protein 1 (HP1). So far this antinuclear antibody specificity has been mainly reported in patients with the CREST syndrome. METHODS: We screened the sera of 199 individuals, including patients suffering from various autoimmune disorders (Group I, n=145) and non autoimmune diseases (Group II, n=44 patients) as well as healthy individuals (Group III, n=30). The sera were systematically tested by Western blot and ELISA using a GST-HP1α fusion protein as an antigen. RESULTS: Anti-HP1 antibodies were detected in 32% of patients in Group I, 11.3% in Group II and 3.3% of individuals in Group III. They could be detected in sera containing or not antinuclear antibodies detectable by indirect immunofluorescence. Anti-HP1 antibodies were mostly associated with the CREST and Sjogren's syndromes (70% and 44.4%, respectively). They could also be detected in 22.2% of patients suffering from various other autoimmune diseases. However, their negative predictive value was 94% in the CREST syndrome. CONCLUSION: Anti-HP1 autoantibodies are associated with a large spectrum of disorders. However, they have a diagnostic value in the CREST syndrome.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Síndrome CREST/inmunología , Proteínas Cromosómicas no Histona/inmunología , Síndrome de Sjögren/inmunología , Adolescente , Adulto , Anciano , Síndrome Antifosfolípido/diagnóstico , Síndrome Antifosfolípido/inmunología , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/inmunología , Síndrome CREST/diagnóstico , Estudios de Casos y Controles , Homólogo de la Proteína Chromobox 5 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Esclerodermia Sistémica/diagnóstico , Esclerodermia Sistémica/inmunología , Síndrome de Sjögren/diagnóstico , Adulto JovenRESUMEN
The nuclear lamina is implicated in the organization of the eukaryotic nucleus. Association of nuclear lamins with the genome occurs through large chromatin domains including mostly, but not exclusively, repressed genes. How lamin interactions with regulatory elements modulate gene expression in different cellular contexts is unknown. We show here that in human adipose tissue stem cells, lamin A/C interacts with distinct spatially restricted subpromoter regions, both within and outside peripheral and intra-nuclear lamin-rich domains. These localized interactions are associated with distinct transcriptional outcomes in a manner dependent on local chromatin modifications. Down-regulation of lamin A/C leads to dissociation of lamin A/C from promoters and remodels repressive and permissive histone modifications by enhancing transcriptional permissiveness, but is not sufficient to elicit gene activation. Adipogenic differentiation resets a large number of lamin-genome associations globally and at subpromoter levels and redefines associated transcription outputs. We propose that lamin A/C acts as a modulator of local gene expression outcome through interaction with adjustable sites on promoters, and that these position-dependent transcriptional readouts may be reset upon differentiation.
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Tejido Adiposo/citología , Cromatina/metabolismo , Lamina Tipo A/metabolismo , Regiones Promotoras Genéticas , Células Madre/metabolismo , Transcripción Genética , Adipogénesis , Tejido Adiposo/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica , Sitios Genéticos , Humanos , Lamina Tipo A/genética , Lámina Nuclear/genética , Lámina Nuclear/metabolismo , Células Madre/citología , Activación TranscripcionalRESUMEN
Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (∆607-656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.
Asunto(s)
Lamina Tipo A/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Células HeLa , Humanos , Lamina Tipo A/genética , Lipodistrofia Parcial Familiar/genética , Lipodistrofia Parcial Familiar/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Péptidos/metabolismo , Progeria/genética , Progeria/metabolismo , Unión Proteica , Precursores de Proteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Mutations in the lamin A/C (LMNA) gene that cause Hutchinson-Gilford progeria syndrome (HGPS) lead to expression of a protein called progerin with 50 amino acids deleted from the tail of prelamin A. In cells from patients with HGPS, both the amount and distribution of heterochromatin are altered. We designed in vitro assays to ask whether such alterations might reflect changes in chromatin, DNA and/or histone binding properties of progerin compared to wild-type lamin C-terminal tails. We show that progerin tail has a reduced DNA/chromatin binding capacity and modified trimethylated H3K27 binding pattern, offering a molecular mechanism for heterochromatin alterations related to HGPS.
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Progeria/genética , Progeria/metabolismo , Animales , Sitios de Unión/genética , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Genotipo , Heterocromatina/metabolismo , Humanos , Lamina Tipo A , Laminas/genética , Laminas/metabolismo , Ratones , Ratones Transgénicos , Mutación , Proteínas Nucleares , Precursores de Proteínas , Eliminación de SecuenciaRESUMEN
We have studied myoblasts from a patient with a severe autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) caused by an arginine 545 to cystein point mutation (p.R545C) in the carboxy-terminal domain of the lamin A/C gene. This mutation has pleiotropic cellular effects on these myoblasts as demonstrated by nuclear structural defects, exhibiting lobulations which increase with cell passages in culture. The organization of both lamin A/C and its inner nuclear membrane partner emerin are altered, eventually showing a honeycomb pattern upon immunofluorescence microscopy. In addition, the distribution of histone H3 trimethylated at lysine 27 and of phosphorylated RNA polymerase II, markers of inactive and active chromatin domains, respectively, are altered suggesting an impact on gene expression. Patient myoblasts also presented a high index of senescence in ex vivo culture. Moreover, our data show for the first time in an AD-EDMD context that the 20S core particle of the proteasome was inactivated. With cell passages, the 20S core protein progressively accumulated into discrete nuclear foci that largely colocalized with promyelocytic leukemia (PML) bodies while p21 accumulated throughout the nuclear compartment. Proteasome inactivation has been linked to normal cellular ageing. Our data indicate that it may also contribute to premature senescence in AD-EDMD patient myoblasts. Finally, when transferred to low-serum medium, patient myoblasts were deficient in ex vivo differentiation, as assessed by the absence of myotube formation and myogenin induction. Altogether, these data suggest that the LMNA mutation p.R545C impairs both proliferation and differentiation capacities of myoblasts as part of the pathogenesis of AD-EDMD.
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Envejecimiento/genética , Diferenciación Celular/genética , Núcleo Celular/metabolismo , Lamina Tipo A/genética , Distrofia Muscular de Emery-Dreifuss/genética , Mioblastos/metabolismo , Sustitución de Aminoácidos , Anticuerpos/metabolismo , Bisbenzimidazol/metabolismo , Carbocianinas/metabolismo , Estudios de Casos y Controles , Técnicas de Cultivo de Célula , Núcleo Celular/patología , Núcleo Celular/ultraestructura , Células Cultivadas , Cisteína/metabolismo , Femenino , Fluoresceína-5-Isotiocianato/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Masculino , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/patología , Mutación Missense , Mioblastos/citología , Mioblastos/ultraestructura , Mutación Puntual , Xantenos/metabolismoRESUMEN
Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is characterized by muscle wasting and is caused by mutations in the LMNA gene encoding A-type lamins. Overexpression of the EDMD lamin A R453W mutation in C2C12 myoblasts impairs myogenic differentiation. We show here the influence of stable expression of the R453W and of the Dunnigan-type partial lipodystrophy R482W mutation of lamin A in C2C12 cells on transcription and epigenetic regulation of the myogenin (Myog) gene and on global chromatin organization. Expression of R453W-, but not R482W-lamin A, impairs activation of Myog and maintains a repressive chromatin state on the Myog promoter upon induction of differentiation, marked by H3 lysine (K) 9 dimethylation and failure to hypertrimethylate H3K4. Cells expressing WT-LaA also fail to hypertrimethylate H3K4. No defect occurs at the level of Myog promoter DNA methylation in any of the clones. Expression of R453W-lamin A and to a lesser extent R482W-lamin A in undifferentiated C2C12 cells redistributes H3K9me3 from pericentric heterochromatin. R453W-lamin A also elicits a redistribution of H3K27me3 from inactive X (Xi) and partial decondensation of Xi, but maintains Xist expression and coating of Xi, indicating that Xi remains inactivated. Our results argue that gene-specific and genome-wide chromatin rearrangements may constitute a molecular basis for laminopathies.
Asunto(s)
Epigénesis Genética , Lamina Tipo A/genética , Distrofia Muscular de Emery-Dreifuss/genética , Mutación Missense , Mioblastos/metabolismo , Miogenina/genética , Sustitución de Aminoácidos , Animales , Arginina/genética , Diferenciación Celular , Línea Celular , Núcleo Celular/enzimología , Metilación de ADN , Histonas/química , Histonas/metabolismo , Humanos , Lamina Tipo A/metabolismo , Metilación , Ratones , Mioblastos/citología , Miogenina/biosíntesis , Regiones Promotoras Genéticas , Triptófano/genética , Regulación hacia Arriba , Cromosoma X/enzimologíaRESUMEN
Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process.
Asunto(s)
Diferenciación Celular , Lamina Tipo A/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Distrofia Muscular de Emery-Dreifuss/enzimología , Mioblastos/citología , Mioblastos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Antígenos CD1/metabolismo , Complejo CD3/metabolismo , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Proteínas Fluorescentes Verdes/metabolismo , Factor II del Crecimiento Similar a la Insulina/farmacología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Distrofia Muscular de Emery-Dreifuss/patología , Proteínas Mutantes/metabolismo , Mutación/genética , Mioblastos/efectos de los fármacos , Miogenina/metabolismo , Lámina Nuclear/efectos de los fármacos , Lámina Nuclear/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteína de Retinoblastoma/metabolismoRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a dominant autosomal premature aging syndrome caused by the expression of a truncated prelamin A designated progerin (Pgn). A-type and B-type lamins are intermediate filament proteins that polymerize to form the nuclear lamina network apposed to the inner nuclear membrane of vertebrate somatic cells. It is not known if in vivo both type of lamins assemble independently or co-assemble. The blebbing and disorganization of the nuclear envelope and adjacent heterochromatin in cells from patients with HGPS is a hallmark of the disease, and the ex vivo reversal of this phenotype is considered important for the development of therapeutic strategies. Here, we investigated the alterations in the lamina structure that may underlie the disorganization caused in nuclei by Pgn expression. We studied the polymerization of enhanced green fluorescent protein- and red fluorescent protein-tagged wild-type and mutated lamins in the nuclear envelope of living cells by measuring fluorescence resonance energy transfer (FRET) that occurs between the two fluorophores when tagged lamins interact. Using time domain fluorescence lifetime imaging microscopy that allows a quantitative analysis of FRET signals, we show that wild-type lamins A and B1 polymerize in distinct homopolymers that further interact in the lamina. In contrast, expressed Pgn co-assembles with lamin B1 and lamin A to form a mixed heteropolymer in which A-type and B-type lamin segregation is lost. We propose that such structural lamina alterations may be part of the primary mechanisms leading to HGPS, possibly by impairing functions specific for each lamin type such as nuclear membrane biogenesis, signal transduction, nuclear compartmentalization and gene regulation.
Asunto(s)
Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Proteínas Nucleares/metabolismo , Progeria/metabolismo , Precursores de Proteínas/metabolismo , Animales , Células Cultivadas , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lamina Tipo A/genética , Lamina Tipo B/genética , Ratones , Microscopía Fluorescente/métodos , Modelos Biológicos , Modelos Genéticos , Mioblastos/citología , Mioblastos/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Precursores de Proteínas/genética , TransfecciónRESUMEN
Autosomal dominantly inherited missense mutations in lamins A and C cause several tissue-specific diseases, including Emery-Dreifuss muscular dystrophy (EDMD) and Dunnigan-type familial partial lipodystrophy (FPLD). Here we analyze myoblast-to-myotube differentiation in C2C12 clones overexpressing lamin A mutated at arginine 453 (R453W), one of the most frequent mutations in EDMD. In contrast with clones expressing wild-type lamin A, these clones differentiate poorly or not at all, do not exit the cell cycle properly, and are extensively committed to apoptosis. These disorders are correlated with low levels of expression of transcription factor myogenin and with the persistence of a large pool of hyperphosphorylated retinoblastoma protein. Since clones mutated at arginine 482 (a site responsible for FPLD) differentiate normally, we conclude that C2C12 clones expressing R453W-mutated lamin A represent a good cellular model to study the pathophysiology of EDMD. Our hypothesis is that lamin A mutated at arginine 453 fails to build a functional scaffold and/or to maintain the chromatin compartmentation required for differentiation of myoblasts into myocytes.