RESUMEN
The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.
Asunto(s)
Distrofia Muscular Facioescapulohumeral , Humanos , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/metabolismo , Transcriptoma/genética , Proteínas de Homeodominio/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Estrés Oxidativo/genética , Apoptosis , Músculo Esquelético/metabolismo , Regulación de la Expresión Génica/genéticaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is caused by the epigenetic derepression of the 4q-linked D4Z4 macrosatellite repeat resulting in inappropriate expression of the D4Z4 repeat-encoded DUX4 gene in skeletal muscle. In 5% of FSHD cases, D4Z4 chromatin relaxation is due to germline mutations in one of the chromatin modifiers SMCHD1, DNMT3B or LRIF1. The mechanism of SMCHD1- and LRIF1-mediated D4Z4 repression is not clear. We show that somatic loss-of-function of either SMCHD1 or LRIF1 does not result in D4Z4 chromatin changes and that SMCHD1 and LRIF1 form an auxiliary layer of D4Z4 repressive mechanisms. We uncover that SMCHD1, together with the long isoform of LRIF1, binds to the LRIF1 promoter and silences LRIF1 expression. The interdependency of SMCHD1 and LRIF1 binding differs between D4Z4 and the LRIF1 promoter, and both loci show different transcriptional responses to either early developmentally or somatically perturbed chromatin function of SMCHD1 and LRIF1.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Distrofia Muscular Facioescapulohumeral , Humanos , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Epigenómica , Genes Homeobox , Músculo Esquelético , Distrofia Muscular Facioescapulohumeral/genética , Proteínas de Ciclo Celular/genéticaRESUMEN
With several therapeutic strategies for facioscapulohumeral muscular dystrophy (FSHD) entering clinical testing, outcome measures are becoming increasingly important. Considering the spatiotemporal nature of FSHD disease activity, clinical trials would benefit from non-invasive imaging-based biomarkers that can predict FSHD-associated transcriptome changes. This study investigated two FSHD-associated transcriptome signatures (DUX4 and PAX7 signatures) in FSHD skeletal muscle biopsies, and tested their correlation with a variety of disease-associated factors, including Ricci clinical severity score, disease duration, D4Z4 repeat size, muscle pathology scorings and functional outcome measures. It establishes that DUX4 and PAX7 signatures both show a sporadic expression pattern in FSHD-affected biopsies, possibly marking different stages of disease. This study analyzed two imaging-based biomarkers-Turbo Inversion Recovery Magnitude (TIRM) hyperintensity and fat fraction-and provides insights into their predictive power as non-invasive biomarkers for FSHD signature detection in clinical trials. Further insights in the heterogeneity of-and correlation between-imaging biomarkers and molecular biomarkers, as provided in this study, will provide important guidance to clinical trial design in FSHD. Finally, this study investigated the role of infiltrating non-muscle cell types in FSHD signature expression and detected potential distinct roles for two fibro-adipogenic progenitor subtypes in FSHD.
Asunto(s)
Proteínas de Homeodominio/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Factor de Transcripción PAX7/genética , Transcriptoma , Biomarcadores/metabolismo , Biopsia , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/patología , Distrofia Muscular Facioescapulohumeral/diagnóstico por imagen , Distrofia Muscular Facioescapulohumeral/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Factor de Transcripción PAX7/metabolismo , Índice de Severidad de la Enfermedad , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
Structural Maintenance of Chromosomes Hinge Domain Containing 1 (SMCHD1) is a chromatin repressor, which is mutated in > 95% of Facioscapulohumeral dystrophy (FSHD) type 2 cases. In FSHD2, SMCHD1 mutations ultimately result in the presence of the cleavage stage transcription factor DUX4 in muscle cells due to a failure in epigenetic repression of the D4Z4 macrosatellite repeat on chromosome 4q, which contains the DUX4 locus. While binding of SMCHD1 to D4Z4 and its necessity to maintain a repressive D4Z4 chromatin structure in somatic cells are well documented, it is unclear how SMCHD1 is recruited to D4Z4, and how it exerts its repressive properties on chromatin. Here, we employ a quantitative proteomics approach to identify and characterize novel SMCHD1 interacting proteins, and assess their functionality in D4Z4 repression. We identify 28 robust SMCHD1 nuclear interactors, of which 12 are present in D4Z4 chromatin of myocytes. We demonstrate that loss of one of these SMCHD1 interacting proteins, RuvB-like 1 (RUVBL1), further derepresses DUX4 in FSHD myocytes. We also confirm the interaction of SMCHD1 with EZH inhibitory protein (EZHIP), a protein which prevents global H3K27me3 deposition by the Polycomb repressive complex PRC2, providing novel insights into the potential function of SMCHD1 in the repression of DUX4 in the early stages of embryogenesis. The SMCHD1 interactome outlined herein can thus provide further direction into research on the potential function of SMCHD1 at genomic loci where SMCHD1 is known to act, such as D4Z4 repeats, the inactive X chromosome, autosomal gene clusters, imprinted loci and telomeres.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/genética , Proteínas Portadoras/genética , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Proteínas de Homeodominio/genética , Distrofia Muscular Facioescapulohumeral/genética , Proteómica/métodos , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Unión ProteicaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent skeletal muscle dystrophies. Skeletal muscle pathology in individuals with FSHD is caused by inappropriate expression of the transcription factor DUX4, which activates different myotoxic pathways. At the moment there is no molecular therapy that can delay or prevent skeletal muscle wasting in FSHD. In this study, a systemically delivered antisense oligonucleotide (ASO) targeting the DUX4 transcript was tested in vivo in ACTA1-MCM;FLExDUX4 mice that express DUX4 in skeletal muscles. We show that the DUX4 ASO was well tolerated and repressed the DUX4 transcript, DUX4 protein, and mouse DUX4 target gene expression in skeletal muscles. In addition, the DUX4 ASO alleviated the severity of skeletal muscle pathology and partially prevented the dysregulation of inflammatory and extracellular matrix genes. DUX4 ASO-treated ACTA1-MCM;FLExDUX4 mice performed better on a treadmill; however, the hanging grid and four-limb grip strength tests were not improved compared to control ASO-treated ACTA1-MCM;FLExDUX4 mice. This study shows that systemic delivery of ASOs targeting DUX4 is a promising therapeutic strategy for FSHD and strategies that further improve the ASO efficacy in skeletal muscle are warranted.
RESUMEN
BACKGROUND: FSHD is caused by specific genetic mutations resulting in activation of the Double Homeobox 4 gene (DUX4). DUX4 targets hundreds of downstream genes eventually leading to muscle atrophy, oxidative stress, abnormal myogenesis, and muscle inflammation. We hypothesized that DUX4-induced aberrant expression of genes triggers a sustained autoimmune response against skeletal muscle cells. OBJECTIVE: This study aimed at the identification of autoantibodies directed against muscle antigens in FSHD. Moreover, a possible relationship between serum antibody reactivity and DUX4 expression was also investigated. METHODS: FSHD sera (Nâ=â138, 48±16 years, 48% male) and healthy control sera (Nâ=â20, 47±14 years, 50% male) were analyzed by immunoblotting for antibodies against several skeletal muscle protein extracts: healthy muscle, FSHD muscle, healthy and FSHD myotubes, and inducible DUX4 expressing myoblasts. In addition, DUX4 expressing myoblasts were analyzed by immunofluorescence with FSHD and healthy control sera. RESULTS: The results showed that the reactivity of FSHD sera did not significantly differ from that of healthy controls, with all the tested muscle antigen extracts. Besides, the immunofluorescent staining of DUX4-expressing myoblasts was not different when incubated with either FSHD or healthy control sera. CONCLUSION: Since the methodology used did not lead to the identification of disease-specific autoantibodies in the FSHD cohort, we suggest that autoantibody-mediated pathology may not be an important disease mechanism in FSHD. Nevertheless, it is crucial to further unravel if and which role the immune system plays in FSHD pathogenesis. Other innate as well as adaptive immune players could be involved in the complex DUX4 cascade of events and could become appealing druggable targets.
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Autoanticuerpos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/sangre , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Mioblastos/metabolismoRESUMEN
Microfluidic atomic force microscopy (AFM) cantilever probes have all the functionalities of a standard AFM cantilever along with fluid pipetting. They have a channel inside the cantilever and an aperture at the tip. Such probes are useful for precise fluid manipulation at a desired location, for example near or inside cells. They are typically made by complex microfabrication process steps, resulting in expensive probes. Here, we used two different 3D additive manufacturing techniques, stereolithography and two-photon polymerization, to directly print ready-to-use microfluidic AFM cantilever probes. This approach has considerably reduced the fabrication time and increased the design freedom. One of the probes, 564 µm long, 30 µm wide, 30 µm high, with a 25 µm diameter channel and 2.5 µm wall thickness had a spring constant of 3.7 N m-1 and the polymer fabrication material had an elastic modulus of 4.2 GPa. Using these 3D printed probes, AFM imaging of a surface, puncturing of the cell membrane, and aspiration at the single cell level have been demonstrated.
Asunto(s)
Dispositivos Laboratorio en un Chip , Sondas Moleculares/química , Impresión Tridimensional , Microscopía de Fuerza Atómica/instrumentación , Tamaño de la Partícula , Impresión Tridimensional/instrumentación , Propiedades de SuperficieRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is characterized by sporadic de-repression of the transcription factor DUX4 in skeletal muscle. DUX4 activates a cascade of muscle disrupting events, eventually leading to muscle atrophy and apoptosis. Yet, how sporadic DUX4 expression leads to the generalized muscle wasting remains unclear. Transcriptome analyses have systematically been challenged by the majority of nuclei being DUX4neg, weakening the DUX4 transcriptome signature. Moreover, DUX4 has been shown to be expressed in a highly dynamic burst-like manner, likely resulting in the detection of the downstream cascade of events long after DUX4 expression itself has faded. Identifying the FSHD transcriptome in individual cells and unraveling the cascade of events leading to FSHD development may therefore provide important insights in the disease process. We employed single-cell RNA sequencing, combined with pseudotime trajectory modeling, to study FSHD disease etiology and cellular progression in human primary myocytes. We identified a small FSHD-specific cell population in all tested patient-derived cultures and detected new genes associated with DUX4 de-repression. We furthermore generated an FSHD cellular progression model, reflecting both the early burst-like DUX4 expression as well as the downstream activation of various FSHD-associated pathways, which allowed us to correlate DUX4 expression signature dynamics with that of regulatory complexes, thereby facilitating the prioritization of epigenetic targets for DUX4 silencing. Single-cell transcriptomics combined with pseudotime modeling thus holds valuable information on FSHD disease etiology and progression that can potentially guide biomarker and target selection for therapy.
Asunto(s)
Distrofia Muscular Facioescapulohumeral/etiología , Distrofia Muscular Facioescapulohumeral/genética , Adulto , Secuencia de Bases , Núcleo Celular/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Humanos , Masculino , Células Musculares , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Cultivo Primario de Células , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodosRESUMEN
Despite its importance during viral or bacterial infections, transcriptional regulation of the interferon-ß gene (Ifnb1) in activated macrophages is only partially understood. Here we report that TRIM33 deficiency results in high, sustained expression of Ifnb1 at late stages of toll-like receptor-mediated activation in macrophages but not in fibroblasts. In macrophages, TRIM33 is recruited by PU.1 to a conserved region, the Ifnb1 Control Element (ICE), located 15 kb upstream of the Ifnb1 transcription start site. ICE constitutively interacts with Ifnb1 through a TRIM33-independent chromatin loop. At late phases of lipopolysaccharide activation of macrophages, TRIM33 is bound to ICE, regulates Ifnb1 enhanceosome loading, controls Ifnb1 chromatin structure and represses Ifnb1 gene transcription by preventing recruitment of CBP/p300. These results characterize a previously unknown mechanism of macrophage-specific regulation of Ifnb1 transcription whereby TRIM33 is critical for Ifnb1 gene transcription shutdown.
Asunto(s)
Regulación de la Expresión Génica , Interferón beta/genética , Macrófagos/citología , Macrófagos/metabolismo , Factores de Transcripción/metabolismo , Animales , Femenino , Interferón beta/metabolismo , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Hematopoietic stem/progenitor cells (HSPCs) are regulated through numerous molecular mechanisms that have not been interconnected. The transcription factor stem cell leukemia/T-cell acute leukemia 1 (TAL1) controls human HSPC but its mechanism of action is not clarified. In this study, we show that knockdown (KD) or short-term conditional over-expression (OE) of TAL1 in human HSPC ex vivo, respectively, blocks and maintains hematopoietic potentials, affecting proliferation of human HSPC. Comparative gene expression analyses of TAL1/KD and TAL1/OE human HSPC revealed modifications of cell cycle regulators as well as previously described TAL1 target genes. Interestingly an inverse correlation between TAL1 and DNA damage-induced transcript 4 (DDiT4/REDD1), an inhibitor of the mammalian target of rapamycin (mTOR) pathway, is uncovered. Low phosphorylation levels of mTOR target proteins in TAL1/KD HSPC confirmed an interplay between mTOR pathway and TAL1 in correlation with TAL1-mediated effects of HSPC proliferation. Finally chromatin immunoprecipitation experiments performed in human HSPC showed that DDiT4 is a direct TAL1 target gene. Functional analyses showed that TAL1 represses DDiT4 expression in HSPCs. These results pinpoint DDiT4/REDD1 as a novel target gene regulated by TAL1 in human HSPC and establish for the first time a link between TAL1 and the mTOR pathway in human early hematopoietic cells. Stem Cells 2015;33:2268-2279.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Choque Térmico/metabolismo , Células Madre Hematopoyéticas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Choque Térmico/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos NOD , Proteínas Proto-Oncogénicas/genética , Factor 1 de Transcripción de Linfocitos T , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Transcripción/genética , TransfecciónRESUMEN
An intimate relationship exists between nuclear architecture and gene activity. Unraveling the fine-scale three-dimensional structure of the genome and its impact on gene regulation is a major goal of current epigenetic research, one with direct implications for understanding the molecular mechanisms underlying human phenotypic variation and disease susceptibility. In this context, the novel revolutionary genome editing technologies and emerging new ways to manipulate genome folding offer new promises for the treatment of human disorders.
Asunto(s)
Regulación de la Expresión Génica , Elementos de Facilitación Genéticos , Terapia Genética/métodos , Genoma Humano , Humanos , Edición de ARN , Elementos Reguladores de la TranscripciónRESUMEN
Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1-8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.
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Cromatina/metabolismo , Mapeo Cromosómico/métodos , Animales , Línea Celular , Cromatina/química , Cromosomas/química , Cromosomas/metabolismo , Biología Computacional , Formaldehído/química , Biblioteca Genómica , Genómica/métodos , Humanos , RatonesRESUMEN
Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system.
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Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Transcripción Genética , Animales , Ensamble y Desensamble de Cromatina , Genoma , Humanos , Elongación de la Transcripción GenéticaRESUMEN
The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping.