RESUMEN
Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.
Asunto(s)
COVID-19 , Enfermedades Cardiovasculares , Humanos , Hematopoyesis Clonal/genética , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/genéticaRESUMEN
Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.
Asunto(s)
Reparación del ADN , Anemia de Fanconi , Genómica , Neoplasias de Cabeza y Cuello , Humanos , Aldehídos/efectos adversos , Aldehídos/metabolismo , Reparación del ADN/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Neoplasias de Cabeza y Cuello/inducido químicamente , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Infecciones por Papillomavirus , Carcinoma de Células Escamosas de Cabeza y Cuello/inducido químicamente , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Daño del ADN/efectos de los fármacosRESUMEN
DNA double-strand breaks (DSBs) are rare, but highly toxic, lesions requiring orchestrated and conserved machinery to prevent adverse consequences, such as cell death and cancer-causing genome structural mutations. DSBs trigger the DNA damage response (DDR) that directs a cell to repair the break, undergo apoptosis, or become senescent. There is increasing evidence that the various endpoints of DSB processing by different cells and tissues are part of the aging phenotype, with each stage of the DDR associated with specific aging pathologies. In this Perspective, we discuss the possibility that DSBs are major drivers of intrinsic aging, highlighting the dynamics of spontaneous DSBs in relation to aging, the distinct age-related pathologies induced by DSBs, and the segmental progeroid phenotypes in humans and mice with genetic defects in DSB repair. A model is presented as to how DSBs could drive some of the basic mechanisms underlying age-related functional decline and death.
Asunto(s)
Envejecimiento/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN/genética , Regulación de la Expresión Génica , Progeria/genética , Ácido Anhídrido Hidrolasas , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Senescencia Celular , ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Proteína Homóloga de MRE11 , Ratones , Modelos Genéticos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Progeria/metabolismo , Progeria/patología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.
Asunto(s)
Aneuploidia , Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Cromatina/metabolismo , Cromosomas Humanos/metabolismo , Replicación del ADN , Línea Celular , Centrómero/genética , Proteína A Centromérica/genética , Cromatina/genética , Cromosomas Humanos/genética , Humanos , Fase SRESUMEN
BACKGROUND: Mammalian aging is a highly complex process, a full mechanistic understanding of which is still lacking. One way to help understand the molecular changes underlying aging is through a comprehensive analysis of the transcriptome, the primary determinant of age-related phenotypic diversity. Previous studies have relied on microarray analysis to examine gene expression profiles in different tissues of aging organisms. However, studies have shown microarray-based transcriptional profiling is less accurate and not fully capable of capturing certain intricacies of the global transcriptome. METHODS: Here, using directional whole transcriptome RNA-sequencing of aged mouse liver we have identified a comprehensive high-resolution profile of differentially expressed liver transcripts comprised of canonical protein-coding transcripts, transcript isoforms, and non-coding RNA transcripts, including pseudogenes, long non-coding RNAs and small RNA species. RESULTS: Results show extensive age-related changes in every component of the mouse liver transcriptome and a pronounced increase in inter-individual variation. Functional annotation of the protein-coding mRNAs and isoforms indicated broad alterations in immune response, cell activation, metabolic processes, and RNA modification. Interestingly, multiple lncRNAs (Meg3, Rian, Mirg) from the Dlk-Dio3 microRNA locus were found up-regulated in aging liver, classifying this locus as a putative regulatory hotspot locus in aging liver. Moreover, integration of the altered non-coding RNAs and protein-coding transcripts into interaction networks of age-related change revealed inflammation, cellular proliferation, and metabolism as the dominant aging phenotypes in mouse liver. CONCLUSIONS: Our analyses provide the first comprehensive dissection of the transcriptional landscape in aging mouse liver.
Asunto(s)
Hígado/metabolismo , Transcriptoma/genética , Envejecimiento/genética , Envejecimiento/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Freshwater polyps of the genus Hydra do not age. However, temperature stress induces aging and a shift from reproduction by asexual budding to sexual gamete production in a cold-sensitive (CS) strain of H. oligactis. We sequenced the transcriptome of a male CS strain before and after this life history shift and compared changes in gene expression relative to those seen in a cold-resistant (CR) strain that does not undergo a life history shift in response to altered temperature. We found that the switch from non-aging asexual reproduction to aging and sexual reproduction involves upregulation of genes not only involved in gametogenesis but also genes involved in cellular senescence, apoptosis, and DNA repair accompanied by a downregulation of genes involved in stem cell maintenance. These results suggest that aging is a byproduct of sexual reproduction-associated cellular reprogramming and underscore the power of these H. oligactis strains to identify intrinsic mechanisms of aging.
Asunto(s)
Hydra , Envejecimiento/genética , Animales , Masculino , Reproducción/genética , Reproducción Asexuada , Células MadreRESUMEN
Genomic instability is one of the hallmarks of aging, and both DNA damage and mutations have been found to accumulate with age in different species. Certain gene families, such as sirtuins and the FoxO family of transcription factors, have been shown to play a role in lifespan extension. However, the mechanism(s) underlying the increased longevity associated with these genes remains largely unknown and may involve the regulation of responses to cellular stressors, such as DNA damage. Here, we report that FOXO3a reduces genomic instability in cultured mouse embryonic fibroblasts (MEFs) treated with agents that induce DNA double-strand breaks (DSBs), that is, clastogens. We show that DSB treatment of both primary human and mouse fibroblasts upregulates FOXO3a expression. FOXO3a ablation in MEFs harboring the mutational reporter gene lacZ resulted in an increase in genome rearrangements after bleomycin treatment; conversely, overexpression of human FOXO3a was found to suppress mutation accumulation in response to bleomycin. We also show that overexpression of FOXO3a in human primary fibroblasts decreases DSB-induced γH2AX foci. Knocking out FOXO3a in mES cells increased the frequency of homologous recombination and non-homologous end-joining events. These results provide the first direct evidence that FOXO3a plays a role in suppressing genome instability, possibly by suppressing genome rearrangements.
Asunto(s)
Roturas del ADN de Doble Cadena , Daño del ADN/genética , Proteína Forkhead Box O3/genética , Factores de Edad , Humanos , MutaciónRESUMEN
Standard methods for mitochondrial DNA (mtDNA) extraction do not provide the level of enrichment for mtDNA sufficient for direct sequencing and must be followed by long-range-PCR amplification, which can bias the sequence results. Here, we describe a reliable method for the preparation of mtDNA-enriched samples from eukaryotic cells ready for direct sequencing. This protocol utilizes a conventional miniprep kit, in conjunction with a paramagnetic bead-based purification step.
Asunto(s)
ADN Mitocondrial/genética , ADN Mitocondrial/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Animales , Células Cultivadas , HumanosRESUMEN
The advent of next generation sequencing (NGS) technology has provided the means to directly analyze the genetic material in primary cells or tissues of any species in a high throughput manner for mutagenic effects of potential genotoxic agents. In principle, direct, genome-wide sequencing of human primary cells and/or tissue biopsies would open up opportunities to identify individuals possibly exposed to mutagenic agents, thereby replacing current risk assessment procedures based on surrogate markers and extrapolations from animal studies. NGS-based tests can also precisely characterize the mutation spectra induced by genotoxic agents, improving our knowledge of their mechanism of action. Thus far, NGS has not been widely employed in genetic toxicology due to the difficulties in measuring low-abundant somatic mutations. Here, we review different strategies to employ NGS for the detection of somatic mutations in a cost-effective manner and discuss the potential applicability of these methods in testing the mutagenicity of genotoxic agents.
Asunto(s)
Daño del ADN , Estudio de Asociación del Genoma Completo/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Animales , Análisis Mutacional de ADN/métodos , HumanosRESUMEN
DNA damage has been implicated in ageing, but direct evidence for a causal relationship is lacking, owing to the difficulty of inducing defined DNA lesions in cells and tissues without simultaneously damaging other biomolecules and cellular structures. Here we directly test whether highly toxic DNA double-strand breaks (DSBs) alone can drive an ageing phenotype using an adenovirus-based system based on tetracycline-controlled expression of the SacI restriction enzyme. We deliver the adenovirus to mice and compare molecular and cellular end points in the liver with normally aged animals. Treated, 3-month-old mice display many, but not all signs of normal liver ageing as early as 1 month after treatment, including ageing pathologies, markers of senescence, fused mitochondria and alterations in gene expression profiles. These results, showing that DSBs alone can cause distinct ageing phenotypes in mouse liver, provide new insights in the role of DNA damage as a driver of tissue ageing.
Asunto(s)
Adenoviridae/genética , Envejecimiento/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN/genética , Envejecimiento/patología , Animales , Biomarcadores/metabolismo , ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Tetraciclina/farmacología , TransgenesRESUMEN
Differences in DNA repair capacity have been hypothesized to underlie the great range of maximum lifespans among mammals. However, measurements of individual DNA repair activities in cells and animals have not substantiated such a relationship because utilization of repair pathways among animals--depending on habitats, anatomical characteristics, and life styles--varies greatly between mammalian species. Recent advances in high-throughput genomics, in combination with increased knowledge of the genetic pathways involved in genome maintenance, now enable a comprehensive comparison of DNA repair transcriptomes in animal species with extreme lifespan differences. Here we compare transcriptomes of liver, an organ with high oxidative metabolism and abundant spontaneous DNA damage, from humans, naked mole rats, and mice, with maximum lifespans of ~120, 30, and 3 years, respectively, with a focus on genes involved in DNA repair. The results show that the longer-lived species, human and naked mole rat, share higher expression of DNA repair genes, including core genes in several DNA repair pathways. A more systematic approach of signaling pathway analysis indicates statistically significant upregulation of several DNA repair signaling pathways in human and naked mole rat compared with mouse. The results of this present work indicate, for the first time, that DNA repair is upregulated in a major metabolic organ in long-lived humans and naked mole rats compared with short-lived mice. These results strongly suggest that DNA repair can be considered a genuine longevity assurance system.
Asunto(s)
Reparación del ADN/fisiología , Longevidad/genética , Longevidad/fisiología , Animales , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Ratas , Especificidad de la Especie , TranscriptomaRESUMEN
We found that a neuron-specific isoform of LSD1, LSD1n, which results from an alternative splicing event, acquires a new substrate specificity, targeting histone H4 Lys20 methylation, both in vitro and in vivo. Selective genetic ablation of LSD1n led to deficits in spatial learning and memory, revealing the functional importance of LSD1n in neuronal activity-regulated transcription that is necessary for long-term memory formation. LSD1n occupied neuronal gene enhancers, promoters and transcribed coding regions, and was required for transcription initiation and elongation steps in response to neuronal activity, indicating the crucial role of H4K20 methylation in coordinating gene transcription with neuronal function. Our results indicate that this alternative splicing of LSD1 in neurons, which was associated with altered substrate specificity, serves as a mechanism acquired by neurons to achieve more precise control of gene expression in the complex processes underlying learning and memory.
Asunto(s)
Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Memoria a Largo Plazo/fisiología , Transcripción Genética/fisiología , Animales , Células Cultivadas , Corteza Cerebral/metabolismo , Femenino , Eliminación de Gen , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G(2)). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes-two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit-granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.
Asunto(s)
Cromosomas de los Mamíferos/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Recombinación Genética/fisiología , Animales , Cromosomas de los Mamíferos/genética , Ratones , Ratones Transgénicos , Especificidad de Órganos/fisiologíaRESUMEN
Standard methods for mitochondrial DNA (mtDNA) extraction do not provide the level of enrichment for mtDNA sufficient for direct sequencing and must be followed by long-range-PCR amplification, which can bias the sequencing results. Here, we describe a fast, cost-effective, and reliable method for preparation of mtDNA enriched samples from eukaryotic cells ready for direct sequencing. Our protocol utilizes a conventional miniprep kit, paramagnetic bead-based purification, and an optional, limited PCR amplification of mtDNA. The first two steps alone provide more than 2000-fold enrichment for mtDNA when compared with total cellular DNA (~200-fold in comparison with current commercially available kits) as demonstrated by real-time PCR. The percentage of sequencing reads aligned to mtDNA was about 22% for non-amplified samples and greater than 99% for samples subjected to 10 cycles of long-range-PCR with mtDNA specific primers.
Asunto(s)
ADN Mitocondrial/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Animales , Biotecnología/métodos , Línea Celular , ADN Mitocondrial/química , ADN Mitocondrial/genética , Ratones , Reacción en Cadena de la Polimerasa , Alineación de SecuenciaRESUMEN
Steady-state levels of spontaneous DNA damage, the by-product of normal metabolism and environmental exposure, are controlled by DNA repair pathways. Incomplete repair or an age-related increase in damage production and/or decline in repair could lead to an accumulation of DNA damage, increasing mutation rate, affecting transcription, and/or activating programmed cell death or senescence. These consequences of DNA damage metabolism are highly conserved, and the accumulation of lesions in the DNA of the genome could therefore provide a universal cause of aging. An important corollary of this hypothesis is that defects in DNA repair cause both premature aging and accelerated DNA damage accumulation. While the former has been well-documented, the reliable quantification of the various lesions thought to accumulate in DNA during aging has been a challenge. Here, we quantified inhibition of long-distance PCR as a measure of DNA damage in liver and brain of both normal and prematurely aging, DNA repair defective mice. The results indicate a marginal, but statistically significant, increase in spontaneous DNA damage with age in normal mouse liver but not in brain. Increased levels of DNA damage were not observed in the DNA repair defective mice. We also show that oxidative lesions do not increase with age. These results indicate that neither normal nor premature aging is accompanied by a dramatic increase in DNA damage. This suggests that factors other than DNA damage per se, for example, cellular responses to DNA damage, are responsible for the aging phenotype in mice.