RESUMO
Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.
Assuntos
Envelhecimento , Senescência Celular , Humanos , Envelhecimento/genética , Senescência Celular/genéticaRESUMO
Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1-4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin-IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures.
Assuntos
Envelhecimento , Longevidade , Elongação da Transcrição Genética , Animais , Humanos , Camundongos , Ratos , Envelhecimento/genética , Insulina/metabolismo , Longevidade/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transdução de Sinais , Drosophila melanogaster/genética , Caenorhabditis elegans/genética , RNA Circular , Somatomedinas , Nucleossomos , Histonas , Divisão Celular , Restrição CalóricaRESUMO
Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Elementos Facilitadores Genéticos/genética , CoesinasRESUMO
Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.
Assuntos
Senescência Celular , Inversão Cromossômica , Cromossomos/ultraestrutura , Transição Epitelial-Mesenquimal , Neoplasias/genética , Oncogenes , Recombinação Genética , Animais , Brônquios/metabolismo , Sistemas CRISPR-Cas , Ciclo Celular , Transformação Celular Neoplásica , Ritmo Circadiano , Biologia Computacional , Células Epiteliais/metabolismo , Citometria de Fluxo , Genômica , Humanos , Cariotipagem , Camundongos , Camundongos SCID , Neoplasias/metabolismo , Fenótipo , Ligação Proteica , Domínios Proteicos , Fenótipo Secretor Associado à SenescênciaRESUMO
Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome-wide in two primary cell lines. We integrated ChIP-seq and Hi-C with graph theory to uncover clustering of HMGB1-marked topological domains that harbor genes involved in paracrine senescence. Using simplified Cross-Linking and Immuno-Precipitation and functional tests, we show that HMGB1 is also a bona fide RNA-binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate availability of SASP-relevant transcripts. Our findings reveal a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell-autonomous and paracrine senescence.
Assuntos
Proteína HMGB1 , RNA , Animais , Senescência Celular/genética , Cromatina/genética , Proteína HMGB1/genética , Homeostase/genéticaRESUMO
Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.
Assuntos
Autofagia/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Autofagia/genética , Cromatina , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Epigenômica , Edição de Genes , Expressão Gênica , Síndrome de Hallermann/genética , Humanos , Mutação , FenótipoRESUMO
Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Genoma Humano , Proteína HMGB2/metabolismo , Fator de Ligação a CCCTC/genética , Proliferação de Células , Senescência Celular , Cromatina/genética , Proteína HMGB2/genética , Células Endoteliais da Veia Umbilical Humana , HumanosRESUMO
Over time eukaryotic genomes have evolved to host genes carrying multiple exons separated by increasingly larger intronic, mostly non-protein-coding, sequences. Initially, little attention was paid to these intronic sequences, as they were considered not to contain regulatory information. However, advances in molecular biology, sequencing, and computational tools uncovered that numerous segments within these genomic elements do contribute to the regulation of gene expression. Introns are differentially removed in a cell type-specific manner to produce a range of alternatively-spliced transcripts, and many span tens to hundreds of kilobases. Recent work in human and fruitfly tissues revealed that long introns are extensively processed cotranscriptionally and in a stepwise manner, before their two flanking exons are spliced together. This process, called "recursive splicing," often involves non-canonical splicing elements positioned deep within introns, and different mechanisms for its deployment have been proposed. Still, the very existence and widespread nature of recursive splicing offers a new regulatory layer in the transcript maturation pathway, which may also have implications in human disease.