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.
DNA damage signaling and repair machineries operate in a nuclear environment where DNA is wrapped around histone proteins and packaged into chromatin. Understanding how chromatin structure is restored together with the DNA sequence during DNA damage repair has been a topic of intense research. Indeed, chromatin integrity is central to cell functions and identity. However, chromatin shows remarkable plasticity in response to DNA damage. This review presents our current knowledge of chromatin dynamics in the mammalian cell nucleus in response to DNA double strand breaks and UV lesions. I provide an overview of the key players involved in regulating histone dynamics in damaged chromatin regions, focusing on histone chaperones and their concerted action with histone modifiers, chromatin remodelers and repair factors. I also discuss how these dynamics contribute to reshaping chromatin and, by altering the chromatin landscape, may affect the maintenance of epigenetic information.
Histone deposition onto DNA assisted by specific chaperones forms the chromatin basic unit and serves to package the genome within the cell nucleus. The resulting chromatin organization, often referred to as the epigenome, contributes to a unique transcriptional program that defines cell identity. Importantly, during cellular life, substantial alterations in chromatin structure may arise due to cell stress, including DNA damage, which not only challenges the integrity of the genome but also threatens the epigenome. Considerable efforts have been made to decipher chromatin dynamics in response to genotoxic stress, and to assess how it affects both genome and epigenome stability. Here, we review recent advances in understanding the mechanisms of DNA damage-induced chromatin plasticity in mammalian cells. We focus specifically on the dynamics of histone H3 variants in response to UV irradiation, and highlight the role of their dedicated chaperones in restoring both chromatin structure and function. Finally, we discuss how, in addition to restoring chromatin integrity, the cellular networks that signal and repair DNA damage may also provide a window of opportunity for modulating the information conveyed by chromatin.
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.
Chromosomal deletions and rearrangements in tumors are often associated with common fragile sites, which are specific genomic loci prone to gaps and breaks in metaphase chromosomes. Common fragile sites appear to arise through incomplete DNA replication because they are induced after partial replication inhibition by agents such as aphidicolin. Here, we show that in G1 cells, large nuclear bodies arise that contain p53 binding protein 1 (53BP1), phosphorylated H2AX (?H2AX), and mediator of DNA damage checkpoint 1 (MDC1), as well as components of previously characterized OPT (Oct-1, PTF, transcription) domains. Notably, we find that incubating cells with low aphidicolin doses increases the incidence and number of 53BP1-OPT domains in G1 cells, and by chromatin immunoprecipitation and massively parallel sequencing analysis of ?H2AX, we demonstrate that OPT domains are enriched at common fragile sites. These findings invoke a model wherein incomplete DNA synthesis during S phase leads to a DNA damage response and formation of 53BP1-OPT domains in the subsequent G1.
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.
Chromatin assembly factor-1 (CAF-1), whose function is critical for maintaining chromatin stability during DNA replication and repair, has been identified as a proliferation marker in breast cancer. The aim was to investigate CAF-1 as a proliferation marker in a wide variety of solid tumours, and to assess its potential value in predicting clinical outcome.
DNA double-strand break (DSB) repair occurs within chromatin and can be modulated by chromatin-modifying enzymes. Here we identify the related human histone deacetylases HDAC1 and HDAC2 as two participants in the DNA-damage response. We show that acetylation of histone H3 Lys56 (H3K56) was regulated by HDAC1 and HDAC2 and that HDAC1 and HDAC2 were rapidly recruited to DNA-damage sites to promote hypoacetylation of H3K56. Furthermore, HDAC1- and 2-depleted cells were hypersensitive to DNA-damaging agents and showed sustained DNA-damage signaling, phenotypes that reflect defective DSB repair, particularly by nonhomologous end-joining (NHEJ). Collectively, these results show that HDAC1 and HDAC2 function in the DNA-damage response by promoting DSB repair and thus provide important insights into the radio-sensitizing effects of HDAC inhibitors that are being developed as cancer therapies.
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.
Posttranslational modifications play key roles in regulating chromatin plasticity. Although various chromatin-remodeling enzymes have been described that respond to specific histone modifications, little is known about the role of poly[adenosine 5-diphosphate (ADP)-ribose] in chromatin remodeling. Here, we identify a chromatin-remodeling enzyme, ALC1 (Amplified in Liver Cancer 1, also known as CHD1L), that interacts with poly(ADP-ribose) and catalyzes PARP1-stimulated nucleosome sliding. Our results define ALC1 as a DNA damage-response protein whose role in this process is sustained by its association with known DNA repair factors and its rapid poly(ADP-ribose)-dependent recruitment to DNA damage sites. Furthermore, we show that depletion or overexpression of ALC1 results in sensitivity to DNA-damaging agents. Collectively, these results provide new insights into the mechanisms by which poly(ADP-ribose) regulates DNA repair.
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.
It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair.
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.
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.
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