ZNF281 Contributes to the DNA Damage Response by Controlling the Expression of XRCC2 and XRCC4

ORIGINAL ARTICLE ZNF281 contributes to the DNA damage response by controlling the expression of XRCC2 and XRCC4

M Pieraccioli1,4, S Nicolai1,4, A Antonov2, J Somers2, M Malewicz2, G Melino1,2 and G Raschellà1,3

ZNF281 is a zinc-finger factor involved in the control of cellular stemness and epithelial–mesenchymal transition (EMT). Here, we report that ZNF281 expression increased after genotoxic stress caused by DNA-damaging drugs. Comet assays demonstrated that DNA repair was delayed in cells silenced for the expression of ZNF281 and treated with etoposide. Furthermore, the expression of 10 DNA damage response genes was downregulated in cells treated with etoposide and silenced for ZNF281. In line with this finding, XRCC2 and XRCC4, two genes that take part in homologous recombination and non-homologous end joining, respectively, were transcriptionally activated by ZNF281 through a DNA-binding-dependent mechanism, as demonstrated by luciferase assays and Chromatin crosslinking ImmunoPrecipitation experiments. c-Myc, which also binds to the promoters of XRCC2 and XRCC4, was unable to promote their transcription or to modify ZNF281 activity. Of interest, bioinformatic analysis of 1971 breast cancer patients disclosed a significant correlation between the expression of ZNF281 and that of XRCC2. In summary, our data highlight, for the first time, the involvement of ZNF281 in the cellular response to genotoxic stress through the control exercised on the expression of genes that act in different repair mechanisms.

Oncogene (2016) 35, 2592–2601; doi:10.1038/onc.2015.320; published online 24 August 2015

INTRODUCTION stemness, ZNF281 is a significant player of the EMT-regulating ZNF281 (also known as ZBP-99 or ZNP-99) is a 99 kDa zinc-finger transcriptional network, and, therefore, this gene may have a transcriptional regulator that binds to GC-rich regions located in crucial role in controlling migration, invasion, stemness and the promoters of a variety of genes,1 among which ornithine metastasis in tumors. decarboxylase was the first target identified.2 More recently, Eukaryotic organisms have evolved complex DNA damage ZNF281 was recognized as a transcriptional repressor of Nanog, a responses to detect DNA lesions, signal their presence and 7 relevant gene for cellular stemness.3 Furthermore, the autoinhi- organize their repair. Nevertheless, if DNA lesions are too severe bitory role that Nanog exerts on its own transcription appears to to be repaired, mechanisms of cellular demise are activated to 8 be ZNF281-dependent.4 limit damage. Cells defective in these mechanisms generally The transcription of ZNF281 is controlled by SOX4, which is display increased sensitivity towards DNA-damaging agents and expressed in many human malignancies where it affects key growth become inclined to develop a variety of diseases, among which factors, developmental pathways and influences cancer progression.5 are several types of cancer.7 DNA lesions are repaired by a A ChIP-Seq analysis demonstrated that SOX4 binds to DNA sequence of catalytic events mediated by multiple proteins,9 sequences in the proximity of ZNF281 gene,5 suggesting that the which, in many instances, require a finely tuned expression control positive regulation exerted by SOX4 on ZNF281 could occur through by transcription factors.10,11 A central issue in this field, which direct transcriptional control. Conversely, ZNF281 is posttranscrip- remains to be understood in greater detail, is the identification of tionally repressed by miR34a through a p53-dependent the controllers of the expression of DNA damage response mechanism.1 Of note, the ZNF281 protein is phosphorylated by proteins. ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia- We show here that ZNF281 is induced upon treatment with mutated- and Rad3-related (ATR) kinases upon DNA damage.6 DNA damage-inducing drugs in cancer cell lines, normal A step towards understanding the biological function of keratinocytes and in mouse skin in vivo and that silencing of ZNF281 in tumors has been made by a comprehensive study of ZNF281 perturbs the execution of DNA repair, as measured by ZNF281 role in epithelial–mesenchymal transition (EMT).1 Snail, an Comet assays. Furthermore, we demonstrate that ZNF281 acts in EMT inducer, promotes the expression of ZNF281, which in turn promoting the transcription of XRCC2 and XRCC4, two highly activates the transcription of Snail.1 ZNF281 itself induces EMT and relevant DNA damage response genes. In addition, ZNF281 works controls the expression of several EMT-associated genes. Notably, as a c-Myc co-factor to stimulate the expression of nucleolin and c-Myc-induced EMT is dependent on ZNF281 expression.1 Taken cyclin B1. Our data strongly suggest a role of ZNF281 in the DNA together, these results show that beside being a controller of damage response and highlight the flexibility of this protein in

1Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata', Rome, Italy; 2Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK and 3Radiation Biology and Human Health Unit, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA) Research Center Casaccia, Rome, Italy. Correspondence: Dr G Raschellà, Radiation Biology and Human Health Unit, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA) Research Center Casaccia, Via Anguillarese, 301, Rome 00123, Lazio, Italy or Professor G Melino, Department of Experimental Medicine and Surgery, University of Rome ‘Tor Vergata’, Via Montpellier, 1, Rome 00133, Italy. E-mail: [email protected] or [email protected] 4These authors contributed equally to this work. Received 18 May 2015; revised 2 July 2015; accepted 6 July 2015; published online 24 August 2015 ZNF281 in DNA damage response M Pieraccioli et al 2593 controlling the expression of its targets by itself or as a co-factor of genes in cells treated with etoposide (25 μM, 24 h) after silencing other regulators. ZNF281 by specific siRNA. The silencing efficiency (480%) was assessed at the transcript and protein level (Figures 3a and b). The array analysis showed that the expression of 10 genes was RESULTS significantly (⩾40%) downregulated in etoposide-treated cells ZNF281 expression increases after DNA damage and ZNF281 after ZNF281 silencing compared with cells treated with scramble silencing perturbs DNA repair in Comet assays siRNA (Figure 3c). Downregulated genes are involved in ataxia The current knowledge on ZNF281 would predict that this gene telangiectasia-mutated/ataxia telangiectasia-mutated- and Rad3- should be reduced in steady-state protein expression level upon related signaling (FANCD2, RAD9A), DSB repair (BLM, XRCC2, DNA damage. Indeed, as the expression of ZNF281 is repressed by XRCC3), cell cycle (MDC1) and other DNA damage responses p53 through its target miR34a in SW480 colon cancer cells and in (FANCA, FANCG, GADD45A, GADD45G) (see the complete list of MiaPaCa2 pancreatic cancer cells,1 we confirmed in HEK293 cells the genes in the array in Supplementary Figure S3). These genes that miR34a binds to a site in the 3′-untranslated region of the were significantly repressed after DNA damage in cells treated ZNF281 mRNA causing its repression in a luciferase assay with etoposide for 24 h (Figures 3d–f) in independent (Supplementary Figure S1A). Accordingly, ZNF281 was down- RT–qPCR (quantitative reverse transcription PCR) experiments. regulated upon transfection of miR34a at the transcript At earlier time points (3 and 6 h), the same genes were (Supplementary Figures S1B and C) and protein level significantly downregulated, with the exception of GADD45A (Supplementary Figure S1D). We therefore set out to verify a and GADD45G at 6 h of treatment (Supplementary Figure S4). potential downregulation of ZNF281 during DNA damage. Taken together, these data suggest that ZNF281 controls the As the activation of p53 is implicated in many cellular responses expression of genes involved in several DNA damage responses. including stress caused by DNA-damaging agents and DNA 12 repair, we analyzed the expression of ZNF281 upon treatment ZNF281 controls its target genes as an autonomous transcription with DNA-damaging agents that cause double- or single-strand factor or as a co-factor of other regulators breaks (DSB or SSB), activate p53 response and induce histone In silico analysis of the promoters of all genes whose expression H2A.X phosphorylation (γH2A.X). Unexpectedly, ZNF281 increased – was downregulated by ZNF281 silencing detected one or more at the RNA (Figures 1a and d) and protein level (Figures 1b e) after potential binding sites for ZNF281 (GC-rich sequences) in regions etoposide (25 μM), doxorubicin (1 μM) and camptothecin (1 μM) − fi – from 2000 to +500 nt from the transcription start site (TSS) (not treatment (24 h) in p53-pro cient U2OS (Figures 1a c) and shown). In particular, in silico analysis of the promoter of XRCC2 HCT-116 cells (data not shown), as well as in p53-deficient – highlighted two potential ZNF281 binding sites in the proximity of H1299 cancer cells (Figures 1d f). To test whether upregulation of the E-box bound by c-Myc at short distance from the TSS ZNF281 was part of the response to DNA-damaging agents also in (Figure 4a). c-Myc protein has been implicated in proliferation, cell normal cells, we used human primary keratinocytes treated with μ μ cycle and apoptosis control, as well as in the response to DNA etoposide (100 and 200 M) and doxorubicin (1 M). Both damage.13 Accordingly, c-Myc has been shown to bind to the compounds induced an increase in ZNF281 protein expression promoter of several DNA damage response genes including that paralleled that of p53 (Figures 1g and h). We also evaluated XRCC2.13 These findings suggested that XRCC2 could be a the expression of ZNF281 in vivo in the skin of newborn CD1 mice μ transcriptional target of both ZNF281 and c-Myc. To broaden after subcutaneous injection of 50 l of etoposide at different the study on the possible dual control mediated by c-Myc and concentrations (0.736, 1.472, 2.945 and 5.890 mg/kg). Western blot ZNF281, we therefore included in our analysis three additional analysis detected an increase of ZNF281 expression in the skin of genes (not present in PCR array) that shared with XRCC2 a similar mice injected with 1.472, 2.945 and 5.890 mg/kg etoposide promoter organization. XRCC4 gene,14 a close relative of XRCC2, compared with controls (injected with solvent) (Figure 1i). Further, shows a potential ZNF281 binding site close to an E-box bound by we analyzed the course of ZNF281 expression in U2OS cells 13 μ μ c-Myc in its promoter (Figure 4a). As in the case of XRCC2, it is treated with etoposide (25 M) and doxorubicin (1 M). The unknown whether c-Myc binding to XRCC4 promoter is increase of ZNF281 protein started after 3 h and continued till transcriptionally relevant. Cyclin B1 is a regulatory protein 24 h of etoposide treatment (Supplementary Figures S2A and B). fi involved in mitosis; it complexes with p34(cdc2) to form the Signi cant increase was detected after 1 h up to 24 h with maturation-promoting factor.15 Nucleolin, a nucleolar multitask doxorubicin (Supplementary Figures S2C and D). phosphoprotein, (i) acts in the synthesis and maturation of To understand whether ZNF281 could affect the execution of ribosomes,16 (ii) possesses histone chaperone activity,17 DNA repair, we run Comet assays in HCT-116 cells silenced for (iii) interacts with RAD50 and is recruited to DSBs in an MRE11– ZNF281 expression by small interfering RNA (siRNA) and treated – 18 μ NBS1 RAD50 complex-dependent manner. Cyclin B1 and with etoposide (50 M) for 1 h. Tail moment measurement of cells nucleolin are known transcriptional targets of c-Myc19,20 and at different times of recovery (3, 6 and 18 h) indicated that DNA fi show potential ZNF281 binding sites close to c-Myc E-box repair was signi cantly delayed after 3 h of recovery in cells (Figure 4a). To assess whether ZNF281 alone or in association silenced for ZNF281 expression compared with controls with c-Myc was able to control the transcription of XRCC2, XRCC4, (Figures 2a and b). Of note, tail moment of ZNF281-silenced cells cyclin B1 and nucleolin, we cloned in pGL3 reporter vectors the returned to normal after 18 h of recovery (Figure 2a). The extent of promoter regions of these four genes containing ZNF281 and ZNF281 silencing in Comet experiments was evaluated by western c-Myc binding sites. Luciferase assays were performed in HEK293 blot analysis (Figure 2c). and U2OS cells by co-transfection of the reporter vectors with Altogether, these data suggest that upregulation of ZNF281 expression vectors for c-Myc and ZNF281. Results highlighted protein could be part of the DNA damage response evoked by transcriptional activation of XRCC2 and XRCC4 promoters by both DSB- and SSB-inducing drugs. ZNF281, whereas no significant increase in transcription was caused by c-Myc (Figures 4b and c). Furthermore, there was not an ZNF281 modulates the expression of DNA damage response additive/synergistic effect in transcriptional activation by co- genes transfecting ZNF281 and c-Myc (Figures 4b and c). Nucleolin and To test whether ZNF281 could affect the expression of genes cyclin B1 promoters were slightly activated by c-Myc as expected, involved in the cellular DNA damage response, we used a PCR whereas there was not a significant effect by ZNF281 alone array system to detect the expression of 84 DNA damage signaling (Figures 4d and e). Notably, when ZNF281 and c-Myc were

Figure 1. ZNF281 expression increases after genotoxic stress. Real-time PCR to detect ZNF281 and p53 expression in U2OS (a) and H1299 (d) cells treated with etoposide (Eto), doxorubicin (Doxo) and camptothecin (Cpt) at the indicated concentrations for 24 h. β-Actin was used to normalize. WB analyses for ZNF281, histone γH2A.X and p53 in U2OS (b) and H1299 (e) cells treated as for real-time experiments. β-Actin was used as a loading control. (c and f) Densitometric analyses of the blots in (b)and(e). (g) WB analyses of ZNF281, histone γH2A.X and p53 expression in human primary keratinocytes (HEKn) after Eto and Doxo treatments at the indicated concentrations for 24 h. (h) Densitometric analyses of the blots in (g). (i) WB analyses of Zfp281, histone γH2A.X and p53 expression in the skin of CD1 newborn mice after subcutaneous injection of Eto at the indicated concentrations for 24 h. Experiments were repeated three times for cancer cell lines and two times for HEKn cells and CD1 mice. Statistical signiﬁcance was calculated by two-tailed Student’s t-test. **P ⩽ 0.01. ND, not detected.

co-transfected, nucleolin and cyclin B1 transcription increased at by siRNA to c-Myc but not by siRNA to ZNF281 (Figures 5a–d). In significantly higher levels compared with those induced by c-Myc brief, the combined c-Myc and ZNF281 silencing produced an alone (Figures 4d and e). These data suggest that ZNF281 acts as additive effect on cyclin B1 and nucleolin downregulation autonomous transcription factor in controlling the expression of (Figures 5a–d). XRCC2 and XRCC4, whereas it works in association with c-Myc to augment c-Myc-dependent transcription of cyclin B1 and nucleo- ZNF281 binds to the promoters of XRCC2, XRCC4, nucleolin and lin. To further confirm the regulatory role of ZNF281 alone or in cyclin B1 association with c-Myc, we silenced ZNF281 and c-Myc by siRNA in We analyzed whether ZNF281 affects the transcription of XRCC2, p53-proficient HCT-116 and p53-deficient H1299 cells and XRCC4, nucleolin and cyclin B1 by binding to their promoters. To measured XRCC2, XRCC4, cyclin B1 and nucleolin expression by this aim, we carried out Chromatin crosslinking ImmunoPrecipita- western blot (WB) analysis. Results indicated that silencing ZNF281 tion (ChIP) analyses in H1299 cells. We detected endogenous significantly decreased XRCC2 and XRCC4, whereas siRNA to c-Myc ZNF281 binding to XRCC2, XRCC4, nucleolin and cyclin B1 had negligible effects on the same genes (Figures 5a–d). promoters at positions where potential ZNF281 sites were Conversely, cyclin B1 and nucleolin expression was downregulated detected by in silico analysis (Figure 6a). A known ZNF281 target

Figure 2. Silencing of ZNF281 delays the execution of DNA repair. (a) neutral Comet assays were carried out in HCT-116 colon cancer cells exposed to 50 μM etoposide (Eto) for 1 h, washed two times in phosphate-buffered saline (PBS) and allowed to recover in complete medium (no Eto) for the indicated times. Mean tail moment was calculated from three independent experiments. (b) Representative images of the Comet assay of cells after 3 h of recovery. (c) The efﬁciency of ZNF281 silencing was evaluated in HCT-116 cells used in Comet assays by western blotting. Statistical signiﬁcance was calculated by two-tailed Student’s t-test. *P ⩽ 0.05.

Figure 3. Silencing of ZNF281 causes downregulation of DNA damage response genes in cells treated with etoposide. Control of ZNF281 silencing efficiency at the transcript (a) and protein (b) levels in U2OS cells treated with etoposide (25 μM) for 24 h. ZNF281 silencing started 24 h before etoposide treatment. (c) Array results indicating the relative expression levels of 10 genes that were downregulated in cells treated with siRNA to ZNF281 (siZNF281) compared with scramble siRNA (scr). (d–f) Representative validation experiment in U2OS cells treated as above: control of ZNF281 silencing efficiency at transcript (d) and protein (e) level. (f) Real-time PCR of the 10 genes indicated in (c). Experiments were repeated three times. Statistical significance was calculated by Student’s t-test. *P ⩽ 0.05 and **P ⩽ 0.01.

(Axin2) and a genomic region lacking potential ZNF281 binding when the ZNF281 binding site was mutated (Mut1) (Figure 7d). sites (16q22) were used as positive and negative controls, These data suggest that ZNF281 binding to XRCC2 promoter respectively1 (Figures 6b and c). depends on the presence of a speciﬁc ZNF281 binding site, To understand whether ZNF281 binding was required for its whereas binding to the cyclin B1 promoter is indirect and possibly regulatory activity, we generated mutants of the ZNF281 potential occurs through interaction with c-Myc. binding sites in the XRCC2 and cyclin B1 promoters. Luciferase assays carried out in HEK293 cells by co-transfecting c-Myc and ZNF281 with reporter vectors, in which the mutated promoters Expression of ZNF281 and XRCC2 is positively correlated in breast (schematized in Figures 7a and c) were cloned, demonstrated that cancer patients deletion of the ZNF281 binding site (Δ1) abolished activation of To expand the translational meaning of our results using a XRCC2 promoter (Figure 7b). Conversely, ZNF281 continued to bioinformatics approach, we used the METABRIC data set21 activate cyclin B1 transcription in association with c-Myc even covering 1971 breast cancer samples of various subtypes. This

Figure 4. Effects of ZNF281 on the transcription of XRCC2, XRCC4, nucleolin and cyclin B1. (a) Schemes of XRCC2, XRCC4, nucleolin and cyclin B1 promoters. Arrowheads indicate the regions cloned in pGL3-basic reporter vector. Luciferase assays carried out in HEK293 cells co-transfecting ZNF281 and/or c-Myc expression vectors with reporter vectors for XRCC2 (b), XRCC4 (c), nucleolin (d) and cyclin B1 (e). Experiments were repeated three times in triplicate. Statistical signiﬁcance was calculated by Student’s t-test. *P ⩽ 0.05 and **P ⩽ 0.01. NS, not signiﬁcant; EV, empty vector.

would provide an additional independent evidence that the observed in p53-proficient and -deficient tumor cells, as well as in expression of XRCC2 is non-randomly correlated with expression normal primary keratinocytes and in mouse skin in vivo. This result levels of ZNF281. The observed correlation between ZNF281 indicates that although we confirmed the ability of miR34a to (probe id: 'ILMN_1683127') and XRCC2 (probe id: 'ILMN_2204909') downregulate ZNF281, and that p53 is indeed able to down- is 0.36 (Figure 8). We also computed the correlation between regulate the expression of ZNF281 via its posttranscriptional ZNF281 and all other probes on the chip (total of 36 156 probes). repression by miR34a,1 this mechanism does not occur during From this distribution, we can figure out that the correlation value DNA damage. Consequently, a dominant, p53-independent 0.36 belongs to the top 1 percentile: only 226 probes on the chip mechanism causes the increase of ZNF281 following DNA have similar or better correlation with ZNF281. The P-value of damage. correlation estimated using this distribution is 0.0064. This A further indication that the increase of ZNF281 expression after suggests that the expression of XRCC2 is non-randomly correlated DNA damage could have functional implications in DNA repair with expression levels of ZNF281 in the METABRIC data set. was given by the Comet assays, which highlighted a significant, albeit transient, delay in DNA repair. A role for ZNF281 in the cellular response to treatment with DNA-damaging drugs was DISCUSSION further suggested by the downregulation of 10 DNA repair genes We report an unexpected increase of ZNF281 expression after in ZNF281-silenced cells treated with etoposide. Among these genotoxic stress by DNA damage-inducing drugs. This finding was genes, ZNF281 promoted the transcription of XRCC2, a paralog of

Figure 5. Effects of ZNF281 and c-Myc silencing on the expression of XRCC2, XRCC4, nucleolin and cyclin B1. Western blot analyses in HCT-116 (a) and H1299 (c) cells treated with siRNA for ZNF281 and for c-Myc as indicated. (b and d) Densitometric analyses of the blots in (a) and (c). Experiments were repeated two times. Statistical significance of the differences is referred to the control samples transfected with the scramble siRNA (first column on the left in each graph), unless otherwise indicated. Statistical significance was calculated by Student’s t-test. *P ⩽ 0.05 and **P ⩽ 0.01. NS, not significant.

RAD51,22 by binding to speciﬁc DNA sequences in the proximal Nevertheless, in other instances, it has been observed that region of XRCC2 promoter. c-Myc suppression of DSB repair and V(D)J recombination possibly Cells exposed to genotoxic insults, such as ionizing radiations occurs through the inhibition of non-homologous end joining.33 and DNA-damaging drugs, activate a signaling cascade to repair Of interest, proteomic analysis demonstrated the physical inter- the damaged DNA.23 Among the players of this complex and action between c-Myc and ZNF281.34 Thus, we explored the coordinated response, several transcription regulators take part in possibility that c-Myc could modulate the transcription of XRCC2 setting the stage for the DNA repair factors.24 Binding of different alone or in association with ZNF281. Functional assays and loss-of- transcription factors to the same promoter can cause synergic or function experiments failed to show any variation in XRCC2 opposite effect on transcription.25 In the promoter region of the transcription/expression caused by c-Myc. Furthermore, coexpres- XRCC2 gene, an E-box for c-Myc binding is located close to the sion of c-Myc and ZNF281 did not cause any c-Myc-dependent sequence recognized by ZNF281. c-Myc is a basic helix–loop–helix variation in XRCC2 transcription. Similar results were obtained with leucine zipper transcription factor that binds in complex with XRCC4, another DNA damage response gene,14 for which we could MAX26 to promoters/enhancers containing the E-box motifs demonstrate ZNF281-binding-dependent transcription activation, CACGTG or CACATG to induce gene transcription.27 c-Myc is but no activation by c-Myc despite the presence of an E-box in its involved in the control of a wide spectrum of functions including promoter. proliferation and cell cycle, differentiation, sensitization to The number of potential c-Myc targets in the human genome is apoptotic stimuli and genomic instability.28,29 In many solid extremely high as inferred by ChIP-Seq analyses that highlighted tumors30 and leukemias,31 c-Myc expression is deregulated by a c-Myc binding to regions preferentially located between − 1 and variety of mechanisms including gene ampliﬁcation, insertional +1 kb from the TSSs of the putative target genes.35,36 Never- mutations or chromosomal translocation of the myc gene.32 theless, our data suggest that, at least in the cellular models used Extensive experimental evidence highlights the involvement of in this study, c-Myc is not a transcriptional activator of XRCC2 and c-Myc in DNA repair. Chromatin immunoprecipitation showed that XRCC4 despite the presence of c-Myc binding sites in the c-Myc associates with several DSB repair gene promoters.13 promoter of these genes. Induction of c-Myc in G0–G1 and S–G2–M cells resulted in the To study other genes whose promoters had ZNF281 and c-Myc increase of Rad51 expression.13 In addition, c-Myc is required for binding sites, we analyzed cyclin B1 and nucleolin, which are both p53-induced apoptosis in response to DNA damage.28 known targets of c-Myc.19,20 ZNF281 alone was unable to activate

© 2016 Macmillan Publishers Limited Oncogene (2016) 2592 – 2601 ZNF281 in DNA damage response M Pieraccioli et al 2598 transcription from these promoters. Conversely, ZNF281 operated Mutant analysis of XRCC2 and cyclin B1 promoters, on which as a co-factor of c-Myc signiﬁcantly increasing the c-Myc-dependent ZNF281 acted as an independent factor or a co-factor of c-Myc, transcription of cyclin B1 and nucleolin. Of interest, it has been respectively, demonstrated that mutation of the ZNF281 binding recently demonstrated that nucleolin is involved in DNA repair site in the XRCC2 promoter abolished transcription activation by through its recruitment to sites of DNA breaks via binding to RAD50 ZNF281. On the contrary, when ZNF281 binding site was mutated of the MRE11–NBS1–RAD50 complex, where it removes histone in the cyclin B1 promoter, the additive effect of c-Myc and ZNF281 proteins H2A and H2B from the nucleosome at the break site.18

Figure 6. ZNF281 binds to the promoters of XRCC2, XRCC4, nucleolin and cyclin B1. (a, left) Schemes of XRCC2, XRCC4, nucleolin and cyclin B1 promoters. Arrows define the position of the primers used for PCR. (a, right) Results of the ChIP experiments carried out Figure 8. Positive correlation between the expression of ZNF281 and by immunoprecipitating chromatin with a specific antibody to XRCC2 in breast cancer. Expression profiles of ZNF281 and XRCC2 in ZNF281 in H1299 cells. Rabbit immunoglobulins (IgG) were used as samples of the METABRIC data set. Observed correlation between negative controls of IP. Inp, Input; − , negative control of PCR (no ZNF281 (probe id 'ILMN_1683127') and XRCC2 (probe id DNA). Axin2 and 16q22 were used as positive (b) and negative 'ILMN_2204909') is 0.36. The P-value of correlation estimated using (c) controls of ZNF281 ChIP, respectively. Experiments were repeated this distribution is 0.0064. See Material and methods for details of two times. the statistical calculation used.

Figure 7. Effects of the mutation of ZNF281 sites in the promoters of XRCC2 and cyclin B1. Schemes of XRCC2 (a) and cyclin B1 (c) promoters in the WT and mutant forms (Δ1 and Mut1). Crosses indicate the mutations. Arrowheads define the regions cloned in the reporter vectors. Luciferase assays carried out in HEK293 cells co-transfecting ZNF281 and/or c-Myc expression vectors with reporter vectors for XRCC2 (b) and cyclin B1 (d). Experiments were repeated three times in triplicate. Statistical significance was calculated by Student’s t-test. *P ⩽ 0.05 and **P ⩽ 0.01. NS, not significant; EV, empty vector.

Oncogene (2016) 2592 – 2601 © 2016 Macmillan Publishers Limited ZNF281 in DNA damage response M Pieraccioli et al 2599 was fully maintained. The latter result suggests that ZNF281 Supplementary Table S1. The sequence of all plasmids generated for this binding to the cyclin B1 promoter is indirect, possibly through its paper was checked by dideoxysequencing. interaction with c-Myc.34 Altogether, these findings suggest the ability of ZNF281 protein to work as a transcriptional regulator Transfection and silencing alone as for XRCC2 and XRCC4 or in association with c-Myc as for Transfections were carried out using Effectene Transfection Reagent cyclin B1 and nucleolin. (Qiagen, Venlo, The Netherlands) according to the manufacturer’s Our results expand the knowledge on the role of ZNF281. instruction. For silencing, cells were transfected with a pool of siRNA Indeed, ZNF281 was originally recognized as a factor contributing (20 nM) against human ZNF281 or c-Myc mRNA or with a pool of non- to the regulation of stemness through its inhibitory activity of targeting siRNA as a control (Dharmacon, Lafayette, CO, USA), using Nanog expression.3 More recently, ZNF281 was demonstrated to Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, ’ be a central regulator of EMT.1 The function of ZNF281 in USA) according to the manufacturer s instructions. controlling the transcription of XRCC2 and nucleolin implicated in homologous recombination18,37 and XRCC4 involved in Comet assay non-homologous end joining14 strongly suggests that ZNF281 Neutral Comet assays were carried out in HCT-116 colon cancer cells may have a role in modulating the cellular response to DNA- silenced for ZN281 by siRNA and treated with etoposide (50 μM for 1 h) as 39,40 damaging agents. In addition, bioinformatic analysis performed described, with some modifications. Briefly, slides with agarose- on a large data set of ~ 2000 breast cancer patients disclosed a embedded cells were immersed 1 h at 4 °C in lysis solution (30 mM EDTA, fi 0.5% sodium dodecyl sulfate, pH 8.0). Slides were then incubated in cold signi cant positive correlation between the expression of ZNF281 TBE (Tris-borate-EDTA) for 2 h before electrophoresis. Electrophoresis was and that of XRCC2, further hinting to a link between these carried out at 4 °C for 25 min at 8 mA. Slides were rinsed in water, two genes. transferred to ice-cold methanol for 10 min and air-dried at room In summary, these data demonstrate, for the first time, that temperature. Immediately before scoring, slides were stained with 12 µg/ml ZNF281 controls the expression of DNA damage response genes. ethidium bromide (Sigma) and examined at × 20 magnification, with an Dysfunction of ZNF281 may have detrimental consequences for Olympus fluorescence microscope (Olympus, Center Valley, PA, USA). Cells the maintenance of genomic stability, thus contributing to human were analyzed with a computerized image analysis system (Delta Sistemi, pathologies. Additional studies are needed to evaluate the Rome, Italy). One hundred cells were scored for each experimental point prognostic value of the correlation between ZNF281 and XRCC2 from two different slides. Tail moment measurements were performed at 3, 6 and 18 h of recovery. Three independent experiments were carried out. in breast cancer and other malignancies. Statistical significance (P) was analyzed by two-tailed Student’s t-test.

MATERIALS AND METHODS Array analysis Cell lines U2OS cells transfected with siRNA as indicated after 24 h were treated with etoposide, and after additional 24 h were harvested and array analysis was H1299 lung cancer, HCT-116 colon cancer, U2OS bone cancer and HEK293 performed using RT2 Profiler PCR Array (cat. no.: PAHS-029Z; Qiagen) kidney cell lines (ATCC, Manassas, VA, USA) were cultured in Dulbecco's according to the manufacturer’s instructions in Applied Biosystems 7500 modified Eagle's medium/high glucose with L-glutamine (Lonza, Basel, Fast Real-Time PCR Systems (Applied Biosystems, Life Technologies, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum Monza, Italy). (Gibco, Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin, 0.1 mg/ml streptomycin at 37 °C in 5% CO2 and 100% humidity. Neonatal human epidermal keratinocytes were cultured in EpiLife (Gibco, Life Technologies) Functional assays supplemented by human keratinocyte growth supplement (Gibco, Life Cells were co-transfected with wild-type and mutant reporters and the Technologies) at 37 °C in 5% CO2 and 100% humidity. expression vectors pcDNA3- -ZNF281FLAG (for ZNF281) or pcDNA3-c-Myc Cells were treated with etoposide (Sigma, Saint Louis, MO, USA), (for c-Myc) using Effectene Transfection Reagent (Qiagen). Luciferase doxorubicin (Sigma) and camptothecin (Sigma) for the indicated times and assays were carried out using the Dual Luciferase Reporter assay concentrations. Control cells were incubated in the presence of solvent (Promega). Each experimental point was analyzed in triplicate in three (dimethyl sulfoxide for etoposide and camptothecin and water for independent experiments. Statistical significance (P) was calculated by doxorubicin). Cell lines used in this study were tested for mycoplasma two-tailed Student’s t-test. contamination using MycoAlert Mycoplasma Detection Kit (Lonza) every 3 months. Chromatin immunoprecipitation A total of 1.5 × 106 cells were seeded and transfected with FLAG-tagged Animal experiments vectors for ZNF281 and c-Myc (where indicated). Crosslinking was Male and female newborn CD1 mice were subcutaneously injected with performed 24 h posttransfection. The ChIP procedure was carried out 50 μl of etoposide at different concentrations (0.736, 1.472, 2.945 and using a Commercial Kit MAGnify Chromatin Immunoprecipitation System 5.890 mg/kg). Etoposide stock solution (50 mM in dimethyl sulfoxide) was (Invitrogen) and ChIP-grade antibody anti-Zfp281 ab101318 (Abcam, diluted in phosphate-buffered saline at the appropriate concentrations. Cambridge, UK). Primers used for amplification of the regions including Control animals were injected with 0.4% dimethyl sulfoxide in phosphate- the potential ZNF281 binding sites in the promoter regions of XRCC2, buffered saline. Animals were killed after 24 h from injection. Skin was XRCC4, cyclin B1, nucleolin, Axin2 (positive control) and 16q22 (negative removed and proteins were extracted as described.38 Five animals for each control) are listed in the Supplementary Table S1. PCR was carried out experimental group were used. Experiments were repeated two times. using Q5 High-Fidelity DNA Polymerase (New England Biolabs Inc., Ipswich, Experiments were approved by the Ethical Committee of the Tor Vergata MA, USA) according to the manufacturer’s instructions. University. RNA extraction and real-time qPCR analyses Cloning RNA was extracted from 106 cells using the mirVana RNA Isolation Kit Promoter regions of XRCC2, XRCC4, cyclin B1 and nucleolin were amplified (Ambion Inc., Life Technologies, Monza, Italy) or the RNeasy Mini Kit 50 using the primers listed in Supplementary Table S1 and cloned in pGL3- (Qiagen) according to the manufacturer’s instructions. Reverse transcrip- basic vector (Promega, Madison, WI, USA) by standard cloning procedures. tion was carried out by using GoScript Reverse Transcription System Deletion mutant of pGL3-prom-XRCC2 were generated by amplification of (Promega) according to the manufacturer’s instructions. qPCR was carried the pGL3-prom-XRCC2 plasmid used as a template using the primers listed out in an Applied Biosystems 7500 and 7500 Fast Real-Time PCR Systems in Supplementary Table S1. Site-directed mutants of pGL3-prom-cyclin B1 (Applied Biosystems, Life Technologies) using GoTaq qPCR Master Mix were generated using the QuikChange® II XL Site-Directed Mutagenesis Kit (Promega) and primers were listed in Supplementary Table S1. The miRNA (Stratagene, La Jolla, CA). Primers for site-directed mutagenesis are listed in reverse transcription was carried out by using 10 ng of RNA using TaqMan

© 2016 Macmillan Publishers Limited Oncogene (2016) 2592 – 2601 ZNF281 in DNA damage response M Pieraccioli et al 2600 MicroRNA Reverse Transcription Kit (Applied Biosystems, Life Technologies) 4 Fidalgo M, Faiola F, Pereira CF, Ding J, Saunders A, Gingold J et al. Zfp281 and TaqMan MicroRNA Assay Kits for miR34a and miR203, using U6 as an mediates Nanog autorepression through recruitment of the NuRD complex and endogenous control (Applied Biosystems, Life Technologies), and Fast inhibits somatic cell reprogramming. Proc Natl Acad Sci USA 2012; 109: Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, Life 16202–16207. Technologies). Applied Biosystems software was used to calculate relative 5 Scharer CD, McCabe CD, Ali-Seyed M, Berger MF, Bulyk ML, Moreno CS. Genome- RNA amounts. In real-time qPCR experiments, each sample was run in wide promoter analysis of the SOX4 transcriptional network in prostate triplicate in three independent experiments. Statistical significance (P) was cancer cells. Cancer Res 2009; 69: 709–717. calculated by two-tailed Student’s t-test. 6 Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER III, Hurov KE, Luo J et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to 316 – Western blot analysis DNA damage. Science 2007; :1160 1166. 7 Jackson SP, Bartek J. The DNA-damage response in human biology and disease. 41 Western blot analyses were carried out as described. The following Nature 2009; 461: 1071–1078. β antibodies were used: anti-Zfp281 (Abcam; cat. no.: ab101318), anti- -actin 8 Bartek J, Lukas J. DNA damage checkpoints: from initiation to recovery or (AC-15) (Sigma; cat. no.: A5441), anti-XRCC2 (Abcam; cat. no.: ab97316), adaptation. Curr Opin Cell Biol 2007; 19: 238–245. anti-XRCC4 (Abcam; cat. no.: ab145), anti-cyclin B1 (BD Biosciences, 9 Kakarougkas A, Jeggo PA. DNA DSB repair pathway choice: an orchestrated Franklin Lakes, NJ, USA; cat. no.: 610220), anti-nucleolin (Abcam; cat. no.: handover mechanism. Br J Radiol 2014; 87: 20130685. ab22758), anti-p53 (DO-1) (Santa Cruz Biotechnology, Dallas, TX, USA; cat. 10 Biswas AK, Johnson DG. Transcriptional and nontranscriptional functions of E2F1 number: sc-126), anti-phospho-histone H2A.X (Ser139) (Merck Millipore, in response to DNA damage. Cancer Res 2012; 72:13–17. Billerica, MA, USA; cat. number: 05-636), anti-mouse-HRP-conjugated 11 Sabatel H, Pirlot C, Piette J, Habraken Y. Importance of PIKKs in NF-kappaB acti- (Bio-Rad, Hercules, CA, USA; cat. no.: 170-5047) and anti-rabbit-HRP- vation by genotoxic stress. Biochem Pharmacol 2011; 82:1371–1383. conjugated (Bio-Rad; cat. no.: 170-6515). Band intensities were analyzed by 12 Rufini A, Tucci P, Celardo I, Melino G. Senescence and aging: the critical roles of the Image J software (ImageJ, Bethesda, MD, USA). p53. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)