Promotes DNA Double-strand Break Repair Research Articles

Roles of the SUMO-related enzymes, PIAS1, PIAS4, and RNF4, in DNA double-strand break repair by homologous recombination

Post-translational modification of proteins by small ubiquitin-like modifier (SUMO) is known to be involved in a variety of cellular events. This modification, called SUMOylation, is carried out by the E1 activating enzyme, the E2 conjugating enzyme, and multiple E3 ligases. Previous studies have demonstrated that the SUMO E3 ligases, protein inhibitors of activated STAT 1 (PIAS1) and 4 (PIAS4), and the SUMO-targeted ubiquitin ligase, RING finger protein 4 (RNF4), play important roles in the repair of DNA double-strand breaks (DSBs). However, the mechanism by which these SUMO-related enzymes promote DSB repair is still poorly understood. In the present study, we focused on homologous recombination (HR), the most accurate DSB repair pathway, and aimed to elucidate the mechanism by which PIAS1, PIAS4, and RNF4 promote HR. In γ-ray-irradiated normal human fibroblasts, DSB end resection and RAD51 loading, the two essential steps of HR, were significantly impaired by small interfering RNA (siRNA)-mediated depletion of PIAS1, PIAS4, or RNF4. The recruitment of BRCA1, a major HR factor, to DSB sites was reduced in cells depleted of these SUMO-related enzymes. Consistent with the role of BRCA1 in counteracting the p53-binding protein 1 (53BP1)-mediated resection blockade, 53BP1 depletion rescued the reduced resection and RAD51 loading in the cells depleted of PIAS1, PIAS4, or RNF4. Moreover, Rap1-interacting factor 1 (RIF1), a resection inhibitor downstream of 53BP1, became more abundant at DSBs when PIAS1, PIAS4, RNF4, or BRCA1 was depleted. Importantly, the concomitant depletion of BRCA1 with either one of the SUMO-related enzymes did not further increase RIF1 at DSBs, when compared to single depletion of BRCA1. Collectively, these results suggest that PIAS1, PIAS4, RNF4, and BRCA1 work epistatically to counteract 53BP1/RIF1-mediated resection blockade, thereby promoting resection.

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5'-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.

Nuclear Transglutaminase 2 interacts with topoisomerase II\u237a to promote DNA damage repair in lung cancer cells

BackgroundTo block repairs of DNA damages, especially the DNA double strand break (DSB) repair, can be used to induce cancer cell death. DSB repair depends on a sequential activation of DNA repair factors that may be potentially targeted for clinical cancer therapy. Up to now, many protein components of DSB repair complex remain unclear or poorly characterized. In this study, we discovered that Transglutaminase 2 (TG2) acted as a new component of DSB repair complex.MethodsA bioinformatic analysis was performed to identify DNA damage relative genes from dataset from The Cancer Genome Atlas. Immunofluorescence and confocal microscopy were used to monitor the protein localization and recruitment kinetics. Furthermore, immunoprecipitation and mass spectrometry analysis were performed to determine protein interaction of both full-length and fragments or mutants in distinct domain. In situ lung cancer model was used to study the effects cancer therapy in vivo.ResultsAfter DSB induction, cytoplasmic TG2 was extensively mobilized and translocated into nucleus after phosphorylated at T162 site by DNA-PKcs. Nuclear TG2 quickly accumulated at DSB sites and directly interacting with Topoisomerase IIα (TOPOIIα) with its TGase domain to promote DSB repair. TG2 deficient cells lost capacity of DSB repair and become susceptible to ionizing radiation. Specific inhibition of TG2-TOPOIIα interaction by glucosamine also significantly inhibited DSB repair, which increased sensitivity in lung cancer cells and engrafted lung cancers.ConclusionsThese findings elucidate new mechanism of TG2 in DSB repair trough directly interacting with TOPOIIα, inhibition of which provided potential target for overcoming cancer resistance.

Abstract 2308: The protein interaction landscape of breast cancer

Abstract Cancers have been associated with a diverse array of genomic alterations, many of which are rare with unknown significance. To understand the cellular mechanisms impacted by such alterations in breast invasive carcinoma, we have applied affinity-purification mass spectrometry to delineate comprehensive biophysical interaction networks for 40 frequently altered breast cancer proteins across three human breast cell lines, providing a novel resource of context-specific and shared protein-protein interaction networks in breast cancer cells. These networks interconnect and enrich for common and rare cancer mutations, and are substantially rewired by mutations. Our analysis identifies novel PIK3CA-interacting proteins which repress AKT signaling, and UBE2N emerges as a BRCA1 interactor predictive of clinical response to PARP inhibition. We also show that Spinophilin interacts with and dephosphorylates BRCA1 to promote DNA double-strand break repair. Thus, cancer protein interaction landscapes provide a framework for recognizing oncogenic drivers and drug vulnerabilities. Citation Format: Minkyu Kim, Jisoo Park, Mehdi Bouhaddou, Kyumin Kim, Ajda Rojc, Maya Modak, Margaret Soucheray, Patrick O'Leary, Denise Wolf, Dominique C. Mitchell, Fan Zheng, John D. Gordan, Jean-Philippe Coppé, Danielle L. Swaney, Laura van' t Veer, Alan Ashworth, Trey Ideker, Nevan J. Krogan. The protein interaction landscape of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2308.

Zinc finger protein E4F1 cooperates with PARP-1 and BRG1 to promote DNA double-strand break repair

Zinc finger (ZnF) proteins represent one of the largest families of human proteins, although most remain uncharacterized. Given that numerous ZnF proteins are able to interact with DNA and poly(ADP ribose), there is growing interest in understanding their mechanism of action in the maintenance of genome integrity. We now report that the ZnF protein E4F transcription factor 1 (E4F1) is an actor in DNA repair. Indeed, E4F1 is rapidly recruited, in a poly(ADP ribose) polymerase (PARP)-dependent manner, to DNA breaks and promotes ATR/CHK1 signaling, DNA-end resection, and subsequent homologous recombination. Moreover, we identify E4F1 as a regulator of the ATP-dependent chromatin remodeling SWI/SNF complex in DNA repair. E4F1 binds to the catalytic subunit BRG1/SMARCA4 and together with PARP-1 mediates its recruitment to DNA lesions. We also report that a proportion of human breast cancers show amplification and overexpression of E4F1 or BRG1 that are mutually exclusive with BRCA1/2 alterations. Together, these results reveal a function of E4F1 in the DNA damage response that orchestrates proper signaling and repair of double-strand breaks and document a molecular mechanism for its essential role in maintaining genome integrity and cell survival.

The function of RAD52 N-terminal domain is essential for viability of BRCA-deficient cells

RAD52 is a member of the homologous recombination pathway that is important for survival of BRCA-deficient cells. Inhibition of RAD52 leads to lethality in BRCA-deficient cells. However, the exact mechanism of how RAD52 contributes to viability of BRCA-deficient cells remains unknown. Two major activities of RAD52 were previously identified: DNA or RNA pairing, which includes DNA/RNA annealing and strand exchange, and mediator, which is to assist RAD51 loading on RPA-covered ssDNA. Here, we report that the N-terminal domain (NTD) of RAD52 devoid of the potential mediator function is essential for maintaining viability of BRCA-deficient cells owing to its ability to promote DNA/RNA pairing. We show that RAD52 NTD forms nuclear foci upon DNA damage in BRCA-deficient human cells and promotes DNA double-strand break repair through two pathways: homology-directed repair (HDR) and single-strand annealing (SSA). Furthermore, we show that mutations in the RAD52 NTD that disrupt these activities fail to maintain viability of BRCA-deficient cells.

Relationship among DNA double-strand break (DSB), DSB repair, and transcription prevents genome instability and cancer.

DNA double‐strand break (DSB) is a serious type of DNA damage and is known to trigger multiple responses within cells. In these responses, novel relationships among DSB, DSB repair, and transcription machineries are created. First, transcription is repressed if DSB occurs near or at the transcription site, termed DSB‐induced transcriptional repression, which contributes to DSB repair with the aid of DNA damage‐signaling pathways, ATM‐ or DNA‐PKcs‐signaling pathways. DSB‐induced transcriptional repression is also regulated by transcriptional factors TLP1, NELF, and ENL, as well as chromatin remodeling and organizing factors ZMYND8, CDYL1, PBAF, and cohesin. Second, transcription and RNA promote DSB repair for genome integrity. Transcription factors such as LEDGF, SETD2, and transcriptionally active histone modification, H3K36, facilitate homologous recombination to overcome DSB. At transcriptional active sites, DNA:RNA hybrids, termed R‐loops, which are formed by DSB, are processed by RAD52 and XPG leading to an activation of the homologous recombination pathway. Even in a transcriptionally inactive non‐genic sites, noncoding RNAs that are produced by RNA polymerase II, DICER, and DROSHA, help to recruit DSB repair proteins at the DSB sites. Third, transcriptional activation itself, however, can induce DSB. Transcriptional activation often generates specific DNA structures such as R‐loops and topoisomerase‐induced DSBs, which cause genotoxic stress and may lead to genome instability and consequently to cancer. Thus, transcription and DSB repair machineries interact and cooperate to prevent genome instability and cancer.

Topological DNA-binding of structural maintenance of chromosomes-like RecN promotes DNA double-strand break repair in Escherichia coli

Bacterial RecN, closely related to the structural maintenance of chromosomes (SMC) family of proteins, functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Here we show that the purified Escherichia coli RecN protein topologically loads onto both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) that has a preference for ssDNA. RecN topologically bound to dsDNA slides off the end of linear dsDNA, but this is prevented by RecA nucleoprotein filaments on ssDNA, thereby allowing RecN to translocate to DSBs. Furthermore, we found that, once RecN is recruited onto ssDNA, it can topologically capture a second dsDNA substrate in an ATP-dependent manner, suggesting a role in synapsis. Indeed, RecN stimulates RecA-mediated D-loop formation and subsequent strand exchange activities. Our findings provide mechanistic insights into the recruitment of RecN to DSBs and sister chromatid interactions by RecN, both of which function in RecA-mediated DSB repair.

Abstract 2561: DNA-PKCS deacetylation by SIRT2 promotes DNA double-strand break repair by non-homologous end joining

Abstract DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. How DNA-PK is activated in response to DSBs has remained elusive. Here, we show that the SIRT2 sirtuin deacetylase and tumor suppressor directs the activation of DNA-PK through deacetylation of its catalytic subunit (DNA-PKcs). SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR, which facilitates its interaction with Ku and recruitment to DSBs, thereby leading to DNA-PKcs autophosphorylation and DNA-PK signaling to downstream NHEJ substrates. Moreover, SIRT2 inhibitor sensitizes resistant cancer cells and tumors to IR. Our findings define a mechanism for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ to promote genome integrity and govern IR resistance, which can be exploited for improvements in cancer therapy. Citation Format: PamelaSara E. Head, Nagaraju P. Ganji, Shi-Ya Wang, Duc Duong, Hui Zhang, Waaqo Daddacha, Shuyi Li, Nicholas T. Seyfried, David M. Smalley, Ya Wang, Xingming Deng, William S. Dynan, Bassel El-Rayes, Anthony J. Davis, David S. Yu. DNA-PKCS deacetylation by SIRT2 promotes DNA double-strand break repair by non-homologous end joining [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 2561.

WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks.

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.

SIRT6 Is Responsible for More Efficient DNA Double-Strand Break Repair in Long-Lived Species

DNA repair has been hypothesized to be a longevity determinant, but the evidence for it is based largely on accelerated aging phenotypes of DNA repair mutants. Here, using a panel of 18 rodent species with diverse lifespans, we show that more robust DNA double-strand break (DSB) repair, but not nucleotide excision repair (NER), coevolves with longevity. Evolution of NER, unlike DSB, is shaped primarily by sunlight exposure. We further show that the capacity of the SIRT6 protein to promote DSB repair accounts for a major part of the variation in DSB repair efficacy between short- and long-lived species. We dissected the molecular differences between a weak (mouse) and a strong (beaver) SIRT6 protein and identified five amino acid residues that are fully responsible for their differential activities. Our findings demonstrate that DSB repair and SIRT6 have been optimized during the evolution of longevity, which provides new targets for anti-aging interventions.

The BRUCE-ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development.

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

Radiation-induced astrocyte senescence is rescued by Δ133p53.

Cellular senescence and the senescence-associated secretory phenotype (SASP) may contribute to the development of radiation therapy-associated side effects in the lung and blood vessels by promoting chronic inflammation. In the brain, inflammation contributes to the development of neurologic disease, including Alzheimer's disease. In this study, we investigated the roles of cellular senescence and Δ133p53, an inhibitory isoform of p53, in radiation-induced brain injury. Senescent cell types in irradiated human brain were identified with immunohistochemical labeling of senescence-associated proteins p16INK4A and heterochromatin protein Hp1γ in 13 patient cases, including 7 irradiated samples. To investigate the impact of radiation on astrocytes specifically, primary human astrocytes were irradiated and examined for expression of Δ133p53 and induction of SASP. Lentiviral expression of ∆133p53 was performed to investigate its role in regulating radiation-induced cellular senescence and astrocyte-mediated neuroinflammation. Astrocytes expressing p16INK4A and Hp1γ were identified in all irradiated tissues, were increased in number in irradiated compared with untreated cancer patient tissues, and had higher labeling intensity in irradiated tissues compared with age-matched controls. Human astrocytes irradiated in vitro also experience induction of cellular senescence, have diminished Δ133p53, and adopt a neurotoxic phenotype as demonstrated by increased senescence-associated beta-galactosidase activity, p16INK4A, and interleukin (IL)-6. In human astrocytes, Δ133p53 inhibits radiation-induced senescence, promotes DNA double-strand break repair, and prevents astrocyte-mediated neuroinflammation and neurotoxicity. Restoring expression of the endogenous p53 isoform, ∆133p53, protects astrocytes from radiation-induced senescence, promotes DNA repair, and inhibits astrocyte-mediated neuroinflammation.

Bre1-dependent H2B ubiquitination promotes homologous recombination by stimulating histone eviction at DNA breaks.

Repair of DNA double-strand breaks (DSBs) requires eviction of the histones around DNA breaks to allow the loading of numerous repair and checkpoint proteins. However, the mechanism and regulation of this process remain poorly understood. Here, we show that histone H2B ubiquitination (uH2B) promotes histone eviction at DSBs independent of resection or ATP-dependent chromatin remodelers. Cells lacking uH2B or its E3 ubiquitin ligase Bre1 exhibit hyper-resection due to the loss of H3K79 methylation that recruits Rad9, a known negative regulator of resection. Unexpectedly, despite excessive single-strand DNA being produced, bre1Δ cells show defective RPA and Rad51 recruitment and impaired repair by homologous recombination and response to DNA damage. The HR defect in bre1Δ cells correlates with impaired histone loss at DSBs and can be largely rescued by depletion of CAF-1, a histone chaperone depositing histones H3-H4. Overexpression of Rad51 stimulates histone eviction and partially suppresses the recombination defects of bre1Δ mutant. Thus, we propose that Bre1 mediated-uH2B promotes DSB repair through facilitating histone eviction and subsequent loading of repair proteins.

Local activation of mammalian separase in interphase promotes double-strand break repair and prevents oncogenic transformation.

Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double-strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology-directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1, and Mms21 represent the corresponding kinase, methyltransferase, and SUMO ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation.

Abstract A12: Characterization of the mitronic tsmiR miR6855-3p as a DNA damage repair regulator in the SLUG-high metastatic breast cancer cells

Abstract The precursor of the human microRNA hsa-miR-6855-3p is a 5-tailed mitronic pre-miRNA that is coded by intron #13 of the deubiquitinase USP20 gene. Our preliminary data suggest that the biosynthesis of this miRNA is upregulated by oxidative stress in the SLUG-high metastatic breast cancer cells, promoting DNA double-strand break repair (DSBR) through the indirect enhancement of BRCA2 gene expression. We found that miR-6855-3p favors BRCA2 biosynthesis through the enhancement of the level of an alternately translated form of the BRCA2 gene enhancer protein SPRDX5. We hypothesize that BRCA2 gene silencer acts as a scaffolding to co-recruit SLUG and SPRDX5 and promote SPRDX5-mediated oxidative inactivation of the BRCA2 negative regulator protein SLUG. Here, we present evidence that (a) the expression of miR-6855-3p is essential for the expression of BRCA2 in the SLUG-high metastatic breast cancer cells; (b) knockdown of miR-6855-3p affects DSBR in these cells; and (c) the miR-6855-3p knocked-down cells are susceptible to the synthetic lethality by the PARP inhibitors. Our study reveals a novel mechanism of regulation of BRCA2 gene expression in the aggressive breast cancer cells, offering an alternative strategy to chemically intervene in the progression of hard-to-cure, highly aggressive SLUG-high breast cancer. We propose that the inhibition of miR-6855-3p will induce BRCA2 deficiency in the BLBC cells, thus increasing their susceptibility to standard chemotherapy as well as to PARP inhibitors as has been described for other BRCA2-deficient cells. Supported in part by DOD-CDMRP IDEA Expansion Grant #BC103645 and NIH/NCI grant 1R21CA181920-01 to GC and 1U54RR026140 to SM. Note: This abstract was not presented at the conference. Citation Format: Marshall JD Ellison, Smita Misra, Gautam Chaudhuri. Characterization of the mitronic tsmiR miR6855-3p as a DNA damage repair regulator in the SLUG-high metastatic breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A12.

FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange

FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNAdouble-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.

Somatic role of SYCE2: an insulator that dissociates HP1α from H3K9me3 and potentiates DNA repair.

The synaptonemal complex is a proteinaceous structure essential for meiotic recombination, and its components have been assumed to play a role exclusively in the germ line. However, SYCE2, a component constituting the synaptonemal complex, is expressed at varying levels in somatic cells. Considering its potent protein-binding activities, it may be possible that SYCE2 plays a somatic role by affecting nuclear functions. Here, we show that SYCE2 constitutively insulates HP1α from trimethylated histone H3 lysine 9 (H3K9me3) to promote DNA double-strand break repair. Unlike other HP1α-binding proteins, which use the canonical PXVXL motifs for their bindings, SYCE2 interacts with the chromoshadow domain of HP1α through its N-terminal hydrophobic sequence. SYCE2 reduces HP1α-H3K9me3 binding without affecting H3K9me3 levels and potentiates ataxia telangiectasia mutated-mediated double-strand break repair activity even in the absence of exogenous DNA damage. Such a somatic role of SYCE2 is ubiquitously observed even if its expression levels are low. These findings suggest that SYCE2 plays a somatic role in the link between the nuclear microenvironment and the DNA damage response potentials as a scaffold of HP1α localization.

NELF‐E and CDYL1: Two novel players for switching off transcription at DNA damage sites

Transcription activity is rapidly and transiently paused in response to DNA double‐strand breaks (DSBs) to eliminate production of abnormal transcripts and to avoid deleterious collisions between transcription and repair machineries. Little is known about the mechanisms that ensure transcription repression following DNA damage. Here we describe two novel DNA‐damage responsive proteins, regulated by PARP1 activity, that foster DSB‐induced transcription silencing and promote DSB repair. First, using I‐SceI and CRISPR‐Cas9 endonucleases, we showed that the negative elongation factor member E (NELF‐E), subunit of the NELF complex, is preferentially recruited to DSBs induced upstream transcriptionally active rather than inactive genes. The preferential recruitment of NELF‐E to DNA breakage sites is dependent on the presence of RNA polymerase II [1]. Second, we showed that the chromodomain Y‐like (CDYL1) protein is rapidly recruited to damaged euchromatic regions. In addition, CDYL1 promotes the recruitment of EZH2 methyltransferase, stimulates local increase of the repressive methyl mark H3K27me3 and promotes transcription silencing at DSB sites. Furthermore, the “traffic‐light reporter” system revealed that CDYL1 mainly promotes homology‐directed repair of DSBs. Consequently, CDYL1 knockout cells display synthetic lethality with the chemotherapeutic agent, cisplatin [2]. In the meeting, I will also discuss unpublished data implicating CDYL1 in regulating histone crotonylation at DNA damage sites. Altogether, our findings identify NELF‐E and CDYL1 as novel components that play an essential role in the cellular response to DNA damage.Support or Funding InformationThis work was supported by grants from the Israel Science Foundation (ISF) (grant no. 2021242), the Israel Cancer association (grant no. 2019404), the Binational Science Foundation (grant no. 2023065), the Israel Cancer Research Fund (ICRF) (grant no. 2021762), Volkswagen Foundation (grant no. 2020594). Enas R. Abu‐Zhaiya and Samah W. Awwad are supported by the Council for Higher Education 19 fellowship for outstanding minority MSc and PhD students, respectively. N. Ayoub is supported by the Neubauer Family Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

XRCC3 contributes to temozolomide resistance of glioblastoma cells by promoting DNA double-strand break repair

Glioblastoma is the most frequent and aggressive form of high-grade malignant glioma. Due to the dismal prognosis faced by patients suffering from this disease, there is a need for identifying new targets that might improve therapy. The aim of this study was to determine the contribution of the DNA double-strand break (DSB) repair protein X-ray repair cross-complementing 3 (XRCC3) to the resistance of glioma cells to the chemotherapeutic drug temozolomide. Analysis of a publicly available database, E-GEOD-4290, showed that gliomas overexpress XRCC3 (NM_005432) compared to normal brain tissue. Using an isogenic glioma cell system, in which XRCC3 was downregulated by interference RNA, we demonstrate that XRCC3 protects glioma cells against temozolomide-induced reproductive cell death, apoptosis and cell cycle inhibition. Furthermore, XRCC3 knockdown significantly reduced the rate of repair of DSBs following TMZ treatment, which results in increased drug sensitivity. This study confirms the importance of homologous recombination in the resistance of glioma cells to the methylating drug temozolomide and adds XRCC3 to the list of homology-directed DNA repair proteins as possible targets for therapeutic intervention.