ABSTRACT
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.
Subject(s)
Chromatin , DNA Breaks, Double-Stranded , CRISPR-Cas Systems , Chromatin/genetics , DNA , DNA Repair , HumansABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin , DNA Damage/radiation effects , Histone Chaperones/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Cell Line, Tumor , DNA Repair , HeLa Cells , Histones/metabolism , Humans , Ubiquitination , Ultraviolet RaysABSTRACT
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.
Subject(s)
DNA Repair , DNA-Binding Proteins/genetics , DNA/genetics , High Mobility Group Proteins/genetics , Histones/genetics , Transcriptional Elongation Factors/genetics , Alpha-Amanitin/pharmacology , Animals , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line, Tumor , Chromatin Assembly and Disassembly/drug effects , DNA/metabolism , DNA Damage , DNA-Binding Proteins/metabolism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression Regulation , High Mobility Group Proteins/metabolism , Histones/metabolism , Humans , Mice , Morpholines/pharmacology , NIH 3T3 Cells , Nucleosomes/chemistry , Nucleosomes/drug effects , Nucleosomes/metabolism , Poisons/pharmacology , Pyrimidines/pharmacology , Pyrones/pharmacology , Signal Transduction , Transcriptional Elongation Factors/metabolismABSTRACT
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.
Subject(s)
Brain Neoplasms , Glioma , Histones , Child , Humans , Brain Neoplasms/pathology , DNA Repair/genetics , DNA Repair Enzymes/metabolism , Glioma/pathology , Histones/genetics , Histones/metabolism , Mutation , Phosphotransferases (Alcohol Group Acceptor)/geneticsABSTRACT
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.
Subject(s)
Histones/genetics , Schizosaccharomyces pombe Proteins/genetics , Chromatin , Heterochromatin , Schizosaccharomyces/geneticsABSTRACT
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.
Subject(s)
Chromatin , DNA Repair , DNA Breaks, Double-Stranded , HumansABSTRACT
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.
Subject(s)
Cell Nucleus/metabolism , Chromatin Assembly and Disassembly , DNA Damage , DNA Repair , DNA/genetics , Epigenesis, Genetic , Animals , Cell Nucleus/ultrastructure , Cell Plasticity , DNA/biosynthesis , DNA/chemistry , DNA Methylation , DNA Replication , Epigenomics/methods , Genomic Instability , Genotype , Heterochromatin/genetics , Heterochromatin/metabolism , Histones/metabolism , Humans , Nucleic Acid Conformation , Phenotype , Transcription, GeneticABSTRACT
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.
Subject(s)
Chromatin/radiation effects , DNA Repair , Fluorescent Antibody Technique/methods , Histones/genetics , Osteoblasts/radiation effects , Cell Line, Tumor , Chromatin/chemistry , Chromatin/metabolism , Chromatin Assembly and Disassembly , DNA Damage , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genomic Instability , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Histones/antagonists & inhibitors , Histones/metabolism , Humans , Osteoblasts/cytology , Osteoblasts/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ultraviolet RaysABSTRACT
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.
Subject(s)
Chromatin/metabolism , DNA Damage/genetics , Histones/metabolism , HumansABSTRACT
Transmission of extracellular signals by G protein-coupled receptors typically relies on a cascade of intracellular events initiated by the activation of heterotrimeric G proteins or ß-arrestins followed by effector activation/inhibition. Here, we report an alternative signal transduction mode used by the orphan GPR50 that relies on the nuclear translocation of its carboxyl-terminal domain (CTD). Activation of the calcium-dependent calpain protease cleaves off the CTD from the transmembrane-bound GPR50 core domain between Phe-408 and Ser-409 as determined by MALDI-TOF-mass spectrometry. The cytosolic CTD then translocates into the nucleus assisted by its 'DPD' motif, where it interacts with the general transcription factor TFII-I to regulate c-fos gene transcription. RNA-Seq analysis indicates a broad role of the CTD in modulating gene transcription with ~ 8000 differentially expressed genes. Our study describes a non-canonical, direct signaling mode of GPCRs to the nucleus with similarities to other receptor families such as the NOTCH receptor.
Subject(s)
Nerve Tissue Proteins/genetics , Protein Transport/genetics , Receptors, G-Protein-Coupled/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , Humans , Protein Binding/genetics , Receptors, Notch , Signal Transduction/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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.
Subject(s)
Chromatin/metabolism , DNA/metabolism , Animals , DNA Damage , DNA Repair , Genome , Histones/metabolism , Humans , Models, BiologicalABSTRACT
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.
Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair , Heterogeneous-Nuclear Ribonucleoproteins/physiology , Nuclear Proteins/physiology , Transcription Factors/physiology , Acid Anhydride Hydrolases , Carrier Proteins/genetics , Carrier Proteins/metabolism , Carrier Proteins/physiology , Cell Cycle Proteins/metabolism , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , MRE11 Homologue Protein , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
DNA Damage , Heterochromatin/genetics , Animals , Cell Nucleus/genetics , Cell Nucleus/metabolism , DNA Repair , Heterochromatin/metabolism , Histones/metabolism , Humans , Signal TransductionABSTRACT
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.
Subject(s)
DNA Breaks , DNA Repair/physiology , Protein Processing, Post-Translational , Proteins/metabolism , Animals , Humans , Mammals/genetics , Mammals/metabolism , Saccharomyces/genetics , Saccharomyces/metabolism , Signal TransductionABSTRACT
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.
Subject(s)
Chromatin/genetics , DNA Damage/genetics , DNA Repair/genetics , Transcription, Genetic , Animals , Chromosome Structures , HumansABSTRACT
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , Protein Inhibitors of Activated STAT/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Animals , BRCA1 Protein/metabolism , Cell Line , Cell Line, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fluorescence Recovery After Photobleaching , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Models, Biological , Phosphorylation , Protein Inhibitors of Activated STAT/chemistry , Protein Inhibitors of Activated STAT/genetics , Protein Structure, Tertiary , Replication Protein A/metabolism , Small Ubiquitin-Related Modifier Proteins/genetics , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
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.
Subject(s)
Autoantigens/metabolism , Cell Cycle/physiology , Chromatin Assembly and Disassembly , DNA Damage , DNA Helicases/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Autoantigens/genetics , Blotting, Western , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Helicases/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fluorescent Antibody Technique , Histones/physiology , Humans , Immunoprecipitation , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Mice , Mice, Knockout , Phosphorylation , Poly Adenosine Diphosphate Ribose/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53/physiology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
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.
Subject(s)
Chromatin , DNA Damage , DNA Repair , Genomic Instability , Histones , Protein Processing, Post-Translational , Humans , Chromatin/metabolism , Histones/metabolism , Animals , Chromosome SegregationABSTRACT
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.