RESUMEN
BACKGROUND: DNA methylation is an important epigenetic modification which has numerous roles in modulating genome function. Its levels are spatially correlated across the genome, typically high in repressed regions but low in transcription factor (TF) binding sites and active regulatory regions. However, the mechanisms establishing genome-wide and TF binding site methylation patterns are still unclear. RESULTS: Here we use a comparative approach to investigate the association of DNA methylation to TF binding evolution in mammals. Specifically, we experimentally profile DNA methylation and combine this with published occupancy profiles of five distinct TFs (CTCF, CEBPA, HNF4A, ONECUT1, FOXA1) in the liver of five mammalian species (human, macaque, mouse, rat, dog). TF binding sites are lowly methylated, but they often also have intermediate methylation levels. Furthermore, biding sites are influenced by the methylation status of CpGs in their wider binding regions even when CpGs are absent from the core binding motif. Employing a classification and clustering approach, we extract distinct and species-conserved patterns of DNA methylation levels at TF binding regions. CEBPA, HNF4A, ONECUT1, and FOXA1 share the same methylation patterns, while CTCF's differ. These patterns characterize alternative functions and chromatin landscapes of TF-bound regions. Leveraging our phylogenetic framework, we find DNA methylation gain upon evolutionary loss of TF occupancy, indicating coordinated evolution. Furthermore, each methylation pattern has its own evolutionary trajectory reflecting its genomic contexts. CONCLUSIONS: Our epigenomic analyses indicate a role for DNA methylation in TF binding changes across species including that specific DNA methylation profiles characterize TF binding and are associated with their regulatory activity, chromatin contexts, and evolutionary trajectories.
Asunto(s)
Metilación de ADN , Evolución Molecular , Factores de Transcripción , Animales , Sitios de Unión , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ratones , Ratas , Islas de CpG , Perros , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Unión Proteica , Hígado/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genéticaRESUMEN
DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.
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Daño del ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN , ADN , Mutagénesis , Mutación , Animales , Humanos , Ratones , Alquilación/efectos de la radiación , Línea Celular , ADN/química , ADN/genética , ADN/metabolismo , ADN/efectos de la radiación , Aductos de ADN/química , Aductos de ADN/genética , Aductos de ADN/metabolismo , Aductos de ADN/efectos de la radiación , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Reparación del ADN/genética , Reparación del ADN/fisiología , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Mutagénesis/genética , Mutagénesis/efectos de la radiación , Mutación/genética , Mutación/efectos de la radiación , Neoplasias/genética , Transcripción Genética , Rayos Ultravioleta/efectos adversosRESUMEN
DNA base damage is a major source of oncogenic mutations and disruption to gene expression. The stalling of RNA polymerase II (RNAP) at sites of DNA damage and the subsequent triggering of repair processes have major roles in shaping the genome-wide distribution of mutations, clearing barriers to transcription, and minimizing the production of miscoded gene products. Despite its importance for genetic integrity, key mechanistic features of this transcription-coupled repair (TCR) process are controversial or unknown. Here, we exploited a well-powered in vivo mammalian model system to explore the mechanistic properties and parameters of TCR for alkylation damage at fine spatial resolution and with discrimination of the damaged DNA strand. For rigorous interpretation, a generalizable mathematical model of DNA damage and TCR was developed. Fitting experimental data to the model and simulation revealed that RNA polymerases frequently bypass lesions without triggering repair, indicating that small alkylation adducts are unlikely to be an efficient barrier to gene expression. Following a burst of damage, the efficiency of transcription-coupled repair gradually decays through gene bodies with implications for the occurrence and accurate inference of driver mutations in cancer. The reinitation of transcription from the repair site is not a general feature of transcription-coupled repair, and the observed data is consistent with reinitiation never taking place. Collectively, these results reveal how the directional but stochastic activity of TCR shapes the distribution of mutations following DNA damage.
Asunto(s)
Daño del ADN , Reparación del ADN , ARN Polimerasa II , Transcripción Genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Animales , Procesos Estocásticos , Ratones , ADN/metabolismo , ADN/genética , Humanos , Alquilación , Mutación , Reparación por EscisiónRESUMEN
How chronic mutational processes and punctuated bursts of DNA damage drive evolution of the cancer genome is poorly understood. Here, we demonstrate a strategy to disentangle and quantify distinct mechanisms underlying genome evolution in single cells, during single mitoses and at single-strand resolution. To distinguish between chronic (reactive oxygen species (ROS)) and acute (ultraviolet light (UV)) mutagenesis, we microfluidically separate pairs of sister cells from the first mitosis following burst UV damage. Strikingly, UV mutations manifest as sister-specific events, revealing mirror-image mutation phasing genome-wide. In contrast, ROS mutagenesis in transcribed regions is reduced strand agnostically. Successive rounds of genome replication over persisting UV damage drives multiallelic variation at CC dinucleotides. Finally, we show that mutation phasing can be resolved to single strands across the entire genome of liver tumors from F1 mice. This strategy can be broadly used to distinguish the contributions of overlapping cancer relevant mutational processes.
Asunto(s)
Daño del ADN , Reparación del ADN , Mitosis , Mutagénesis , Rayos Ultravioleta , Animales , Ratones , Reparación del ADN/genética , Rayos Ultravioleta/efectos adversos , Daño del ADN/genética , Mitosis/genética , Especies Reactivas de Oxígeno/metabolismo , Mutación , HumanosRESUMEN
BACKGROUND & AIMS: Polyploidy in hepatocytes has been proposed as a genetic mechanism to buffer against transcriptional dysregulation. Here, we aim to demonstrate the role of polyploidy in modulating gene regulatory networks in hepatocytes during ageing. METHODS: We performed single-nucleus RNA sequencing in hepatocyte nuclei of different ploidy levels isolated from young and old wild-type mice. Changes in the gene expression and regulatory network were compared to three independent strains that were haploinsufficient for HNF4A, CEBPA or CTCF, representing non-deleterious perturbations. Phenotypic characteristics of the liver section were additionally evaluated histologically, whereas the genomic allele composition of hepatocytes was analysed by BaseScope. RESULTS: We observed that ageing in wild-type mice results in nuclei polyploidy and a marked increase in steatosis. Haploinsufficiency of liver-specific master regulators (HFN4A or CEBPA) results in the enrichment of hepatocytes with tetraploid nuclei at a young age, affecting the genomic regulatory network, and dramatically suppressing ageing-related steatosis tissue wide. Notably, these phenotypes are not the result of subtle disruption to liver-specific transcriptional networks, since haploinsufficiency in the CTCF insulator protein resulted in the same phenotype. Further quantification of genotypes of tetraploid hepatocytes in young and old HFN4A-haploinsufficient mice revealed that during ageing, tetraploid hepatocytes lead to the selection of wild-type alleles, restoring non-deleterious genetic perturbations. CONCLUSIONS: Our results suggest a model whereby polyploidisation leads to fundamentally different cell states. Polyploid conversion enables pleiotropic buffering against age-related decline via non-random allelic segregation to restore a wild-type genome. IMPACT AND IMPLICATIONS: The functional role of hepatocyte polyploidisation during ageing is poorly understood. Using single-nucleus RNA sequencing and BaseScope approaches, we have studied ploidy dynamics during ageing in murine livers with non-deleterious genetic perturbations. We have identified that hepatocytes present different cellular states and the ability to buffer ageing-associated dysfunctions. Tetraploid nuclei exhibit robust transcriptional networks and are better adapted to genomically overcome perturbations. Novel therapeutic interventions aimed at attenuating age-related changes in tissue function could be exploited by manipulation of ploidy dynamics during chronic liver conditions.
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Envejecimiento , Hepatocitos , Poliploidía , Animales , Hepatocitos/metabolismo , Hepatocitos/fisiología , Ratones , Envejecimiento/fisiología , Envejecimiento/genética , Redes Reguladoras de Genes , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Haploinsuficiencia , Senescencia Celular/genética , Senescencia Celular/fisiología , Masculino , Ratones Endogámicos C57BL , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Hígado/metabolismo , Hígado Graso/genética , Hígado Graso/patologíaRESUMEN
In placental females, one copy of the two X chromosomes is largely silenced during a narrow developmental time window, in a process mediated by the non-coding RNA Xist1. Here, we demonstrate that Xist can initiate X-chromosome inactivation (XCI) well beyond early embryogenesis. By modifying its endogenous level, we show that Xist has the capacity to actively silence genes that escape XCI both in neuronal progenitor cells (NPCs) and in vivo, in mouse embryos. We also show that Xist plays a direct role in eliminating TAD-like structures associated with clusters of escapee genes on the inactive X chromosome, and that this is dependent on Xist's XCI initiation partner, SPEN2. We further demonstrate that Xist's function in suppressing gene expression of escapees and topological domain formation is reversible for up to seven days post-induction, but that sustained Xist up-regulation leads to progressively irreversible silencing and CpG island DNA methylation of facultative escapees. Thus, the distinctive transcriptional and regulatory topologies of the silenced X chromosome is actively, directly - and reversibly - controlled by Xist RNA throughout life.
RESUMEN
Cis-genetic effects are key determinants of transcriptional divergence in discrete tissues and cell types. However, how cis- and trans-effects act across continuous trajectories of cellular differentiation in vivo is poorly understood. Here, we quantify allele-specific expression during spermatogenic differentiation at single-cell resolution in an F1 hybrid mouse system, allowing for the comprehensive characterisation of cis- and trans-genetic effects, including their dynamics across cellular differentiation. Collectively, almost half of the genes subject to genetic regulation show evidence for dynamic cis-effects that vary during differentiation. Our system also allows us to robustly identify dynamic trans-effects, which are less pervasive than cis-effects. In aggregate, genetic effects were strongest in round spermatids, which parallels their increased transcriptional divergence we identified between species. Our approach provides a comprehensive quantification of the variability of genetic effects in vivo, and demonstrates a widely applicable strategy to dissect the impact of regulatory variants on gene regulation in dynamic systems.
Asunto(s)
Regulación de la Expresión Génica , Espermátides , Masculino , Animales , RatonesRESUMEN
The female reproductive tract (FRT) undergoes extensive remodeling during reproductive cycling. This recurrent remodeling and how it shapes organ-specific aging remains poorly explored. Using single-cell and spatial transcriptomics, we systematically characterized morphological and gene expression changes occurring in ovary, oviduct, uterus, cervix, and vagina at each phase of the mouse estrous cycle, during decidualization, and into aging. These analyses reveal that fibroblasts play central-and highly organ-specific-roles in FRT remodeling by orchestrating extracellular matrix (ECM) reorganization and inflammation. Our results suggest a model wherein recurrent FRT remodeling over reproductive lifespan drives the gradual, age-related development of fibrosis and chronic inflammation. This hypothesis was directly tested using chemical ablation of cycling, which reduced fibrotic accumulation during aging. Our atlas provides extensive detail into how estrus, pregnancy, and aging shape the organs of the female reproductive tract and reveals the unexpected cost of the recurrent remodeling required for reproduction.
Asunto(s)
Envejecimiento , Genitales Femeninos , Animales , Femenino , Ratones , Embarazo , Genitales Femeninos/citología , Genitales Femeninos/metabolismo , Inflamación/metabolismo , Útero/citología , Vagina/citología , Análisis de la Célula IndividualRESUMEN
Low-grade chronic inflammation is a hallmark of ageing, associated with impaired tissue function and disease development. However, how cell-intrinsic and -extrinsic factors collectively establish this phenotype, termed inflammaging, remains poorly understood. We addressed this question in the mouse intestinal epithelium, using mouse organoid cultures to dissect stem cell-intrinsic and -extrinsic sources of inflammaging. At the single-cell level, we found that inflammaging is established differently along the crypt-villus axis, with aged intestinal stem cells (ISCs) strongly upregulating major histocompatibility complex class II (MHC-II) genes. Importantly, the inflammaging phenotype was stably propagated by aged ISCs in organoid cultures and associated with increased chromatin accessibility at inflammation-associated loci in vivo and ex vivo, indicating cell-intrinsic inflammatory memory. Mechanistically, we show that the expression of inflammatory genes is dependent on STAT1 signaling. Together, our data identify that intestinal inflammaging in mice is promoted by a cell-intrinsic mechanism, stably propagated by ISCs, and associated with a disbalance in immune homeostasis.
Asunto(s)
Mucosa Intestinal , Intestinos , Ratones , Animales , Células Madre , Fenotipo , InflamaciónRESUMEN
The mammalian body plan is shaped by rhythmic segmentation of mesoderm into somites, which are transient embryonic structures that form down each side of the neural tube. We have analyzed the genome-wide transcriptional and chromatin dynamics occurring within nascent somites, from early inception of somitogenesis to the latest stages of body plan establishment. We created matched gene expression and open chromatin maps for the three leading pairs of somites at six time points during mouse embryonic development. We show that the rate of somite differentiation accelerates as development progresses. We identified a conserved maturation program followed by all somites, but somites from more developed embryos concomitantly switch on differentiation programs from derivative cell lineages soon after segmentation. Integrated analysis of the somitic transcriptional and chromatin activities identified opposing regulatory modules controlling the onset of differentiation. Our results provide a powerful, high-resolution view of the molecular genetics underlying somitic development in mammals.
Asunto(s)
Desarrollo Embrionario , Somitos , Embarazo , Femenino , Ratones , Animales , Desarrollo Embrionario/genética , Mesodermo , Diferenciación Celular/genética , Cromatina/genética , MamíferosRESUMEN
BACKGROUND: To investigate the mechanisms driving regulatory evolution across tissues, we experimentally mapped promoters, enhancers, and gene expression in the liver, brain, muscle, and testis from ten diverse mammals. RESULTS: The regulatory landscape around genes included both tissue-shared and tissue-specific regulatory regions, where tissue-specific promoters and enhancers evolved most rapidly. Genomic regions switching between promoters and enhancers were more common across species, and less common across tissues within a single species. Long Interspersed Nuclear Elements (LINEs) played recurrent evolutionary roles: LINE L1s were associated with tissue-specific regulatory regions, whereas more ancient LINE L2s were associated with tissue-shared regulatory regions and with those switching between promoter and enhancer signatures across species. CONCLUSIONS: Our analyses of the tissue-specificity and evolutionary stability among promoters and enhancers reveal how specific LINE families have helped shape the dynamic mammalian regulome.
Asunto(s)
Evolución Molecular , Regulación de la Expresión Génica , Elementos de Nucleótido Esparcido Largo , Mamíferos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Retroelementos , Animales , Mapeo Cromosómico , Secuencia Conservada , Elementos de Facilitación Genéticos , Humanos , Especificidad de Órganos/genética , Regiones Promotoras GenéticasRESUMEN
BACKGROUND: The introduction of novel CTCF binding sites in gene regulatory regions in the rodent lineage is partly the effect of transposable element expansion, particularly in the murine lineage. The exact mechanism and functional impact of evolutionarily novel CTCF binding sites are not yet fully understood. We investigated the impact of novel subspecies-specific CTCF binding sites in two Mus genus subspecies, Mus musculus domesticus and Mus musculus castaneus, that diverged 0.5 million years ago. RESULTS: CTCF binding site evolution is influenced by the action of the B2-B4 family of transposable elements independently in both lineages, leading to the proliferation of novel CTCF binding sites. A subset of evolutionarily young sites may harbour transcriptional functionality as evidenced by the stability of their binding across multiple tissues in M. musculus domesticus (BL6), while overall the distance of subspecies-specific CTCF binding to the nearest transcription start sites and/or topologically associated domains (TADs) is largely similar to musculus-common CTCF sites. Remarkably, we discovered a recurrent regulatory architecture consisting of a CTCF binding site and an interferon gene that appears to have been tandemly duplicated to create a 15-gene cluster on chromosome 4, thus forming a novel BL6 specific immune locus in which CTCF may play a regulatory role. CONCLUSIONS: Our results demonstrate that thousands of CTCF binding sites show multiple functional signatures rapidly after incorporation into the genome.
Asunto(s)
Factor de Unión a CCCTC/genética , Evolución Molecular , Genoma , Animales , Sitios de Unión/genética , Factor de Unión a CCCTC/metabolismo , Perfilación de la Expresión Génica , Masculino , Ratones , Familia de Multigenes/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Especificidad de la EspecieRESUMEN
Pseudogenes are ideal markers of genome remodelling. In turn, the mouse is an ideal platform for studying them, particularly with the recent availability of strain-sequencing and transcriptional data. Here, combining both manual curation and automatic pipelines, we present a genome-wide annotation of the pseudogenes in the mouse reference genome and 18 inbred mouse strains (available via the mouse.pseudogene.org resource). We also annotate 165 unitary pseudogenes in mouse, and 303, in human. The overall pseudogene repertoire in mouse is similar to that in human in terms of size, biotype distribution, and family composition (e.g. with GAPDH and ribosomal proteins being the largest families). Notable differences arise in the pseudogene age distribution, with multiple retro-transpositional bursts in mouse evolutionary history and only one in human. Furthermore, in each strain about a fifth of all pseudogenes are unique, reflecting strain-specific evolution. Finally, we find that ~15% of the mouse pseudogenes are transcribed, and that highly transcribed parent genes tend to give rise to many processed pseudogenes.
Asunto(s)
Seudogenes/genética , Transcripción Genética , Animales , Secuencia Conservada/genética , Evolución Molecular , Ontología de Genes , Genoma , Humanos , Ratones Endogámicos C57BL , Anotación de Secuencia Molecular , Especificidad de la EspecieRESUMEN
Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.
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Segregación Cromosómica/genética , Evolución Molecular , Genoma/genética , Neoplasias/genética , Alelos , Animales , Reparación del ADN , Replicación del ADN , Receptores ErbB/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Mutación , Neoplasias/patología , Selección Genética , Transducción de Señal , Intercambio de Cromátides Hermanas , Transcripción Genética , Quinasas raf/metabolismo , Proteínas ras/metabolismoRESUMEN
Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.
Asunto(s)
División Celular/genética , División Celular/fisiología , ARN Largo no Codificante/genética , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Mitosis/genética , Mitosis/fisiología , Proteínas/genética , Interferencia de ARN/fisiologíaRESUMEN
Transcription factor binding to enhancer and promoter regions critical for homeostatic adult gene activation is established during development. To understand how cell-specific gene expression patterns are generated, we study the developmental timing of association of two prominent hepatic transcription factors with gene regulatory regions. Most individual binding events display extraordinarily high temporal variations during liver development. Early and persistent binding is necessary, but not sufficient, for gene activation. Stable gene expression patterns are the result of combinatorial activity of multiple transcription factors, which mark regulatory regions long before activation and promote progressive broadening of active chromatin domains. Both temporally stable and dynamic, short-lived binding events contribute to the developmental maturation of active promoter configurations. The results reveal a developmental bookmarking function of master regulators and illuminate remarkable parallels between the principles employed for gene activation during development, during evolution, and upon mitotic exit.
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Hígado/embriología , Hígado/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
BACKGROUND: CTCF binding contributes to the establishment of a higher-order genome structure by demarcating the boundaries of large-scale topologically associating domains (TADs). However, despite the importance and conservation of TADs, the role of CTCF binding in their evolution and stability remains elusive. RESULTS: We carry out an experimental and computational study that exploits the natural genetic variation across five closely related species to assess how CTCF binding patterns stably fixed by evolution in each species contribute to the establishment and evolutionary dynamics of TAD boundaries. We perform CTCF ChIP-seq in multiple mouse species to create genome-wide binding profiles and associate them with TAD boundaries. Our analyses reveal that CTCF binding is maintained at TAD boundaries by a balance of selective constraints and dynamic evolutionary processes. Regardless of their conservation across species, CTCF binding sites at TAD boundaries are subject to stronger sequence and functional constraints compared to other CTCF sites. TAD boundaries frequently harbor dynamically evolving clusters containing both evolutionarily old and young CTCF sites as a result of the repeated acquisition of new species-specific sites close to conserved ones. The overwhelming majority of clustered CTCF sites colocalize with cohesin and are significantly closer to gene transcription start sites than nonclustered CTCF sites, suggesting that CTCF clusters particularly contribute to cohesin stabilization and transcriptional regulation. CONCLUSIONS: Dynamic conservation of CTCF site clusters is an apparently important feature of CTCF binding evolution that is critical to the functional stability of a higher-order chromatin structure.
Asunto(s)
Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Evolución Molecular , Ratones/genética , Animales , Secuenciación de Inmunoprecipitación de Cromatina , GenomaRESUMEN
How the age-associated decline of immune function leads to increased cancer incidence is poorly understood. Here, we have characterised the cellular composition of the γδ T-cell pool in peripheral lymph nodes (pLNs) upon ageing. We find that ageing has minimal cell-intrinsic effects on function and global gene expression of γδ T cells, and γδTCR diversity remains stable. However, ageing alters TCRδ chain usage and clonal structure of γδ T-cell subsets. Importantly, IL-17-producing γδ17 T cells dominate the γδ T-cell pool of aged mice-mainly due to the selective expansion of Vγ6+ γδ17 T cells and augmented γδ17 polarisation of Vγ4+ T cells. Expansion of the γδ17 T-cell compartment is mediated by increased IL-7 expression in the T-cell zone of old mice. In a Lewis lung cancer model, pro-tumourigenic Vγ6+ γδ17 T cells are exclusively activated in the tumour-draining LN and their infiltration into the tumour correlates with increased tumour size in aged mice. Thus, upon ageing, substantial compositional changes in γδ T-cell pool in the pLN lead to an unbalanced γδ T-cell response in the tumour that is associated with accelerated tumour growth.
Asunto(s)
Envejecimiento/genética , Carcinoma Pulmonar de Lewis/genética , Regulación Neoplásica de la Expresión Génica , Interleucina-7/genética , Ganglios Linfáticos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Subgrupos de Linfocitos T/inmunología , Envejecimiento/inmunología , Animales , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/patología , Diferenciación Celular , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Inmunofenotipificación , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-7/inmunología , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T gamma-delta/clasificación , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Transducción de Señal , Subgrupos de Linfocitos T/clasificación , Subgrupos de Linfocitos T/patología , Carga Tumoral/genética , Carga Tumoral/inmunologíaRESUMEN
Hyperdiploidy, i.e. gain of whole chromosomes, is one of the most common genetic features of childhood acute lymphoblastic leukemia (ALL), but its pathogenetic impact is poorly understood. Here, we report a proteogenomic analysis on matched datasets from genomic profiling, RNA-sequencing, and mass spectrometry-based analysis of >8,000 genes and proteins as well as Hi-C of primary patient samples from hyperdiploid and ETV6/RUNX1-positive pediatric ALL. We show that CTCF and cohesin, which are master regulators of chromatin architecture, display low expression in hyperdiploid ALL. In line with this, a general genome-wide dysregulation of gene expression in relation to topologically associating domain (TAD) borders were seen in the hyperdiploid group. Furthermore, Hi-C of a limited number of hyperdiploid childhood ALL cases revealed that 2/4 cases displayed a clear loss of TAD boundary strength and 3/4 showed reduced insulation at TAD borders, with putative leukemogenic effects.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transcripción Genética , Adolescente , Aneuploidia , Factor de Unión a CCCTC/genética , Proteínas de Ciclo Celular/genética , Niño , Preescolar , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Aberraciones Cromosómicas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Femenino , Dosificación de Gen , Perfilación de la Expresión Génica , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Lactante , Recién Nacido , Masculino , Proteogenómica/métodos , Proteoma/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Represoras/genética , Análisis de Secuencia de ARN , Cohesinas , Proteína ETS de Variante de Translocación 6RESUMEN
Male gametes are generated through a specialised differentiation pathway involving a series of developmental transitions that are poorly characterised at the molecular level. Here, we use droplet-based single-cell RNA-Sequencing to profile spermatogenesis in adult animals and at multiple stages during juvenile development. By exploiting the first wave of spermatogenesis, we both precisely stage germ cell development and enrich for rare somatic cell-types and spermatogonia. To capture the full complexity of spermatogenesis including cells that have low transcriptional activity, we apply a statistical tool that identifies previously uncharacterised populations of leptotene and zygotene spermatocytes. Focusing on post-meiotic events, we characterise the temporal dynamics of X chromosome re-activation and profile the associated chromatin state using CUT&RUN. This identifies a set of genes strongly repressed by H3K9me3 in spermatocytes, which then undergo extensive chromatin remodelling post-meiosis, thus acquiring an active chromatin state and spermatid-specific expression.