Defective DNA Repair and Chromatin Organization in Patients with Quiescent Systemic Lupus Erythematosus Vassilis L

Home , Base excision repair, DDB1, H2AFX, RAD51, TP53BP1

Souliotis et al. Arthritis Research & Therapy (2016) 18:182 DOI 10.1186/s13075-016-1081-3

RESEARCH ARTICLE Open Access Defective DNA repair and chromatin organization in patients with quiescent systemic lupus erythematosus Vassilis L. Souliotis1,2*, Konstantinos Vougas3, Vassilis G. Gorgoulis3,4 and Petros P. Sfikakis2

Abstract Background: Excessive autoantibody production characterizing systemic lupus erythematosus (SLE) occurs irrespective of the disease’s clinical status and is linked to increased lymphocyte apoptosis. Herein, we tested the hypothesis that defective DNA damage repair contributes to increased apoptosis in SLE. Methods: We evaluated nucleotide excision repair at the N-ras locus, DNA double-strand breaks repair and apoptosis rates in peripheral blood mononuclear cells from anti-dsDNA autoantibody-positive patients (six with quiescent disease and six with proliferative nephritis) and matched healthy controls following ex vivo treatment with melphalan. Chromatin organization and expression levels of DNA repair- and apoptosis-associated genes were also studied in quiescent SLE. Results: Defective nucleotide excision repair and DNA double-strand breaks repair were found in SLE, with lupus nephritis patients showing higher DNA damage levels than those with quiescent disease. Melphalan-induced apoptosis rates were higher in SLE than control cells and correlated inversely with DNA repair efficiency. Chromatin at the N-ras locus was more condensed in SLE than controls, while treatment with the histone deacetylase inhibitor vorinostat resulted in hyperacetylation of histone H4, chromatin decondensation, amelioration of DNA repair efficiency and decreased apoptosis. Accordingly, genes involved in DNA damage repair and signaling pathways, such as DDB1, ERCC2, XPA, XPC, MRE11A, RAD50, PARP1, MLH1, MLH3, and ATM were significantly underexpressed in SLE versus controls, whereas PPP1R15A, BARD1 and BBC3 genes implicated in apoptosis were significantly overexpressed. Conclusions: Epigenetically regulated functional abnormalities of DNA repair machinery occur in SLE, regardless of clinical disease activity, and may promote lymphocyte apoptosis. Approaches to correct these abnormalities may be of therapeutic value in SLE. Keywords: Systemic lupus erythematosus, Nucleotide excision repair, DNA double-strand breaks repair, Chromatin organization, Apoptosis, Histone deacetylase inhibitor

Background presentation and autoantibody production is considered The prototypic systemic autoimmune disease, systemic a central pathogenetic mechanism of SLE [2]. Significant lupus erythematosus (SLE), is characterized by loss of diversity in organ involvement, disease activity, severity immunological tolerance to self-nuclear antigens and ab- and responsiveness to treatment are common [3]. Of normal T- and B-cell responses, which are attributed to note, the production of myriads of autoantibodies in genetic and epigenetic causes [1]. Excessive autoantigen human SLE may precede the onset of clinical disease by many years and is facilitated by accumulation of dead * Correspondence: [email protected] cells due to dysregulated apoptosis [1, 4]. 4 1Institute of Biology, Medicinal Chemistry and Biotechnology, National Every day, each of our cells is faced with at least 10 Hellenic Research Foundation, Athens, Greece DNA lesions and their efficient repair is a mandatory 2Joint Rheumatology Program and First Department of Propaedeutic Internal Medicine, National and Kapodistrian University of Athens Medical School, biological process to preserve cell survival and function Athens, Greece [5]. The genomic integrity is maintained by a network of Full list of author information is available at the end of the article

© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 2 of 12

DNA damage response and repair pathways, a compre- Rheumatology criteria for SLE and had high serum titers hensive signaling process that determines the cell's abil- of anti-double-stranded DNA autoantibodies at the time ity to repair DNA damage or to undergo mutation(s) or of sampling were studied. Six patients (all women, aged apoptosis [6]. The wide diversity of DNA lesion types between 24 and 45 years, with disease duration ranging necessitates multiple, largely distinct DNA repair mecha- between 2 and 11 years) were selected on the basis of nisms. For example, nucleotide excision repair executes the presence of clinically quiescent disease for at least 6 the repair of DNA lesions induced by UV light, polycyc- months prior to sampling (SLE disease activity index lic aromatic hydrocarbons, heavy metals such as plat- Selena modification score of 6 or less, in the absence of inum and cadmium, and by other sources that form any lupus-related clinical manifestation) under daily bulky DNA lesions that distort the DNA helix [7]. On therapy with hydroxychloroquine only (n = 3), or in the other hand, for DNA double-strand breaks, which combination with 5 mg or less of prednisolone (n = 3). are introduced by exogenous agents such as ionizing ra- Six other patients (four women, aged between 21 and 59 diation and therapeutic drugs as well as by cellular pro- years, with disease duration ranging between 1 and 15 cesses such as V(D)J recombination, class switch years) were selected on the basis of the presence of pro- recombination, stalled replication forks and reactions liferative glomerulonephritis; their characteristics, dis- that generate reactive oxygen species [8], two principal ease status, and medications have been described in repair mechanisms are used. The first is the homologous detail elsewhere [13]. Twelve apparently healthy individ- recombination, an error-free repair pathway that uses uals, who were antinuclear antibody negative and DNA homology to direct DNA repair, and the second is matched 1:1 for age and gender to the patients, were the nonhomologous end joining, a straightforward reli- studied in parallel. gation of broken DNA ends without any requirement for a template [5]. Defective nucleotide excision repair Cell cultures has been indeed reported in SLE [9–11], whereas the Freshly isolated PBMCs were stimulated into prolifera- majority of B lymphoblastoid cell lines derived from tion using phytohemagglutinin (PHA; 10 μg/ml) for 48 children with SLE gave evidence of inefficient or delayed hours and then treated with 100 μg/ml melphalan for 5 kinetics of DNA double-strand breaks repair [12]. Our min at 37 °C in complete RPMI-1640 medium supple- recent study has shown that circulating mononuclear mented with 10 % fetal bovine serum (FBS), 100 units/ cells from patients with a severe SLE complication, such ml penicillin, 100 μg/ml streptomycin and 2 mmol/l L- as lupus nephritis, are characterized by higher intrinsic glutamine. Cells were subsequently incubated in drug- DNA double-strand breaks accumulation compared to free medium for various times (0–48 h), harvested, and healthy controls, possibly due to increased susceptibility stored at −70 °C. In some experiments, PBMCs were ex- of these cells to develop these DNA lesions and/or an posed to 2.5 μg/ml of vorinostat (suberoylanilide hydro- impaired repair [13]. xamic acid; Millipore Sigma, St. Louis, MO, USA) for 24 Herein, we considered that increased apoptosis in SLE h [14], or 10 μg/ml a-amanitin (Millipore Sigma) for 6 h could be due, at least in part, to defective mechanisms [15], treated with melphalan as above, harvested and of DNA damage repair, thus providing an additional pu- stored at −70 °C. tative pathogenetic mechanism in systemic autoimmunity. Indeed, we found that nucleotide excision repair and Nucleotide excision repair measurement DNA double-strand breaks repair mechanisms are de- The N-ras gene-specific nucleotide excision repair fective in patients with either clinically quiescent disease was evaluated by the monofunctional binding of the or proliferative lupus nephritis, and strongly correlate drug melphalan to a single site in the DNA molecule with increased apoptosis. Notably, treatment of lupus (monoadducts) at various time points, as described mononuclear cells with the histone deacetylase inhibitor elsewhere [16]. vorinostat resulted in amelioration of DNA repair efficiency and decreased apoptosis. In line with these find- DNA double-strand breaks repair measurement ings, genes involved in DNA repair and signaling For the evaluation of DNA double-strand breaks the im- pathways were found downregulated, whereas genes munofluorescence antigen staining and confocal laser implicated in apoptosis were found upregulated in quies- scanning microscope analysis was used as described pre- cent SLE patients. viously [13]. Briefly, aliquots of 2 × 104 PBMCs were stimulated into proliferation using PHA, treated as Methods described above, adhered to coverslip, fixed and stored Patients at −70 °C until the analysis of γH2AX and Rad51. Cells Peripheral blood mononuclear cells (PBMCs) from 12 were incubated with antibodies against γH2AX (serine- patients (10 women) who fulfilled American College of 139, Cell Signaling, Danvers, MA, USA) or Rad51 (Santa Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 3 of 12

Cruz Biotechnology, Dallas, TX, USA), washed, incu- Venlo, the Netherlands) according to the manufacturer’s bated with fluorescent secondary antibodies (Alexa Fluor instructions and stored at −80 °C until use. For PCR 488 goat anti-mouse IgG; Abcam, Cambridge, UK) and array analysis, the RT2 Profiler™ PCR Array (Qiagen) of images were visualized with a Leica TCS SP-1 confocal 84 genes related to the DNA damage signaling pathway laser scanning microscope (Leica, Wetzlar, Germany). was utilized. Total RNA (200 ng) was reverse transcribed Foci were manually counted in 200 cells/treatment con- using the RT2 First Strand kit following the manufac- dition and results were expressed as the mean γH2AX turer’s instructions. Resulting cDNA was diluted in or Rar51 foci per cell [mean ± standard deviation (SD)] nuclease-free water and added to RT2 SYBR Green from three independent experiments. Fluor qPCR Mastermix (Qiagen), which was subsequently aliquoted to each well of the PCR array for Micrococcal nuclease digestion-based analysis of quantitative PCR. Thermal cycling and fluorescence chromatin condensation detection were performed using a Biorad iQ™5(Bio- Nuclei were isolated from freshly isolated, untreated Rad Laboratories, Hemel Hempstead, UK). Cycling PBMCs (5 × 104) and digested with 1 unit of micrococcal conditions were: 10 min denaturation at 95 °C nuclease (Millipore Sigma) for 0, 1, 2, 5 and 10 min at followed by 40 cycles of 15 sec at 95 °C and 1 min at 37 °C. DNA was purified, separated in 1.5 % agarose gel, 60 °C. Six healthy control samples and six quiescent transferred onto nitrocellulose, and hybridized with the SLE patients samples were run on individual plates appropriate labeled probe [17]. anddatawereanalyzedusingtheRT2ProfilerPCR Array Data Analysis Webportal (http://pcrdataanaly Western blot analysis sis.sabiosciences.com/pcr/arrayanalysis.php). Two inde- Cells were harvested after treatment with various doses pendent experiments were performed for each sample. of vorinostat for 24 hours at 37 °C, rinsed in ice-cold phosphate-buffered saline (PBS), and lysed in buffer con- Systems biology statistical analysis taining 50 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM The transcripts presenting statistically significant differ- EDTA, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 5 μg/ml ential expression (p < 0.05) were analyzed through the benzamidine, 0.5 mM phenylmethylsulfonyl fluoride use of Qiagen’s Ingenuity® Pathway Analysis (IPA®, (PMSF), and 1 % NP-40, followed by SDS-PAGE on Qiagen, Redwood City, CA, USA, www.qiagen.com/in 12 % polyacrylamide gels. Subsequently, the proteins genuity) considering all direct and indirect relationships were transferred to nitrocellulose membranes, which in the Ingenuity Knowledge base obtained only from were incubated for 40 min at room temperature with experimentally verified information. Canonical path- nonfat dry milk (5 %) in 1 × Tris-buffered saline ways underwent enrichment analysis and the corre- (TBS) followed by incubation with specific primary sponding p values were calculated (p values < 0.05 antibodies to nonacetylated histone H4, acetylated his- were considered significant). tone H4, and beta actin (Cell Signaling) overnight at 4 °C. Then, the membranes were washed with 0.3 % bovine Statistical analysis serum albumin in 1 × TBS and incubated with horseradish The efficiency of DNA repair and the induction of apop- peroxidase-conjugated secondary antibodies for 2 h at tosis were compared between groups of individuals using room temperature. After washing with 1 × TBS, the the Wilcoxon rank sum test. Correlations between bound antibody complexes were visualized using the melphalan-induced DNA damage and induction of apop- Western Blotting Detection Kit ECL Plus (Amersham, tosis were assessed by the linear regression analysis. A p Little Chalfont, UK) and XOMAT-AR film (Kodak, value less than 0.05 was considered statistically significant. Rochester, NY, USA). P values indicating statistical significance of the gene expression levels where corrected for multiple comparisons Apoptosis assay (FDR correction; Additional file 1: Table S1) [18]. Cells were treated with various doses of melphalan (0–200 μg/ml) for 5 min, followed by 24 h post- Results incubation time. Then, the Cell Death Detection Nucleotide excision repair and DNA double-strand breaks ELISA-PLUS kit (Roche Diagnostics Corp., Basel, repair are defective in SLE, regardless of disease activity Switzerland) was used to determine apoptosis accord- level ing to the protocol provided by the manufacturer. In the present study, two major DNA repair mechanisms were evaluated in SLE patients, i.e., nucleotide excision RT2 Profiler™ PCR Array repair and DNA double-strand breaks repair. Based on Total RNA was extracted from freshly isolated untreated our previous experience, we used the genotoxic drug PBMCs using the Qiagen RNeasy Mini Kit (Qiagen, melphalan as a tool to induce (a) DNA lesions such as Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 4 of 12

the monofunctional binding of this drug to a single site in the DNA molecule (monoadducts), which are almost exclusively repaired by nucleotide excision repair [16], and (b) DNA double-strand breaks, which are indirectly formed as a consequence of melphalan-induced oxidative stress [19] and as intermediates in the repair of melphalan-induced DNA damage [20, 21]. First, the efficiency of the nucleotide excision repair was measured in six patients with quiescent disease and their matched controls. Since we have previously shown that the efficiency of nucleotide excision repair at the N- ras locus strongly correlates with melphalan-induced apoptosis in PBMCs from healthy controls [22], the repair kinetics of N-ras-specific monoadducts were followed for up to 48 h using Southern blot analysis (Fig. 1a). In all subjects, similar formation of monoadducts was found at the end of the 5-min melphalan treatment. Thereafter, their levels were decreased, with the removal capacity being higher in healthy controls than in quiescent SLE patients and lowest in patients with active proliferative nephritis (Fig. 1b). In accordance to these data, monoadducts burden (expressed as the area under the curve for DNA adducts during the whole experiment), a parameter strongly correlating with the cytotoxicity of genotoxic agents [23], was significantly higher in quiescent SLE patients compared to healthy controls; maximal values were observed in patients with active proliferative nephritis (Fig. 1c). Then, to study the formation and repair of DNA double-strand breaks, γH2AX foci as a marker of DNA damage and the formation of Rad51 foci as a marker of homologous recombination were determined. In all subjects analyzed, γH2AX foci reached maximal levels within 8 h; thereafter, their levels were decreased, with the removal capacity being higher in healthy controls than in SLE patients (Fig. 2a, b). Increased γH2AX levels were found in lupus nephritis compared to quiescent SLE patients; statistical significance was observed at 2 h and 48 h following melphalan treatment (Fig. 2b). In accordance to these results, higher γH2AX foci burden, expressed as AUC, was observed in quiescent SLE patients compared to healthy controls, whereas patients with active proliferative nephritis showed higher γH2AX burden than quiescent SLE patients (Fig. 2c). The Rad51 response followed the same time course as the γH2Ax Fig. 1 Impaired nucleotide excision repair in SLE patients. response, peaking at 8 h and declining thereafter, with a Representative autoradiograms for the Southern blot analysis healthy controls showing lower Rad51 foci levels com- of melphalan-induced N-ras-specific DNA adducts. 0/0, no treatment. pared to quiescent SLE patients (Fig. 2d-f). The kinetics of monoadducts (b) and total amounts of monoadducts expressed as AUC (c) in quiescent systemic lupus erythematosus (QSLE) and lupus nephritis (LN) patients as well as in 12 healthy controls (HC) are shown. The asterisks indicate significant differences (p < 0.05) Apoptosis rates of quiescent SLE-derived mononuclear between QSLE and LN patients. The experiments shown were based cells correlate inversely with DNA repair efficiencies on a minimum of three independent repeats and the data reported The melphalan-induced apoptosis rates in PBMCs from are the mean ± SD of all subjects analyzed quiescent SLE patients and their matched healthy Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 5 of 12

Fig. 2 Impaired double-strand breaks repair in quiescent SLE patients. a Typical images showing the γH2AX staining at different time points after treatment of PBMCs from one representative healthy control (HC) and one quiescent systemic lupus erythematosus (QSLE) patient after treatment with melphalan. Cell nuclei labeled with DAPI, γH2AX staining, as well as merged DAPI and γH2AX staining are shown. 0/0 no treatment. Formation of γH2AX foci (b) and total amounts of γH2AX foci expressed as AUC (c) after treatment of PBMCs from QSLE and lupus nephritis (LN) patients as well as from 12 HC with melphalan. The asterisks indicate significant differences (p < 0.05) between QSLE and LN patients. NSD no significant difference. d Typical images showing the Rad51 staining at different time points after treatment of PBMCs from one representative HC and one QSLE patient with melphalan. Cell nuclei labeled with DAPI, Rad51 staining, as well as merged DAPI and Rad51 staining are shown. 0/0 no treatment. Formation of Rad51 foci (e) and total amounts of Rad51 foci expressed as AUC (f) after treatment of PBMCs from QSLE patients and their matched HC with melphalan. The experiments shown were based on a minimum of three independent repeats and the data reported are the mean ± SD of all subjects analyzed controls were also measured. We found that the lowest by increased apoptotic rates. Interestingly, an inverse concentrations of melphalan required for the induction correlation was observed between the melphalan con- of apoptosis were significantly higher in healthy controls centration required for the induction of apoptosis and than in quiescent SLE patients (Fig. 3a), indicating that the levels of both types of DNA damage examined in the PBMCs from quiescent SLE patients are characterized same quiescent SLE patients (linear regression analysis; Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 6 of 12

Fig. 3 Increased apoptosis rates of quiescent SLE-derived mononuclear cells correlate with defective DNA repair efficiencies. a The induction of apoptosis after treatment of PBMCs from quiescent systemic lupus erythematosus (SLE) patients and their matched healthy controls (HC) with melphalan. Correlation between the melphalan doses required for the induction of apoptosis and the drug-induced monoadducts (b) or double- strand breaks levels (c) in the same QSLE patients or healthy controls. d Correlation between individual efficiencies of nucleotide excision repair versus double-strand breaks repair (expressed as AUC) in all SLE patients (six with quiescent disease and six with proliferative nephritis) and 12 healthy controls. The experiments shown were based on a minimum of three independent repeats and the data reported are the mean ± SD of all the subjects analyzed monoadducts: r2 = 0.831 for quiescent SLE patients and gene locus following treatment of PBMCs from quies- 0.920 for their matched controls, Fig. 3b; double-strand cent SLE with vorinostat, a histone deacetylase inhibitor breaks: r2 = 0.878 for quiescent SLE patients and 0.907 which has been approved by the US Food and Drug for healthy controls, Fig. 3c). Notably, a strong correl- Administration for the treatment of refractory cutaneous ation was found between the individual efficiencies of T-cell lymphoma [24]. First, we determined the degree the two DNA repair mechanisms under study in all sub- of H4 histone acetylation in PBMCs from quiescent SLE jects analyzed (linear regression analysis: r2 = 0.767 for patients after treatment with vorinostat using Western SLE patients and 0.567 for healthy controls, Fig. 3d). blot analysis; nonacetylated histone H4 was included as a control for acetylated histone H4. We found that be- Defective chromatin organization and nucleotide excision fore incubation with vorinostat, the levels of acetylated repair in quiescent SLE are partially corrected by histone H4 were low and did not increase remarkably vorinostat with up to 1 μM vorinostat for 24 hours. However, incu- We next analyzed chromatin condensation in the N-ras bation with 2.5 μM vorinostat for 24 h resulted in the gene locus, using micrococcal nuclease digestion of un- maximal accumulation of acetylated histone H4 (Fig. 4d) treated PBMCs from quiescent SLE patients and their associated with chromatin decondensation (Fig. 4c). In matched controls. As shown in Fig. 4a and b, patients line with previous data showing that the rate of repair of are characterized by more condensed chromatin at the UV-induced DNA damage increased following histone N-ras locus than their matched controls. That is, in hyperacetylation by sodium butyrate [25], we found that healthy controls the N-ras gene locus gave rise mostly to vorinostat treatment of PBMCs from quiescent SLE mono- and di-nucleosome structures, with a significant patients resulted in increased efficiency of nucleotide portion in mono-nucleosomes, while the same locus in excision repair (Fig. 4e) and decreased DNA damage quiescent SLE patients gave rise to di-, tri-nucleosomes burden of these cells (Fig. 4f). Conversely, following and higher structures. treatment of PBMCs from quiescent SLE patients Chromatin organization and the efficiency of nucleo- with a-amanitin, an inhibitor of RNA polymerase II, tide excision repair were also evaluated in the N-ras which causes conversion of chromatin into a more Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 7 of 12

condensed form [16], chromatin compaction of the significant involvement (enrichment p value < 0.05) of N-ras gene locus (Additional file 1: Figure S1a, b) several DNA damage response and repair pathways, was accompanied by reduction of the efficiency of including nucleotide excision repair (p = 1.00E-007; nucleotide excision repair and increased DNA damage Additional file 1: Figure S2), homologous recombin- burden (Additional file 1: Figure S1c, d). ation (p = 5.01E-020; Additional file 1: Figure S3), nonhomologous end joining (p = 2.51E-017; Additional Vorinostat ameliorates DNA double-strand breaks repair file 1: Figure S4), base excision repair (p = 2.63E-010; efficiency and decreases apoptosis rates in quiescent SLE Additional file 1: Figure S5), ATM signaling (p = The effects of the chromatin organization modifiers on 1.58E-043; Additional file 1: Figure S6), mismatch the efficiency of double-strand breaks repair and the repair (p = 3.16E-014; Additional file 1: Figure S7), as apoptosis rates were also evaluated. In line with previous well as apoptosis pathway signaling (p = 3.16E-003). data showing that the compaction of chromatin influ- Given that the enrichment analysis was performed on ences the efficiency of the DNA damage response and the differentially expressed genes only, the fact that the sensitivity of cells to DNA double-strand breaks most of the preselected genes were differentially [26], we found that vorinostat treatment of PBMCs expressed enhances the initial working hypothesis. from quiescent SLE patients increased the repair efficiency of double-strand breaks (Fig. 4g), reduced Discussion double-strand breaks burden (Fig. 4h), and suppressed Herein, two major DNA repair mechanisms were evalu- apoptosis rates of these cells (Fig. 4i). Conversely, in ated ex vivo in patients with quiescent SLE and active all subjects, treatment of PBMCs with a-amanitin lupus nephritis. The first mechanism, namely nucleotide resulted in higher double-strand breaks levels and excision repair was found to be less efficient in SLE pa- increased apoptosis rates (Additional file 1: Figure tients compared to healthy controls, whereas patients S1e-g). Under the conditions used in this study, vori- with quiescent disease showed higher repair efficiency nostat or a-amanitin alone did not induce apoptosis than those with active nephritis. Since the specific chro- in PBMCs from all subjects examined (data not matin environment of a DNA lesion is considered an shown). important factor that may affect recognition of lesions and repair rates [16, 27], a possible explanation for the Altered expression of DNA repair- and apoptosis-related lower efficiency of nucleotide excision repair could be genes in quiescent SLE related to a defective chromatin organization in our pa- The expression of genes involved in DNA damage repair tients. Indeed, we found that quiescent SLE patients are and apoptosis pathways was analyzed in untreated characterized by more condensed local chromatin than PBMCs by RT2 Profiler™ PCR Array (Fig. 5a). Of 84 healthy controls, which possibly impedes the access of genes examined, a total of 37 genes representing several DNA repair proteins to sites of DNA damage, thus redu- non-mutually exclusive categories demonstrated at least cing DNA repair capacity [28]. twofold difference in gene expression between healthy Whether a defective chromatin organization in SLE is as- controls and quiescent SLE patients (Additional file 1: sociated with aberrations in histone acetylation processes Tables S2 and S3). Particularly, the downregulated genes remains unclear. In general, histone acetyltransferases and were categorized into nucleotide excision repair (DDB1, deacetylases regulate chromatin accessibility by adding or ERCC2, XPA, XPC), double-strand breaks repair (BLM, removing, respectively, acetyl groups to lysine residues in CHEK1, HUS1, MRE11A, NBN, RAD50, RAD51, RPA1, the N-terminal histone domains [29]. Acetylation neutral- TP53BP1, XRCC2, XRCC6), base excision repair (LIG1, izes the positive charge of histone lysines, weakening elec- PARP1, XRCC1), mismatch repair (MLH1, MLH3) and trostatic DNA-histone interactions and increasing DNA signaling pathways (ATM, RAD1) (Fig. 5b). The expres- accessibility, while deacetylation strengthens DNA-histone sion of the DNA repair XRCC3 gene was also found interactions, thus decreasing DNA accessibility. Regarding decreased in SLE patients. On the other hand, several the patterns of histone modifications in SLE patients, con- overexpressed genes are implicated in the apoptosis troversial results have been reported. For example, Zhou et pathway, including PPP1R15A, CDKN1A, BARD1, al. [30] reported that histone H3 acetylation of the TNFSF7 RAD21, RAD9A, PRKDC, CIB1, BRCA1, ABL1, CHEK2 (CD70) promoter in SLE CD4 + T cells is significantly in- and BBC3 (Fig. 5c). Also, GADD45A, involved in DNA creased, and may contribute to the development of lupus repair, and H2AFX, a DNA damage response gene, were by increasing the expression of the CD70 gene. In addition, overexpressed in lupus PBMCs. in monocytes isolated from SLE patients, Zhang et al. [31] Finally, we performed a systems biology analysis of the identified a group of 179 genes with significantly enriched significantly differentially expressed transcripts on acetylation of H4 promoter compared to controls. Other canonical pathways through IPA®, which revealed data showed significantly increased total histone H4 Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 8 of 12

Fig. 4 Effects of vorinostat treatment of quiescent SLE-derived cells. Autoradiograms showing micrococcal nuclease sensitivity of the N-ras locus in untreated PBMCs from six healthy controls (HC) (a) and six quiescent systemic lupus erythematosus (QSLE) patients (b), as well as in PBMCs from the same QSLE patients after treatment with 2.5 μM vorinostat for 24 h (c). Symbols M, D and T represent the positions of nucleosome monomer, dimer and trimer respectively. d Western blot analysis of PBMCs from a representative quiescent SLE patient treated with various doses of vorinostat for 24 h. Nonacetylated histone H4 was used as a control for acetylated histone H4, and beta actin as a loading control. The kinetics of monoadducts (e) and γH2AX foci (g) as well as total amounts of monoadducts (f) and γH2AX foci expressed as AUC (h) after treatment of PBMCs from QSLE patients and their matched healthy controls with melphalan in the presence or not of vorinostat. The asterisks indicate significant differences (p < 0.05) between QSLE and LN patients. i The induction of apoptosis after treatment of PBMCs from the same QSLE patients and their matched HC with melphalan in the presence or not of vorinostat. The experiments shown were based on a minimum of three independent repeats and the data reported are the mean ± SD of all the subjects analyzed. acetylation in SLE monocytes [32]. In contrast to the with the DNA double-strand breaks burden being higher above, and in line with our findings, global hypoacetylation in patients with active nephritis compared to those with of histone H3 and H4 was observed in active lupus CD4+ quiescent SLE. These results are in line with recent data T cells compared with controls [33]. Notably, the degree of showing that both T cells and monocytes in SLE patients histone H3 acetylation correlated inversely with SLE dis- have increased sensitivity to acquire double-strand ease activity [33]. breaks following oxidative stress, which associates with The second major mechanism studied herein, namely disease activity [34]. Although we did not address the DNA double-strand breaks repair, was also found less mechanistic basis for the decreased double-strand breaks efficient in SLE patients compared to healthy controls, repair efficiency of our patients, previous studies have Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 9 of 12

Fig. 5 Expression of 84 genes involved in DNA damage response pathways. a Hierarchical clustergram of the 84 genes under study in untreated PBMCs from six quiescent systemic lupus erythematosus (QSLE) patients versus their matched healthy controls (HC). b, c Genes demonstrating at least twofold difference in the transcription activity between QSLE patients and their matched HC. Gene acronyms are explained in Additional file 1: Tables S2 and S3 shown that many repair proteins implicated in this Following our assumption that the more condensed repair mechanism are targets for autoantibodies. For chromatin in our quiescent lupus patients is linked to example, autoantibodies against DNA repair proteins in- deficient DNA repair, we considered that chemical mod- volved in nonhomologous end joining, including the two ifiers of chromatin organization would affect repair effi- subunits of Ku protein, the poly(ADP-ribose) polymerase ciency. Indeed, we found that treatment of PBMCs from 1 (PARP-1) protein, the catalytic subunit of the DNA- quiescent SLE patients with the histone deacetylase dependent protein kinase (DNA-PK) and the DNA ligase inhibitor vorinostat resulted in hyperacetylation of his- IV were all identified in patients with systemic auto- tone H4, chromatin decondensation, amelioration of immune diseases [35–38]. Moreover, a cell-penetrating both nucleotide excision repair and DNA double-strand lupus autoantibody, 3E10, was found to preferentially bind breaks repair efficiencies, and decreased apoptosis. Pre- DNA single-strand tails and inhibit key steps in DNA vious studies have shown that the in vivo administration single- and double-strand breaks repair process [39]. of several histone deacetylase inhibitors including vori- Interestingly, we found that the individual capacities of nostat, corrected the site-specific hypoacetylation states both nucleotide excision repair and double-strand breaks on H3 and H4 lupus mouse models with marked repair mechanisms strongly correlated with the corre- improvement of disease phenotype [40–42]. sponding PBMCs apoptosis rates, in either SLE patients or In line with the data presented above, gene expression healthy controls, supporting our hypothesis that accumu- results showed that many genes involved in DNA dam- lation of DNA damage contributes to increased apoptosis. age repair and signaling pathways were downregulated in quiescent SLE patients, whereas, several genes Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 10 of 12

implicated in the apoptosis pathway were upregulated. since total PBMCs were used in the present study, we To the best of our knowledge, this is the first study in cannot exclude the possibility that differences in cell SLE patients showing underexpression of the XPA, XPC, subsets constituents between patients and controls HUS1, MRE11A, NBN, RAD50, RAD51, RPA1, TP53BP1 contribute to some of the differences observed. and ATM genes involved in DNA damage repair and Notably, a recent report provides evidence for defect- signaling pathways, as well as overexpression of ive repair of oxidative stress-induced double-strand PPP1R15A, BARD1, BBC3, BRCA1 genes involved in breaks in all peripheral blood mononuclear cell sub- the apoptosis pathway. Moreover, in accordance to sets (CD4+ T cells, CD8+ T cells and monocytes) de- previous studies, we found downregulation of the ex- rived from SLE patients compared with those from cision repair cross-complementing ERCC2 and ERCC5 healthy controls [34]. Also, previous studies have also genes [43, 44], the X-ray repair cross-complementing shown that both lymphocytes and neutrophils from XRCC6gene[44],theDDB1genecodingforthe SLE patients display increased DNA damage and, damage-specific DNA binding protein 1 [45], the additionally, may demonstrate defective repair of oxi- Bloom syndrome RecQ helicase-like gene (BLM) [46], dative DNA damage [9, 10]. the XRCC1 gene [11] and the poly (ADP-ribose) polymerase 1 gene (PARP-1) [47]. Also, we found that Conclusions SLE cells showed higher GADD45A mRNA levels as Our findings support the hypothesis that increased compared with healthy controls [48]. Overexpression apoptosis in SLE could be due, at least in part, to defect- of several other apoptosis-related genes has also been ive DNA repair and chromatin organization (Fig. 6). reported in PBMCs from SLE patients [49]. Based on our findings we propose that SLE patients, for Regarding the pathogenetic mechanisms of auto- reasons currently unknown, are characterized by con- immunity, an important link between DNA damage densed chromatin in certain loci, resulting in lower repair and the activation of the type I interferon repair efficiency of important DNA repair pathways (IFN) characterizing SLE emerges [50, 51]. Indeed, (nucleotide excision repair and DNA double-strand Gunther et al. [52] reported recently that rare vari- breaks repair) and, thus, increased DNA damage bur- antsofthegenomesurveillanceenzymeribonuclease den, which in turn triggers the induction of the apop- H2 gene are associated with systemic autoimmunity totic pathway. Although not tested in vivo, i.e., in and that these variants resulted in impaired ribonu- lupus mouse models, treatment of human SLE- cleotide removal. They also showed that such embed- ded ribonucleotides caused increased UV-induced cyclobutane pyrimidine dimer formation and enhanced type I IFN signaling, thus linking mutations in a DNA repair enzyme with systemic autoimmunity. Furthermore, Härtlova et al. [53] showed that accumulation of spontaneous DNA lesions stimulates type I IFN system via the activation of the STING pathway by self-DNA released into the cytoplasm, implying that the DNA repair network emerges as an important new player in the pathogenesis of autoimmunity. Moreover, a polymorphism in the DNA repair gene RAD51B has been associated with increased risk of SLE [54]. A limitation of the present work is the small sample size of patients with quiescent SLE; increasing the number of patients would increase confidence in the data generated. However, the main conclusion of this study, i.e., that both nucleotide excision repair and DNA double-strand breaks repair were defective in SLE patients (a) was confirmed in both quiescent SLE and in lupus nephritis patients (12 SLE patients versus 12 healthy controls) and (b) is in line with previous data showing that SLE T cells and monocytes have in- Fig. 6 Epigenetically regulated functional abnormalities of DNA creased sensitivity to acquiring DNA double-strand repair machinery may promote lymphocyte apoptosis in SLE: a proposed model (for details see “Conclusions”) breaks in response to oxidative stress [34]. Moreover, Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 11 of 12

derived cells with the histone deacetylase inhibitor PPS: Professor, First Department of Prop. Internal Medicine and vorinostat results in chromatin decondensation, ameli- Rheumatology Unit, Joint Academic Rheumatology Program, National Kapodistrian University of Athens Medical School, Athens, Greece. oration of DNA repair efficiency, and decreased apoptosis. Therefore, further work on the mechanisms of Competing interests the epigenetic imbalance in DNA damage repair may The authors declare that they have no competing interests. reveal potential therapeutic targets in SLE. Consent for publication Additional file Not applicable.

Ethical approval and consent to participate Additional file 1: Table S1. Gene expression levels corrected for The study was approved by the Laikon Hospital ethics committee and all multiple comparisons (FDR correction). Table S2. Genes that were participants provided informed consent according to the Declaration of downregulated in quiescent SLE patients. Table S3. Genes that were Helsinki. upregulated in quiescent SLE patients. Figure S1. Effect of a-amanitin treatment of quiescent SLE-derived cells. Figure S2. Canonical pathways Author details related to DNA damage response and repair from IPA® presenting 1Institute of Biology, Medicinal Chemistry and Biotechnology, National p statistically significant ( < 0.05) enrichment: nucleotide excision repair Hellenic Research Foundation, Athens, Greece. 2Joint Rheumatology Program p ( = 1.00E-007). Figure S3. Canonical pathways related to DNA damage and First Department of Propaedeutic Internal Medicine, National and p response and repair from IPA® presenting statistically significant ( <0.05) Kapodistrian University of Athens Medical School, Athens, Greece. enrichment: DNA double-strand breaks repair by homologous 3Biomedical Research Foundation, Academy of Athens, Athens, Greece. p recombination ( = 5.01E-020). Figure S4. Canonical pathways 4Molecular Carcinogenesis Group, Department of Histology and Embryology, related to DNA damage response and repair from IPA® presenting National and Kapodistrian University of Athens Medical School, Athens, p statistically significant ( < 0.05) enrichment: DNA double-strand Greece. breaks repair by nonhomologous end joining (p =2.51E-017).Figure S5. Canonical pathways related to DNA damage response and repair from IPA® Received: 16 May 2016 Accepted: 21 July 2016 presenting statistically significant (p < 0.05) enrichment: base excision repair (p = 2.63E-010). Figure S6. Canonical pathways related to DNA damage response and repair from IPA® presenting statistically significant (p <0.05) enrichment: ATM signaling (p = 1.58E-043). Figure S7. Canonical pathways References related to DNA damage response and repair from IPA® presenting 1. Tsokos GC. Systemic lupus erythematosus. N Engl J Med. 2011;365:2110–21. statistically significant (p < 0.05) enrichment: mismatch repair (p = 3.16E-014). 2. Nagata S, Hanayama R, Kawane K. Autoimmunity and the clearance of dead (DOC 7236 kb) cells. Cell. 2010;140:619–30. 3. Bertsias GK, Salmon JE, Boumpas DT. Therapeutic opportunities in systemic lupus erythematosus: state of the art and prospects for the new decade. Abbreviations Ann Rheum Dis. 2010;69:1603–11. FBS, fetal bovine serum; IFN, interferon; PBMCs, peripheral blood 4. Arbuckle MR, Mc Clain MT, Rubertone MV, Scofield RH, Dennis GJ, James JA, mononuclear cells; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; et al. Development of autoantibodies before the clinical onset of systemic PMSF, phenylmethylsulfonyl fluoride; SLE, systemic lupus erythematosus; TBS, lupus erythematosus. N Engl J Med. 2003;349:1526–33. Tris-buffered saline 5. Ohnishi T, Mori E, Takahashi A. DNA double-strand breaks: their production, recognition, and repair in eukaryotes. Mutat Res. 2009;669:8–12. Acknowledgements 6. Sirbu BM, Cortez D. DNA damage response: three levels of DNA repair We would like to thank Prof. George C. Tsokos (Division of Rheumatology, regulation. Cold Spring Harb Perspect Biol. 2013;5:a012724. Harvard Medical School) for critical review of the manuscript. 7. Friedberg EC. How nucleotide excision repair protects against cancer. Nat Rev Cancer. 2001;1:22–33. Funding 8. Helleday T, Lo J, van Gent DC, Engelward BP. DNA double-strand break The study was funded by the Special Account for Research Grants of the repair: from mechanistic understanding to cancer treatment. DNA Repair National and Kapodistrian University of Athens, Athens, Greece (ELKE Grant (Amst). 2007;6:923–35. number 974). 9. Bashir S, Harris G, Denman MA, Blake DR, Winyard PG. Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune Availability of supporting data diseases. Ann Rheum Dis. 1993;52:659–66. The datasets supporting the conclusions of this article are available in the 10. McConnell JR, Crockard AD, Cairns AP, Bell AL. Neutrophils from systemic Helios Digital Repository: http://helios-eie.ekt.gr/EIE/handle/10442/15330. lupus erythematosus patients demonstrate increased nuclear DNA damage. Clin Exp Rheumatol. 2002;20:653–60. Authors’ contributions 11. Bassi C, Xavier DJ, Palomino G, Nicolucci P, Soares C, Sakamoto-Hojo E, et al. VLS and PPS directed and coordinated the project, performed data Efficiency of the DNA repair and polymorphisms of the XRCC1, XRCC3 and acquisition and data analysis, and drafted the manuscript. KV and VGG XRCC4 DNA repair genes in systemic lupus erythematosus. Lupus. 2008;17: contributed to data analysis and revised the manuscript. All authors 988–95. read and approved the final manuscript. 12. Davies RC, Pettijohn K, Fike F, Wang J, Nahas SA, Tunuguntla R, et al. Defective DNA double-strand break repair in pediatric systemic lupus Authors’ information erythematosus. Arthritis Rheum. 2012;64:568–78. VLS: Research Associate Professor, Institute of Biology, Medicinal Chemistry 13. Souliotis VL, Sfikakis PP. Increased DNA double-strand breaks and enhanced and Biotechnology (IBMCB), National Hellenic Research Foundation (NHRF), apoptosis in patients with lupus nephritis. Lupus. 2015;24:804–15. 48 Vassileos Constantinou Ave., 11635 Athens, Greece. 14. Munshi A, Tanaka T, Hobbs ML, Tucker SL, Richon VM, Meyn RE. Vorinostat, KV: Staff Research Scientist-Associate Professor Level, Biomedical Research a histone deacetylase inhibitor, enhances the response of human tumor Foundation of the Academy of Athens (BRFAA), 4 Soranou Ephessiou St., cells to ionizing radiation through prolongation of γ-H2AX foci. Mol Cancer 115 27 Athens, Greece. Ther. 2006;5:1967–74. VGG: Professor, Laboratory of Histology-Embryology, Molecular Carcinogenesis 15. Souliotis VL, Dimopoulos MA, Episkopou HG, Kyrtopoulos SA, Sfikakis PP. Group, Medical School National and Kapodistrian University of Athens and Preferential in vivo DNA repair of melphalan-induced damage in human Sciences, University of Manchester, Manchester Academic Health Science genes is greatly affected by the local chromatin structure. DNA Repair Centre, Manchester, UK. (Amst). 2006;5:972–85. Souliotis et al. Arthritis Research & Therapy (2016) 18:182 Page 12 of 12

16. Episkopou H, Kyrtopoulos SA, Sfikakis PP, Fousteri M, Dimopoulos MA, 39. Hansen JE, Chan G, Liu Y, Hegan DC, Dalal S, Dray E, et al. Targeting cancer Mullenders LH, et al. Association between transcriptional activity, local with a lupus autoantibody. Sci Transl Med. 2012;4:157ra142. chromatin structure, and the efficiencies of both subpathways of nucleotide 40. Mishra N, Reilly CM, Brown DR, Ruiz P, Gilkeson GS. Histone deacetylase excision repair of melphalan adducts. Cancer Res. 2009;69:4424–33. inhibitors modulate renal disease in the MRL-lpr/lpr mouse. J Clin Invest. 17. Episkopou H, Kyrtopoulos SA, Sfikakis PP, Dimopoulos MA, Souliotis VL. 2003;111:539–52. The repair of melphalan-induced DNA adducts in the transcribed strand 41. Waibel M, Christiansen AJ, Hibbs ML, Shortt J, Jones SA, Simpson I, et al. of active genes is subject to a strong polarity effect. Mutat Res. 2011; Manipulation of B-cell responses with histone deacetylase inhibitors. Nat 714:78–87. Commun. 2015;6:6838. 18. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical 42. Regna NL, Chafin CB, Hammond SE, Puthiyaveetil AG, Caudell DL, Reilly CM. and powerful approach to multiple testing. J Roy Statist Soc Ser B Class I and II histone deacetylase inhibition by ITF2357 reduces SLE (Methodological). 1995;57:289–300. pathogenesis in vivo. Clin Immunol. 2014;151:29–42. 19. Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer 43. Frangou EA, Bertsias GK, Boumpas DT. Gene expression and regulation in strategy. Nat Rev Drug Discov. 2013;12:931–47. systemic lupus erythematosus. Eur J Clin Invest. 2013;43:1084–96. 20. Simonelli V, Narciso L, Dogliotti E, Fortini P. Base excision repair 44. Lee HM, Sugino H, Aoki C, Nishimoto N. Underexpression of mitochondrial- intermediates are mutagenic in mammalian cells. Nucleic Acids Res. 2005; DNA encoded ATP synthesis-related genes and DNA repair genes in 33:4404–11. systemic lupus erythematosus. Arthritis Res Ther. 2011;13:R63. 21. Deans AJ, West SC. DNA interstrand crosslink repair and cancer. Nat Rev 45. O’Hanlon TP, Rider LG, Gan L, Fannin R, Paules RS, Umbach DM, et al. Cancer. 2011;11:467–80. Gene expression profiles from discordant monozygotic twins suggest 22. Gkotzamanidou M, Terpos E, Bamia C, Kyrtopoulos SA, Sfikakis PP, that molecular pathways are shared among multiple systemic Dimopoulos MA, et al. Progressive changes in chromatin structure and DNA autoimmune diseases. Arthritis Res Ther. 2011;13:R69. damage response signals in bone marrow and peripheral blood during 46. Kaneko H, Fukao T, Kasahara K, Yamada T, Kondo N. Augmented cell death myelomagenesis. Leukemia. 2014;28:1113–21. with Bloom syndrome helicase deficiency. Mol Med Rep. 2011;4:607–9. 23. Hansson J, Lewensohn R, Ringborg U, Nilsson B. Formation and removal of 47. Rapoport MJ, Bloch O, Amit-Vasina M, Yona E, Molad Y. Constitutive DNA cross-links induced by melphalan and nitrogen mustard in relation to abnormal expression of RasGRP-1 isoforms and low expression drug-induced cytotoxicity in human melanoma cells. Cancer Res. 1987;47: of PARP-1 in patients with systemic lupus erythematosus. Lupus. 2631–7. 2011;20:1501–9. 24. Hopfinger G, Nösslinger T, Lang A, Linkesch W, Melchardt T, Weiss L, et al. 48. Li Y, Zhao M, Yin H, Gao F, Wu X, Luo Y, et al. Overexpression of the Lenalidomide in combination with vorinostat and dexamethasone for the growth arrest and DNA damage-induced 45alpha gene contributes to treatment of relapsed/refractory peripheral T cell lymphoma (PTCL): report autoimmunity by promoting DNA demethylation in lupus T cells. – of a phase I/II trial. Ann Hematol. 2014;93:459–62. Arthritis Rheum. 2010;62:1438 47. 25. Smerdon MJ, Lan SY, Calzal RE, Reeve R. Sodium butyrate stimulates DNA 49. Eggleton P, Harries LW, Alberigo G, Wordsworth P, Viner N, Haigh R, et al. repair in UV-irradiated normal and xeroderma pigmentosum human Changes in apoptotic gene expression in lymphocytes from rheumatoid fibroblasts. J Biol Chem. 1982;257:13441–7. arthritis and systemic lupus erythematosus patients compared with healthy – 26. Murga M, Jaco I, Fan Y, Soria R, Martinez-Pastor B, Cuadrado M, et al. Global lymphocytes. J Clin Immunol. 2010;30:649 58. chromatin compaction limits the strength of the DNA damage response. 50. Baccala R, Hoebe K, Kono DH, Beutler B, Theofilopoulos AN. TLR-dependent J Cell Biol. 2007;178:1101–8. and TLR-independent pathways of type I interferon induction in systemic – 27. Feng Z, Hu W, Chasin LA, Tang MS. Effects of genomic context and autoimmunity. Nat Med. 2007;13:543 51. chromatin structure on transcription-coupled and global genomic repair in 51. Pateras IS, Havaki S, Nikitopoulou X, Vougas K, Townsend PA, Panayiotidis mammalian cells. Nucleic Acids Res. 2003;31:5897–906. MI, et al. The DNA damage response and immune signaling alliance: is it 28. Gong F, Kwon Y, Smerdon MJ. Nucleotide excision repair in chromatin and good or bad? Nature decides when and where. Pharmacol Ther. 2015;154: – the right of entry. DNA Repair (Amst). 2005;4(10):884–96. 36 56. 29. Patel DR, Richardson BC. Epigenetic mechanisms in lupus. Curr Opin 52. Gunther C, Kind B, Reijns MA, Berndt N, Martinez-Bueno M, Wolf C, et al. Rheumatol. 2010;22:478–82. Defective removal of ribonucleotides from DNA promotes systemic autoimmunity. J Clin Invest. 2015;125:413–24. 30. Zhou Y, Qiu X, Luo Y, Yuan J, Li Y, Zhong Q, et al. Histone modifications 53. Härtlova A, Erttmann SF, Raffi FA, Schmalz AM, Resch U, Anugula S, et al. and methyl-CpG-binding domain protein levels at the TNFSF7 (CD70) DNA damage primes the type I interferon system via the cytosolic DNA promoter in SLE CD4 + T cells. Lupus. 2011;20:1365–71. sensor STING to promote anti-microbial innate immunity. Immunity. 2015; 31. Zhang Z, Song L, Maurer K, Petri MA, Sullivan KE. Global H4 acetylation 42:332–43. analysis by ChIP-chip in systemic lupus erythematosus monocytes. Genes 54. Bentham J, Morris DL, Cunninghame Graham DS, Pinder CL, Tombleson P, Immun. 2010;11:124–33. Behrens TW, et al. Genetic association analyses implicate aberrant regulation 32. Leung YT, Shi L, Maurer K, Song L, Zhang Z, Petri M, et al. Interferon of innate and adaptive immunity genes in the pathogenesis of systemic regulatory factor 1 and histone H4 acetylation in systemic lupus lupus erythematosus. Nat Genet. 2015;47:1457–64. erythematosus. Epigenetics. 2015;10:191–9. 33. Hu N, Qiu X, Luo Y, Yuan J, Li Y, Lei W, et al. Abnormal histone modification patterns in lupus CD4+ T-cells. J Rheumatol. 2008;35:804–10. 34. Namas R, Renauer P, Ognenovski M, Tsou PS, Sawalha AH. Histone H2AX phosphorylation as a measure of DNA double-strand breaks and a marker of environmental stress and disease activity in lupus. Lupus Sci Med. 2016;3: e000148. Submit your next manuscript to BioMed Central 35. Takeda Y, Dynan WS. Autoantibodies against DNA double strand break and we will help you at every step: repair proteins. Front Biosci. 2001;6:1412–22. 36. Reeves WH. Antibodies to the p70/p80 (Ku) antigens in systemic lupus • We accept pre-submission inquiries – erythematosus. Rheum Dis Clin North Am. 1992;18:391 414. • Our selector tool helps you to ﬁnd the most relevant journal 37. Muller S, Briand JP, Barakat S, Lagueux J, Poirier GG, De Murcia G, et al. • We provide round the clock customer support Autoantibodies reacting with poly(ADP-ribose) and with a zinc-finger functional domain of poly(ADP-ribose) polymerase involved in the • Convenient online submission recognition of damaged DNA. Clin Immunol Immunopathol. 1994;73: • Thorough peer review 187–96. • Inclusion in PubMed and all major indexing services 38. Lee KJ, Dong X, Wang J, Takeda Y, Dynan WS. Identification of human autoantibodies to the DNA ligase IV/XRCC4 complex and mapping of an • Maximum visibility for your research autoimmune epitope to a potential regulatory region. J Immunol. 2002; 169:3413–21. Submit your manuscript at www.biomedcentral.com/submit