Global DNA Damage Research Articles

Post-translational modification-dependent oligomerization switch in regulation of global transcription and DNA damage repair during genotoxic stress

Mechanisms of functional cross-talk between global transcriptional repression and efficient DNA damage repair during genotoxic stress are poorly known. In this study, using human AF9 as representative of Super Elongation Complex (SEC) components, we delineate detailed mechanisms of these processes. Mechanistically, we describe that Poly-Serine domain-mediated oligomerization is pre-requisite for AF9 YEATS domain-mediated TFIID interaction-dependent SEC recruitment at the promoter-proximal region for release of paused RNA polymerase II. Interestingly, during genotoxic stress, CaMKII-mediated phosphorylation-dependent nuclear export of AF9-specific deacetylase HDAC5 enhances concomitant PCAF-mediated acetylation of K339 residue. This causes monomerization of AF9 and reduces TFIID interaction for transcriptional downregulation. Furthermore, the K339 acetylation-dependent enhanced AF9-DNA-PKc interaction leads to phosphorylation at S395 residue which reduces AF9-SEC interaction resulting in transcriptional downregulation and efficient repair of DNA damage. After repair, nuclear re-entry of HDAC5 reduces AF9 acetylation and restores its TFIID and SEC interaction to restart transcription.

Novel insights into bulky DNA damage formation and nucleotide excision repair from high-resolution genomics

DNA damages compromise cell function and fate. Cells of all organisms activate a global DNA damage response that includes a signaling stress response, activation of checkpoints, and recruitment of repair enzymes. Especially deleterious are bulky, helix-distorting damages that block transcription and replication. Due to their miscoding nature, these damages lead to mutations and cancer. In human cells, bulky DNA damages are repaired by nucleotide excision repair (NER). To date, the basic mechanism of NER in naked DNA is well defined. Still, there is a fundamental gap in our understanding of how repair is orchestrated despite the packaging of DNA in chromatin, and how it is coordinated with active transcription and replication. The last decade has brought forth huge advances in our ability to detect and assay bulky DNA damages and their repair at single nucleotide resolution across the human genome. Here we review recent findings on the effect of chromatin and DNA-binding proteins on the formation of bulky DNA damages, and novel insights on NER, provided by the recent application of genomic methods.

The FHA domain protein ArnA functions as a global DNA damage response repressor in the hyperthermophilic archaeon Saccharolobus islandicus.

Forkhead-associated (FHA) domain proteins specifically recognize phosphorylated threonine via the FHA domain and are involved in signal transduction in various processes especially DNA damage response (DDR) and cell cycle regulation in eukaryotes. Although FHA domain proteins are found in prokaryotes, archaea, and bacteria, their functions are far less clear as compared to the eukaryotic counterparts, and it has not been studied whether archaeal FHA proteins play a role in DDR. Here, we have characterized an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) by genetic, biochemical, and transcriptomic approaches. We find that ΔSisarnA exhibits higher resistance to DNA damage agent 4-nitroquinoline 1-oxide (NQO). The transcription of ups genes, encoding the proteins for pili-mediated cell aggregation and cell survival after DDR, is elevated in ΔSisarnA. The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro. ΔSisarnB displays higher resistance to NQO than the wild type. In addition, the interaction between SisArnA and SisArnB, which is reduced in the NQO-treated cells, is indispensable for DNA binding in vitro. These indicate that SisArnA and SisArnB work together to inhibit the expression of ups genes in vivo. Interestingly, ΔSisarnE is more sensitive to NQO than the wild type, and the interaction between SisArnA and SisArnE is strengthened after NQO treatment, suggesting a positive role of SisArnE in DDR. Finally, transcriptomic analysis reveals that SisArnA represses a number of genes, implying that archaea apply the FHA/phospho-peptide recognition module for extensive transcriptional regulation. IMPORTANCE Cellular adaption to diverse environmental stresses requires a signal sensor and transducer for cell survival. Protein phosphorylation and its recognition by forkhead-associated (FHA) domain proteins are widely used for signal transduction in eukaryotes. Although FHA proteins exist in archaea and bacteria, investigation of their functions, especially those in DNA damage response (DDR), is limited. Therefore, the evolution and functional conservation of FHA proteins in the three domains of life is still a mystery. Here, we find that an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) represses the transcription of pili genes together with its phosphorylated partner SisArnB. SisArnA derepression facilitates DNA exchange and repair in the presence of DNA damage. The fact that more genes including a dozen of those involved in DDR are found to be regulated by SisArnA implies that the FHA/phosphorylation module may serve as an important signal transduction pathway for transcriptional regulation in archaeal DDR.

Embryonic abnormalities and genotoxicity induced by 2,4-dichlorophenoxyacetic acid during indirect somatic embryogenesis in Coffea

Indirect somatic embryogenesis (ISE) is a morphogenetic pathway in which somatic cells form callus and, later, somatic embryos (SE). 2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin that promotes the proliferation and dedifferentiation of somatic cells, inducing the ISE. However, 2,4-D can cause genetic, epigenetic, physiological and morphological disorders, preventing the regeneration and/or resulting abnormal somatic embryos (ASE). We aimed to evaluate the toxic 2,4-D effect during the Coffea arabica and C. canephora ISE, assessing the SE morphology, global 5-methylcytosine levels (5-mC%) and DNA damage. Leaf explants were inoculated in media with different 2,4-D concentrations. After 90 days, the friable calli were transferred to the regeneration medium, and the number of normal and abnormal SE was monthly counted. The increase of the 2,4-D concentration increased the number of responsive explants in both Coffea. At 9.06, 18.08 and 36.24 μM 2,4-D, C. arabica presented the highest values of responsive explants, differing from C. canephora. Normal and abnormal SE regeneration increased in relation to the time and 2,4-D concentration. Global 5-mC% varied at different stages of the ISE in both Coffea. Furthermore, the 2,4-D concentration positively correlated with global 5-mC%, and with the mean number of ASE. All ASE of C. arabica and C. canephora exhibited DNA damage and showed higher global 5-mC%. The allotetraploid C. arabica exhibited greater tolerance to the toxic effect of 2,4-D than the diploid C. canephora. We conclude that synthetic 2,4-D auxin promotes genotoxic and phytotoxic disorders and promotes epigenetic changes during Coffea ISE.

Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion.

The oncolytic autonomous parvovirus Minute Virus of Mice (MVM) establishes infection in the nuclear environment by usurping host DNA damage signaling proteins in the vicinity of cellular DNA break sites. MVM replication induces a global cellular DNA Damage Response (DDR) that is dependent on signaling by the ATM kinase and inactivates the cellular ATR-kinase pathway. However, the mechanism of how MVM generates cellular DNA breaks remains unknown. Using single molecule DNA Fiber Analysis, we have discovered that MVM infection leads to a shortening of host replication forks as infection progresses, as well as induction of replication stress prior to the initiation of virus replication. Ectopically expressed viral non-structural proteins NS1 and NS2 are sufficient to cause host-cell replication stress, as is the presence of UV-inactivated non-replicative MVM genomes. The host single-stranded DNA binding protein Replication Protein A (RPA) associates with the UV-inactivated MVM genomes, suggesting MVM genomes might serve as a sink for cellular stores of RPA. Overexpressing RPA in host cells prior to UV-MVM infection rescues DNA fiber lengths and increases MVM replication, confirming that MVM genomes deplete RPA stores to cause replication stress. Together, these results indicate that parvovirus genomes induce replication stress through RPA exhaustion, rendering the host genome vulnerable to additional DNA breaks.

Human papillomavirus E6/E7 oncoproteins promote radiotherapy-mediated tumor suppression by globally hijacking host DNA damage repair.

Rationale: Whatever the mucosa primary infected, HPV-positive cancers are traditionally associated with a favorable outcome, attributable to a high sensitivity to radiation therapy. However, the direct impact of viral E6/E7 oncoproteins on the intrinsic cellular radiosensitivity (and, globally, on host DNA repair) remains mostly speculative. Methods: Using several isogenic cell models expressing HPV16 E6 and/or E7, the effect of viral oncoproteins on global DNA damage response was first investigated by in vitro/in vivo approaches. The binary interactome of each individual HPV oncoprotein with factors involved in the various host DNA damage/repair mechanisms was then precisely mapped by Gaussia princeps luciferase complementation assay (and validated by co-immunoprecipitation). The stability/half-life of protein targets for HPV E6 and/or E7 as well as their subcellular localizations were determined. At last, the host genome integrity following E6/E7 expression and the synergy between radiotherapy and compounds targeting DNA repair were analyzed. Results: We first showed that the sole expression of one viral oncoprotein from HPV16 was able to significantly increase the sensitivity to irradiation of cells without affecting their basal viability parameters. In total, 10 novel targets (CHEK2, CLK2, CLK2/3, ERCC3, MNAT1, PER1, RMI1, RPA1, UVSSA and XRCC6) for E6 and 11 (ALKBH2, CHEK2, DNA2, DUT, ENDOV, ERCC3, PARP3, PMS1, PNKP, POLDIP2 and RBBP8) for E7 were identified. Importantly, not degraded following their interaction with E6 or E7, these proteins have been shown to be less linked to host DNA and to colocalize with HPV replication foci, denoting their crucial implication in viral life cycle. Finally, we found that E6/E7 oncoproteins globally jeopardize host genome integrity, increase the cellular sensitivity to DNA repair inhibitors and enhance their synergy with radiotherapy. Conclusion: Taken together, our findings provide a molecular insight into the direct hijacking of host DNA damage/repair responses by HPV oncoproteins, demonstrate the significant impact of this phenomenon on both intrinsic cellular radiosensitivity and host DNA integrity and suggest novel connected therapeutic vulnerabilities.

Synergistic Leukaemia Cell Death Induced with PARP and TiP60 Inhibitors Is a Prospective Therapeutic Strategy for AML

Background: We previously identified pharmacological inhibition of lysine demethylase 4A (KDM4A) sensitises AML cells to inhibition of poly ADP ribose polymerase (PARP), one of the major DNA damage response (DDR) mediators. We hypothesise that synergy between inhibition of PARP and histone acetyltransferase (HAT) family member, Tip60 that is involved in DSB repair, may drive effective AML leukemic cell death sparing normal cells. We aim to unravel molecular mechanisms of action of the targeted inhibitors and downstream targets of Tip60 and PARP in AML. Methods: Phenotypic changes, such as in cell viability, apoptosis, cell cycle arrest by flow cytometry, colony forming capacity, and DNA damage protein markers by western blotting, in AML cells following drug treatments were determined by standard protocols. Enrichment of biological pathways in which genes with transcriptional changes in AML cells upon the drug treatment were discovered using gene set enrichment analysis (GSEA) of RNA-seq dataset generated from treated cell lines. Downstream target genes of Tip60 and PARP together in common were identified via cross-validation using least absolute shrinkage and selection operator (LASSO) linear regression in clinical datasets. Results: Loss of PARP and Tip60 enzymatic activities was driven by pharmacological inhibition of PARP and Tip60 individually and in combination in AML cell lines. As a result, a significant synergistic effect of the combination treatment (PARPi plus Tip60i) was confirmed in THP-1 and MOLM-13 cells with combination indices (CI) < 1, which was further validated via the Bliss independent model and isobolograms. Simultaneous combination of PARPi together with Tip60i resulted in significant phenotypic changes with increased early and late apoptotic cell populations, cell cycle arrest at S phase, decreased colony forming ability, and accumulation of DNA lesion markers, γH2A.X and cleaved PARP proteins, which contributed to inhibition of cell growth and ultimately significant increase (p<0.0001) in AML leukemic cell death. GSEA revealed that combination treatment of PARPi and Tip60i induced significant enrichment of genes (adjusted p-value<0.05) involved in DDR sub-pathways, mismatch repair (MMR; NES=-1.54; FDR=0.088) and interstrand crosslink repair (ICLR; NES=-1.25; FDR=0.22), in parallel, suggesting an inhibitory role of PARPi and Tip60i in impairing DDR in AML cells. Furthermore, global GSEA further demonstrated that synergistic effect of PARPi plus Tip60i induced significant upregulation of biological pathways of hypoxia, unfolded protein response, p53 pathway, and apoptosis; and significant downregulation of oxidative phosphorylation, glycolysis, and Myc pathway. A minimum subset of 13 genes mediated by inhibition of Tip60 and PARP ('TiP13') in AML cells was constructed in relation to patient survival. Kaplan-Meier survival analysis showed that the gene signature score of each patient in four patient cohorts, calculated as the sum of the weighted gene expression for each gene in TiP13, was highly correlated with patient survival in four test clinical datasets (GSE12417, FDR=0.00091; GSE37642, FDR=0.042; GSE6891, FDR=0.044; Beat AML, FDR<0.0001). Discussion: Failure to repair DNA damage at transcriptional level by blocking of MMR and ICLR pathways led to constant accumulation of DNA lesions and thereby effective cell killing in AML cells treated with PARPi and Tip60i in combination. This study further demonstrates that transcriptional outcomes driven by synergising PARPi and Tip60i activities contribute to dysregulation of oxidative phosphorylation for oxidation of nutrients and production of ATP in glycolysis in AML cells, leading to genomic instability, accumulated global DNA damage, and increased response to stress or unfolded protein. Consequently, the accumulated DNA lesions alongside impaired capacity of AML cells to repair their damaged DNA, thereby stimulated apoptosis, facilitated with downregulation of an oncogenic Myc pathway and upregulation of tumour suppressor p53 pathway. In addition, this study has uncovered a novel TiP13 gene signature (downstream targets of PARP and Tip60) with prognostic significance in AML that successfully stratifies outcomes of AML patients.

Determination of double- and single-stranded DNA breaks in bovine sperm is predictive of their fertilizing capacity

BackgroundThe analysis of chromatin integrity has become an important determinant of sperm quality. In frozen-thawed bovine sperm, neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood. The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics, and to assess if this parameter can predict pregnancy rates in cattle.ResultsA total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods (0-2 h and 2-4 h), analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays, chromatin deprotamination and decondensation, sperm motility, viability, acrosomal status, and intracellular levels of total ROS, superoxides and calcium. Insemination of 59,605 females was conducted using sperm from the same bulls, thus obtaining the non-return to estrus rates after 90 d (NRR). Results showed an increased rate of double-stranded breaks in the first period (0-2 h: 1.29 ± 1.01%/h vs. 2-4 h: 0.13 ± 1.37%/h; P < 0.01), whereas the rate of sperm with moderate + high single-stranded breaks was higher in the second period (0-2 h: 3.52 ± 7.77 %/h vs. 2-4h: 21.06 ± 11.69 %/h; P < 0.0001). Regarding sperm physiology, viability decrease rate was different between the two periods (0-2 h: − 4.49 ± 1.79%/h vs. 2-4 h: − 2.50 ± 3.39%/h; P = 0.032), but the progressive motility decrease rate was constant throughout post-thawing incubation (0-2 h: − 4.70 ± 3.42%/h vs. 2-4 h: − 1.89 ± 2.97%/h; P > 0.05). Finally, whereas no correlations between bull fertility and any dynamic parameter were found, there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet (Rs = − 0.563, P = 0.003), between NRR and basal progressive motility (Rs = 0.511, P = 0.009), and between NRR and sperm with high ROS at 4 h post-thaw (Rs = 0.564, P = 0.003).ConclusionThe statistically significant correlations found between intracellular ROS, sperm viability, sperm motility, DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress, where viability and motility would be affected first and sperm chromatin would be altered at a later stage, thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw. Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.

Seminal Microbiota of Idiopathic Infertile Patients and Its Relationship With Sperm DNA Integrity.

The development of new biomarkers for human male infertility is crucial to improve the diagnosis and the prognosis of this disease. Recently, seminal microbiota was shown to be related to sperm quality parameters, suggesting an effect in human fertility and postulating it as a biomarker candidate. However, its relationship to sperm DNA integrity has not been studied yet. The aim of the present study is to characterize the seminal microbiota of a western Mediterranean population and to evaluate its relationship to sperm chromatin integrity parameters, and oxidative stress. For that purpose, 14 samples from sperm donors and 42 samples from infertile idiopathic patients were obtained and were analyzed to assess the composition of the microbiota through full-length 16S rRNA gene sequencing (Illumina MiSeq platform). Microbial diversity and relative abundances were compared to classic sperm quality parameters (macroscopic semen parameters, motility, morphology and concentration), chromatin integrity (global DNA damage, double-stranded DNA breaks and DNA protamination status) and oxidative stress levels (oxidation-reduction potential). The seminal microbiota observed of these samples belonged to the phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. The most abundant genera were Finegoldia, Peptoniphilus, Anaerococcus, Campylobacter, Streptococcus, Staphylococcus, Moraxella, Prevotella, Ezakiella, Corynebacterium and Lactobacillus. To our knowledge, this is the first detection of Ezakiella genus in seminal samples. Two clusters of microbial profiles were built based on a clustering analysis, and specific genera were found with different frequencies in relation to seminal quality defects. The abundances of several bacteria negatively correlate with the sperm global DNA fragmentation, most notably Moraxella, Brevundimonas and Flavobacterium. The latter two were also associated with higher sperm motility and Brevundimonas additionally with lower oxidative-reduction potential. Actinomycetaceae, Ralstonia and Paenibacillus correlated with reduced chromatin protamination status and increased double-stranded DNA fragmentation. These effects on DNA integrity coincide in many cases with the metabolism or enzymatic activities of these genera. Significant differences between fertile and infertile men were found in the relative presence of the Propionibacteriaceae family and the Cutibacterium, Rhodopseudomonas and Oligotropha genera, which supports its possible involvement in male fertility. Our findings sustain the hypothesis that the seminal microbiome has an effect on male fertility.

Sperm DNA damage compromises embryo development, but not oocyte fertilisation in pigs

BackgroundThe assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays.ResultsThe results showed that global DNA damage negatively correlates (P < 0.05) with normal sperm morphology (R = − 0.460) and progressive motility (R = − 0.419), and positively with the percentage of non-viable sperm (R = 0.507). Multiple regression analyses showed that non-viable sperm were related to SSB (β = − 0.754). In addition, while fertilisation did not seem to be affected by sperm DNA integrity, global DNA damage, DSB and SSB were found to be correlated to embryo development outcomes. Specifically, whereas global DNA damage and DSB negatively affected (P < 0.05) the later preimplantation embryo stages (percentage of early blastocyst/blastocyst D6: for global DNA damage, R = − 0.458, and for DSB, R = − 0.551; and percentage of hatching/hatched blastocyst D6: for global DNA damage, R = − 0.505, and for DSB, R = − 0.447), global DNA damage and SSB had a negative impact (P < 0.05) on the developmental competency of fertilised embryos (R = − 0.532 and R = − 0.515, respectively). Remarkably, multiple regression analyses supported the associations found in correlation analyses. Finally, the present work also found that the inclusion of Comet assays to the conventional sperm quality tests improves the prediction of blastocyst formation (AUC = 0.9021, P < 0.05), but not fertilisation rates (P > 0.05).ConclusionConsidering all these findings, this work sets a useful model to study how SDF negatively influences fertility.

Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage

BackgroundThe main function of telomerase is at the telomeres but under adverse conditions telomerase can bind to internal regions causing deleterious effects as observed in cancer cells.ResultsBy mapping the global occupancy of the catalytic subunit of telomerase (Est2) in the budding yeast Saccharomyces cerevisiae, we reveal that it binds to multiple guanine-rich genomic loci, which we termed “non-telomeric binding sites” (NTBS). We characterize Est2 binding to NTBS. Contrary to telomeres, Est2 binds to NTBS in G1 and G2 phase independently of Est1 and Est3. The absence of Est1 and Est3 renders telomerase inactive at NTBS. However, upon global DNA damage, Est1 and Est3 join Est2 at NTBS and telomere addition can be observed indicating that Est2 occupancy marks NTBS regions as particular risks for genome stability.ConclusionsOur results provide a novel model of telomerase regulation in the cell cycle using internal regions as “parking spots” of Est2 but marking them as hotspots for telomere addition.

Evidence for age-related contributions of DNA damage and epigenetics in brain tumorigenesis.

Glioblastoma (GBM) is a highly malignant primary brain tumour displaying rapid cell proliferation and infiltration. GBM primarily occurs at older age; however, younger populations have also been affected. In GBM and other cancers, genetic and epigenetic alterations promote tumorigenesis causing increased cell proliferation and invasiveness. This investigation explored epigenetic events as contributing factors, especially in gliomas that arise in patients aged 40-60years. Furthermore, DNA damage in tumours with respect to age was assessed. Archival fixed tissues from 88 cases of glioblastoma and adjacent non-malignant tissues were tested. Global methylation and DNA damage were measured using ELISA detection of 5-methyl cytosine and 8-hydroxy guanine, respectively. IDH mutations and CDKN2 promoter hypermethylation were analysed by pyrosequencing. Tumour tissue was hypomethylated compared with non-malignant tissue (P=.001), and there was a trend towards increased methylation with increasing age. There was a significant increase in DNA damage in patients older than forty years compared with those aged forty years or younger (P=.035). CDKN2 promoter methylation levels followed the age trends of global methylation in this patient group. Patients younger than 60 had more frequently mutated IDH (P=.004). Conclusions: The data support the potential of epigenetic factors in promoting tumorigenesis in younger patients, while increased DNA damage contributes to tumorigenesis in the older patients.

From single-molecule to genome-wide mapping of DNA lesions: repair-assisted damage detection sequencing

Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, such as cancer, and assists in monitoring their progression and prognosis. Developments in recent years have enabled unprecedented sensitivity in quantifying the global DNA damage dose in cells via fluorescence-based analysis down to the single-molecule level. However, genome-wide maps of DNA damage distribution are challenging to produce. Here, we describe the localization of DNA damage and repair loci by repair-assisted damage detection sequencing (RADD-seq). Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next-generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of the oxidation DNA damage lesion 8-oxo-7,8-dihydroguanine before and after repair. Oxidation lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and is capable of identifying genomic hotspots prone to mutation.

AKT-dependent signaling of extracellular cues through telomeres impact on tumorigenesis.

The telomere-bound shelterin complex is essential for chromosome-end protection and genomic stability. Little is known on the regulation of shelterin components by extracellular signals including developmental and environmental cues. Here, we show that human TRF1 is subjected to AKT-dependent regulation. To study the importance of this modification in vivo, we generate knock-in human cell lines carrying non-phosphorylatable mutants of the AKT-dependent TRF1 phosphorylation sites by CRISPR-Cas9. We find that TRF1 mutant cells show decreased TRF1 binding to telomeres and increased global and telomeric DNA damage. Human cells carrying non-phosphorylatable mutant TRF1 alleles show accelerated telomere shortening, demonstrating that AKT-dependent TRF1 phosphorylation regulates telomere maintenance in vivo. TRF1 mutant cells show an impaired response to proliferative extracellular signals as well as a decreased tumorigenesis potential. These findings indicate that telomere protection and telomere length can be regulated by extracellular signals upstream of PI3K/AKT activation, such as growth factors, nutrients or immune regulators, and this has an impact on tumorigenesis potential.

MiR-490 suppresses telomere maintenance program and associated hallmarks in glioblastoma.

Glioblastoma (GBM) is the most aggressive cancer of central nervous system with worst patient outcome. Telomere maintenance is a crucial mechanism governing GBM initiation and progression making it an attractive target. microRNAs (miRNAs) have shown therapeutic potential in GBM. Earlier, we showed miR-490 is downregulated in GBM patients and plays a tumor suppressive role. Here, we show that miR-490 regulates telomere maintenance program in GBM by directly targeting Telomeric Repeat-binding Factor 2 (TERF2) of the shelterin complex, Tankyrase 2 (TNKS2) and Serine/Threonine-protein kinase, SMG1. Overexpression of miR-490 resulted in effects characteristic to hampered telomere maintenance via TERF2 inhibition. These include induction of telomere dysfunction-induced foci and global DNA damage (53BP1 foci), along with an increase in p-γH2AX levels. Further, it led to inhibition of telomere maintenance hallmarks via reduced stemness (SOX2 and SOX4 downregulation) and induction of senescence (H3K9me3 marks gain and SIRT1 downregulation). It also initiated downstream DNA damage response (DDR) leading to p53 pathway activation. Moreover, microarray data analysis highlighted an overlap between miR-490expression and REST-inhibition responses in GBM. Thus, miR-490-mediated targeting of telomere maintenance could be therapeutically important in GBM.

Abstract 1646: Beyond BRCA: Discovery of novel drivers of homologous recombination deficiencies in cancer

Abstract Since the discovery of BRCA1 and BRCA2 mutations as cancer risk factors, we have gained much understanding of their role in maintaining genomic stability through homologous recombination (HR) DNA repair. However, mutations in BRCA1/2 and other classical HR proteins such as RAD51 and PALB2 only identify 10-20% of TCGA patients who display HR deficiencies, indicating that we do not understand the vast majority of HR defects. Here, we leveraged the abundance of molecular characterization from TCGA patients for network analysis to fill this knowledge void. We discovered that over half of HR deficiencies originate outside of canonical DNA damage response genes, with particular enrichment for RNA binding proteins (RBPs). Experimental techniques validated over 90% of our predictions in a panel of 50 genes tested by siRNA, as well as 30/31 additional engineered mutations identified in TCGA patients. We further cross-validated these findings in independent patient cohorts, finding that the identified RNA processing mutations again enriched for HR deficient patients to an equal or greater degree than mutations in DNA damage genes. Using a series of experimental approaches, including protein interactome screening, RNA sequencing, and quantitative imaging cytometry, we probed how loss of RBP function induced global DNA damage response rewiring, including changes in RNA splicing, protein-protein interactions, and recruitment of repair factors to DNA damage sites. Clinically, defects in HR are known to promote cancer initiation, but also sensitize cells to targeted therapies such as PARP inhibition. We found that depletion RBPs from PARP-resistant triple-negative breast cancer cells induced sensitivity to multiple PARP inhibitors, indicating that this could identify new cohorts of patients who may benefit from PARP inhibition, beyond the small number of BRCA1/2-mutant patients. Moreover, we find that the identified RBP genes are significantly enriched for genes associated with cancer risk identified through GWAS. In patients, HR deficiencies were not equally distributed across all demographics, so preferential screening of those most at risk could further heighten this benefit. Thus, these novel drivers may show clinical relevance both for treatment stratification and for identifying individuals at high risk for cancer development to improve patient outcomes. Taken together, this exhaustive study greatly expands our repertoire of known drivers of HR deficiencies and mechanisms of damage repair, which may impact research from the most basic biology studies to clinical screening and stratification. Citation Format: Nidhi Sahni, Daniel McGrail, Yongsheng Li, Gordon Mills, Song (Stephen) Yi. Beyond BRCA: Discovery of novel drivers of homologous recombination deficiencies in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1646.

Modulation of the ATM/autophagy pathway by a G-quadruplex ligand tips the balance between senescence and apoptosis in cancer cells.

G-quadruplex ligands exert their antiproliferative effects through telomere-dependent and telomere-independent mechanisms, but the inter-relationships among autophagy, cell growth arrest and cell death induced by these ligands remain largely unexplored. Here, we demonstrate that the G-quadruplex ligand 20A causes growth arrest of cancer cells in culture and in a HeLa cell xenografted mouse model. This response is associated with the induction of senescence and apoptosis. Transcriptomic analysis of 20A treated cells reveals a significant functional enrichment of biological pathways related to growth arrest, DNA damage response and the lysosomal pathway. 20A elicits global DNA damage but not telomeric damage and activates the ATM and autophagy pathways. Loss of ATM following 20A treatment inhibits both autophagy and senescence and sensitizes cells to death. Moreover, disruption of autophagy by deletion of two essential autophagy genes ATG5 and ATG7 leads to failure of CHK1 activation by 20A and subsequently increased cell death. Our results, therefore, identify the activation of ATM by 20A as a critical player in the balance between senescence and apoptosis and autophagy as one of the key mediators of such regulation. Thus, targeting the ATM/autophagy pathway might be a promising strategy to achieve the maximal anticancer effect of this compound.

Quantifying site-specific chromatin mechanics and DNA damage response

DNA double-strand breaks pose a direct threat to genomic stability. Studies of DNA damage and chromatin dynamics have yielded opposing results that support either increased or decreased chromatin motion after damage. In this study, we independently measure the dynamics of transcriptionally active or repressed chromatin regions using particle tracking microrheology. We find that the baseline motion of transcriptionally repressed regions of chromatin are significantly less mobile than transcriptionally active chromatin, which is statistically similar to the bulk motion of chromatin within the nucleus. Site specific DNA damage using KillerRed tags induced in loci within repressed chromatin causes an increased motion, while loci within transcriptionally active regions remains unchanged at similar time scales. We also observe a time-dependent response associated with a further increase in chromatin decondensation. Global induction of damage with bleocin displays similar trends of chromatin decondensation and increased mobility only at 53BP1-labeled damage sites but not at non-damaged sites, indicating that chromatin dynamics are tightly regulated locally after damage. These results shed light on the evolution of the local and global DNA damage response associated with chromatin remodeling and dynamics, with direct implications for their role in repair.

Distribution of DNA damage in the sperm nucleus: A study of zebrafish as a model of histone-packaged chromatin

Reproductive defects can occur when the integrity of the male gamete genome is affected. Sperm chromatin is not homogeneous, having relaxed regions which are more accessible to the transcription machinery in the embryo, and thought to be specially sensitive to DNA damage. The level of damage in specific genes located in these sensitive regions could represent an early biomarker of damage. Our objective is to test the hypothesis that these more relaxed regions show greater susceptibility to damage in zebrafish, a species lacking protamines and whose sperm chromatin is compacted with histones. After sperm UV irradiation, treatment with H2O2 and cryopreservation, global chromatin fragmentation was evaluated using the TUNEL assay, and the number of lesions per 10 Kb in specific genes (hoxa3a, hoxb5b, sox2, accessible for early transcription and rDNA 18S and rDNA 28S) was quantified by using a qPCR approach. Additionally, oxidative damage within the sperm nucleus and the potential colocalization of this injury with histone H3 and TOPO IIα+β were located by using immunofluorescence. UV irradiation produced the highest degree of fragmentation (p = 0.041) and the highest number of lesions per 10 Kb in all the genes, but no differences were observed in sensitivity to damage in the studied genes (ranging from 14.93 to 8.03 lesions per 10 Kb in hoxb5b and 28S, respectively). In contrast, H2O2 and cryopreservation caused varying levels of damage in the analyzed genes which was not related to their accessibility, ranging from 0.00 to 1.65 lesions per 10 Kb in 28S and hoxb5b, respectively, after H2O2 treatment, and from 0.073 to 5.51 in 28S and sox2, respectively, after cryopreservation. Immunodetection near oxidative lesions also revealed different spatial patterns depending on the treatments used, these being mostly homogeneous with UV irradiation or cryopreservation, and peripherally located around the nucleus after H2O2 treatment. Oxidative lesions did not colocalize with histone H3 or TOPO IIα+β, thus demonstrating that the relaxed DNA regions associated with these proteins were not more vulnerable to oxidative damage. Results suggest that accessibility of each agent to the nucleus could be the main factor responsible for the distribution of sperm DNA damage rather than the organization of the chromatin. Lesions in these genes important to early embryo development assayed in this study cannot be used as biomarkers of global DNA damage.

APE1 deficiency promotes cellular senescence and premature aging features.

Base excision repair (BER) handles many forms of endogenous DNA damage, and apurinic/apyrimidinic endonuclease 1 (APE1) is central to this process. Deletion of both alleles of APE1 (a.k.a. Apex1) in mice leads to embryonic lethality, and deficiency in cells can promote cell death. Unlike most other BER proteins, APE1 expression is inversely correlated with cellular senescence in primary human fibroblasts. Depletion of APE1 via shRNA induced senescence in normal human BJ fibroblasts, a phenotype that was not seen in counterpart cells expressing telomerase. APE1 knock-down in primary fibroblasts resulted in global DNA damage accumulation, and the induction of p16INK4a and p21WAF1 stress response pathways; the DNA damage response, as assessed by γ-H2AX, was particularly pronounced at telomeres. Conditional knock-out of Apex1 in mice at post-natal day 7/12 resulted in impaired growth, reduced organ size, and increased cellular senescence. The effect of Apex1 deletion at post-natal week 6 was less obvious, other than cellular senescence, until ∼8-months of age, when premature aging characteristics, such as hair loss and impaired wound healing, were seen. Low APE1 expression in patient cancer tissue also correlated with increased senescence. Our results point to a key role for APE1 in regulating cellular senescence and aging features, with telomere status apparently affecting the outcome.