Search

The long interspersed element 1 (LINE-1 or L1) integration is affected by many cellular factors through various mechanisms. Some of these factors are required for L1 amplification, while others either suppress or enhance specific steps during L1 propagation. Previously, TRIM28 has been identified to suppress transposable elements, including L1 expression via its canonical role in chromatin remodeling. Here, we report that TRIM28 through its B box domain increases L1 retrotransposition and facilitates shorter cDNA and L1 insert generation in cultured cells. Consistent with the latter, we observe that tumor specific L1 inserts are shorter in endometrial, ovarian, and prostate tumors with higher TRIM28 mRNA expression than in those with lower TRIM28 expression. We determine that three amino acids in the B box domain that are involved in TRIM28 multimerization are critical for its effect on both L1 retrotransposition and cDNA synthesis. We provide evidence that B boxes from the other two members in the Class VI TRIM proteins, TRIM24 and TRIM33, also increase L1 retrotransposition. Our findings could lead to a better understanding of the host/L1 evolutionary arms race in the germline and their interplay during tumorigenesis.

Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

Genome integrity is constantly challenged by various processes including DNA damage, structured DNA, transcription, and DNA-protein crosslinks. During DNA replication, active replication forks that encounter these obstacles can result in their stalling and collapse. Accurate DNA replication requires the ability of forks to navigate these threats, which is aided by DNA repair proteins. Histone acetylation participates in this process through an ability to signal and recruit proteins to regions of replicating DNA. For example, the histone acetyltransferase PCAF promotes the recruitment of the DNA repair factors MRE11 and EXO1 to stalled forks by acetylating histone H4 at lysine 8 (H4K8ac). These highly dynamic processes can be detected and analyzed using a modified proximity ligation assay (PLA) method, known as SIRF (in situ protein interactions with nascent DNA replication forks). This single-cell assay combines PLA with EdU-coupled Click-iT chemistry reactions and fluorescence microscopy to detect these interactions at sites of replicating DNA. Here we provide a detailed protocol utilizing SIRF that detects the HAT PCAF and histone acetylation at replication forks. This technique provides a robust methodology to determine protein recruitment and modifications at the replication fork with single-cell resolution.

Post-translational modifications of proteins are well-established participants in DNA damage response (DDR) pathways, which function in the maintenance of genome integrity. Emerging evidence is starting to reveal the involvement of modifications on RNA in the DDR. RNA modifications are known regulators of gene expression but how and if they participate in DNA repair and genome maintenance has been poorly understood. Here, we review several studies that have now established RNA modifications as key components of DNA damage responses. RNA modifying enzymes and the binding proteins that recognize these modifications localize to and participate in the repair of UV-induced and DNA double-strand break lesions. RNA modifications have a profound effect on DNA–RNA hybrids (R-loops) at DNA damage sites, a structure known to be involved in DNA repair and genome stability. Given the importance of the DDR in suppressing mutations and human diseases such as neurodegeneration, immunodeficiencies, cancer and aging, RNA modification pathways may be involved in human diseases not solely through their roles in gene expression but also by their ability to impact DNA repair and genome stability.

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.

Cells encounter a multitude of external and internal stress-causing agents that can ultimately lead to DNA damage, mutations and disease. A cascade of signaling events counters these challenges to DNA, which is termed as the DNA damage response (DDR). The DDR preserves genome integrity by engaging appropriate repair pathways, while also coordinating cell cycle and/or apoptotic responses. Although many of the protein components in the DDR are identified, how chemical modifications to DNA impact the DDR is poorly understood. This review focuses on our current understanding of DNA methylation in maintaining genome integrity in mammalian cells. DNA methylation is a reversible epigenetic mark, which has been implicated in DNA damage signaling, repair and replication. Sites of DNA methylation can trigger mutations, which are drivers of human diseases including cancer. Indeed, alterations in DNA methylation are associated with increased susceptibility to tumorigenesis but whether this occurs through effects on the DDR, transcriptional responses or both is not entirely clear. Here, we also highlight epigenetic drugs currently in use as therapeutics that target DNA methylation pathways and discuss their effects in the context of the DDR. Finally, we pose unanswered questions regarding the interplay between DNA methylation, transcription and the DDR, positing the potential coordinated efforts of these pathways in genome integrity. While the impact of DNA methylation on gene regulation is widely understood, how this modification contributes to genome instability and mutations, either directly or indirectly, and the potential therapeutic opportunities in targeting DNA methylation pathways in cancer remain active areas of investigation.

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis–trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25’s catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

International audience; Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.

Histone variants represent chromatin components that diversify the structure and function of the genome. The variants of H2A, primarily H2A.X, H2A.Z and macroH2A, are well-established participants in DNA damage response (DDR) pathways, which function to protect the integrity of the genome. Through their deposition, post-translational modifications and unique protein interaction networks, these variants guard DNA from endogenous threats including replication stress and genome fragility as well as from DNA lesions inflicted by exogenous sources. A growing body of work is now providing a clearer picture on the involvement and mechanistic basis of H2A variant contribution to genome integrity. Beyond their well-documented role in gene regulation, we review here how histone H2A variants promote genome stability and how alterations in these pathways contribute to human diseases including cancer.

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here, APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in human cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts overexpressed genes and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B mediates R-loop homeostasis and contributes to R-loop mutagenesis in cancer.HighlightsUnbiased proteomics link antiviral APOBEC3B to R-loop regulationSystematic alterations of APOBEC3B levels trigger corresponding changes in R-loopsAPOBEC3B binds R-loops in living cells and in vitroBioinformatics analyses support an R-loop deamination and mutation model

Endogenous DNA damage is a major contributor to mutations, which are drivers of cancer development. Bromodomain (BRD) proteins are well-established participants in chromatin-based DNA damage response (DDR) pathways, which maintain genome integrity from cell-intrinsic and extrinsic DNA-damaging sources. BRD proteins are most well-studied as regulators of transcription, but emerging evidence has revealed their importance in other DNA-templated processes, including DNA repair and replication. How BRD proteins mechanistically protect cells from endogenous DNA damage through their participation in these pathways remains an active area of investigation. Here, we review several recent studies establishing BRD proteins as key influencers of endogenous DNA damage, including DNA–RNA hybrid (R-loops) formation during transcription and participation in replication stress responses. As endogenous DNA damage is known to contribute to several human diseases, including neurodegeneration, immunodeficiencies, cancer, and aging, the ability of BRD proteins to suppress DNA damage and mutations is likely to provide new insights into the involvement of BRD proteins in these diseases. Although many studies have focused on BRD proteins in transcription, evidence indicates that BRD proteins have emergent functions in DNA repair and genome stability and are participants in the etiology and treatment of diseases involving endogenous DNA damage., DNA repair: multitasking proteins Bromodomain (BRD) proteins, known to regulate gene expression, switching particular genes on and off, also play key roles in repairing DNA damage, and studying them may help identify treatments for various diseases, including cancer. DNA damage can occur during normal cellular metabolism, for example, during copying DNA and gene expression. DNA damage is implicated in tumor formation as well as in neurodegeneration, immunodeficiency, and aging. Seo Yun Lee and colleagues at The University of Texas at Austin, USA, have reviewed new results showing how BRD proteins function in repairing DNA damage. They report that when DNA is damaged during copying in BRD-deficient cells, tumors can result. They also report that defects in BRD proteins are often present in cancers. Studying how BRD proteins function in both healthy and diseased cells could help to identify new therapies.

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically ‘recognizes’ H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1’s activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability., How histone modifications crosstalk with DNA methylation to regulate epigenomic patterning and genome stability in mammals remains elusive. Here, the authors show that DNA methyltransferase DNMT1 is a reader for histone H4K20 trimethylation via its BAH1 domain, which leads to optimal maintenance of DNA methylation at repetitive LINE-1 elements.

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

The histone demethylase KDM5A removes histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDR). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a non-canonical poly(ADP-ribose), (PAR), binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi) blocks KDM5A-PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and functions at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR-binding and macroH2A1.2 in KDM5A recognition of damage sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.SummaryThe histone demethylase KDM5A demethylates H3K4 to promote repair and transcriptional responses at DNA breaks. We identified poly(ADP-ribose)-binding and macroH2A1.2 as modulators of KDM5A association with DNA damage sites, revealing how KDM5A engages DNA breaks within chromatin.

Summary The p53 tumor suppressor pathway is frequently inactivated in human cancers. However, there are some cancer types without commonly recognized alterations in p53 signaling. Here we report that histone demethylase KDM5A is involved in the regulation of p53 activity. KDM5A is significantly amplified in multiple types of cancers, an event that tends to be mutually exclusive to p53 mutation. We show that KDM5A acts as a negative regulator of p53 signaling through inhibition of p53 translation via suppression of a subgroup of eukaryotic translation initiation genes. Genetic deletion of KDM5A results in upregulation of p53 in multiple lineages of cancer cells and inhibits tumor growth in a p53-dependent manner. In addition, we have identified a regulatory loop between p53, miR-34, and KDM5A, whereby the induction of miR-34 leads to suppression of KDM5A. Thus, our findings reveal a mechanism by which KDM5A inhibits p53 translation to modulate cancer progression., Graphical Abstract, Highlights • Genetic amplification of KDM5A tends to be negatively correlated with TP53 alterations • KDM5A inhibits p53 protein translation by suppressing a subset of translation genes • KDM5A regulates tumor growth in a p53-dependent manner • miR-34 targets KDM5A expression, Molecular Mechanism of Gene Regulation; Cancer

Defective arginine synthesis, due to the silencing of argininosuccinate synthase 1 (ASS1), is a common metabolic vulnerability in cancer, known as arginine auxotrophy. Understanding how arginine depletion kills arginine-auxotrophic cancer cells will facilitate the development of anti-cancer therapeutic strategies. Here we show that depletion of extracellular arginine in arginine-auxotrophic cancer cells causes mitochondrial distress and transcriptional reprogramming. Mechanistically, arginine starvation induces asparagine synthetase (ASNS), depleting these cancer cells of aspartate, and disrupting their malate-aspartate shuttle. Supplementation of aspartate, depletion of mitochondria, and knockdown of ASNS all protect the arginine-starved cells, establishing the causal effects of aspartate depletion and mitochondrial dysfunction on the arginine starvation-induced cell death. Furthermore, dietary arginine restriction reduced tumor growth in a xenograft model of ASS1-deficient breast cancer. Our data challenge the view that ASNS promotes homeostasis, arguing instead that ASNS-induced aspartate depletion promotes cytotoxicity, which can be exploited for anti-cancer therapies., Chun-Ting Cheng et al. demonstrate that arginine starvation kills arginine-auxotrophic cancer cells by depleting them of aspartate through asparagine synthetase (ASNS) and disrupting their mitochondrial metabolism. This study presents ASNS-induced aspartate depletion as an anti-cancer therapeutic strategy.

Upon DNA damage, histone modifications are reshaped to accommodate DNA damage signaling and repair. Gong et al. report that the histone demethylase KDM5A promotes loading of the chromatin remodeling complex ZMYND8–NuRD to double-strand DNA breaks through H3K4me3 demethylation, thereby allowing repair of the lesion., Upon DNA damage, histone modifications are dynamically reshaped to accommodate DNA damage signaling and repair within chromatin. In this study, we report the identification of the histone demethylase KDM5A as a key regulator of the bromodomain protein ZMYND8 and NuRD (nucleosome remodeling and histone deacetylation) complex in the DNA damage response. We observe KDM5A-dependent H3K4me3 demethylation within chromatin near DNA double-strand break (DSB) sites. Mechanistically, demethylation of H3K4me3 is required for ZMYND8–NuRD binding to chromatin and recruitment to DNA damage. Functionally, KDM5A deficiency results in impaired transcriptional silencing and repair of DSBs by homologous recombination. Thus, this study identifies a crucial function for KDM5A in demethylating H3K4 to allow ZMYND8–NuRD to operate within damaged chromatin to repair DSBs.

Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.

Although long overlooked, it is now well understood that DNA does not systematically assemble into a canonical double helix, known as B-DNA, throughout the entire genome but can also accommodate other structures including DNA hairpins, G-quadruplexes and RNA:DNA hybrids. Notably, these non-canonical DNA structures form preferentially at transcriptionally active loci. Acting as replication roadblocks and being targeted by multiple machineries, these structures weaken the genome and render it prone to damage, including DNA double-strand breaks (DSB). In addition, secondary structures also further accumulate upon DSB formation. Here we discuss the potential functions of pre-existing or de novo formed nucleic acid structures, as bona fide repair intermediates or repair roadblocks, especially during Transcription-Coupled DNA Double-Strand Break repair (TC-DSBR), and provide an update on the specialized protein complexes displaying the ability to remove these structures to safeguard genome integrity.

Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.

Modulation of chromatin templates in response to cellular cues, including DNA damage, relies heavily on the post-translation modification of histones. Numerous types of histone modifications including phosphorylation, methylation, acetylation, and ubiquitylation occur on specific histone residues in response to DNA damage. These histone marks regulate both the structure and function of chromatin, allowing for the transition between chromatin states that function in undamaged condition to those that occur in the presence of DNA damage. Histone modifications play well-recognized roles in sensing, processing, and repairing damaged DNA to ensure the integrity of genetic information and cellular homeostasis. This review highlights our current understanding of histone modifications as they relate to DNA damage responses (DDRs) and their involvement in genome maintenance, including the potential targeting of histone modification regulators in cancer, a disease that exhibits both epigenetic dysregulation and intrinsic DNA damage.

Maintenance of genomic integrity depends on the spatiotemporal recruitment and regulation of DNA damage response and repair proteins at DNA damage sites. These highly dynamic processes have been widely studied using laser microirradiation coupled with fluorescence microscopy. Laser microirradiation has provided a powerful methodology to identify and determine mechanisms of DNA damage response pathways. Here we describe methods used to analyze protein recruitment dynamics of fluorescently tagged or endogenous proteins to laser-induced DNA damage sites using laser scanning and fluorescence microscopy. We further describe multiple applications employing these techniques to study additional processes at DNA damage sites including transcription. Together, we aim to provide robust visualization methods employing laser-microirradiation to detect and determine protein behavior, functions and dynamics in response to DNA damage in mammalian cells.

2019 Klein et al., Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies., HLK was supported by NIH grant R01-CA146940. AA was supported by the European research Council (ERC2014- ADG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2016-75058-P), and the Junta de Andalucía (BIO1238). JLA was supported by NIH grant R35G-GM119788. CHF was supported by NIH grants P01- GM105473 and R01-GM122880. SMG was supported by the Swiss national Science Foundation and the Novartis Research Foundation. DAG was supported by National Institutes of Health (Intramural Research Program Project Z1AES103266. JEH was supported by NIH grants R35- GM127029 and P01-GM105473. GI was supported by NIH grants R01-GM125650 and R01-GM080600. SJ-R was supported by NIH grant R35-GM118077. MCK was supported by National Science Foundation Graduate Research Fellowship DGE-1122492, NIH training grant T32-GM007223 and NIH grant DP20D008429. RDK and CDP were supported by NIH grants R01-GM26017 and R01-GM50006. AK was supported by NIH grant R01-GM073115. SAEL was supported by grants from Agence Nationale de la Recherche ANR-14- CE10-0010-01 and the Foundation pour la Recherche Médicale Equipe FRM DEQ20160334889. SEL was supported by NIH grant R01-GM071011. SMM was supported by NIH grants R01-GM60987 and P01-GM105473. TDP was supported by NIH grant R35-GM118020. SMR was supported by a gift from WM Keck Foundation, NIH Director’s Pioneer Award DP1-CA174424 and NIH grant R35-GM122598. DMF was supported by the Cancer Prevention and Research Institute of Texas, Baylor College of Medicine Comprehensive Cancer Training Program Postdoctoral Fellowship RP160283. RR was supported by NIH grant R35-GM118180 and JM-H was supported by a Marie Curie International Outgoing Fellowship and the ANR-12-PDOC-0035-01. LSS was supported by NIH grant R35-GM126997. PZ was supported by National Science Centre Poland 2015/19/103859 and FNP First TEAM/2016-1/9. NK was supported by NIH grant R01-GM116007 and the Welch Foundation (AU1875). ML was supported by grants from the Danish Agency for Science, Technology and Innovation, the Villum Foundation and the Danish National Research Foundation (DNRF115). AM was supported by NIH grants R35-GM127006 and R03- ES029306.

The Sae2/CtIP protein is required for efficient processing of DNA double-strand breaks that initiate homologous recombination in eukaryotic cells. Sae2/CtIP is also important for survival of single-stranded Top1-induced lesions and CtIP is known to associate directly with transcription-associated complexes in mammalian cells. Here we investigate the role of Sae2/CtIP at single-strand lesions in budding yeast and in human cells and find that depletion of Sae2/CtIP promotes the accumulation of stalled RNA polymerase and RNA-DNA hybrids at sites of highly expressed genes. Overexpression of the RNA-DNA helicase Senataxin suppresses DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells, and a catalytic mutant of CtIP fails to complement this sensitivity, indicating a role for CtIP nuclease activity in the repair process. Based on this evidence, we propose that R-loop processing by 5’ flap endonucleases is a necessary step in the stabilization and removal of nascent R-loop initiating structures in eukaryotic cells.

International audience; Anti-cancer drugs often increase reactive oxygen species (ROS) and cause DNA damage. Here, we highlight a new cross talk between chronic oxidative stress and the histone variant H2AX, a key player in DNA repair. We observe that persistent accumulation of ROS, due to a deficient JunD-/Nrf2-antioxidant response, reduces H2AX protein levels. This effect is mediated by an enhanced interaction of H2AX with the E3 ubiquitin ligase RNF168, which is associated with H2AX poly-ubiquitination and promotes its degradation by the proteasome. ROS-mediated H2AX decrease plays a crucial role in chemosensitivity. Indeed, cycles of chemotherapy that sustainably increase ROS reduce H2AX protein levels in Triple-Negative breast cancer (TNBC) patients. H2AX decrease by such treatment is associated with an impaired NRF2-antioxidant response and is indicative of the therapeutic efficiency and survival of TNBC patients. Thus, our data describe a novel ROS-mediated regulation of H2AX turnover, which provides new insights into genetic instability and treatment efficacy in TNBC patients.

The Sae2/CtIP protein is required for efficient processing of DNA double-strand breaks that initiate homologous recombination in eukaryotic cells. Sae2/CtIP is also important for survival of single-stranded Top1-induced lesions and CtIP is known to associate directly with transcription-associated complexes in mammalian cells. Here we investigate the role of Sae2/CtIP at single-strand lesions in budding yeast and in human cells and find that depletion of Sae2/CtIP promotes the accumulation of stalled RNA polymerase and RNA-DNA hybrids at sites of highly expressed genes. Overexpression of the RNA-DNA helicase Senataxin suppresses DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells, and a catalytic mutant of CtIP fails to complement this sensitivity, indicating a role for CtIP nuclease activity in the repair process. Based on this evidence, we propose that R-loop processing by 5’ flap endonucleases is a necessary step in the stabilization and removal of nascent R-loop initiating structures in eukaryotic cells.

Histone H2A variants play important roles in maintaining the integrity of the genome. For example, the histone variant H2AX is phosphorylated on Ser139 (called γH2AX) at DNA double-strand breaks (DSB) and serves as a signal for the initiation of downstream DNA damage response (DDR) factor recruitment and DNA repair activities within damaged chromatin. For decades, genetic studies in human cells involving DNA damage signaling and repair factors have relied mostly on either knockdown by RNA interference (i.e., shRNA and siRNA) or the use of mouse embryonic fibroblasts derived from knockout (KO) mice. Recent advances in gene editing using ZNF nuclease, TALEN, and CRISPR/Cas9 have allowed the generation of human KO cell lines, allowing genetic models for studying the DDR, including histone H2A variants in human cells. Here, we describe a detailed protocol for generating and verifying KO of H2AX and macroH2A histone H2A variants using CRISPR/Cas9 gene editing in human cancer cell lines. This protocol allows the use and development of genetic systems in human cells to study histone variants and their functions, including within the DDR.

Zinc finger (ZnF) domains are present in at least 5% of human proteins. First characterized as binding to DNA, ZnFs display extraordinary binding plasticity and can bind to RNA, lipids, proteins, and protein post-translational modifications (PTMs). The diverse binding properties of ZnFs have made their functional characterization challenging. While once confined to large and poorly characterized protein families, proteomic, cellular, and molecular studies have begun to shed light on their involvement as protectors of the genome. We focus here on the emergent roles of ZnF domain-containing proteins in promoting genome integrity, including their involvement in telomere maintenance and DNA repair. These findings have highlighted the need for further characterization of ZnF proteins, which can reveal the functions of this large gene class in normal cell function and human diseases, including those involving genome instability such as aging and cancer.

Histone H2A variants play important roles in maintaining the integrity of the genome. For example, the histone variant H2AX is phosphorylated on Ser139 (called γH2AX) at DNA double-strand breaks (DSB) and serves as a signal for the initiation of downstream DNA damage response (DDR) factor recruitment and DNA repair activities within damaged chromatin. For decades, genetic studies in human cells involving DNA damage signaling and repair factors have relied mostly on either knockdown by RNA interference (i.e., shRNA and siRNA) or the use of mouse embryonic fibroblasts derived from knockout (KO) mice. Recent advances in gene editing using ZNF nuclease, TALEN, and CRISPR/Cas9 have allowed the generation of human KO cell lines, allowing genetic models for studying the DDR, including histone H2A variants in human cells. Here, we describe a detailed protocol for generating and verifying KO of H2AX and macroH2A histone H2A variants using CRISPR/Cas9 gene editing in human cancer cell lines. This protocol allows the use and development of genetic systems in human cells to study histone variants and their functions, including within the DDR.

We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ--a histone protein involved in DNA repair--as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

How chromatin shapes pathways that promote genome–epigenome integrity in response to DNA damage is an issue of crucial importance. We report that human bromodomain (BRD)-containing proteins, the primary “readers” of acetylated chromatin, are vital for the DNA damage response (DDR). We discovered that more than one-third of all human BRD proteins change localization in response to DNA damage. We identified ZMYND8 (zinc finger and MYND [myeloid, Nervy, and DEAF-1] domain containing 8) as a novel DDR factor that recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to damaged chromatin. Our data define a transcription-associated DDR pathway mediated by ZMYND8 and the NuRD complex that targets DNA damage, including when it occurs within transcriptionally active chromatin, to repress transcription and promote repair by homologous recombination. Thus, our data identify human BRD proteins as key chromatin modulators of the DDR and provide novel insights into how DNA damage within actively transcribed regions requires chromatin-binding proteins to orchestrate the appropriate response in concordance with the damage-associated chromatin context.

Preserving genome function and stability are paramount for ensuring cellular homeostasis, an imbalance in which can promote diseases including cancer. In the presence of DNA lesions, cells activate pathways referred to as the DNA damage response (DDR). As nuclear DNA is bound by histone proteins and organized into chromatin in eukaryotes, DDR pathways have evolved to sense, signal and repair DNA damage within the chromatin environment. Histone proteins, which constitute the building blocks of chromatin, are highly modified by post-translational modifications (PTMs) that regulate chromatin structure and function. An essential histone PTM involved in the DDR is histone methylation, which is regulated by histone methyltransferase (HMT) and histone demethylase (HDM) enzymes that add and remove methyl groups on lysine and arginine residues within proteins respectively. Methylated histones can alter how proteins interact with chromatin, including their ability to be bound by reader proteins that recognize these PTMs. Here, we review histone methylation in the context of the DDR, focusing on DNA double-strand breaks (DSBs), a particularly toxic lesion that can trigger genome instability and cell death. We provide a comprehensive overview of histone methylation changes that occur in response to DNA damage and how the enzymes and reader proteins of these marks orchestrate the DDR. Finally, as many epigenetic pathways including histone methylation are altered in cancer, we discuss the potential involvement of these pathways in the etiology and treatment of this disease.

Cisplatin derivatives can form various types of DNA lesions (DNA‐Pt) and trigger pleiotropic DNA damage responses. Here, we report a strategy to visualize DNA‐Pt with high resolution, taking advantage of a novel azide‐containing derivative of cisplatin we named APPA, a cellular pre‐extraction protocol and the labeling of DNA‐Pt by means of click chemistry in cells. Our investigation revealed that pretreating cells with the histone deacetylase (HDAC) inhibitor SAHA led to detectable clusters of DNA‐Pt that colocalized with the ubiquitin ligase RAD18 and the replication protein PCNA. Consistent with activation of translesion synthesis (TLS) under these conditions, SAHA and cisplatin cotreatment promoted focal accumulation of the low‐fidelity polymerase Polη that also colocalized with PCNA. Remarkably, these cotreatments synergistically triggered mono‐ubiquitination of PCNA and apoptosis in a RAD18‐dependent manner. Our data provide evidence for a role of chromatin in regulating genome targeting with cisplatin derivatives and associated cellular responses.

DNA damage from endogenous and exogenous sources threatens the genome and the epigenome, requiring chromatin-based DNA damage response pathways (DDR) to operate within structurally and functionally diverse chromatin environments. DDR pathways are essential for sensing, processing and repairing DNA damage. We describe how chromatin dynamics, including those regulated by histone modifications, histone variants and chromatin-associated proteins, promote DNA repair within chromatin. We will also discuss how DNA damage regulates transcription, alters the chromatin environment and affects the mobility of chromosomes to promote the DDR, and how genome organization impacts DNA repair and chromosome translocations. Finally, we explore the use of site-specific DNA damage systems and methodologies to measure histone dynamics to study the DDR and chromatin. This chapter provides an overview of how the DDR operates within the chromatin landscape to maintain genome and epigenome integrity, functions critical for cellular homeostasis and averting diseases including cancer.

Small molecules--including various approved and novel cancer therapeutics--can operate at the genomic level by targeting the DNA and protein components of chromatin. Emerging evidence suggests that functional interactions between small molecules and the genome are non-stochastic and are influenced by a dynamic interplay between DNA sequences and chromatin states. The establishment of genome-wide maps of small-molecule targets using unbiased methodologies can help to characterize and exploit drug responses. In this Review, we discuss how high-throughput sequencing strategies, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing) and Chem-seq (chemical affinity capture and massively parallel DNA sequencing), are enabling the comprehensive identification of small-molecule target sites throughout the genome, thereby providing insights into unanticipated drug effects.

SUMMARYDNA damage provokes mutations and cancer, and results from external carcinogens or endogenous cellular processes. Yet, the intrinsic instigators of DNA damage are poorly understood. Here we identify proteins that promote endogenous DNA damage when overproduced: the DNA-damaging proteins (DDPs). We discover a large network of DDPs inEscherichia coliand deconvolute them into six DNA-damage-causing function clusters, demonstrating DDP mechanisms in three: reactive-oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their expression in tumors predicts heavy mutagenesis and poor prognosis. Half of tested human homologs, when overproduced in human cells, promote DNA damage and mutation, with DNA-damaging mechanisms like those inE. coli. Together, our work reveals DDP networks that provoke endogenous DNA damage and may indicate functions of many human known and newly implicated cancer-promoting proteins.

DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

The occurrence of a double-strand break in DNA activates a complex series of events that recruit to the break many proteins involved in its repair. A number of these proteins are modified by addition of a small protein, SUMO; this modification is performed SUMO ligases. In this work, Jackson and colleagues show that two such ligases, PIAS1 and PIAS4, add various SUMOs onto DNA repair proteins at double-strand breaks. The PIAS ligases are recruited via their SAP domains, and their activity is required for effective repair. SUMOylation by PIAS1 and PIAS4 is also necessary for the further modification of certain repair factors by ubiquitin, a somewhat larger protein adduct related to SUMO. The successive SUMOylation and ubiquitylation of repair proteins regulates their targeting to, and repair of, DNA breaks. Following the formation of a DNA double-strand break (DSB), cells activate the DNA-damage response and recruit a number of proteins to the lesion. Some of these proteins are modified by the attachment of small ubiquitin-related modifier (SUMO). Here, SUMO1, SUMO2 and SUMO3 are shown to accumulate at DSB sites in mammalian cells. SUMO1 and SUMO2/3 accrual requires the E3 ligase enzymes PIAS4 and PIAS1, which promote DSB 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 repair1,2. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions3,4,5,6. 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 damage7,8,9,10,11. 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.

Genome stability depends upon the RecQ helicases, which are conserved from bacteria to man, but little is known about how their myriad activities are regulated. Fission yeast lacking the telomere protein Taz1 (mammalian TRF1/TRF2 ortholog) lose many hallmarks of telomeres, including accurate replication and local protection from DNA repair reactions. Here we show that the RecQ homolog, Rqh1, is sumoylated. Surprisingly, Rqh1 acts on taz1Delta telomeres in a deleterious way, promoting telomere breakage and entanglement. Mutation of Rqh1 sumoylation sites rescues taz1Delta cells from these hazards without dramatically affecting nontelomeric Rqh1 functions. The prominence of Rqh1 in the etiology of several different telomere defects supports the idea that they originate from a common underlying lesion--aberrant processing of the stalled telomeric replication forks that accumulate in the absence of Taz1. Our work underscores the principle that RecQ helicases are "double-edged swords" whose activity, while necessary for maintaining genome-wide stability, must be vigilantly controlled.