RESUMEN
Using a barcoded reporter introduced within a thousand different chromatin locations in human cells, (Schep et al., 2021) characterize repair outcomes of Cas9-induced DNA double-strand breaks (DSBs) and the relative use of DSB repair pathways depending on the local chromatin context.
Asunto(s)
Cromatina , Roturas del ADN de Doble Cadena , Sistemas CRISPR-Cas , Cromatina/genética , ADN , Reparación del ADN , HumanosRESUMEN
Understanding how to recover fully functional and transcriptionally active chromatin when its integrity has been challenged by genotoxic stress is a critical issue. Here, by investigating how chromatin dynamics regulate transcriptional activity in response to DNA damage in human cells, we identify a pathway involving the histone chaperone histone regulator A (HIRA) to promote transcription restart after UVC damage. Our mechanistic studies reveal that HIRA accumulates at sites of UVC irradiation upon detection of DNA damage prior to repair and deposits newly synthesized H3.3 histones. This local action of HIRA depends on ubiquitylation events associated with damage recognition. Furthermore, we demonstrate that the early and transient function of HIRA in response to DNA damage primes chromatin for later reactivation of transcription. We propose that HIRA-dependent histone deposition serves as a chromatin bookmarking system to facilitate transcription recovery after genotoxic stress.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina , Daño del ADN/efectos de la radiación , Chaperonas de Histonas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Línea Celular Tumoral , Reparación del ADN , Células HeLa , Histonas/metabolismo , Humanos , Ubiquitinación , Rayos UltravioletaRESUMEN
Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/genética , ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Histonas/genética , Factores de Elongación Transcripcional/genética , Alfa-Amanitina/farmacología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/efectos de los fármacos , ADN/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/metabolismo , Humanos , Ratones , Morfolinas/farmacología , Células 3T3 NIH , Nucleosomas/química , Nucleosomas/efectos de los fármacos , Nucleosomas/metabolismo , Venenos/farmacología , Pirimidinas/farmacología , Pironas/farmacología , Transducción de Señal , Factores de Elongación Transcripcional/metabolismoRESUMEN
Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.
Asunto(s)
Neoplasias Encefálicas , Glioma , Histonas , Niño , Humanos , Neoplasias Encefálicas/patología , Reparación del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Glioma/patología , Histonas/genética , Histonas/metabolismo , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genéticaRESUMEN
In this issue of Molecular Cell, Taneja et al. (2017) uncover a dual role for the conserved chromatin remodeler Fft3 in the maintenance of silent heterochromatin and the suppression of replication barriers at euchromatic loci through controlled histone turnover.
Asunto(s)
Histonas/genética , Proteínas de Schizosaccharomyces pombe/genética , Cromatina , Heterocromatina , Schizosaccharomyces/genéticaRESUMEN
DNA double-strand breaks (DSBs) elicit major chromatin changes. Using super-resolution microscopy in human cells, Ochs et al. unveil that the DSB response protein 53BP1 and its effector RIF1 organize DSB-flanking chromatin into circular micro-domains. These structures control the spatial distribution of DSB repair factors safeguarding genome integrity.
Asunto(s)
Cromatina , Reparación del ADN , Roturas del ADN de Doble Cadena , HumanosRESUMEN
Organism viability relies on the stable maintenance of specific chromatin landscapes, established during development, that shape cell functions and identities by driving distinct gene expression programs. Yet epigenome maintenance is challenged during transcription, replication, and repair of DNA damage, all of which elicit dynamic changes in chromatin organization. Here, we review recent advances that have shed light on the specialized mechanisms contributing to the restoration of epigenome structure and function after DNA damage in the mammalian cell nucleus. By drawing a parallel with epigenome maintenance during replication, we explore emerging concepts and highlight open issues in this rapidly growing field. In particular, we present our current knowledge of molecular players that support the coordinated maintenance of genome and epigenome integrity in response to DNA damage, and we highlight how nuclear organization impacts genome stability. Finally, we discuss possible functional implications of epigenome plasticity in response to genotoxic stress.
Asunto(s)
Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Daño del ADN , Reparación del ADN , ADN/genética , Epigénesis Genética , Animales , Núcleo Celular/ultraestructura , Plasticidad de la Célula , ADN/biosíntesis , ADN/química , Metilación de ADN , Replicación del ADN , Epigenómica/métodos , Inestabilidad Genómica , Genotipo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Conformación de Ácido Nucleico , Fenotipo , Transcripción GenéticaRESUMEN
Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.
Asunto(s)
Cromatina/efectos de la radiación , Reparación del ADN , Técnica del Anticuerpo Fluorescente/métodos , Histonas/genética , Osteoblastos/efectos de la radiación , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Daño del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Rayos UltravioletaRESUMEN
DNA damage challenges both genome integrity and its organization with histone proteins into chromatin, with prominent alterations in histone variant dynamics and histone modifications. While these alterations jeopardize epigenome stability, they are also instrumental for an efficient and timely response to DNA damage. Here, we review recent findings illustrating how histone variants and post-translational modifications actively contribute to and control the DNA damage response. We present accumulating evidence that histone protein changes help relieve the chromatin barrier to DNA repair by regulating chromatin compaction and mobility. We also highlight how histone modifications and variants control transcriptional silencing at damage sites, and we describe both pre-existing and DNA damage-induced chromatin features that govern DNA damage signaling and guide DNA repair pathway choice. We discuss how histone dynamics ultimately participate to the restoration of epigenome integrity and present our current knowledge of key molecular players involved in these critical processes.
Asunto(s)
Cromatina/metabolismo , Daño del ADN/genética , Histonas/metabolismo , HumanosRESUMEN
The view of DNA packaging into chromatin as a mere obstacle to DNA repair is evolving. In this review, we focus on histone variants and heterochromatin proteins as chromatin components involved in distinct levels of chromatin organization to integrate them as real players in the DNA damage response (DDR). Based on recent data, we highlight how some of these chromatin components play active roles in the DDR and contribute to the fine-tuning of damage signaling, DNA and chromatin repair. To take into account this integrated view, we revisit the existing access-repair-restore model and propose a new working model involving priming chromatin for repair and restoration as a concerted process. We discuss how this impacts on both genomic and epigenomic stability and plasticity.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Animales , Daño del ADN , Reparación del ADN , Genoma , Histonas/metabolismo , Humanos , Modelos BiológicosRESUMEN
DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Ribonucleoproteínas Nucleares Heterogéneas/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Ácido Anhídrido Hidrolasas , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Proteína Homóloga de MRE11 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Eukaryotic genomes are organized into chromatin, divided into structurally and functionally distinct euchromatin and heterochromatin compartments. The high level of compaction and the abundance of repeated sequences in heterochromatin pose multiple challenges for the maintenance of genome stability. Cells have evolved sophisticated and highly controlled mechanisms to overcome these constraints. Here, we summarize recent findings on how the heterochromatic state influences DNA damage formation, signaling, and repair. By focusing on distinct heterochromatin domains in different eukaryotic species, we highlight the heterochromatin contribution to the compartmentalization of DNA damage repair in the cell nucleus and to the repair pathway choice. We also describe the diverse chromatin alterations associated with the DNA damage response in heterochromatin domains and present our current understanding of their regulatory mechanisms. Finally, we discuss the biological significance and the evolutionary conservation of these processes.
Asunto(s)
Daño del ADN , Heterocromatina/genética , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Reparación del ADN , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Transducción de SeñalRESUMEN
Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed the DNA damage response (DDR). A common feature of DDR proteins is their mobilization in response to genotoxic stress. Here, we outline how the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells. Considerable advances have also been made in understanding the underlying molecular mechanisms for these events, with post-translational modifications of DDR factors being shown to play prominent roles in controlling the formation of foci in response to DNA-damaging agents. We review these regulatory mechanisms and discuss their biological significance to the DDR.
Asunto(s)
Roturas del ADN , Reparación del ADN/fisiología , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Animales , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Transducción de SeñalRESUMEN
DNA damage interferes with the progression of transcription machineries. A tight coordination of transcription with signaling and repair of DNA damage is thus critical for safeguarding genome function. This coordination involves modulations of chromatin organization. Here, we focus on the central role of chromatin dynamics, in conjunction with DNA Damage Response (DDR) factors, in controlling transcription inhibition and restart at sites of DNA damage in mammalian cells. Recent work has identified chromatin modifiers and histone chaperones as key regulators of transcriptional activity in damaged chromatin regions. Conversely, the transcriptional state of chromatin before DNA damage influences both DNA damage signaling and repair. We discuss the importance of chromatin plasticity in coordinating the interplay between the DDR and transcription, with major implications for cell fate maintenance.
Asunto(s)
Cromatina/genética , Daño del ADN/genética , Reparación del ADN/genética , Transcripción Genética , Animales , Estructuras Cromosómicas , HumanosRESUMEN
The chromatin remodelling factor chromodomain helicase DNA-binding protein 4 (CHD4) is a catalytic subunit of the NuRD transcriptional repressor complex. Here, we reveal novel functions for CHD4 in the DNA-damage response (DDR) and cell-cycle control. We show that CHD4 mediates rapid poly(ADP-ribose)-dependent recruitment of the NuRD complex to DNA-damage sites, and we identify CHD4 as a phosphorylation target for the apical DDR kinase ataxia-telangiectasia mutated. Functionally, we show that CHD4 promotes repair of DNA double-strand breaks and cell survival after DNA damage. In addition, we show that CHD4 acts as an important regulator of the G1/S cell-cycle transition by controlling p53 deacetylation. These results provide new insights into how the chromatin remodelling complex NuRD contributes to maintaining genome stability.
Asunto(s)
Autoantígenos/metabolismo , Ciclo Celular/fisiología , Ensamble y Desensamble de Cromatina , Daño del ADN , ADN Helicasas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Autoantígenos/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Histonas/fisiología , Humanos , Inmunoprecipitación , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Ratones Noqueados , Fosforilación , Poli Adenosina Difosfato Ribosa/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The maintenance of genome integrity by DNA damage response machineries is key to protect cells against pathological development. In cell nuclei, these genome maintenance machineries operate in the context of chromatin, where the DNA wraps around histone proteins. Here, we review recent findings illustrating how the chromatin substrate modulates genome maintenance mechanisms, focusing on the regulatory role of histone variants and post-translational modifications. In particular, we discuss how the pre-existing chromatin landscape impacts DNA damage formation and guides DNA repair pathway choice, and how DNA damage-induced chromatin alterations control DNA damage signaling and repair, and DNA damage segregation through cell divisions. We also highlight that pathological alterations of histone proteins may trigger genome instability by impairing chromosome segregation and DNA repair, thus defining new oncogenic mechanisms and opening up therapeutic options.
Asunto(s)
Cromatina , Daño del ADN , Reparación del ADN , Inestabilidad Genómica , Histonas , Procesamiento Proteico-Postraduccional , Humanos , Cromatina/metabolismo , Histonas/metabolismo , Animales , Segregación CromosómicaRESUMEN
Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.
Asunto(s)
Proteínas de Unión al ADN , Chaperonas de Histonas , Animales , Humanos , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Cromatina/metabolismo , Nucleosomas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
In the cell nucleus, DNA damage signaling and repair machineries operate on a chromatin substrate, the integrity of which is critical for cell function and viability. Here, we review recent advances in deciphering the tight coordination between chromatin maintenance and the DNA damage response (DDR). We discuss how the DDR impacts chromatin marks, organization and mobility, and, in turn, how chromatin alterations actively contribute to the DDR, providing additional levels of regulation. We present our current knowledge of the molecular bases of these critical processes in physiological and pathological conditions, and also highlight open questions that emerge in this expanding field.
Asunto(s)
Cromatina , Reparación del ADN , Cromatina/genética , Daño del ADN , Núcleo Celular/genética , Transducción de SeñalRESUMEN
The faithful segregation of intact genetic material and the perpetuation of chromatin states through mitotic cell divisions are pivotal for maintaining cell function and identity across cell generations. However, most exogenous mutagens generate long-lasting DNA lesions that are segregated during mitosis. How this segregation is controlled is unknown. Here, we uncover a mitotic chromatin-marking pathway that governs the segregation of UV-induced damage in human cells. Our mechanistic analyses reveal two layers of control: histone ADP-ribosylation, and the incorporation of newly synthesized histones at UV damage sites, that both prevent local mitotic phosphorylations on histone H3 serines. Functionally, this chromatin-marking pathway drives the asymmetric segregation of UV damage in the cell progeny with potential consequences on daughter cell fate. We propose that this mechanism may help preserve the integrity of stem cell compartments during asymmetric cell divisions.