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DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.
The DNA-binding and chromatin-regulating DEK oncogene is expressed across multicellular eukaryotes and is highly conserved in mammals. Strong sequence conservation of the ψSAP-SAP and C-terminal DNA binding domains, as well as the lack of any known DEK paralogs, suggest stringent evolutionary pressure on this gene1,2. However, despite extensive biochemical, cellular, and clinical investigations into the DEK protein, molecular functions that explain this selective pressure remain poorly understood, as does the frequent over-expression of DEK in human cancers3,4,5,6,7,8.
In cultured cells, DEK has functions in chromatin remodeling9,10,11, DNA replication9,12, and mRNA splicing13. Depending on the experimental system chosen, DEK loss attenuates distinct oncogenic phenotypes such as proliferation14,15,16, survival17,18, and chemoresistance14,17,19. Dek knockout mice are viable and resistant to macroscopic tumor development8,20. Biochemically, there are no known enzymatic functions associated with DEK, but the protein self-multimerizes, induces positive supercoils in DNA through the ψ-SAP-SAP and C-terminal DNA binding domains, and preferentially binds cruciform DNA structures21,22,23,24. DEK also has roles in maintaining chromatin architecture9,11 and interacts with histones10,25 and chromatin modifiers11. Since the discovery of the DEK gene as a DEK-NUP214 fusion protein in AML26 and the discovery of elevated DEK expression in breast3,27, colorectal5,28, lung4,29, and several other types of cancer1, many systems have been used to investigate the pathological consequences of DEK over-expression. A prominent phenotype in cell models is the requirement of DEK for chemotherapy and radiation resistance. For example, expression of the DEK C-terminal domain in ataxia-telangiectasia fibroblasts partially restored radiation resistance, and the loss of DEK conferred sensitivity to DNA damaging agents in multiple cell types14,19,30. Mechanistically, our prior report found that DEK was required for optimal kinase activity of DNA-PK. This kinase is a key mediator of canonical non-homologous end joining (NHEJ), which repairs DNA double strand breaks (DSBs)31, and DEK loss correspondingly suppressed NHEJ19.
The observed NHEJ defects in DEK-deficient cells are unlikely to fully account for the severe sensitivity to genotoxic agents, especially DNA interstrand cross linkers and topoisomerase inhibitors14,32. This suggests additional roles for DEK in genotoxic drug tolerance and DNA repair. A common mechanism by which genotoxic agents induce cell death is through perturbation of replication fork progression33. A recent study determined that DEK attenuates DNA replication stress12 in a manner similar to RAD51 and FANCD2, factors well known for their function in both homologous recombination (HR) DSB repair and activities at arrested replication forks34,35,36,37.
HR requires the presence of a homologous template, often the sister-chromatid, to be used for repair, and is therefore largely confined to S/G2 phases of the cell cycle. By copying a homologous DNA sequence, HR is considered an error-free repair process that preserves genome integrity38. This signal cascade is initiated by the DSB sensor, ATM kinase. After localizing to a DSB, ATM autophosphorylates39,40, pATM catalyzes the phosphorylation of CHK2 to inhibit cell cycle progression41 and the H2AX histone to produce gamma-H2AX (γH2AX) epigenetic marks on both sides of the DSB. The γH2AX mark supports DSB repair by enhancing the recruitment of BRCA1 and key nucleases including the MRN complex and CtIP38,42,43,44. These factors coordinate DNA end processing into single strand DNA (ssDNA) 3′ tails38,42,43. The resulting ssDNA is initially coated by RPA, which is then efficiently replaced with a RAD51 filament through the combined activities of BRCA1, BRCA2, the RAD51 paralogs, and other factors38,42. This RAD51 filament catalyzes strand invasion, complementary strand annealing, and the formation of a stable synaptic complex with a homologous sequence on the sister chromatid45.
While the cellular regulation and choice between DSB-repair pathways remains incompletely understood46,47,48,49,50, NHEJ and HR factors have been shown to be mutually antagonistic as one pathway tends to compensate when the other is compromised42,51. For example, loss of critical HR gene functions in Fanconi Anemia mutant cells results in enhanced dependence upon NHEJ factors52,53,54 while HR repair efficiency is increased following the disruption of essential NHEJ factors by chemical inhibition or mutation of DNA-PK51,55,56. Compromising both the NHEJ and HR repair pathways may be synthetic lethal to cancer cells.
In this report, we demonstrate that DEK is required for the repair of DSBs by HR. Investigation of potential DEK functions in the HR pathway revealed that the protein was important for γH2AX activation, promoted the co-recruitment of BRCA1 and RAD51 to resected ssDNA, and formed a complex with RAD51 in a BRCA1-independent manner. The dependence of HR on DEK expression was underscored by the synthetic lethal relationship between DEK loss and NHEJ inhibition. Together, these results reveal a novel, multifunctional role for DEK in HR.
We have previously shown that DEK knockout (DEK−/−) mouse embryonic fibroblasts (MEFs) harbor decreased NHEJ activity19. To determine the contribution of DEK to NHEJ activity in vivo, we measured the concentrations of immunoglobulins in DEK−/− mouse serum. Class switch recombination (CSR) from IgM to the IgA and IgG classes of immunoglobulins is an NHEJ-dependent process31, but DEK−/− mice were fully competent in producing all classes of immunoglobulins (Fig. 1a). This data suggests the residual NHEJ activity in the knockout mice is sufficient for CSR under physiologically normal in vivo conditions, despite the sensitivity of DEK-deficient cells to DNA damaging agents14,19. To determine if DEK-deficient cells require NHEJ for survival, we examined the need for NHEJ by inhibiting the upstream kinase, DNA-PK. We utilized two well-established DNA-PK inhibitors, NU7026 and NU7441, to prevent DNA-PKcs autophosphorylation and activation57,58,59,60. DEK−/− MEFs treated with the DNA-PK inhibitor NU7026 displayed dramatically more cell death than wild-type (DEK+/+) or untreated samples (Fig. 1b). Analysis of cleaved caspase 3 positive cells by flow cytometry revealed that the inhibitors were sufficient to induce a significant and specific 2–3 fold increase in apoptosis in the DEK−/− cells in the absence of exogenous DNA damaging agents (Fig. 1c). Similar experiments in HeLa cells infected with a well-characterized DEK knockdown versus control adenovirus vector (AdDEKsh versus AdGFP)18 revealed a similar 2-fold increase in apoptosis after treatment with either DNA-PK inhibitor, NU7026 or NU7441 (Fig. 1d,e). These data suggest that DEK-deficient cells, despite their attenuated NHEJ activity19, rely significantly on the remaining NHEJ for survival.
Several reports have described an antagonistic relationship between HR and NHEJ mechanisms of DSB repair46,55,61, and it is generally thought that loss of one pathway will result in compensation by the remaining repair modality53,55,56. Given the dependence of DEK-deficient cells on NHEJ and their inherent sensitivity to chemotherapeutics, we hypothesized that HR may be compromised in DEK−/− cells. To test this we first utilized two established episomal HR reporter systems56,62. These vectors harbor two defunct EGFP genes, the first bearing an I-SceI meganuclease cleavage site and the second bearing a truncated gene sequence (Fig. 2a). Co-transfection with an I-SceI expression vector induces a DSB that, when repaired by HR, generates a functional EGFP gene. Both the HR-EGFP/5′EGFP and pHPRT-DR-GFP reporters demonstrated a remarkable and severe loss of HR efficiency in DEK−/− MEFs (Fig. 2b). To compare the effects of DEK loss on chromosomal HR repair following direct or replication-dependent DSB induction, we utilized the recently published 6xTer-HR reporter technology63. The 11CO/47 mouse embryonic stem (mES) cell line bearing a single copy of the 6xTer-I-SceI-GFP reporter cassette integrated at the Rosa26 locus quantifies HR either at an I-SceI endonuclease-induced DSB or at an adjacent Tus/Ter-induced stalled replication fork. Fork stalling is induced by Tus protein binding to an array of six Ter elements, forming a physical barrier that impedes approaching replication fork progression63 (Fig. 2c). Using this system, we quantified the impact of DEK depletion on error-free short tract gene conversion (STGC), producing GFP+RFP− cells, and error-prone long tract gene conversion (LTGC), producing GFP+RFP+ cells through duplication and thus proper alignment of two synthetic exons of RFP. After co-transfecting Dek siRNA (Fig. 2D) and I-SceI, we found that siDek treated cells were significantly compromised for both total HR and STGC, and LTGC followed the same trend (Fig. 2e). This decrease was similar in magnitude to loss of the BRCA proteins64, but not as severe as RAD51 deficiency63. However, there was no significant decrease in STGC, LTGC, or total HR with Tus-induced DNA replication fork stalling (Fig. 2f). This suggests that DEK, unlike most HR factors63, is dispensable for replication fork associated repair but required for efficient repair of DSBs.
To understand how DEK functions in HR, we examined the consequences of DEK loss at successive steps in the repair pathway following irradiation (IR) mediated DSB induction. For these studies we used DEK−/− MEFs and AdDEKsh-infected HeLa cell systems, whose cell cycle progression was reported comparable to that of their respective DEK-proficient controls18,19. Beginning with the relevant upstream DNA damage sensor kinase, we found that ATM was strongly autophosphorylated in AdDEKsh-treated HeLa cells (Supplementary Fig. S1a) and functional as indicated by phosphorylation of pCHK2 T68, an ATM substrate (Supplementary Fig. S1b). Surprisingly, γH2AX phosphorylation was not enhanced in the AdDEKsh HeLa cells despite robustly activated pATM (Supplementary Fig. S1a). This held true and was even more severe in the DEK−/− MEF system where DEK was required to increase γH2AX phosphorylation above baseline by immunofluorescence (IF) (Fig. 3a). Both the total number of cells harboring γH2AX foci and the average number of foci per cell were severely attenuated from 3–24hours after irradiation (Fig. 3b,c). These results were also confirmed by western blot analysis in MEFs and in DEK knockdown C33A cancer cells (Fig. 3d,e), suggesting that DEK is an important general contributor to γH2AX phosphorylation in IR-treated cells.
DSB end-processing occurs downstream of the pATM-γH2AX signal amplification feedback loop and generates ssDNA overhangs that are bound and protected by RPA. End processing is coordinated by several factors, including the MRN complex and BRCA143,44. Chromatin fractionation of HeLa cells revealed that chromatin recruitment of MRE11 and NBS1, components of the MRN complex, as well as BRCA1 were not affected by DEK status following IR (Supplementary Fig. S2a,b). Following end-processing, BRCA1 both co-localizes with and is necessary for robust RAD51 IF foci development43. Through assessing the quality and quantity of these foci, we determined whether DEK loss disrupts RAD51 loading onto RPA-bound ssDNA43. Since our BRCA1 antibody does not detect murine BRCA1, we performed these experiments in HeLa cells. DEK knockdown in HeLa cells had a mild, but statistically significant delay in BRCA1-RAD51 foci formation at 3hours following IR (Fig. 4a,b) with no difference in individual BRCA1 or RAD51 foci formation at any time point (Supplementary Fig. S3). By 6hours the co-localization of BRCA1 and RAD51 foci was indistinguishable from control cells. To determine if the delay in RAD51-BRCA1 co-localization correlated with impaired RAD51 loading onto RPA-protected ssDNA, we also determined the kinetics of RAD51 localization to RPA foci. In line with the delay with BRCA1-RAD51 co-localization, we found a significant decrease of RAD51-RPA2 co-localized foci at 3 and 6hours post irradiation (Fig. 4c). A 24-hour time course in DEK+/+ and DEK−/− MEFs revealed that RAD51 loading was similarly attenuated (Fig. 4d,e). There was no difference in RAD51 foci quantity or quality (Supplementary Fig. S4a), but RPA2 foci were generally smaller in DEK−/− cells. DEK−/− cells also retained persistent RPA2 foci at later time points, suggesting a DNA repair defect (Supplementary Fig. S4b). In summary, DEK-deficient cells have a small reduction in the recruitment of RAD51 to BRCA1 foci, which results in a mild attenuation of RAD51 loading on RPA-coated ssDNA.
While RAD51 loading was not dramatically affected by DEK loss, physical DEK interactions with the RAD51 recombinase were possible. To test this, pMIEG His-FLAG-DEK expressing HeLa cells were left untreated or received 1mM hydroxyurea (HU) for 15hrs to induce replication fork stalling and a low level of DSB generation as published previously65, prior to FLAG immunoprecipitation (IP). Under these conditions, we found that RAD51 complex formation occurred with His-FLAG DEK (Fig. 5a). Validation of the complex was performed by IP of endogenous DEK and reverse IP of RAD51 in parental HeLa cells (Fig. 5b,c). No BRCA1 interaction with DEK was observed (Fig. 5b), suggesting that DEK forms a separate complex from the standard BRCA-RAD51 homologue apparatus. The DEK-RAD51 complex was also observed in untreated and irradiated cells. Thus, DEK interacts with the HR recombinase RAD51 in the presence or absence of exogenous DNA damage (Supplementary Fig. S5).
Herein we demonstrate that DEK is required for the repair of DSBs by HR, and regulates multiple steps in the HR cascade by promoting γH2AX activation, enabling robust RAD51 loading, and forming a complex with RAD51 (Fig. 5d). This importance of DEK in HR was further underscored by the exquisite sensitivity of DEK-deficient cells to NHEJ loss through DNA-PK inhibition. In summary, this is the first report to describe DEK as a HR factor and to identify a synthetic lethal relationship that exists between DNA-PK inhibition and DEK loss.
According to the 6xTer reporter assays (Fig. 2c–f), DEK appears to be required for HR only in the context of conventional DSBs, wherein DEK-deficient cells had a similar HR deficiency to BRCA1 loss63, but not in the context of stalled replication forks. However, this selectivity differs when DEK activities are compared to those of BRCA1, BRCA2, and RAD51, factors which are required for the HR repair triggered by both I-SceI induced DSBs and Tus-stalled replication forks63. Based on these observations, it is likely that DEK is expendable for replication-associated HR repair, but this by no means precludes non-HR functions at stalled replication forks. Indeed, published DNA fiber assays have elegantly shown that DEK can promote stalled replication fork restart and reduce γH2AX response to DNA replication stress12.
Differential γH2AX responses to irradiation (Fig. 3) versus chemotherapy-induced damage12,19 lends further support to a model where DEK is specifically required for HR repair of DSBs. Previous studies have found that loss of DEK enhances γH2AX activation following treatment with replication fork stalling agents12,19. While similar pATM activation was observed in response to stalling or irradiation, γH2AX phosphorylation was specifically not engaged after IR exposure. This suggests that DEK is specifically required for ATM-H2AX signal transduction under conditions of DSBs. While it is known that γH2AX is not strictly required for HR, its loss can slow the kinetics of the pathway and lead to a moderate decrease in repair efficiency44,66. Thus, the attenuation of BRCA1-RAD51 and RAD51-RPA2 foci co-localization (Fig. 4) is likely a consequence of failed γH2AX activation. These pulldown results, in which DEK interacted with RAD51 in a BRCA1-free complex (Fig. 5), further support this hypothesis as they suggest DEK is not part of the BRCA1-Palb2-BRCA2-RAD51 complex that facilitates RAD51 loading. However, we cannot presently rule out weak interactions or a role for BRCA in promoting the DEK-RAD51 interaction. Also, since these interactions were identified and confirmed in cervical cancer cells, it is uncertain if the DEK-RAD51 complex is a component of normal cell biology or specific to cancer cells.
Taken together these data support a model in which DEK has two distinct roles in the repair of DSBs by HR. First, DEK has an important function in γH2AX activation by ATM, but this is unlikely to be sufficient for the degree of HR deficiency observed in DEK-deficient cells44,66. Therefore, DEK must have a second function involving the DEK-RAD51 complex. With regard to a possible mechanism of action, DEK might play an early role in homologous recombination by supporting the initial formation of the RAD51-DNA filament. However, since BRCA1 and BRCA2 play key roles in this step and, since BRCA1 was not detectable in the DEK-RAD51 complex (Fig. 5b), we do not favor this scenario. Based on previous structural and cell-free studies, DEK binding to DNA stimulates self-multimerization and potential interactions with other proteins22,67,68. Furthermore, DEK preferentially binds cruciform over conventional linear DNA structures24, and harbors three putative DNA binding motifs67,68. Thus, one possible scenario is that DEK interacts with both the invading RAD51 filament and the sister chromatid to stabilize resulting D-loop structures. Such activities can now be readily explored through established biochemical assays69.
Since this is the first report to establish a role for DEK in HR, our work opens up multiple avenues for future study. At the molecular level, mechanisms whereby DEK promotes γH2AX activation specifically at DSBs and the nature and function of the complex with RAD51 remain unclear. Secondly, in the absence of known DEK homologues, detailed studies of the evolutionary history and origin of DEK are lacking despite its strong conservation across mammals and presence in plants. Interestingly, DEK is absent in bacteria and yeast, wherein mechanisms of HR are well described. It is possible that the early multicellular eukaryotes required a novel chromatin regulator to stabilize their increasingly complex genomes during DNA repair. Considering its role in chromatin modification and ATM-mediated γH2AX activation, its affinity for RAD51 and DNA structures similar to Holliday junctions, and its ability to stabilize DNA breaks for in vitro DNA ligation, DEK is a likely candidate to fill this niche1,24.
Still, further investigations into the activities of DEK in HR and NHEJ repair will remain important for deepening our knowledge of the evident evolutionary pressure on DEK and HR, understanding the multiple activities of DEK in HR, and for the development of small molecule inhibitors to target relevant functions of this oncogene. In support of these future therapeutic efforts, we identified a synthetic lethal relationship between DEK loss and canonical NHEJ inhibition, as well as a cellular phenotype that can be assessed for DEK inhibition in vivo. This is important as DEK is largely dispensable for cell division, and DEK−/− mice are healthy and viable12,18,20, suggesting a high therapeutic index for a potential anti-DEK drug. Thus, it is conceivable that DEK overexpressing tumors may regress without a need for adjuvant radiation or traditional chemotherapy if substituted for tumor-specific co-targeting of DEK and DNA-PK.
HeLa and C33A cells were grown in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. MEFs, generated previously19, were cultured in DMEM with 10% heat inactivated FBS, 100μM MEM non-essential amino acids, 0.055mM β-mercaptoethanol (BME), 2mM L-glutamine, and 10μg/ml gentamycin. Mouse embryonic stem (ES) cells were grown in ES media on fibroblast or gelatin substrate as previously described63. To induce DNA damage, cells were treated with γ-IR from a 137Cs source or 1mM hydroxyurea (HU). Both 40μM NU7026 and 2μM NU7441 (Tocris, Bristol, UK) were used to specifically inhibit DNA-PK as previously published59,70. DEK knockdown was accomplished using the adenoviral AdDEKsh as compared to control AdGFP vectors at 10 infectious units (IU) per cell for 48hr prior to IR treatment, as described previously18. The pMIEG His-FLAG-DEK retroviral vector was described recently71, and cells were transduced with virus for 24hrs prior to sorting for GFP positive cells on a BD-FACSAria II flow cytometer.
Dek+/+ and Dek−/− littermate mice were born from heterozygous crosses on a mixed C57Bl6/S129 background as previously described20. Usage and handling of mice was performed with the approval of the Cincinnati Children’s Institutional Animal Care and Use Committee and complied with institutional, state, and federal guidelines and regulations as well as AAALAC accreditation standards. All mice were housed in specific pathogen free housing with ad libitum access to food and water. Blood was obtained by cardiac puncture of three DEK+/+ and three DEK−/− mice. To measure immunoglobulin (Ig) in the blood serum by ELISA, plates were coated with the following Southern Biotechnology Associates antibodies (Birmingham, AL, USA): anti-mouse IgM (no. 406501), IgA (no.556969), or IgG (no. 1030-01), and Ig was detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 (no. 1070-05), IgG3 (no. 1100-05), IgG2a (no.1080-05), IgG2b (no. 1090-05), IgA (no. 1040-05), or IgM (no. 1020-05). In all cases, wells were developed with the Ultra TMB peroxidase substrate system (Thermo Scientific) and OD was measured at 450nm using a Fluostar Omega microplate reader (BMG-Labtech, Ortenberg, Germany).
Cells were trypsinized, fixed in 4% PFA for 10min at 37°C, permeabilized in 90% ice-cold methanol for 30min, and incubated with the cleaved caspase 3 cell signaling antibody for 1hr. Analysis was performed on a BD FACSCanto II instrument.
MEFs were co-transfected with 2μg of HR-EGFP/5′EGFP, pHPRT-DR-GFP, or an EGFP expressing plasmid (transfection control) and 2μg pCMV-I-SceI using Fugene HD transfection reagent (Roche, Penzberg, Germany) prior to a 48hr incubation62. Cells were collected and analyzed for EGFP expression by flow cytometry. Analysis of chromosomal HR repair was conducted using mouse ES cells bearing a single copy knock-in of the 6xTer-HR reporter cassette targeted to the Rosa26 locus63. For the 6xTer-HR reporter cells, 1.6×105 cells were cotransfected in suspension with 0.35μg empty vector, pcDNA3β-myc NLS-Tus, or pcDNA3β-myc NLS-I-SceI, and 20pmol ONTargetPlus-smartpool, (Dharmacon, Lafayette, CO), siLuc (p-002099-01-20), or siDek (M-050260-00-0005) as described previously63. Transfected cells were analyzed by flow cytometry 72hrs post transfection using a Becton Dickinson LSRII. GFP+ frequencies were measured in duplicate samples, 3–6×105 total events were scored. Repair frequencies presented are corrected for background events and for transfection efficiency (60–85%). Transfection efficiency was measured by parallel transfection with 0.05μg wild type GFP expression vector, 0.30μg control vector and 20pmol siRNA.
Cell lysates were generated by lysis in NETN buffer (50mM Tris-HCl pH=7.4, 250mM NaCl, 5mM EDTA, 0.1% NP-40, 10mM NaF, 200μM Na2VO3, 0.5mM PMSF, and 1x protease inhibitor cocktail) on ice for 30min. Non-soluble debris was precipitated by a 5min 12000rpm spin at 4°C and discarded. Lysate was treated with 4x loading buffer (0.5M Tris-HCl pH 6.8, 277mM SDS, 40% glycerol, bromophenol blue, 4% 2-mercaptoethanol), ran on a 10% SDS-PAGE gel, and transferred onto a PVDF membrane for 1–2hr at 500mA. The following Santa Cruz (Dallas, TX, USA) antibodies were used: BRCA1 (sc-6954) and RAD51 (sc-8349 and in-house aliquot B32). The following Cell Signaling (Danvers, MA, USA) antibodies were used: ATM (2873), GAPDH (5174), Chk2 (2662), pChk2 T68 (2661), Chk1 (2345), and pChk1 S345 (2341). Other antibodies included DEK (610948 BD Bioscience, San Jose, CA, USA), DEK (16448-1-AP, Protein Tech, Rosemont, IL, USA), DEK (in-house K-877)72, DNA-PKcs (ab1832, Abcam, Cambridge, MA, USA), pDNA-PKcs S2056 (ab18192), pATM S1981 (AF1655, R&D Systems, Minneapolis, MN), γH2AX (05-636, Millipore, Darmstadt, Germany), MRE11 (GTX70212, genetex, San Antonio, TX, USA), and NBS1 (NB100-143SS, Novus Biologicals, Littleton, CO, USA).
1–5×105 cells were plated onto poly-d-lysine treated coverslips and allowed to attach prior to adenovirus infection and/or drug treatment. To stain, coverslips were fixed in 4% paraformaldehyde, antigens retrieved with a 0.2% Triton x-100 PBS solution, and cells blocked with PBS plus 5% normal goat serum and 0.3% Triton X-100. Antibody dilutions are as follows: BRCA1 (sc-6954), γH2AX 1:1000 (05-3636, Millipore), RPA2 1:300 (ab2175), RAD51 1:500 (sc-8349), goat anti-mouse Alexa Fluor 568 1:500 (A-11004, ThermoFischer Scientific, Waltham, MA), and goat anti-rabbit Alexa Fluor 633 1:500 (A-21070). Nuclei were counterstained with DAPI ProLong Gold (P-36931, Life Technologies). RPA and RAD51 foci were captured on a Carl Zeiss Apotome instrument, and foci within 100 cells counted per time point per replicate. BRCA1, RAD51, and RPA foci counts were performed by eye due to background signal from the antibodies. High resolution images were imaged on an inverted Nikon A1R GaAsP confocal microscope. Whole cell γH2AX foci were also collected on the confocal microscope using z-stacks and were quantified using Imaris 6 (Bitplane AG, Zurich, Switzerland).
Chromatin fractionation was performed as described previously73 according to the schematic in Fig. 3d. Briefly, HeLa cells were infected with AdGFP or AdDEKsh 48hr prior to receiving 10Gy of IR. Six hours following IR, cells were counted to ensure equal numbers of cells per sample. Cell lysis and nuclear isolation was performed in a wash buffer (10mM PIPES pH=7.0, 1mM EGTA, 0.1M NaCl, 0.3M sucrose, 0.5M NaF, 0.5mM Na3VO4, and 1x protease inhibitor cocktail) plus 1% Triton X-100. Serial separation of the precipitated nuclear fraction was performed first by treatment with wash buffer +20U DNAseI (AM2235, ThermoFischer Scientific), followed by a 5min incubation with wash buffer +0.5M (NH4)SO4 on the resulting pellet. The resulting fractions were treated with 4x loading buffer.
Subconfluent cells were treated with or without 1mM HU for 15hrs. Cells were homogenized in lysis buffer (10mM HEPES, pH=7.9, 1.5mM MgCl2, 10mM KCl, 0.5mM PMSF, 0.5mM DTT, 1mM NaVO3, 10mM NaF, and 1x Sigma P5955 protease inhibitor cocktail), and the nuclear pellet was lysed in a nuclear lysis buffer (20mM HEPES pH=7.9, 400mM NaCl, 1% Triton X-100, 0.1% NP-40, 10% Glycerol, 0.5mM PMSF, 0.5mM DTT, 1mM NaVO3, 10mM NaF, and 1x protease inhibitor cocktail) on ice. After ultracentrifugation, pMIEG and pMIEG His-FLAG-DEK supernatants were loaded onto anti-FLAG M2 affinity gel (A2200, Sigma, St. Louis, MO, USA). Parental cell supernatants were pretreated with antibodies for 1hour prior to loading onto Protein A sepharose beads. For both IP experiments, lysates were treated with either 50μg/ml ethidium bromide (EtBr) or 125U of benzonase (E8263, Sigma) plus 2mM MgCl2 as controls. After incubating overnight at 4°C, the beads were washed, and the IP products collected by boiling in 2x Laemmli Buffer (12.5mM Tris-HCl, pH=6.8, 20% Glycerol, 4% SDS, 0.004% Bromophenol Blue, 10% 2-β-mercaptoethanol).
Statistics were performed using Graphpad Prism 6 software (Graphpad Software Inc., San Diego, CA). Significance is indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), and the number of independent biological replicates and type of analysis is indicated in the Fig. legends.
How to cite this article: Smith, E. A. et al. DEK is required for homologous recombination repair of DNA breaks. Sci. Rep. 7, 44662; doi: 10.1038/srep44662 (2017).
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We thank the Cincinnati Children’s Hospital Medical Center Confocal Imaging Core for assistance with fluorescence microscopy, confocal microscopy, and the Imaris quantification software. A portion of the flow cytometry work was performed using equipment from the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children’s Hospital Medical Center. The core is supported by three NIH P30 grants through the Division of Rheumatology’s Center for Rheumatic Disease Research (NIH AR47363), Division of Gastroenterology’s Digestive Health Center (NIH DK78392), and the Division of Experimental Hematology and Cancer Biology’s Center for Excellence in Molecular Hematology (NIH DK90971). We are thankful to Regina Häfele and Patrick Schäfer, Ulm University, for experimental help, and to Allie Adams, Tim Chlon, Marie Matrka, Nicholas Pease, Lisa Privette Vinnedge, Molly Smith, Madeline Niederkorn, and Sonya Ruiz-Torres for helpful discussions and manuscript review. This work was supported by the National Institutes of Health [R01-CA-102357 and R01-CA116316 to S.I.W., R01-CA095175 to R.S., T32 GM063483-13 to University of Cincinnati MSTP, T32-ES007250 University of Cincinnati]; German Research Foundation (DFG) [WI 3099/7-1 and WI 3099/7-2 to L.W., KA 2799/1 to F.K.]; RWTH Aachen START Program of the Faculty of Medicine to F.K.; University of Cincinnati Department of Cancer and Cell Biology Cardell Fellowship for Excellence in Graduate Research to E.S.; Chinese Scholarship Council to H.G.; German Cancer Aid (Deutsche Krebshilfe)  to L.W. Work in the lab of J.A.D. was supported by a grant from the Novo Nordisk Foundation (grant no. NNF14CC0001) and a postdoctoral grant to L.M.S. from the Danish Council for Independent Research in Medical Sciences.
The authors declare no competing financial interests.
Author Contributions E.A.S. carried out the majority of the experimental and intellectual work. IP optimization was conducted by E.F.K., A.R.M., A.G.J., and A.M.A., N.A.W. and R.S. performed the 6xTer reporter experiments; B.G. and L.W. performed the HR-EGFP/5′EGFP HR reporter assay; J.A.D., R.S., L.M.S., and L.M.P.V. performed the CSR experiments; H.G., F.K., L.M.P.V., and P.R.A. contributed significantly to the intellectual development of the project. E.A.S. and S.I.W. developed the project, evaluated and interpreted all data, and wrote the manuscript. All authors discussed data generation and contributed to data interpretation and manuscript preparation.