E2F1 regulates the base excision repair gene XRCC1 and promotes DNA repair.

The E2F1 transcription factor activates S-phase promoting genes, mediates apoptosis, and stimulates DNA repair through incompletely understood mechanisms. X-ray repair cross complementing group 1 (XRCC1) protein is important for efficient single-strand break/base excision repair (SSB/BER). Although both damage and proliferative signals increase XRCC1 levels, the mechanisms regulating XRCC1 transcription remain unclear. To study these upstream mechanisms, the XRCC1 promoter was cloned into a luciferase-reporter.

The E2F1 transcription factor activates Sphase promoting genes, mediates apoptosis, and stimulates DNA repair through incompletely understood mechanisms. Xray repair cross complementing group 1 (XRCC1) protein is important for efficient single-strand break/base excision repair (SSB/BER). Although both damage and proliferative signals increase XRCC1 levels, the mechanisms regulating XRCC1 transcription remain unclear. To study these upstream mechanisms, the XRCC1 promoter was cloned into a luciferasereporter. Ectopic expression of wild-type E2F1, but not an inactive mutant E2F1(132E), activated the XRCC1promoter-luciferase reporter, and deletion of predicted E2F1 binding sites in the promoter attenuated E2F1-induced activation. Endogenous XRCC1 expression increased in cells conditionally expressing wild-type, but not mutant E2F1, and methyl methanesulfonate (MMS)-induced DNA damage stimulated XRCC1 expression in E2F1+/+, but not E2F1-/-, MEFs. Additionally, E2F1-/-MEFs displayed attenuated DNA repair after MMS-induced damage compared to E2F1+/+ MEFs. Moreover, CHO cells with mutant XRCC1 (EM9) were more sensitive to E2F1-induced apoptosis compared to CHO cells with wildtype XRCC1 (AA8). These results provide new mechanistic insight into the role of the E2F pathway in maintaining genomic stability.
DNA SSBs are one of the most common DNA lesions and pose a major threat to genetic stability and survival through accumulation of mutations or through conversion to double-strand breaks (25). SSBs can arise by direct damage to DNA bases or sugar moieties, or indirectly as intermediates during the process of BER (26). SSBR/BER requires highly coordinated overlapping enzymatic steps dependent on the nature and origin of the SSB lesion (25-27).
XRCC1 is a scaffolding protein that promotes efficient repair by interacting with many of the proteins that catalyze SSBR/BER (26,28-30). Although XRCC1 null mice are not viable (31), mouse and Chinese hamster ovary (CHO) cells with mutant or attenuated XRCC1 demonstrate decreased SSBR and hypersensitivity to many different types of DNA damage (26,32-37). In human cells, XRCC1 is necessary for efficient SSBR, genomic stability and survival (38-40). Furthermore, XRCC1 polymorphisms are associated with variable cancer risk which suggests a possible role in cancer development (41). XRCC1-mediated repair includes an Sphase SSBR pathway that operates at replication forks, and a rapid cell cycle independent SSBR/BER pathway (42-45). Posttranslational mechanisms, such as phosphorylation, also modulate XRCC1 function and promote genetic stability (46,47). Additional mechanisms may also regulate XRCC1 since mRNA and protein levels increase after DNA damage in part through a MAPK signaling pathway (48,49). However, although both cell cycle specific and DNA damage signals are upstream of XRCC1, the mechanisms regulating XRCC1 expression remain incompletely characterized.
In this report, we demonstrate that XRCC1 is a direct E2F target gene and that E2F1 enhances SSBR/BER. These results provide new mechanistic insight into the role of E2F1 in the maintenance of genomic stability and cell survival.
Luciferase-reporter assays. Cells at 60% confluence in 6-well plates were transfected with the specified XRCC1 luciferase reporter plasmid, together with indicated amounts of specified E2F expression vector, pRSV β-galactosidase expression vector (0.1 µg), and appropriate amount of empty plasmid vector, for a total of 1.0 µg plasmid DNA per transfection. Luciferase assays were performed 36 hours posttransfection with an AutoLumatB95 and relative luciferase light units were normalized to ß-galactosidase activity as described previously (51).
Western blotting. Total cellular lysates were prepared, quantitated, resolved on 10% SDS-PAGE, immunblotted with the specified primary and HRP-conjugated secondary antibodies, and visualized with chemiluminescence (Pierce SuperSignal) as previously described (51).
Anti-XRCC1 mouse monoclonal antibody was from NeoMarkers, anti-E2F1 antibody was from Santa Cruz, anti-tubulin antibody was from Sigma, and HRP-conjugated secondary antibodies were from Jackson Immunological.
Northern Blotting. Total RNA was prepared using the Total RNA Preparation Kit (Promega, Madison, MI) according manufacturer's instructions. Twenty µg total RNA was resolved by electrophoresis on a 1.0% formaldehyde-agarose gel, transferred and UV cross-linked to a Zeta-ProbeGT membrane (BioRad, Hercules, CA), hybridized to a randomly primed 32 P-labeled XRCC1 cDNA probe, and visualized with autoradiography using standard techniques. RT-PCR. XRCC1 and GAPDH mRNA levels were determined by semiquantitative RT-PCR using standard techniques. Briefly, equivalent amounts of total RNA was prepared from the indicated cells at the specified conditions described in the text, and oligo-dT primed first-strand cDNA synthesis performed with Superscript III reverse transcriptase (Invitrogen). PCR was performed on equivalent amounts of RT product with Taq DNA polymerase for 30 cycles (95 o C for 15 seconds, 65 o C for 20 seconds, 72 o C for 40 seconds). XRCC1 forward primer: 5'-gtatgcaggctccacggatgagaa-3'; reverse primer: 5'aggcttgcggcaccaccccatagagc-3. GAPDH forward primer: 5'-acggccgcatcttcttgtgc-3; reverse primer: 5'-gtgcaggatgcattgctgac-3.
Cross-links were reversed, and then DNA purified by phenolchloroform extraction and ethanol precipitation.
Cell culture. Cells were grown in DMEM or McCoy's media supplemented with 10% heat-treated fetal bovine serum (FBS) and 290 µg of L-glutamine, 100U penicillin, and 100 µg of streptomycin per ml at 37 o C in 5% CO 2 . Saos2 cells stably transfected with various tetracycline-regulated E2F1 expression vectors (a gift from Dr. Karen Vousden, Beatson Institute, UK) were maintained in tetracycline-free FBS (Clontech). E2F1+/+ and E2F1-/-mouse embryonic fibroblasts (MEFs) were gifts from Dr. Joseph Nevins, (Duke University, NC, USA). Chinese hamster ovary (CHO) cell lines AA8 and EM9 were obtained from the American Type Culture Collection (ATCC).
Single-cell agarose alkaline gel electrophoresis was performed per standard methods with minor modifications (52). Equivalent numbers of cells (1 X 10 5 ) were collected at the indicated time points, mixed with low-melt-temperature agarose (Sigma), and then layered onto agarose-coated glass slides. Slides were maintained at 4°C to solidify and for all subsequent steps. Slides were submerged in lysis buffer (2.5 M NaCl, 0.1M EDTA, 10 mM Tris-Cl (pH 7.0), 1% Triton X-100, 1% DMSO) for one hour, washed with nH 2 0, and incubated for 45 minutes in alkaline electrophoresis buffer (50 mM NaOH, 1 mM EDTA, 1% DMSO, pH 12.8). After electrophoresis (25 min, 25 V), air-dried and neutralized slides were stained with 2 µg/ml propidium iodide. Average comet tail moment was scored for duplicate slides (50 cells/slide) in randomly selected fields, and automated calculations were performed using Comet Assay II software (Perceptive Instruments; Suffolk, U.K.).
In situ DNA strand break detection. DNA polymerase I-mediated labeling assay was performed as described previously with minor modifications (53). Briefly, cells grown on cover-slips were fixed in 10% formalin for Cells were rinsed in PBS, permeabilized with 1% Triton X-100 for 20 minutes, and then incubated in a moist-air chamber at 37 o C for 90 min in a labeling mixture containing: 10 µM each of dGTP, dATP, dCTP and 7 µM dTTP, 3µM FITC-dUTP, 20 U/ml of E. coli DNA polymerase I (Sigma, St. Louis, MO) in reaction buffer containing 5 mM MgCl 2 , 10 mM 2mercaptoethanol, and 20 µg/ml bovine serum albumin (BSA). The reaction was terminated by washing the slides twice in PBS. Nonspecific labeling was determined by incubation in the reaction buffer without the enzyme.
Apoptosis assay. Equivalent numbers of indicated cells were plated and allowed to settle for 24 hours at 50% confluency. Adenovirus infections were performed at MOI of 1 x 10 10 (Stratagene AdEasy system). E2F1-expressing and GFP-expressing adenoviruses were a gift from Dr. Rosalie Sears (Oregon Health & Science University, Portland, OR, USA). Forty eight hours after infection, cells were harvested and subjected to Annexin V and flow cytometry as previously described (54). Briefly, attached and floating cells were collected and resuspended in Annexin V Binding Buffer (BD Biosciences) at a concentration of 1 X 10 6 cells per ml. APC-conjugated Annexin V was immediately added, incubated for 15 minutes at room temperature, and then analyzed on a Becton Dickinson FACScan. The percentage of apoptotic cells was determined by the increase in Annexin V-APC signal on stained cells over the baseline signal (defined on unstained cells) per standard flow cytometry convention.  Figure 1). To test this, we utilized a Saos2 stable cell line containing an E2F1 cDNA expression vector under control of a tetracycline-regulated promoter (55).

Results
We found that tetracycline-induced E2F1 expression stimulated an increase in XRCC1 protein ( Figure 1A, lanes 1 and 2) and mRNA levels ( Figure 1B, lanes 1 and 2).
To demonstrate that E2F1 transcription function was required to induce endogenous XRCC1 expression, we additionally utilized a tetracycline-regulated Saos2 stable cell line containing a DNA-binding incompetent mutant E2F1 (132E) (55).
XRCC1 promoter has features suggestive of an E2F target gene. Since transcription competent wild-type E2F1 was capable of stimulating XRCC1 mRNA and protein levels (Figure 1), we examined the genomic sequence encompassing the XRCC1 promoter ( Figure 2A). Sequence analysis from -881 to +158 relative to the putative transcription start site (green sequence) (50) revealed a CpG island (boxed sequence) as well as putative E2F binding sites from -819 to -803 (red sequence). To verify that an active promoter was present in this region, we cloned this genomic fragment into the pGL3-Basic luciferase reporter vector.
After transfection into Saos2 cells, we found that this genomic region stimulated the luciferase reporter nearly six-fold relative to empty vector ( Figure 2B). Sequential deletions from the 5' end attenuated luciferase activation relative to empty vector. This data suggests that a functional XRCC1 promoter is contained within this region although the possibility of other control elements outside of this region cannot be excluded.
The XRCC1 promoter-luciferase reporter is stimulated by E2F1. Since the XRCC1 promoter sequence was typical for an E2F target gene (Figure 2), we reasoned that the XRCC1 promoter-luciferase reporter would be stimulated by E2F. Co-transfection of increasing amounts of E2F1 expression vector with the XRCC1 promoter-luciferase reporter caused a dose-dependent increase in luciferase activation ( Figure 3A). In contrast, DNA-binding incompetent E2F1(132E) could not activate the XRCC1 promoter-luciferase reporter ( Figure 3A). We also found that E2F1 more efficiently stimulated the XRCC1 promoter-reporter relative to other family members (E2F2, E2F3 and E2F4)( Figure 3B). To determine the contribution of the predicted E2F binding sites for their ability to influence XRCC1 promoter activation, we cotransfected E2F1 expression vector with an XRCC1 promoter-luciferase reporter which lacked the E2F binding sites (ΔE2F1-XRCC1) as shown in Figure 3C. We found that in response to E2F1 co-transfection, stimulation of the XRCC1 promoter-luciferase reporter containing the E2F binding sites was over 15fold higher than the ΔE2F1-XRCC1 promoterreporter ( Figure 3C, left hand). As a further control for specificity, mutant E2F1(132E) did not stimulate either promoter ( Figure 3C, right hand). Together, these results suggest that the XRCC1 promoter is stimulated by E2F.
Endogenous XRCC1 promoter binds endogenous E2F1. Since XRCC1 transcription is stimulated by E2F overexpression (Figures 1 and 3), we wanted to demonstrate that endogenous E2F1 could bind the endogenous XRCC1 promoter in its native chromatin context. To examine this, we performed a chromatin immunoprecipitation (ChIP) assay on the proximal XRCC1 promoter region (Figure 4). XRCC1 promoter-specific PCR primers amplified this region after chromatin IP with an anti-E2F1specific antibody, but not non-immune IgG (Figure 4, lanes 1-3). To further demonstrate that this was a specific interaction, XRCC1specific primers which amplify a region that does not contain E2F-binding sites (control primers) could not generate a PCR product after chromatin IP with anti-E2F-1-specific antibody (Figure 4, lanes 4-6). These findings demonstrate that the XRCC1 promoter, in its native chromatin context, can specifically bind E2F1 protein and that XRCC1 is a bonafide E2F target gene.
Endogenous E2F1 stimulates the XRCC1 promoter after DNA damage.
Since XRCC1 is an E2F target gene (Figures  1-4) and E2F1 is a DNA damage-inducible protein (9-12), we wondered whether MMS, which causes heat-labile DNA damage repaired by an XRCC1-mediated BER pathway (56), could stimulate XRCC1 transcription in an E2F1-dependent manner ( Figure 5). To investigate this, we transfected E2F1-/-and E2F1+/+ mouse embryonic fibroblasts (MEFs) with the XRCC1 promoterluciferase reporter and then treated them with MMS. Compared to no treatment, we found the XRCC1 promoter-luciferase reporter was stimulated by MMS in E2F1+/+ but not E2F1-/-cells ( Figure 5A). Consistent with transcriptional activation, an increase in XRCC1 mRNA and protein levels ( Figure 5B, left hand and right hand, respectively) was observed after MMS treatment in E2F1+/+, but not E2F1-/-, cells.
These results demonstrate that MMS-induced DNA damage stimulates XRCC1 transcription at least in part through an E2F1-dependent pathway.
E2F1 enhances DNA repair after MMS-induced damage. Since XRCC1 is a bonafide E2F1 target gene (Figures 1-5), we wished to determine if loss of E2F1 would result in attenuated DNA repair after MMSinduced DNA damage ( Figure 6).
To investigate this, we treated E2F1-/-and E2F1+/+ MEFs with MMS and then measured strand-break repair by single-cell alkaline agarose gel electrophoresis (comet assay) (52) as shown in Figure 6A. The average tail moment was similar in undamaged cells (left hand bars, control).
As expected after incubation in MMS, the average tail moment increased equivalently in both E2F1-/-and E2F1+/+ MEFs (middle bars, 0 hr). However, if cells were allowed to recover in MMS-free media (right hand bars, R), E2F1-/-MEFs had a statistically significant longer average tail moment compared to E2F1+/+ MEFs (33% verses 22% respectively, p = 0.013 unpaired 2tailed t-test). To further confirm that E2F1-/-MEFs had decreased DNA repair capacity, we utilized an in situ DNA polymerase I-mediated FITC-dUTP labeling assay (53) to detect persistent DNA strand breaks after recovery from MMS treatment ( Figure 6B) , 0h). However, if MMS exposure was followed by recovery with incubation in MMS-free media, less FITC-dUTP labeling was detectable in E2F1+/+ MEFs compared to E2F1-/-MEFs (middle panels, R). If XRCC1 was downstream of E2F1 and could mediate E2F1function, we reasoned that loss of XRCC1 would result in increased E2F1-induced apoptosis due to diminished DNA repair. To examine this, we utilized Chinese hamster ovary (CHO) cells containing wild-type XRCC1 (AA8 cell line) and a cell line derived from AA8 cells that is mutant for XRCC1 (EM9 cell line)(26,32). We infected cells with an adenovirus expressing wild-type E2F1, or a control adenovirus expressing green fluorescent protein (GFP), and quantified the level of E2F1-induced apoptosis at 48 hours using Annexin V staining and flow cytometry ( Figure 6C).
Under these conditions, AA8 cells did not have a significant increase in apoptosis after infection with E2F1-expressing adenovirus compared to control GFP-expressing adenovirus (left hand graph). In contrast, EM9 cells demonstrated a statistically significant 1.8-fold increase in apoptosis after infection with E2F1-expressing adenovirus compared to control GFPexpressing adenovirus (right hand graph) (p = 0.04, unpaired 2-tailed t-test). Together, these results suggest a mechanism by which the E2F1-XRCC1 pathway can stimulate DNA repair and promote genomic stability and cell viability. However, the existence of other E2F1-mediated pathways that stimulate BER independent of XRCC1 remains likely.

Discussion.
The E2F1 pathway is centrally involved in the highly complex networks coupling cellular proliferation and apoptosis (1,5,7,8,12,57). We have shown that XRCC1, which plays a critical role in SSBR/BER (26), is a direct E2F1 target gene. Because E2F1 is a damage-inducible protein (9)(10)(11)(12), our data provides novel insight into an important DNA repair function of the E2F pathway and opens new avenues for investigation.
Our finding that E2F1 is upstream of XRCC1 significantly expands on prior observations that E2F plays a role in other repair pathways such as MMR and NER (13)(14)(15)(16)(17)(19)(20)(21)24). Our data is consistent with the report that the BER protein uracil-DNA glycosylase (UDG) is also E2F regulated (58). Intriguingly, although E2F1 is best characterized as a transcription factor, E2F1 protein may have a direct role in DNA repair as suggested by its localization to repair complexes (22,23). Thus, it is likely that multiple E2F regulated mechanisms function in parallel with XRCC1 to stimulate repair.
We found that enforced E2F expression stimulated XRCC1 levels ( Figures  1 and 3) and that MMS, which induces predominantly heat-labile DNA damage repaired by an XRCC1-mediated BER pathway (56), causes an E2F1 dependent increase in XRCC1 expression ( Figure 5). This is consistent with prior reports demonstrating that cellular stress (such as from γ-irradiation) increases endogenous XRCC1 levels (48,49,59) although this may be cell type specific (60). How MMS-induced stress activates the E2F1-XRCC1 axis remains unknown. Cellular sensitivity to MMS may involve an ATR-dependent pathway and genetic evidence suggests that MMS-induced damage activates the yeast Rad53 (Chk2 human homologue) pathway (61,62). Given that the ATM/ATR and Chk2 pathways phosphorylate and activate E2F1 protein (9)(10)(11)(12), it is possible that these kinases stimulate XRCC1 expression through E2F1 activation, although this remains to be demonstrated. Interestingly, Chk2 mediated stabilization of the FoxM1 transcription factor stimulates expression of DNA repair genes including XRCC1 (63). Given that XRCC1 function is complex, it is likely that its control involves multiple levels.
Indeed, posttranslational mechanisms modulate XRCC1 function as evidenced by the ability of DNA-PK to phosphorylate XRCC1 (46), as well as for the requirement of protein kinase CK2 to phosphorylate XRCC1 and enhance SSBR and genetic stability (47). Consistent with the complex control of XRCC1, we found that serum starvation followed by re-feeding stimulated XRCC1 expression (data not shown). This is consistent with cell conditions of high E2F activity, but also suggests that serum/mitogenic factors may be important too. This could be a cell type specific phenomenon since density arrest and release does not alter XRCC1 levels in human T24 cells (64). Nevertheless, our discovery of an E2F1-XRCC1 pathway provides a previously undescribed mechanism for regulating XRCC1 function.
The biological importance of E2F1 regulation of XRCC1 is suggested by the attenuated in vivo DNA repair in E2F1-/verses E2F1+/+ MEFs ( Figure 6A/B). Two different methods demonstrated reduced DNA repair after MMS-induced DNA damage which correlates with the decreased XRCC1 levels observed in E2F1-/-cells. The repair of MMS-damaged DNA still occurs in E2F1-/cells suggesting that the E2F1-XRCC1 axis is not an absolute requirement in these systems. This is not surprising given the complex and overlapping repair pathways involved. However, the significance of even a modestly reduced XRCC1-mediated repair function may have important implications for maintaining genomic stability and cell viability. Consistent with this notion of XRCC1 mediating E2F1 activity is the observation that loss of XRCC1 function resulted in an attenuated E2F1-induced apoptotic response in EM9 cells compared to AA8 cells ( Figure 6C).
Although E2F1 is a damage-response protein, it also plays an important role in promoting the expression of a large number of genes required for replication and proliferation (15,(19)(20)(21). Given the intimate relationship between proliferation and replication/repair, the control of XRCC1 by E2F in undamaged cells further integrates SSBR with cell cycle progression as might be expected if enhanced SSBR were necessary to repair SSBs at replication forks (26,42-45,65). Whether, and in what context, the other E2F family members play a role, as well as what specific SSBR pathways are utilized (e.g. long patch BER) remains to be explored.
The p53-E2F network controls and integrates critical functions such as proliferation, cell cycle checkpoints, apoptosis and DNA repair (7,8,12,66,67). In particular, p53 can promote BER (68)(69)(70)(71)(72), and our discovery that E2F1 may also promote BER expands our understanding of the p53-E2F1 network in regulating DNA repair. Disruption of these cooperative pathways has profound implications for tumorigenesis as evidenced by enhanced tumor formation in knock-out mouse models for both p53 and E2F1; although intriguingly, both oncogenic and tumor suppressor functions for E2F1 are suggested in compound p53-/-;E2F1-/-mice (12,(73)(74)(75). Our findings that E2F1 regulates XRCC1 implies that an intact E2F1 pathway may be important for maintaining genomic stability in response to highly dangerous SSBs. This observation opens new avenues for investigation that may ultimately allow us to develop novel preventive and therapeutic strategies for cancer patients.