RESUMEN
Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1-/- cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1-/- cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging.
Asunto(s)
Senescencia Celular , Citosol , Daño del ADN , Comunicación Paracrina , Telómero , Senescencia Celular/genética , Animales , Telómero/metabolismo , Telómero/genética , Ratones , Citosol/metabolismo , ADN/metabolismo , Transcripción Genética , Ratones Noqueados , Humanos , Vesículas Extracelulares/metabolismo , Inestabilidad Genómica , Envejecimiento/genética , Envejecimiento/metabolismo , Estrés Oxidativo , Ratones Endogámicos C57BLRESUMEN
DNA damage and neurodegenerative disorders are intimately linked but the underlying mechanism remains elusive. Here, we show that persistent DNA lesions in tissue-resident macrophages carrying an XPF-ERCC1 DNA repair defect trigger neuroinflammation and neuronal cell death in mice. We find that microglia accumulate dsDNAs and chromatin fragments in the cytosol, which are sensed thereby stimulating a viral-like immune response in Er1Cx/- and naturally aged murine brain. Cytosolic DNAs are packaged into extracellular vesicles (EVs) that are released from microglia and discharge their dsDNA cargo into IFN-responsive neurons triggering cell death. To remove cytosolic dsDNAs and prevent inflammation, we developed targeting EVs to deliver recombinant DNase I to Er1Cx/- brain microglia in vivo. We show that EV-mediated elimination of cytosolic dsDNAs is sufficient to prevent neuroinflammation, reduce neuronal apoptosis, and delay the onset of neurodegenerative symptoms in Er1Cx/- mice. Together, our findings unveil a causal mechanism leading to neuroinflammation and provide a rationalized therapeutic strategy against age-related neurodegeneration.
Asunto(s)
Vesículas Extracelulares , Microglía , Ratones , Animales , Microglía/metabolismo , Enfermedades Neuroinflamatorias , Neuronas/patología , Daño del ADNRESUMEN
Co-transcriptional RNA-DNA hybrids can not only cause DNA damage threatening genome integrity but also regulate gene activity in a mechanism that remains unclear. Here, we show that the nucleotide excision repair factor XPF interacts with the insulator binding protein CTCF and the cohesin subunits SMC1A and SMC3, leading to R-loop-dependent DNA looping upon transcription activation. To facilitate R-loop processing, XPF interacts and recruits with TOP2B on active gene promoters, leading to double-strand break accumulation and the activation of a DNA damage response. Abrogation of TOP2B leads to the diminished recruitment of XPF, CTCF, and the cohesin subunits to promoters of actively transcribed genes and R-loops and the concurrent impairment of CTCF-mediated DNA looping. Together, our findings disclose an essential role for XPF with TOP2B and the CTCF/cohesin complex in R-loop processing for transcription activation with important ramifications for DNA repair-deficient syndromes associated with transcription-associated DNA damage.
Asunto(s)
Proteínas de Unión al ADN , Estructuras R-Loop , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Cromosomas , Reparación del ADN , CromatinaRESUMEN
The DNA-repair capacity in somatic cells is limited compared with that in germ cells. It has remained unknown whether not only lesion-type-specific, but overall repair capacities could be improved. Here we show that the DREAM repressor complex curbs the DNA-repair capacities in somatic tissues of Caenorhabditis elegans. Mutations in the DREAM complex induce germline-like expression patterns of multiple mechanisms of DNA repair in the soma. Consequently, DREAM mutants confer resistance to a wide range of DNA-damage types during development and aging. Similarly, inhibition of the DREAM complex in human cells boosts DNA-repair gene expression and resistance to distinct DNA-damage types. DREAM inhibition leads to decreased DNA damage and prevents photoreceptor loss in progeroid Ercc1-/- mice. We show that the DREAM complex transcriptionally represses essentially all DNA-repair systems and thus operates as a highly conserved master regulator of the somatic limitation of DNA-repair capacities.
Asunto(s)
Proteínas de Caenorhabditis elegans , Humanos , Animales , Ratones , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Reparación del ADN , Daño del ADN , ADN/metabolismo , Células Germinativas/metabolismoRESUMEN
BACKGROUND: In fluorescence microscopy, co-localization refers to the spatial overlap between different fluorescent labels in cells. The degree of overlap between two or more channels in a microscope may reveal a physical interaction or topological functional interconnection between molecules. Recent advances in the imaging field require the development of specialized computational analysis software for the unbiased assessment of fluorescently labelled microscopy images. RESULTS: Here we present SpotitPy, a semi-automated image analysis tool for 2D object-based co-localization. SpotitPy allows the user to select fluorescent labels and perform a semi-automated and robust segmentation of the region of interest in distinct cell types. The workflow integrates advanced pre-processing manipulations for de-noising and in-depth semi-automated quantification of the co-localized fluorescent labels in two different channels. We validated SpotitPy by quantitatively assessing the presence of cytoplasmic ribonucleoprotein granules, e.g. processing (P) bodies, under conditions that challenge mRNA translation, thus highlighting SpotitPy benefits for semi-automatic, accurate analysis of large image datasets in eukaryotic cells. SpotitPy comes in a command line interface or a simple graphical user interphase and can be used as a standalone application. CONCLUSIONS: Overall, we present a novel and user-friendly tool that performs a semi-automated image analysis for 2D object-based co-localization. SpotitPy can provide reproducible and robust quantifications for large datasets within a limited timeframe. The software is open-source and can be found in the GitHub project repository: ( https://github.com/alexiaales/SpotitPy ).
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Programas Informáticos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodosRESUMEN
Persistent DNA lesions build up with aging triggering inflammation, the body's first line of immune defense strategy against foreign pathogens and irritants. Once established, DNA damage-driven inflammation takes on a momentum of its own, due to the amplification and feedback loops of the immune system leading to cellular malfunction, tissue degenerative changes and metabolic complications. Here, we discuss the use of murine models with inborn defects in genome maintenance and the DNA damage response for understanding how irreparable DNA lesions are functionally linked to innate immune signaling highlighting their relevance for developing novel therapeutic strategies against the premature onset of aging-associated diseases.
RESUMEN
How DNA damage leads to chronic inflammation and tissue degeneration with aging remains to be fully resolved. Here, we show that DNA damage leads to cellular senescence, fibrosis, loss-of-tissue architecture, and chronic pancreatitis in mice with an inborn defect in the excision repair cross complementation group 1 (Ercc1) gene. We find that DNA damage-driven R-loops causally contribute to the active release and buildup of single-stranded DNAs (ssDNAs) in the cytoplasm of cells triggering a viral-like immune response in progeroid and naturally aged pancreata. To reduce the proinflammatory load, we developed an extracellular vesicle (EV)-based strategy to deliver recombinant S1 or ribonuclease H nucleases in inflamed Ercc1−/− pancreatic cells. Treatment of Ercc1−/− animals with the EV-delivered nuclease cargo eliminates DNA damage-induced R-loops and cytoplasmic ssDNAs alleviating chronic inflammation. Thus, DNA damage-driven ssDNAs causally contribute to tissue degeneration, Ercc1−/− paving the way for novel rationalized intervention strategies against age-related chronic inflammation.
Asunto(s)
Reparación del ADN , Estructuras R-Loop , Animales , Citoplasma , Daño del ADN , ADN de Cadena Simple , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Inflamación , RatonesRESUMEN
Progeroid syndromes (PSs) are characterized by the premature onset of age-related pathologies. The genetic mutations underlying PSs are functionally linked to genome maintenance and repair, supporting the causative role of DNA damage accumulation in aging. Recent advances from studies in animal models of PSs have provided new insight into the role of DNA repair mechanisms in human disease and the physiological adaptations to accumulating DNA damage during aging. The molecular pathology of PSs is reminiscent of the natural aging process, highlighting the relevance for a wide range of age-related diseases. Recent progress has led to the development of novel therapeutic strategies against age-related diseases that are relevant to rare diseases as well as the general aging population.
Asunto(s)
Envejecimiento Prematuro , Patología Molecular , Anciano , Envejecimiento/genética , Animales , Daño del ADN , Reparación del ADN/genética , Humanos , Enfermedades RarasRESUMEN
RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Empalme de ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Daño del ADN/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Técnicas de Silenciamiento del Gen , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Células Madre Embrionarias de Ratones , Sesquiterpenos Policíclicos/farmacología , Estructuras R-Loop/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme de ARN/genética , ARN Nuclear Pequeño , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Empalmosomas/metabolismo , Transcripción GenéticaRESUMEN
In mammals, genome instability and aging are intimately linked as illustrated by the growing list of patients with progeroid and animal models with inborn DNA repair defects. Until recently, DNA damage was thought to drive aging by compromising transcription or DNA replication, thereby leading to age-related cellular malfunction and somatic mutations triggering cancer. However, recent evidence suggests that DNA lesions also elicit widespread epigenetic alterations that threaten cell homeostasis as a function of age. In this review, we discuss the functional links of persistent DNA damage with the epigenome in the context of aging and age-related diseases.
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Envejecimiento/genética , Epigénesis Genética , Epigenoma , Acortamiento del Telómero , Animales , Daño del ADN , Reparación del ADN , Inestabilidad Genómica , Humanos , Modelos AnimalesRESUMEN
Regulatory T cells (Tregs) are vital for the maintenance of immune homeostasis, while their dysfunction constitutes a cardinal feature of autoimmunity. Under steady-state conditions, mitochondrial metabolism is critical for Treg function; however, the metabolic adaptations of Tregs during autoimmunity are ill-defined. Herein, we report that elevated mitochondrial oxidative stress and a robust DNA damage response (DDR) associated with cell death occur in Tregs in individuals with autoimmunity. In an experimental autoimmune encephalitis (EAE) mouse model of autoimmunity, we found a Treg dysfunction recapitulating the features of autoimmune Tregs with a prominent mtROS signature. Scavenging of mtROS in Tregs of EAE mice reversed the DDR and prevented Treg death, while attenuating the Th1 and Th17 autoimmune responses. These findings highlight an unrecognized role of mitochondrial oxidative stress in defining Treg fate during autoimmunity, which may facilitate the design of novel immunotherapies for diseases with disturbed immune tolerance.
Asunto(s)
Autoinmunidad/inmunología , Mitocondrias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-ReducciónRESUMEN
Nuclear DNA damage contributes to cellular malfunction and the premature onset of age-related diseases, including cancer. Until recently, the canonical DNA damage response (DDR) was thought to represent a collection of nuclear processes that detect, signal and repair damaged DNA. However, recent evidence suggests that beyond nuclear events, the DDR rewires an intricate network of metabolic circuits, fine-tunes protein synthesis, trafficking, and secretion as well as balances growth with defense strategies in response to genotoxic insults. In this review, we discuss how the active DDR signaling mobilizes extranuclear and systemic responses to promote cellular homeostasis and organismal survival in health and disease.
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Envejecimiento , Reprogramación Celular , Senescencia Celular , Daño del ADN , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Neoplasias/genética , Animales , Enzimas Reparadoras del ADN/genética , Humanos , MutaciónRESUMEN
DNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Here, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1F/-) triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Macrophage-derived EVs accumulate in Er1F/- animal sera and are secreted in macrophage media after DNA damage. The Er1F/- EV cargo is taken up by recipient cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake, higher cellular oxygen consumption rate and greater tolerance to glucose challenge in mice. We find that high glucose in EV-targeted cells triggers pro-inflammatory stimuli via mTOR activation. This, in turn, establishes chronic inflammation and tissue pathology in mice with important ramifications for DNA repair-deficient, progeroid syndromes and aging.
Asunto(s)
Daño del ADN/fisiología , Exosomas/metabolismo , Macrófagos/citología , Animales , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Exosomas/patología , Regulación de la Expresión Génica , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Masculino , Ratones Transgénicos , Neuropéptidos/genética , Neuropéptidos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Transcription is a potential threat to genome integrity, and transcription-associated DNA damage must be repaired for proper messenger RNA (mRNA) synthesis and for cells to transmit their genome intact into progeny. For a wide range of structurally diverse DNA lesions, cells employ the highly conserved nucleotide excision repair (NER) pathway to restore their genome back to its native form. Recent evidence suggests that NER factors function, in addition to the canonical DNA repair mechanism, in processes that facilitate mRNA synthesis or shape the 3D chromatin architecture. Here, these findings are critically discussed and a working model that explains the puzzling clinical heterogeneity of NER syndromes highlighting the relevance of physiological, transcription-associated DNA damage to mammalian development and disease is proposed.
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Reparación del ADN/genética , Inestabilidad Genómica , Transcripción Genética , Animales , Cromatina/química , Cromatina/metabolismo , Daño del ADN/genética , Humanos , ARN Mensajero/biosíntesisRESUMEN
Although the links between defects in DNA repair and cancer are well established, an accumulating body of evidence suggests a series of functional links between genome maintenance pathways, lifespan regulation mechanisms and age-related diseases in mammals. Indeed, the growing number of DNA repair-deficient patients with progeria suggests that persistent DNA damage and genome caretakers are tightly linked to lifespan regulating circuits and age-related diseases. Here, we discuss the impact of irreparable DNA damage events in mammalian physiology highlighting the relevance of DNA repair factors in mammalian development and aging.
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Envejecimiento/genética , Envejecimiento/patología , Núcleo Celular/genética , Núcleo Celular/patología , Daño del ADN , Reparación del ADN , Animales , Humanos , Longevidad , Progeria/genética , Progeria/patologíaAsunto(s)
Envejecimiento , Aberraciones Cromosómicas , Daño del ADN , Reparación del ADN/inmunología , ADN de Neoplasias , Neoplasias , Envejecimiento/genética , Envejecimiento/inmunología , Animales , ADN de Neoplasias/genética , ADN de Neoplasias/inmunología , Humanos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patologíaRESUMEN
Inborn defects in DNA repair are associated with complex developmental disorders whose causal mechanisms are poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the nucleotide excision repair (NER) structure-specific endonuclease ERCC1-XPF complex interacts with the insulator binding protein CTCF, the cohesin subunits SMC1A and SMC3 and with MBD2; the factors co-localize with ATRX at the promoters and control regions (ICRs) of imprinted genes during postnatal hepatic development. Loss of Ercc1 or exposure to MMC triggers the localization of CTCF to heterochromatin, the dissociation of the CTCF-cohesin complex and ATRX from promoters and ICRs, altered histone marks and the aberrant developmental expression of imprinted genes without altering DNA methylation. We propose that ERCC1-XPF cooperates with CTCF and cohesin to facilitate the developmental silencing of imprinted genes and that persistent DNA damage triggers chromatin changes that affect gene expression programs associated with NER disorders.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Silenciador del Gen , Impresión Genómica , Proteínas Represoras/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas Cromosómicas no Histona/genética , Técnicas de Cocultivo , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Fibroblastos/enzimología , Regulación del Desarrollo de la Expresión Génica , Genotipo , Histonas/metabolismo , Hígado/enzimología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteína Nuclear Ligada al Cromosoma X , CohesinasRESUMEN
Nuclear architecture and the chromatin state affect most-if not all- DNA-dependent transactions, including the ability of cells to sense DNA lesions and restore damaged DNA back to its native form. Recent evidence points to functional links between DNA damage sensors, DNA repair mechanisms and the innate immune responses. The latter raises the question of how such seemingly disparate processes operate within the intrinsically complex nuclear landscape and the chromatin environment. Here, we discuss how DNA damage-induced immune responses operate within chromatin and the distinct sub-nuclear compartments highlighting their relevance to chronic inflammation.
Asunto(s)
Cromatina/inmunología , Daño del ADN/inmunología , Reparación del ADN/inmunología , Inmunidad Innata , Animales , Cromatina/genética , Reparación del ADN/genética , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/patologíaRESUMEN
To lessen the "wear and tear" of existence, cells have evolved mechanisms that continuously sense DNA lesions, repair DNA damage and restore the compromised genome back to its native form. Besides genome maintenance pathways, multicellular organisms may also employ adaptive and innate immune mechanisms to guard themselves against bacteria or viruses. Recent evidence points to reciprocal interactions between DNA repair, DNA damage responses and aspects of immunity; both self-maintenance and defense responses share a battery of common players and signaling pathways aimed at safeguarding our bodily functions over time. In the short-term, this functional interplay would allow injured cells to restore damaged DNA templates or communicate their compromised state to the microenvironment. In the long-term, however, it may result in the (premature) onset of age-related degeneration, including cancer. Here, we discuss the beneficial and unrewarding outcomes of DNA damage-driven inflammation in the context of tissue-specific pathology and disease progression.
RESUMEN
DNA damage contributes to the process of aging, as underscored by premature aging syndromes caused by defective DNA repair. Thyroid state changes during aging, but underlying mechanisms remain elusive. Since thyroid hormone (TH) is a key regulator of metabolism, changes in TH signaling have widespread effects. Here, we reveal a significant common transcriptomic signature in livers from hypothyroid mice, DNA repair-deficient mice with severe (Csbm/m/Xpa-/-) or intermediate (Ercc1-/Δ-7) progeria and naturally aged mice. A strong induction of TH-inactivating deiodinase D3 and decrease of TH-activating D1 activities are observed in Csbm/m/Xpa-/- livers. Similar findings are noticed in Ercc1-/Δ-7, in naturally aged animals and in wild-type mice exposed to a chronic subtoxic dose of DNA-damaging agents. In contrast, TH signaling in muscle, heart and brain appears unaltered. These data show a strong suppression of TH signaling in specific peripheral organs in premature and normal aging, probably lowering metabolism, while other tissues appear to preserve metabolism. D3-mediated TH inactivation is unexpected, given its expression mainly in fetal tissues. Our studies highlight the importance of DNA damage as the underlying mechanism of changes in thyroid state. Tissue-specific regulation of deiodinase activities, ensuring diminished TH signaling, may contribute importantly to the protective metabolic response in aging.