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Modulation of chromatin structure plays an important role in the recruitment and function of DNA repair proteins. CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is essential for mammalian development and is an important regulator of chromatin structure. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1-/-) are viable but demonstrate a dramatic decrease in cytosine methylation, altered histone methylation, and an inability to differentiate. We find that ES cells lacking Cfp1 are hypersensitive to a variety of DNA-damaging agents. In addition, CXXC1-/- ES cells accumulate more DNA damage and exhibit decreased protein expression and endonuclease activity of AP endonuclease (Ape1/Ref-1), an enzyme involved in DNA base excision repair. Expression in CXXC1-/- ES cells of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) restores normal Ape1/Ref-1 protein expression and rescues the hypersensitivity to DNA-damaging agents, demonstrating that Cfp1 contains redundant functional domains. Furthermore, retention of either the DNA-binding activity of Cfp1 or interaction with the Setd1A and Setd1B histone H3-Lys4 methyltransferase complexes is required to restore normal sensitivity of CXXC1-/- ES cells to DNA-damaging agents. These results implicate Cfp1 as a regulator of DNA repair processes.
Perturbations of global and gene-specific patterns of cytosine methylation are commonly observed in cancer [1,2]. Hyper-methylation and silencing of tumor suppressor genes contributes to tumorigenesis , and modulation of DNA (cytosine-5)-methyltransferase 1 (Dnmt1) activity alters susceptibility to cancer in otherwise normal mice , in mice lacking DNA mismatch repair , and in mice carrying mutations in the NF1 and p53 genes . Hypo-methylation of DNA has also been linked to chromosome instability and increased chromosomal translocations that are frequently associated with malignancies . Finally, agents, such as azacytidine that inhibit DNA methylation, and histone deacetylase (HDAC) inhibitors such as vorinostat, are being used as chemotherapeutic agents in the treatment of malignancies such as myelodysplastic syndrome, chronic myelogenous leukemia, and acute myelogenous leukemia [8-10]. Both aberrant epigenetic modifications and deficiencies in DNA repair can lead to tumorigenesis, as both processes are linked to genetic instability and play a role in the regulation of genes involved in cell growth and differentiation.
The eukaryotic cell acquires more than 10,000 DNA lesions per day. Failure to repair such lesions can lead to mutations, genomic instability, or cell death . The base excision repair (BER) pathway predominantly recognizes and removes oxidative and alkylation damage, including reactive oxygen species (ROS)-induced damage and spontaneous base loss [12-15]. The damaged base is then removed by one of the DNA glycosylases resulting in a baseless or apurunic/apyrimidinic (AP) site, which is a substrate for the AP endonuclease1/redox effector factor 1 (Ape1/Ref-1). Ape1/Ref-1 is a multi-functional protein with AP endonuclease activity that hydrolyzes the phosphodiester bond immediately 5′ to the AP site, after which β-polymerase and DNA ligase act to complete the repair process. Cells with reduced Ape1/Ref-1 activity are hypersensitive to numerous DNA-damaging agents such as methylmethane sulfonate (MMS), hydrogen peroxide (H2O2), temozolomide (TMZ), ionizing radiation (IR), cisplatin, and other alkylating and oxidative DNA-damaging agents [16-23].
The repair of DNA damage in vivo is complicated by the fact that genomic DNA is packaged with histone and non-histone proteins into chromatin, a highly condensed structure that hinders DNA accessibility and its subsequent repair. The efficiency of DNA repair is affected by chromatin structure, nucleosome accessibility/disruption, and transcriptional activity [24,25]. Chromatin structure is partly controlled by cytosine methylation and covalent modifications of histone proteins including methylation, acetylation, ubiquitination, and phosphorylation [26-29]. Cytosine methylation is catalyzed by Dnmt enzymes in the context of CpG dinucleotides  and is essential for normal mammalian development . Histone variants and modifications also play a major role during DNA repair by marking the lesion, recruiting components of the repair machineries, and facilitating their action .
CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is a protein that intersects with cytosine methylation through Dnmt1  and with histone methylation through the Setd1A and Setd1B histone H3-Lys4 methyltransferase complexes [34,35]. Cfp1 contains several conserved protein domains, including a cysteine-rich CXXC DNA-binding domain that exhibits specificity for unmethylated CpG dinucleotides  and a cysteine-rich Set1 interaction domain (SID), which is required for interaction with the Setd1A and Setd1B histone H3-Lys4 methyltransferase complexes [33,36,37]. Targeted disruption of the CXXC1 gene in mice results in early embryonic lethality at approximately 4.5-6.5 days postcoitus . Interestingly, embryonic stem (ES) cells lacking Cfp1 (CXXC1-/-) are viable but exhibit a variety of defects when compared to wild-type (CXXC1+/+) ES cells. These defects include a lengthened population doubling time due to an increased rate of apoptosis, an inability to achieve in vitro differentiation, and a 70% decrease in global and gene-specific cytosine methylation . CXXC1-/- ES cells also exhibit perturbations in global levels of histone methylation, including increased euchromatin-associated histone H3-Lys4 di- and tri-methylation and decreased heterochromatin-associated histone H3-Lys9 di-methylation . Consequently, Cfp1 plays an important role in the regulation of cytosine and histone methylation, heterochromatin formation, and cellular differentiation.
The purpose of this study was to determine if Cfp1 plays a role in the sensitivity of ES cells to DNA-damaging agents. In these studies, an array of DNA-damaging agents were used, including MMS, TMZ, H2O2, IR, cisplatin, and etoposide, that cause lesions repaired by the BER pathway as well as other pathways (Table 1). Platinum-based drugs such as cisplatin, in addition to causing DNA cross-linking, generate ROS as a byproduct [40-42]. Reduced expression of Ape1/Ref-1 in neuronal cultures enhances cisplatin-induced cell killing as well as apoptosis and ROS generation, indicating that BER and Ape1/Ref-1 are important in the response to these drugs as well . The results reported here demonstrate that CXXC1-/- ES cells exhibit hypersensitivity to these DNA-damaging agents but normal sensitivity to the anti-metabolite/anti-folate agent, methotrexate (MTX), and the microtubule-stabilizing agent, paclitaxel. In addition, CXXC1-/- ES cells express decreased levels of Ape1/Ref-1 protein and AP endonuclease activity and exhibit increased accumulation of DNA damage. Structure-function studies were utilized to determine the domains of Cfp1 that are sufficient to restore normal APE1/Ref-1 protein expression and rescue the hypersensitivity to DNA-damaging agents. These findings reveal that Cfp1 influences sensitivity to DNA-damaging agents and may regulate DNA repair processes.
Generation of CXXC1-/- murine ES cell lines was previously described . DNMT1-/- ES cells were a generous gift from En Li (Novartis Institute for Biomedical Research, Cambridge, MA). ES cell lines were cultured on 0.1% gelatin-coated tissue culture dishes in high-glucose Dulbecco's modified Eagle's medium (GIBCO, BRL, Life Technologies, Grand Island, NY) supplemented with 20% fetal bovine serum (GIBCO, BRL), 100 Units/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen), 0.2% leukemia inhibitory factor-conditioned medium, 100 nM β-mercaptoethanol, 0.025% HEPES pH 7.5, (Invitrogen), and 1% Hank's balanced salt solution (Invitrogen).
To rescue the CXXC1-/- ES cell phenotype, murine Cfp1 cDNA  was subcloned into the pcDNA 3.1/Zeo mammalian expression vector (Invitrogen). The Cfp1 expression vector or the empty expression vector was electroporated into CXXC1-/- ES cells as previously described . For structure-function studies, cDNA constructs encoding full-length FLAG epitope-tagged human Cfp1 (amino acids 1-656) or various Cfp1 truncations and mutations were subcloned into the pcDNA3.1/Hygro mammalian expression vector (Invitrogen). The amino-terminal bipartite nuclear localization signal of Cfp1 (amino acids 109-121) was inserted between the FLAG epitope and Cfp1 sequence for constructs containing amino acids 361-656 and 361-656 C375A. The FLAG epitope was incorporated into each construct to permit convenient evaluation of protein expression levels. Linearized DNA (25 μg) was electroporated into CXXC1-/- ES cells, which were grown in selection medium containing 200 μg/ml hygromycin B (Sigma-Aldrich, St. Louis, MO) for approximately two weeks before single colonies were isolated for expansion, and then maintained in medium containing 50 μg/ml hygromycin B. Expression of FLAG epitope-tagged human Cfp1 was verified by western blot analysis using anti-FLAG (Sigma-Aldrich) or anti-Cfp1 antibodies , as described below. Two or three independent clones for each construct were analyzed, and representative data are shown.
Stock solutions of DNA-damaging and non-genotoxic agents were diluted with ES culture medium to the indicated concentrations, and cells were treated for various amounts of time before being harvested, counted, and plated in triplicate for colony forming assays. Cells were treated with media controls or H2O2 (Sigma-Aldrich) for 2 h, MMS (Sigma Aldrich) for 3 h, or TMZ (LKT Laboratories, St. Paul, MN) for 4 h. Etoposide (Sigma-Aldrich) was prepared as a 50 mM stock solution in dimethyl sulfoxide (DMSO), and cells were treated with etoposide or DMSO (vehicle control) for 3 h. Cis-diammineplatinum (II) dichloride (Cisplatin) (Sigma-Aldrich) was freshly prepared for each experiment as a 20 mM stock solution in DMSO, and cells were treated with cisplatin or DMSO for 3 h. Irradiation was carried out at room temperature using a Co-60 gamma unit (Nordion International, Kanata, Canada) that delivered various doses. After irradiation, the cells were immediately trypsinized, counted, and plated for colony forming assays. Paclitaxel (Sigma-Aldrich) was prepared in DMSO, and cells were treated with paclitaxel or DMSO for 6 h. MTX (Sigma-Aldrich) was freshly prepared in 1 M sodium hydroxide (NaOH), and cells were treated with MTX or NaOH (vehicle control) for 6 h.
Exponentially-growing ES cells were treated with various concentrations of each agent for the indicated times before being trypsinized and counted. For clonogenic survival assays, 400 cells were plated into gelatin-coated, 10 cm tissue culture dishes. Colonies were allowed to form for 7-9 days, and then fixed in methanol:acetic acid (3:1), stained with 0.02% crystal violet, and counted. Colony numbers from the treated samples were normalized to untreated or vehicle control-treated cells and plotted as a function of dose. Data presented represent the average of three independent experiments performed in triplicate.
Global cytosine methylation was assessed utilizing a methyl acceptance assay as previously described . This in vitro methylation reaction quantitates transfer of radiolabled methyl groups from S-adenosyl-L-methionine onto unmethylated genomic CpG dinucleotide sites . Briefly, 500 ng of genomic DNA was incubated with 2 μCi of 3H-methyl-S-adenosyl L-methionine (Amersham, GE Healthcare, Piscataway, NJ; 15 Ci/mmol) and 3 units of SssI (CpG) methylase (New England Biolabs, Ipswich, MA) in 120 mM NaCl, 10 mM Tris-HCl pH 7.9, 10 mM EDTA, and 1 mM dithiothreitol (DTT) for 1 h at 30°C in a total volume of 30 μl. The SssI enzyme was then inactivated by heating the reaction mixture for 10 min at 65°C. In vitro methylated DNA was isolated by filtration through Whatman DE-81 ion-exchange filters (Fisher Scientific, Pittsburgh, PA). The filters were washed five times with 0.5 M sodium phosphate buffer (pH 7.4), air-dried, and the incorporated radioactivity was measured by scintillation counting. Background radioactivity bound to the filter from a reaction mixture lacking DNA was subtracted from the values of the reaction mixtures containing DNA. Methylation reactions were performed in duplicate and the analysis was carried out for three independent experiments.
Cfp1, Dnmt1, and Ape1/Ref-1 protein expression was assessed by harvesting whole cell extracts. Cells were lysed in cytoskeletal buffer (300 mM NaCl, 10% sucrose pH 7.2, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 3% protease inhibitor cocktail [Sigma-Aldrich]) by Dounce homogenization 30 times on ice. Soluble extracts were separated from debris by centrifugation at 16,000 × g for 10 min at 4°C. Protein concentration was quantitated by measuring absorbance at 595 nm using the Bradford Reagent (BioRad, Cambridge, UK) as previously described . In order to separate proteins, 60 μg of whole cell protein was subjected to electrophoresis using PAGEr-Gold pre-cast 4-12% Tris-glycine gradient gels (Lonza Group Ltd., Basel, Switzerland) and transferred to nitrocellulose membranes (Amersham, GE Healthcare). The membranes were probed with rabbit polyclonal Cfp1 , mouse monoclonal Ape1/Ref-1 (Novus Biologicals, Littleton, CA), or β-actin (Lab Vision) antibodies followed by the appropriate horseradish peroxidase-labeled secondary antibody. Proteins were detected with an ECL kit (Amersham, GE Healthcare) and autoradiography, and band intensities were quantitated by densitometry.
Oligonucleotide gel-based Ape1/Ref-1 endonuclease activity assays were modified as a fluorescent assay and performed as previously described . The 26 bp oligonucleotide substrate contained a single THF residue in the middle, yielding a HEX-labeled 13-mer fragment upon excision. Ape1/Ref-1 endonuclease activity was measured by incubating 0.4 pmol of HEX-labeled (2 pmol/ml) excess oligonucleotide substrate with cellular protein in a total volume of 20 μl of assay buffer (50 mM HEPES pH 7.5, 50 mM KCl, 10 mM MgCl2, 1% BSA, and 0.05% Triton X-100) at 37°C for 15 min. Reactions were terminated by adding formamide loading buffer (10 μl) to each sample. The results were detected following electrophoresis and quantitated using the FMBioII fluorescence imaging system and software (Hitachi Genetic Systems, South San Francisco, CA).
Exponentially growing CXXC1+/+ and CXXC1-/- ES cells were treated with 1 μM TMZ (LKT Laboratories) for 4 h. Whole cell protein extracts were collected after treatment, and 10 μg of each extract was utilized in western blotting as previously described [19,48] using mouse monoclonal antibody specific for phosphorylation of H2AX at Ser139 (Upstate, Waltham, MD). Blots were also probed for β-actin (Lab Vision) as a loading control. Bands were detected using a chemiluminescence kit (Pierce, Rockford, IL), visualized using Bio-Rad Chemidoc XRS (Hercules, CA), and quantitated using Chemidoc software, Quantity One 4.6.1.
Atomic absorption spectroscopy was used to quantitate total platinum in DNA as previously described . Exponentially-growing cells were treated with 100, 200, or 300 μM cisplatin or DMSO vehicle controls for 3 h. Genomic DNA was isolated directly after cisplatin treatment by lysing cell pellets with 600 μl of cell lysis buffer (10 mM Tris pH 8.0, 100 mM EDTA, and 0.5% SDS). Cell lysates were then incubated with 4 μl RNAse A (Roche, 10 mg/ml) for 15 min at 37°C, then 200 μl of 7 M ammonium acetate was added before vortexing the sample for 30 sec and incubating on ice for an additional 5 min. Proteins and debris were pelleted by centrifugation, and the resulting supernatants were mixed with 350 μl of isopropanol to precipitate the DNA. DNA pellets were washed with 70% ethanol, air dried, and resuspended in ddH20. Platinum concentration was assessed with a Perkin-Elmer model 1100 flameless atomic absorption spectrometer (Perkin-Elmer, Norwalk, CT) as previously described [49,50].
Statistical significance was assessed by two-tailed student t-tests with equal variance. A P value <0.05 was interpreted as statistically significant.
Wild-type (CXXC1+/+) and CXXC1-/- ES cells were analyzed for clonogenic survival following exposure to increasing amounts of DNA-damaging agents (Table 1). CXXC1-/- ES cells demonstrate significant decreases in cell survival after exposure to IR, MMS, H2O2, TMZ, etoposide, and cisplatin compared to CXXC1+/+ ES cells (Fig. 1A-F). Taken together, these results indicate that CXXC1-/- ES cells are hypersensitive to DNA-damaging agents that cause DNA strand breaks (IR and etoposide), alkylation (MMS, TMZ), DNA cross-links (cisplatin), and oxidative damage (IR, H2O2, cisplatin). Because CXXC1-/- ES cells exhibit an extended population doubling time due to an increase in apoptosis , longer time points for colony formation were additionally analyzed. However, extended incubation time did not alter colony forming numbers for CXXC1-/- ES cells (data not shown).
Due to the variety of chromatin and differentiation defects observed in CXXC1-/- ES cells [34,39], further studies were performed to determine if CXXC1-/- ES cells are hypersensitive to any type of stress. However, there was no significant difference in clonogenic survival between CXXC1+/+ and CXXC1-/- ES cells after treatment with the non-genotoxic agents MTX (Fig. 2A) or paclitaxel (Fig. 2B). These data indicate that the hypersensitivity of CXXC1-/- ES cells is specific to DNA-damaging agents and is not a general sensitivity to cytotoxic agents.
Previous experiments in our laboratory demonstrated that stable expression of full-length wild-type murine Cfp1 in CXXC1-/- ES cells (CXXC1-/cDNA) corrects the epigenetic and differentiation defects observed in CXXC1-/- ES cells . To determine if reconstitution of Cfp1 in CXXC1-/- ES cells additionally rescues the hypersensitivity to DNA-damaging agents, CXXC1+/+, CXXC1-/-, and CXXC1-/cDNA ES cells were analyzed for clonogenic survival after treatment with DNA-damaging agents. For these and the following experiments, TMZ was utilized as a clinically relevant representative alkylating agent, H2O2 was chosen as an oxidizing agent, and cisplatin was utilized as a clinically relevant DNA cross-linking agent. These studies reveal that rescued CXXC1-/cDNA ES cells demonstrate sensitivities to TMZ, cisplatin, and H2O2 similar to those of CXXC1+/+ ES cells (Fig. 3). Therefore, expression of full-length Cfp1 in CXXC1-/- ES cells restores normal sensitivity to DNA-damaging agents, indicating that the observed hypersensitivity is specifically due to loss of Cfp1.
Genomic DNA in CXXC1-/- ES cells may be more accessible to genotoxic agents due to dramatically decreased levels of genomic cytosine methylation, a marker of heterochromatin . To examine whether this could potentially explain the increased sensitivity to DNA-damaging agents, the sensitivity of DNMT1-/- ES cells to DNA-damaging agents was determined. DNMT1-/- ES cells are deficient for maintenance DNA methylation, and suffer a dramatic loss of global cytosine methylation . Using an in vitro methyl acceptance assay, the number of radiolabeled methyl groups incorporated into a genomic DNA sample is inversely related to the native DNA methylation status. These studies reveal that genomic DNA isolated from CXXC1-/- or DNMT1-/- ES cells accept approximately 2.5- and 3-fold more methyl groups than does DNA derived from CXXC1+/+ ES cells, respectively, indicating a ~60% decrease of global cytosine methylation in CXXC1-/- ES cells and a ~67% decrease in DNMT1-/- ES cells (Fig. 4A). Consistent with a previous report , CXXC1-/cDNA ES cells exhibit normal levels of cytosine methylation, indicating that the global loss of cytosine methylation observed in CXXC1-/- ES cell DNA is specifically due to loss of Cfp1.
To determine the sensitivity of DNMT1-/- ES cells to DNA-damaging agents, DNMT1-/-, CXXC1+/+, and CXXC1-/- ES cells were treated with TMZ, cisplatin, or H2O2 and analyzed for clonogenic survival. These studies reveal that DNMT1-/- ES cells are not hypersensitive to TMZ, cisplatin, or H2O2 (Fig. 4 B-D). In addition, DNMT1-/- ES cells exhibit a sensitivity to the non-genotoxic agents MTX and taxol similar to that of CXXC1+/+ ES cells (data not shown). Thus, DNMT1-/- ES cells exhibit a normal sensitivity to DNA-damaging agents, indicating that the observed hypersensitivity of CXXC1-/- ES cells cannot be solely explained by a reduction in global cytosine methylation.
CXXC1-/- ES cells exhibit increased sensitivity to TMZ, MMS, H2O2, cisplatin, and IR, agents that generate lesions that can be repaired by the BER pathway. Furthermore, reduced expression of Ape1/Ref-1 in various cell types results in hypersensitivity to these agents (20-27). Therefore, the expression level and endonuclease activity of Ape1/Ref-1 was evaluated in CXXC1+/+, CXXC1-/-, and DNMT1-/- ES cells. Western blot analysis revealed a ~50% decrease in Ape1/Ref-1 protein expression in CXXC1-/- ES cells, and a slight increase in Ape1/Ref-1 protein expression in DNMT1-/- ES cells (Fig. 5A). In addition, expression of full-length Cfp1 in CXXC1-/- ES cells (CXXC1-/cDNA) is sufficient to reconstitute Ape1/Ref-1 protein expression back to levels observed in CXXC1+/+ ES cells (Fig. 5A). In contrast, expression of another downstream component involved in the BER pathway, XRCC1, is unchanged in CXXC1-/- ES cells (data not shown).
Ape1/Ref-1 endonuclease activity was measured by incubation of cellular protein extracts with a 26-mer HEX-labeled oligonucleotide substrate that contains an AP site. Consistent with Ape1/Ref-1 protein levels, Ape1/Ref-1 endonuclease activity is significantly decreased (40-50%) in CXXC1-/- ES cells compared to CXXC1+/+ ES cells, and slightly increased in DNMT1-/- ES cells (Fig. 5B). Western blot analysis of the protein samples analyzed in the endonuclease activity assay confirmed equal protein loading (Fig. 5B, bottom panel).
Immunoprecipitation was performed to determine if Ape1/Ref-1 physically interacts with Cfp1. Full-length FLAG epitope-tagged Cfp1 was expressed in human embryonic kidney cells (HEK-293), immunoprecipitated, and interacting proteins were detected by western blot analysis. However, Ape1/Ref-1 was not detected as a pull-down product with Cfp1 (data not shown). Co-immunoprecipitation of Cfp1 with the histone methyltransferase Setd1A was used as a positive control . Ape1/Ref-1 sub-cellular localization is controlled by a strictly regulated process and is quite variable between cell types and disease states . However, confocal immunofluorescence microscopy revealed no difference in the sub-cellular localization of Ape1/Ref-1 in CXXC1-/- ES cells compared to CXXC1+/+ ES cells. Each cell line demonstrated a punctate staining of Ape1/Ref-1 distributed mostly within the nucleus with very little cytoplasmic localization (data not shown).
To determine whether the increased sensitivity of CXXC1-/- ES cells to cisplatin correlates with higher levels of DNA damage, atomic absorption spectroscopy was used to evaluate the total amount of platinum in DNA isolated from CXXC1+/+ or CXXC1-/- ES cells treated with cisplatin for three hours. These studies reveal a significant increase in the amount of platinum/μg DNA in CXXC1-/- ES cells compared to CXXC1+/+ ES cells after treatment with cisplatin (Fig. 6A).
As an independent assessment of DNA damage, the level of histone H2AX phosphorylation (H2AX-γ), a marker of double strand breaks (DSBs), was measured in CXXC1+/+ and CXXC1-/- ES cells following TMZ treatment. Repair of DSBs is expected to be important in ES cells due to the high basal level of H2AX-γ  which we also observed (data not shown). Immediately after treatment with TMZ, there is a significant increase in H2AX-γ levels in CXXC1-/- ES cells compared to CXXC1+/+ ES cells (Fig. 6B). The levels of H2AX-γ were normalized to β-actin expression, and the graph presents relative H2AX-γ expression compared to untreated controls. No increase in annexin V staining was detected in cells similarly treated with TMZ (data not shown). Thus, the increased level of H2AX-γ in Cfp1-null ES cells following TMZ exposure is not an indirect consequence of DNA fragmentation associated with apoptosis.
Expression of full-length Cfp1 (aa 1-656) in CXXC1-/- ES cells rescues the hypersensitivity to cisplatin, TMZ, and H2O2 (Fig. 3), thereby providing a convenient method to assess structure-function relationships of Cfp1. Various FLAG epitope-tagged Cfp1 fragments were expressed in CXXC1-/- ES cells to identify functional properties of Cfp1 that are sufficient to restore normal sensitivity to DNA-damaging agents. Stably transfected clones were selected, and clones were screened for protein expression by western blot analysis using an anti-Cfp1 antibody (Fig. 7). Multiple clones were analyzed for each construct and representative data are shown. ES cells heterozygous for the disrupted CXXC1 allele express ~50% of Cfp1 protein compared to CXXC1+/+ ES cells but do not exhibit the epigenetic and differentiation defects observed in CXXC1-/- ES cells . In addition, CXXC1+/- ES cells are not hypersensitive to TMZ, cisplatin, or H2O2 (data not shown). Consequently, clones that express at least 50% of the level of Cfp1 expression observed in CXXC1+/+ ES cells were selected for analysis.
Remarkably, CXXC1-/- ES cells expressing either the amino half of Cfp1 (aa 1-367, containing the PHD1, CXXC, acidic, and basic domains) or the carboxyl half of Cfp1 (aa 361-656, containing the coiled coil, SID, and PHD2 domains) exhibit a sensitivity to TMZ and cisplatin similar to that of wild-type ES cells and CXXC1-/- ES cells expressing full-length Cfp1 1-656 (Fig. 8). These data reveal that Cfp1 contains redundant functional domains capable of rescuing the hypersensitivity phenotype, and indicate that no single Cfp1 domain is required to rescue the hypersensitivity to DNA-damaging agents.
The amino half of Cfp1 (aa 1-367) contains the CXXC DNA-binding domain , and the carboxyl half of Cfp1 (aa 361-656) contains the SID domain that mediates interaction with the Setd1A and Setd1B histone H3-Lys4 methyltransferase complexes . To define further the functional domains required for Cfp1-mediated rescue activity, Cfp1 point mutations that abolish DNA-binding activity of Cfp1 (C169A)  or ablate the interaction of Cfp1 with the Setd1 methyltransferase complexes (C375A)  were analyzed (Fig. 9A).
CXXC1-/- ES cells expressing full-length Cfp1 lacking DNA-binding activity (1-656 C169A) or lacking interaction with the Setd1 histone H3-Lys4 methyltransferase complexes (1-656 C375A) exhibit sensitivity to TMZ and cisplatin similar to that of CXXC1-/- ES cells expressing full-length Cfp1 (Fig. 9B,C). Hence, neither DNA-binding activity of Cfp1 nor association with the Setd1 histone H3-Lys4 methyltransferase complexes is required to rescue hypersensitivity to DNA-damaging agents. In contrast, abolishing DNA-binding activity ablates the rescue activity of the Cfp1 1-367 fragment (1-367 C169A), and abolishing interaction with the Setd1 complexes ablates the rescue activity of the 361-656 Cfp1 fragment (361-656 C375A). Mutation of both DNA-binding and Setd1 interaction domains within full-length Cfp1 also ablates rescue activity (Fig. 9). Therefore, retention of either Cfp1 DNA-binding or Setd1 interaction domains is required to rescue the hypersensitivity of CXXC1-/- ES cells to TMZ and cisplatin.
Western blot analysis was performed to determine the level of Ape1/Ref-1 protein expression in CXXC1-/- ES cells carrying various Cfp1 mutations. Cfp1 mutations that fail to rescue the hypersensitivity to TMZ and cisplatin (Cfp1 1-367 C169A, 361-656 C375A, and 1-656 C169A, C375A) also fail to rescue Ape1/Ref-1 protein expression in CXXC1-/- ES cells (Fig. 10). In contrast, CXXC1-/- ES cells expressing Cfp1 mutations that rescue the hypersensitivity to TMZ and cisplatin (Cfp1 1-656, 1-656 C169A, 1-656 C375A, 1-367, and 361-656) also express normal levels of Ape1/Ref-1 protein (Fig. 10). These results reveal a strong correlation between Ape1/Ref-1 protein expression levels in CXXC1-/- ES cells and sensitivity to DNA-damaging agents.
The studies reported here demonstrate that CXXC1-/- ES cells are hypersensitive to a variety of DNA-damaging agents, including IR, H2O2, TMZ, MMS, cisplatin, and etoposide. CXXC1-/- ES cells also exhibit a ~50% decrease in expression of the DNA repair protein Ape1/Ref-1, and a concomitant decrease in Ape1/Ref-1 endonuclease activity. CXXC1-/- ES cells exhibit increased platinum incorporation into DNA after treatment with cisplatin, and elevated levels of H2AX-γ formation after treatment with TMZ.
The structure of chromatin is a major factor affecting radio-sensitivity and DNA repair efficiency . For example, mitochondrial DNA is approximately 10-fold more sensitive to DNA-damaging agents compared to nuclear DNA [54-56]. Given that mitochondrial DNA is not packaged as chromatin, it has been inferred that chromatin confers protection against DNA-damaging agents. HDAC inhibitors, such as trichostatin A (TSA), increase the acetylation of core histones, resulting in an open chromatin configuration that is more accessible to DNA-damaging agents . TSA increases chromatin accessibility and sensitizes K562 cells to IR [58,59]. HDAC inhibitors also sensitize tumor cells to the induction of cell death by IR, ultraviolet radiation, and several DNA-damaging drugs [57,60-62]. In addition, H2AX-γ persists longer in melanoma cells after IR exposure in combination with sodium butyrate, another HDAC inhibitor. Sodium butyrate also causes an increase in etoposide-induced apoptosis in leukemia cells .
CXXC1-/- ES cells exhibit a ~70% decrease in global cytosine methylation  and altered patterns of histone methylation that correlate with decreased amounts of heterochromatin . Therefore, CXXC1-/- ES cells may exhibit elevated levels of DNA packaged in accessible chromatin, and the increased sensitivity to DNA-damaging agents and increased accumulation of DNA damage observed in CXXC1-/- ES cells may be due, at least in part, to epigenetic alterations that result in decreased heterochromatin. However, previous reports also suggest that the more open configuration of DNA could result in an increased accessibility for DNA repair proteins [64,65]. This does not appear to be the case in this study, as neither Dnmt1 nor Cfp1 deficiency resulted in improved survival.
Structure-function analysis reveals that expression in CXXC1-/- ES cells of either the amino half of Cfp1 (aa 1-367) or carboxyl half of Cfp1 (aa 361-656) is sufficient to restore normal Ape1/Ref-1 protein expression and rescue the hypersensitivity to DNA-damaging agents. Thus, Cfp1 contains redundant functional domains, and no single Cfp1 protein domain is essential for rescue activity. Additional studies revealed that retention of either DNA-binding or Setd1 interaction domains of Cfp1 is required to restore normal Ape1/Ref-1 protein expression and rescue the hypersensitivity of CXXC1-/- ES cells to DNA-damaging agents. Interestingly, other studies in our laboratory reveal an identical requirement for the retention of either the DNA-binding domain or Setd1 complex interaction domain of Cfp1 for rescue of deficient cytosine methylation, aberrant histone methylation, and in vitro differentiation in CXXC1-/- ES cells . The finding of redundant functional Cfp1 domains might be explained by cross-talk between epigenetic modifications. It is thought that cytosine methylation and histone modifications are highly integrated and mutually reinforcing mechanisms that establish and maintain appropriate chromatin structure [67-73]. Thus, rescue of normal cytosine methylation might provide critical epigenetic marks to permit restoration of appropriate histone modifications, and rescue of appropriate histone methyltransferase regulation might permit subsequent restoration of appropriate patterns of cytosine methylation.
Increased sensitivity of CXXC1-/- ES cells to DNA-damaging agents may also be due to a decrease in DNA repair activity. CXXC1-/- ES cells exhibit a ~50% decrease in Ape1/Ref-1 protein expression and endonuclease activity. Ape1/Ref-1 is a critical component of the BER pathway, and provides >95% of the endonuclease activity in mammalian cells . Consistent with the data presented here, reduction of Ape1/Ref-1 protein by specific anti-sense oligonucleotides or siRNA renders mammalian cells hypersensitive to a variety of laboratory and chemotherapeutic agents, including MMS, H2O2, bleomycin, TMZ, and cisplatin [20,22,23,42,75,76]. For example, suppression of Ape1/Ref-1 in rat glioma cells increases sensitivity to MMS and H2O2 , and suppression of Ape1/Ref-1 in a human glioma line and human medulloblastoma cell line results in decreased AP endonuclease activity, increased abasic site content, and increased sensitivity to 1,3-bis(2-chloroethyl)-nitrosourea (BCNU), MMS, and TMZ [75,78]. In these studies, we also observe an increase in sensitivity to the DNA crosslinking agent, cisplatin which creates crosslinks on DNA that are repaired by nucleotide excision repair and homologous recombination. However, cisplatin also generates ROS, [40-42] and Ape1/Ref-1 is well-established as a key enzyme in the repair of oxidative DNA damage . To ensure genomic integrity, there is crossover, interaction and compensation within and between the various DNA repair pathways. This notion is underscored by recent data which illustrates that inhibition of the BER protein PARP (poly(ADP-ribose) polymerase) enhances agents such as cisplatin, doxorubicin, and cyclophosphamide [85,86], as well as data reported here suggesting that decreased Ape1/Ref-1 levels in CXXC1-/- ES cells contribute to the response to a variety of DNA damaging agents. Furthermore, it is probable that BER and Ape1/Ref-1 play a role in repairing the DNA damage induced by the ROS produced secondarily by exposure to these types of agents.
In addition, APE1/REF-1+/- mice exhibit a 40-50% reduction in Ape1/Ref-1 protein and endonuclease activity in all tissues examined [79,80], and display a 35-50% reduction in BER activity [80,81]. APE1/REF-1+/- mice exhibit increased spontaneous mutation frequencies in somatic tissues and spermatogenic cells . APE1/REF-1+/- mice also display hypersensitivity to oxidative stress in vivo, and APE1/REF-1+/- mouse embryonic fibroblasts (MEFs) exhibit diminished survival following exposure to oxidative DNA-damaging agents . Taken together, this literature suggests that the ~50% decrease in Ape1/Ref-1 protein expression and endonuclease activity observed in CXXC1-/- ES cells may contribute to the observed hypersensitivity to DNA-damaging agents. Consistent with this idea, DNMT1-/- ES cells, which exhibit less global cytosine methylation than CXXC1-/- ES cells, express normal levels of Ape1/Ref-1 and exhibit a normal sensitivity to TMZ, H2O2, and cisplatin. Therefore, a global decrease in cytosine methylation cannot solely account for the hypersensitivity to DNA-damaging agents exhibited by CXXC1-/- ES cells. Further work is necessary to ascertain the mechanism of reduced Ape1/Ref-1 expression in CXXC1-/- ES cells. Cfp1 functions as a transcriptional activator in co-transfection assays [36,82], and may play a role in activating transcription of Ape1/Ref-1 through its CpG island-containing promoter [77,83].
In addition to endonuclease activity, Ape1/Ref-1 possesses other activities that are both related and unrelated to its function in DNA repair including 3′phosphodiesterase activity, 3′-phosphatase activity, 3′–5′ exonuclease activity, reduction of key cysteine residues located in the DNA-binding domains of transcription factors such as AP1 (Fos/Jun), p53, and HIF-1α, as well as negatively regulating the Rac1 GTPase to prevent oxidative stress , all of which could contribute to the effects observed here. The causal relationship of reduced Ape1/Ref-1 expression to the observed hypersensitivity of CXXC1-/- ES cells, and the functional domains of Ape1/Ref-1 required for rescue, can be further investigated by introducing into CXXC1-/- ES cells expression vectors containing wild-type Ape1/Ref-1 as well as Ape1/Ref-1 mutants that are deficient in DNA repair or redox function .
Histone modifications are important regulators of BER. For example, in vitro data demonstrate that the ATP-dependent chromatin remodeler SWI/SNF stimulates the processing of 8-oxo-7,8 dihydroguanine during BER . Many chromatin-associated proteins are implicated in DNA repair, and it is well established that DNA becomes more accessible during DNA repair. Nucleosomes are unpackaged, allowing the DNA repair machinery to access the DNA lesion, followed by reassembly of nucleosomes in newly replicated regions, as well as the restoration of the local epigenetic state of the chromatin [11,93,94]. CXXC1-/- ES cells exhibit increased levels of histone H3-Lys4 tri-methylation, suggesting that Cfp1 restricts the activity of the Setd1 complexes . Therefore, the hypersensitivity to DNA-damaging agents and the increased accumulation of DNA damage observed in CXXC1-/- ES cells may be a consequence of aberrant histone methylation and potentially aberrant regulation/targeting of histone methyltransferase complexes which might lead to a reduction in DNA repair.
The studies reported here implicate Cfp1, an epigenetic regulator of cytosine methylation and histone methylation, in DNA repair processes. CXXC1-/- ES cells provide a unique system with which to investigate how chromatin conformation impacts the effectiveness of DNA-damaging or chemotherapeutic agents. Such insights may lead to more efficacious combinations of chemotherapy in cancer patients, resulting in better overall patient outcomes.
We thank Dr. Mark Kelley and Dr. Joe Dynlacht for helpful discussions, and Dr. En Li for providing the DNMT1-/- ES cell line. This work was supported by the Riley Children's Foundation; the Lilly Endowment; the National Science Foundation [grant numbers MCB-0344870, MCB-0641851 (to D.G.S.)]; a pre-doctoral fellowship from National Institutes of Health [grant number T32 AI060519 (to C.M.T.)]; a Department of Education training grant for Graduate Assistance in Areas of National Need (GAANN) (to C.M.T.); and the National Cancer Institute [grant number CA122298 (to M.L.F.)].
Conflict of interest statement: None
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