RESUMEN
Chromatin three-dimensional (3D) organization inside the cell nucleus determines the separation of euchromatin and heterochromatin domains. Their segregation results in the definition of active and inactive chromatin compartments, whereby the local concentration of associated proteins, RNA and DNA results in the formation of distinct subnuclear structures. Thus, chromatin domains spatially confined in a specific 3D nuclear compartment are expected to share similar epigenetic features and biochemical properties, in terms of accessibility and solubility. Based on this rationale, we developed the 4f-SAMMY-seq to map euchromatin and heterochromatin based on their accessibility and solubility, starting from as little as 10 000 cells. Adopting a tailored bioinformatic data analysis approach we reconstruct also their 3D segregation in active and inactive chromatin compartments and sub-compartments, thus recapitulating the characteristic properties of distinct chromatin states. A key novelty of the new method is the capability to map both the linear segmentation of open and closed chromatin domains, as well as their compartmentalization in one single experiment.
Asunto(s)
Eucromatina , Heterocromatina , Heterocromatina/química , Heterocromatina/metabolismo , Eucromatina/química , Eucromatina/metabolismo , Eucromatina/genética , Humanos , Cromatina/química , Cromatina/metabolismo , Cromatina/genética , Núcleo Celular/genética , Núcleo Celular/química , Núcleo Celular/metabolismo , ADN/química , ADN/metabolismo , AnimalesRESUMEN
Enhancers are noncoding regulatory DNA regions that modulate the transcription of target genes, often over large distances along with the genomic sequence. Enhancer alterations have been associated with various pathological conditions, including cancer. However, the identification and characterization of somatic mutations in noncoding regulatory regions with a functional effect on tumorigenesis and prognosis remain a major challenge. Here, we present a strategy for detecting and characterizing enhancer mutations in a genome-wide analysis of patient cohorts, across three lung cancer subtypes. Lung tissue-specific enhancers were defined by integrating experimental data and public epigenomic profiles, and the genome-wide enhancer-target gene regulatory network of lung cells was constructed by integrating chromatin three-dimensional architecture data. Lung cancers possessed a similar mutation burden at tissue-specific enhancers and exons but with differences in their mutation signatures. Functionally relevant alterations were prioritized on the basis of the pathway-level integration of the effect of a mutation and the frequency of mutations on individual enhancers. The genes enriched for mutated enhancers converged on the regulation of key biological processes and pathways relevant to tumor biology. Recurrent mutations in individual enhancers also affected the expression of target genes, with potential relevance for patient prognosis. Together, these findings show that noncoding regulatory mutations have a potential relevance for cancer pathogenesis and can be exploited for patient classification. SIGNIFICANCE: Mapping enhancer-target gene regulatory interactions and analyzing enhancer mutations at the level of their target genes and pathways reveal convergence of recurrent enhancer mutations on biological processes involved in tumorigenesis and prognosis.
Asunto(s)
Redes Reguladoras de Genes , Neoplasias Pulmonares , Humanos , Elementos de Facilitación Genéticos/genética , Neoplasias Pulmonares/genética , Mutación , Carcinogénesis/genéticaRESUMEN
Polycomb-group (PcG) of proteins are evolutionarily conserved transcription factors necessary for the regulation of gene expression during the development and the safeguard of cell identity in adulthood. In the nucleus, they form aggregates whose positioning and dimension are fundamental for their function. We present an algorithm, and its MATLAB implementation, based on mathematical methods to detect and analyze PcG proteins in fluorescence cell image z-stacks. Our algorithm provides a method to measure the number, the size, and the relative positioning of the PcG bodies in the nucleus for a better understanding of their spatial distribution, and thus of their role for a correct genome conformation and function.
Asunto(s)
Núcleo Celular , Imagenología Tridimensional , Proteínas del Grupo Polycomb , Núcleo Celular/metabolismo , Técnicas de Cultivo de Célula , Microscopía Fluorescente/métodosRESUMEN
The Cdkn2a locus is one of the most studied tumor suppressor loci in the context of several cancer types. However, in the last years, its expression has also been linked to terminal differentiation and the activation of the senescence program in different cellular subtypes. Knock-out (KO) of the entire locus enhances the capability of stem cells to proliferate in some tissues and respond to severe physiological and non-physiological damages in different organs, including the heart. Emery-Dreifuss muscular dystrophy (EDMD) is characterized by severe contractures and muscle loss at the level of skeletal muscles of the elbows, ankles and neck, and by dilated cardiomyopathy. We have recently demonstrated, using the LMNA Δ8-11 murine model of Emery-Dreifuss muscular dystrophy (EDMD), that dystrophic muscle stem cells prematurely express non-lineage-specific genes early on during postnatal growth, leading to rapid exhaustion of the muscle stem cell pool. Knock-out of the Cdkn2a locus in EDMD dystrophic mice partially restores muscle stem cell properties. In the present study, we describe the cardiac phenotype of the LMNA Δ8-11 mouse model and functionally characterize the effects of KO of the Cdkn2a locus on heart functions and life expectancy.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Distrofia Muscular de Emery-Dreifuss/patología , Animales , Apoptosis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Modelos Animales de Enfermedad , Sitios Genéticos , Genotipo , Lamina Tipo A/deficiencia , Lamina Tipo A/genética , Longevidad , Ratones , Ratones Noqueados , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/mortalidad , Miocardio/citología , Miocardio/metabolismo , Miocardio/patología , Fenotipo , Células Madre/citología , Células Madre/metabolismo , Tasa de SupervivenciaRESUMEN
Genome architecture and function are strictly related to nuclear structures, which contact chromatin at specific regions, regulating its compaction and three-dimensional higher-order structure, therefore contributing to specialized gene expression programs. Recently, growing evidence uncovers a dynamic role of nuclear structures in the plasticity of transcriptional programs. When the cellular microenvironment changes, external cues are transmitted to the nucleus through complex signalling cascades, finally resulting in a genome reorganization that allows the adjustment of the cell to a new condition. This process can be very rapid, especially in cells whose function is to contain sudden threats to the organism. Some examples are stem cells that switch from a quiescent to an activated state to replace damaged tissues or immune cells that, with a similar dynamic, identify and eliminate pathogens.Experimental treatments often require the isolation of cells from their physiological environment, exposing them to possible sudden changes in their nuclear architecture. Here we propose an early cross-linking on primary cells, a fixing method that can help to minimize the risk of nuclear structure alteration during the isolation process. We also bring some examples of downstream studies on early-fixed cells.
Asunto(s)
Núcleo Celular/química , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Formaldehído/química , Animales , Inmunoprecipitación de Cromatina , HumanosRESUMEN
Hutchinson-Gilford progeria syndrome is a genetic disease caused by an aberrant form of Lamin A resulting in chromatin structure disruption, in particular by interfering with lamina associated domains. Early molecular alterations involved in chromatin remodeling have not been identified thus far. Here, we present SAMMY-seq, a high-throughput sequencing-based method for genome-wide characterization of heterochromatin dynamics. Using SAMMY-seq, we detect early stage alterations of heterochromatin structure in progeria primary fibroblasts. These structural changes do not disrupt the distribution of H3K9me3 in early passage cells, thus suggesting that chromatin rearrangements precede H3K9me3 alterations described at later passages. On the other hand, we observe an interplay between changes in chromatin accessibility and Polycomb regulation, with site-specific H3K27me3 variations and transcriptional dysregulation of bivalent genes. We conclude that the correct assembly of lamina associated domains is functionally connected to the Polycomb repression and rapidly lost in early molecular events of progeria pathogenesis.
Asunto(s)
Heterocromatina/metabolismo , Lamina Tipo A/genética , Proteínas del Grupo Polycomb/metabolismo , Progeria/genética , Células Cultivadas , Niño , Preescolar , Secuenciación de Inmunoprecipitación de Cromatina , Conjuntos de Datos como Asunto , Fibroblastos , Código de Histonas/genética , Histonas/metabolismo , Humanos , Lamina Tipo A/metabolismo , Cultivo Primario de Células , Progeria/patología , RNA-Seq , Piel/citología , Piel/patología , Activación TranscripcionalRESUMEN
Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin Δ8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth. We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin Δ8-11 -/- mutant mice these capabilities are severely impaired. This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.
Asunto(s)
Desarrollo Embrionario/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/embriología , Distrofia Muscular de Emery-Dreifuss/genética , Mioblastos Esqueléticos/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A-dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non-muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C-null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.
Asunto(s)
Epigénesis Genética , Lamina Tipo A/biosíntesis , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Animales , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Lamina Tipo A/genética , Ratones , Ratones Noqueados , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/patología , Proteínas del Grupo Polycomb/genética , Proteínas Represoras/genéticaRESUMEN
The alteration of the several roles that Lamin A/C plays in the mammalian cell leads to a broad spectrum of pathologies that - all together - are named laminopathies. Among those, the Emery Dreifuss Muscular Dystrophy (EDMD) is of particular interest as, despite the several known mutations of Lamin A/C, the genotype-phenotype correlation still remains poorly understood; this suggests that the epigenetic background of patients might play an important role during the time course of the disease. Historically, both a mechanical role of Lamin A/C and a regulative one have been suggested as the driving force of laminopathies; however, those two hypotheses are not mutually exclusive. Recent scientific evidence shows that Lamin A/C sustains the correct gene expression at the epigenetic level thanks to the Lamina Associated Domains (LADs) reorganization and the crosstalk with the Polycomb Group of Proteins (PcG). Furthermore, the PcG-dependent histone mark H3K27me3 increases under mechanical stress, finally pointing out the link between the mechano-properties of the nuclear lamina and epigenetics. Here, we summarize the emerging mechanisms that could explain the high variability seen in Emery Dreifuss muscular dystrophy.