CCAAT/enhancer binding protein delta (C/EBPδ, CEBPD)-mediated nuclear import of FANCD2 by IPO4 augments cellular response to DNA damage

C/EBPδ Interacts with FANCD2.

To understand the molecular functions of C/EBPδ, we explored which proteins it can interact with. Ectopic C/EBPδ was immunoprecipitated from 293T cells, and proteins to which it bound were identified using mass spectrometry (Table S1). This approach suggested that C/EBPδ interacts with FANCD2. Both these proteins are predominantly nuclear, and their expression is down-regulated in breast cancer (19–21). Furthermore, primary C/EBPδ-null MEFs exhibit genomic instability, including triradial chromosomes (18), which are a hallmark of FA-deficient cells (22). For these reasons we decided to pursue the significance of C/EBPδ interaction with FANCD2. Fig. 1A illustrates the various C/EBPδ expression constructs used in this study. The nuclear localization signal of C/EBPδ lies within its DNA-binding domain (23). Therefore, mutants lacking this domain do not enter the nucleus. However, the R198A point mutation, which inhibits sequence-specific DNA binding of C/EBPδ, does not interfere with nuclear localization (24, 25). First, we confirmed the interaction between C/EBPδ and FANCD2 in 293T cells by coimmunoprecipitation (co-IP) assay. As shown in Fig. 1B, endogenous FANCD2 interacted with wild type (WT) C/EBPδ, with a protein truncated at residue 196 (ΔDBD), and with an internal fragment comprising amino acids 140–215 but not with a mutant truncated at residue 151. A mutant with an internal deletion of amino acids 153–199 was unable to coprecipitate FANCD2 (Fig. 1C). We therefore termed this 47-aa region the FANCD2 interaction domain (D2ID). WT C/EBPδ interacted with both the unubiquitinated “S” form of FANCD2 (FANCD2-S) and the monoubiquitinated “L” form (FANCD2-L) in MMC-treated or untreated 293T cells (Fig. 1C), suggesting that potential modifications of either protein in response to DNA damage are not necessary for and do not interfere with their interaction.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Interaction of C/EBPδ and FANCD2 proteins. (A) Schematic representation of mutations in C/EBPδ constructs. R198A is the full-length protein with an R-to-A mutation at residue 198; ΔDBD is a deletion of the DNA-binding basic region (BR) and leucine zipper (LZ); ΔD2ID is a deletion of amino acids 153–199. TAD, transactivation domain. (B) FANCD2 co-IP analysis of 293T cells transfected with Flag-tagged C/EBPδ constructs. Immunoprecipitates (IP) and input samples were analyzed with anti-FANCD2 and anti-Flag antibodies. The multiple bands generated by amino acids 140–215 probably are caused by phosphorylation events because this region harbors several kinase recognition motifs. (C) C/EBPδ co-IP analysis from 293T cells as in B. (D) WT and KO MEFs were treated for 3 h with 5 μg/mL MMC. Fixed cells were incubated with antibodies against C/EBPδ and FANCD2 followed by OLink in situ PLA and fluorescence microscopy using appropriate filters (DAPI, blue; PLA, red). (Fig. S1 shows single-channel images.) (E) FANCD2-deficient cells (PD20) and cells reconstituted with FANCD2 (PD20-D2) were treated for 20 h with 500 ng/mL MMC before processing as described in D. (F) Western blot analysis of whole-cell lysates from WT and C/EBPδ-KO MEFs, PD20-D2 or PD20 cells, or the human breast epithelial cell lines MCF-10A, MCF-7, MDA-MB-468, and SKBR-3; treated for 20 h with 1 μg/mL MMC (100 ng/mL for PD20/-D2 cells). (G) Immunoprecipitation from MDA-MB-468 cells with anti-C/EBPδ antibody (Rockland) and input samples were analyzed with anti-FANCD2 and anti-C/EBPδ (BD Biosciences) antibodies.

To detect interaction of endogenous C/EBPδ and FANCD2, we used the DuoLink in situ proximity ligation assay (PLA). In this assay, two proteins are immunostained with opposite species-specific secondary antibodies that are linked to complementary oligonucleotides. When two different antibody molecules bind in close proximity, the linked DNA can be amplified and visualized with a fluorescent probe as distinct foci. Each signal spot may represent one molecule of each of two interacting proteins. In WT MEFs, the interaction between C/EBPδ and FANCD2 was clearly detectable in both the cytoplasm and the nucleus (Fig. 1D and Fig. S1A). No significant signal was generated in C/EBPδ-null (KO) MEFs because of the absence of C/EBPδ protein (Fig. 1D and Fig. S1A). To verify this interaction further, we obtained human FANCD2-null PD20 fibroblasts, which indeed generated no signal in the PLA assay (Fig. 1E and Fig. S1B). However, in cells reconstituted with FANCD2 (PD20-D2), an interaction with C/EBPδ was again detectable (Fig. 1E and Fig. S1B). Because this assay detects FANCD2 and C/EBPδ only when they are in complex, and C/EBPδ does not localize to DNA-damage foci (SI Results), this assay does not visualize FANCD2 in the DNA-damage foci. As a negative control, we assessed the combination of FANCA and C/EBPδ antibodies in PD20-D2 cells, which did not generate any signal in this assay (Fig. S1C). In summary, this approach demonstrated specific interaction of endogenous C/EBPδ and FANCD2.

In PD20-D2 cells, in particular, there also was a shift of signal from more cytoplasmic to nuclear localization in response to MMC. Moreover, we noted that in MEFs the PLA signal increased with MMC in both the cytoplasm and the nucleus. Given that the co-IP assays with overexpressed C/EBPδ did not indicate an effect of MMC on interaction per se (Fig. 1C), we asked whether expression of endogenous C/EBPδ changed upon DNA damage. Indeed, C/EBPδ protein levels were induced in several different cell types, such as MEFs, PD20/-D2, and human breast tumor cell lines (Fig. 1F). In contrast, MMC did not induce C/EBPδ expression in untransformed human MCF-10A breast epithelial cells, which have higher basal levels of C/EBPδ (25) (Fig. 1F). To assess further the interaction of endogenous C/EBPδ and FANCD2 proteins, we performed co-IP assays in MDA-MB-468 cells and confirmed that both FANCD2-S and FANCD2-L associate with C/EBPδ in the presence and absence of MMC treatment (Fig. 1G).

Taken together, induction of C/EBPδ expression in response to DNA damage and the interaction of C/EBPδ with FANCD2 observed in mouse and human fibroblasts and in human breast cancer cells suggest that C/EBPδ may play a role in the cellular response to DNA damage.

C/EBPδ Augments Cell Survival in Response to DNA Damage.

Because we had found that C/EBPδ interacts with FANCD2, we assessed the role of C/EBPδ in cell survival in response to MMC by comparing cells from WT and C/EBPδ-KO mice. The number of colonies formed by primary mouse bone marrow cells diminished when the cells were cultured in the presence of increasing concentrations of MMC (Fig. 2A). Depending on the MMC concentration, C/EBPδ-null bone marrow cells formed 19–54% fewer colonies than WT cells. In the absence of MMC, there was no difference in colony numbers between genotypes (Fig. 2B). This result shows that C/EBPδ promotes cell survival in the presence of MMC. To test the role of C/EBPδ in response to acute damage, MEFs were treated with MMC for 2 h, and cell numbers were evaluated 1 wk later. Loss of C/EBPδ significantly impaired cell survival after MMC at concentrations of 5 μg/mL or higher (Fig. 2C). Consistent with these observations, lack of C/EBPδ led to 5- to 6-fold increased cell death after 24 h or 72 h of MMC exposure (Fig. 2D). Concomitantly, KO cells were lost more rapidly from the G1 phase of the cell cycle (Fig. 2D). Profiling the distribution of living cells among cell-cycle phases showed that C/EBPδ-null cells were enriched at 4N DNA content (G2) under all conditions (Fig. 2E), a finding that is consistent with the arrest in late S-phase and G2 as seen in FA pathway-deficient cells (26, 27). Interestingly, C/EBPδ-null cells exhibited significantly more cells in S-phase, both untreated and after 8 h MMC treatment, perhaps because of the elevated levels of cyclin D1 in these cells (25). However, after 24 h of MMC exposure, virtually all living WT and KO cells were either in G1 or G2, with C/EBPδ-null cells being enriched at G2 (4N). Collectively, these data show that, like FA cells (5, 28), C/EBPδ-deficient cells are hypersensitive to MMC, although the phenotype is milder than in FA cells.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Analysis of cell survival in response to DNA damage. (A) Bone marrow cell colony formation assay. Primary total bone marrow cells from WT and C/EBPδ-KO mice were cultured in methylcellulose-based medium with MMC at the indicated concentrations. Colonies were counted 10–14 d later. Data are mean ± SEM of four mice per genotype; each experiment was performed in triplicate. Controls were set at 1. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Average number of colonies (mean ± SEM) in dishes without MMC from the experiments in A. (C) Analysis of cell viability. Immortalized WT and KO MEFs were treated for 2 h with MMC as indicated. Medium was changed, and cell viability was assessed 1 wk later. Data are means ± SEM of three experiments; controls were set at 100%. *P < 0.05. (D) Analysis of cell death. Primary WT and KO MEFs were treated with 500 ng/mL MMC for the indicated times. Shown is the fraction of cells at G1 and at sub-G1 DNA content as a measure of cell death from three independent MEF isolates (mean ± SEM) analyzed by flow cytometry. ***P < 0.001. (E) Analysis of cell-cycle distribution in primary WT and KO MEFs. The cellular DNA content was determined by flow cytometry as a measure of cell-cycle phase in cells treated with 1 μg/mL MMC for the indicated times. Shown is the distribution of living cells (%) in the G1, S, and G2 phases from three independent MEF isolates for each genotype (mean ± SEM). *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.00001.

To investigate whether DNA-repair mechanisms are deficient in C/EBPδ-null cells, we assessed the amount of γH2AX, a marker for sites of DNA damage (2). As shown previously (25), untreated KO MEFs contained more γH2AX foci than controls (Fig. S2A). MMC treatment caused an increase in the number of γH2AX foci (Fig. S2A) and in γH2AX levels in cell lysates (Fig. S2B). This response was more pronounced in KO MEFs than in controls. These data are consistent with a DNA-repair defect in C/EBPδ-null cells and support the notion that C/EBPδ interaction with FANCD2 may be functionally relevant.

C/EBPδ Augments Monoubiquitination of FANCD2.

To investigate further the consequence(s) of C/EBPδ interaction with FANCD2, we compared FANCD2 protein in C/EBPδ WT and KO MEFs. There was no significant difference in the steady-state levels of FANCD2 (Fig. 3A). However, upon MMC treatment, KO MEFs exhibited less monoubiquitinated FANCD2-L. This phenotype could be partially rescued by ectopic C/EBPδ (Fig. 3A), suggesting a direct role for C/EBPδ in FANCD2 monoubiquitination. Similar results were obtained in a HEK293 cell line with a tetracycline-inducible C/EBPδ transgene (25) in which C/EBPδ augmented the shift of FANCD2-S to FANCD2-L by MMC (Fig. S3). To assess the functional significance of this observation, we compared the survival response of these cells. Indeed, tetracycline significantly enhanced the survival in the presence of MMC specifically of 293-C/EBPδ cells but not of the parental 293 cells (Fig. 3B). Furthermore, overexpression of WT C/EBPδ improved the survival of MMC-treated, FANCD2-reconstituted PD20-D2 cells by about 30%. However, C/EBPδ did not promote survival of FANCD2-deficient PD20 cells under these conditions (Fig. 3C). Importantly, the C/EBPδ mutant lacking the FANCD2 interaction domain (ΔD2ID) was unable to promote survival of PD20-D2 cells (Fig. 3C). Consistent with these results, FANCD2 monoubiquitination was augmented specifically by WT C/EBPδ but not by ΔD2ID-C/EBPδ in PD20-D2 cells (Fig. 3D). On the other hand, silencing of endogenous C/EBPδ expression reduced the ratio of FANCD2-L to -S isoforms in MMC-treated PD20-D2 cells (Fig. 3E). Taken together, these data demonstrate that C/EBPδ promotes monoubiquitination of FANCD2 and cell survival in response to MMC by interacting directly with FANCD2.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

C/EBPδ augments FANCD2 monoubiquitination and cell survival in response to MMC. (A) C/EBPδ-KO MEFs were transfected with Flag-tagged WT mouse C/EBPδ or vector control for 16 h before treatment with 500 ng/mL MMC for 20 h. Whole-cell lysates were analyzed for expression of C/EBPδ and FANCD2 in comparison with WT MEFs. Tubulin was used as loading control. L/S, quantification of the FANCD2-L/S ratio in lysates of MMC-treated cells. (B) 293-C/EBPδ and 293P cells were seeded in 96-well plates and 16 h later were treated with tetracycline (Tet; 500 ng/mL) and/or MMC (1 μg/mL) for the indicated times before relative quantification of cell numbers. Data represent mean ± SEM of two experiments, each done in six wells per data point, shown relative to untreated controls (set at 1 for each time point). **P < 0.01 comparing MMC-treated cells with or without C/EBPδ induction by tetracycline. (C) PD20-D2 and PD20 cells were transfected with Flag-tagged WT C/EBPδ, the ΔD2ID mutant, or vector control for 8 h before being placed in 96-well dishes at 5,000 cells/well. The next day, MMC (100 ng/mL) was added, and cell viability was assessed 48 h later. Data are mean ± SEM of three experiments, each performed in triplicate, and relative to cells without MMC (set at 100%). **P < 0.01 relative to vector and ΔD2ID transfected cells. (D) Western blot analysis of FANCD2 and C/EBPδ in PD20-D2 and PD20 cells treated as in C. C/EBPδ was detected by anti-Flag antibodies. (E) PD20-D2 cells were transfected with shRNA constructs against C/EBPδ (+) or GFP (-) as control and 24 h later were treated with 500 ng/mL MMC for another 24 h. Whole-cell lysates were analyzed for FANCD2 and C/EBPδ expression.

C/EBPδ Augments Nuclear Import of FANCD2.

To investigate the mechanism by which C/EBPδ promotes FANCD2 monoubiquitination, we introduced various C/EBPδ constructs into HEK293 cells. MMC-induced monoubiquitination was promoted by WT C/EBPδ and the R198A mutant but not by the ΔDBD or ΔD2ID mutants (Fig. 4A). Because monoubiquitination occurs in the nucleus, and our previous results suggested that MMC may cause a shift of C/EBPδ-FANCD2 complexes from the cytoplasm to the nucleus (Fig. 1 D and E), we analyzed the subcellular localization of these proteins by cell fractionation. Although most FANCD2 protein was in the nucleus, MMC triggered further translocation of cytoplasmic FANCD2 to the nucleus (Fig. 4 B and C). Interestingly, WT C/EBPδ and the R198A mutant promoted MMC-induced nuclear translocation of FANCD2, as seen by the loss of cytoplasmic FANCD2-S (Fig. 4B) and increased nuclear FANCD2-L (Fig. 4C). In contrast, C/EBPδ mutants that did not promote monoubiquitination also failed to augment nuclear translocation and were deficient in either nuclear localization (ΔDBD) or FANCD2 interaction (ΔD2ID). These observations suggested that C/EBPδ facilitates the nuclear import of FANCD2 by direct protein interaction. In agreement with this conclusion, analysis of cell fractions revealed reduced FANCD2 levels in the nuclear protein extracts of C/EBPδ-null MEFs (Fig. S4). Additional experiments with FANCA-deficient cells show that FANCD2 monoubiquitination is not required for its nuclear import and that induction of C/EBPδ expression by MMC does not require the FA pathway (SI Results and Fig. S5).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

C/EBPδ augments nuclear import of FANCD2. HEK293 cells were transfected with Flag-tagged C/EBPδ expression constructs and treated with MMC (1 μg/mL) for 24 h. (A) Whole-cell (WE), (B) cytoplasmic (CE), and (C) nuclear (NE) extracts were analyzed for expression of FANCD2 and C/EBPδ (anti-Flag). Tubulin and H2AX are shown as loading controls. The purity of cytoplasmic versus nuclear fractions also is shown in Fig. S7.

Most proteins require active, selective transport mechanisms to enter the nucleus (29). A number of nuclear import complexes have been identified, consisting of either importin α or importin β cargo-recognition molecules. Usually, substrate recognition and binding occurs through nuclear localization signals (NLSs) that are composed of basic residues and reside within DNA- or RNA-binding domains (30). The mass spectrometry results suggested that importin 4 (IPO4) interacts with C/EBPδ (Table S1). Co-IP studies demonstrated that all C/EBPδ variants that contained the NLS (Fig. 1A) could interact with IPO4 in HEK293 cells, whereas the ΔDBD mutant did not (Fig. 5 A and B), consistent with its exclusively cytoplasmic localization (Fig. 4 B and C). As shown previously (Fig. 1 B and C), all mutants except ΔD2ID interacted with FANCD2. On the other hand, IPO4 did not interact with FANCD2 unless WT- or R198A-C/EBPδ was present, as determined by co-IP of either IPO4 (Fig. 5B) or FANCD2 (Fig. 5C). In the presence of ΔDBD or ΔD2ID mutants, IPO4 did not interact with FANCD2. The ΔDBD mutant interacted only with FANCD2, whereas the ΔD2ID mutant interacted only with IPO4. Taken together, these interaction studies show that C/EBPδ mediates the indirect association of FANCD2 with IPO4 through its interaction with both of these proteins via separate domains (Fig. 5D). Although none of the interactions was altered in the presence or absence of MMC, the data suggest that they are a prerequisite for the MMC-induced nuclear translocation of FANCD2 in the presence of C/EBPδ.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 5.

C/EBPδ mediates FANCD2 interaction with IPO4. HEK293 cells were transfected with Flag-tagged C/EBPδ constructs and treated with MMC as indicated. Lysates were immunoprecipitated (IP) with (A) anti-Flag antibody for C/EBPδ, (B) anti-IPO4 antibody, or (C) anti-FANCD2 antibody or respective IgG control. LE, long exposure; SE, short exposure. Western blots of input (shown in A) and immunoprecipitates were analyzed for expression of the indicated proteins. In this experiment, C/EBPδ was detected by anti-C/EBPδ antibody (BD69319; BD Biosciences). (D) Schematic (not drawn to scale) indicating how FANCD2 and IPO4 interact with different domains of C/EBPδ but not with each other (see Fig. 1A for designation of C/EBPδ domains).

IPO4 Participates in the DNA-Damage Response.

To assess directly the role of IPO4 in nuclear import of FANCD2 and C/EBPδ, we silenced IPO4 in MDA-MB-468 cells. These cells were chosen because of the expression levels of endogenous C/EBPδ, FANCD2, and IPO4 and because of the efficient nuclear translocation of FANCD2 in response to MMC (Fig. 6 A and B). Indeed, IPO4 depletion reduced nuclear localization of both FANCD2 and C/EBPδ both before and after MMC treatment (Fig. 6B), and cytoplasmic FANCD2 still was detectable upon MMC treatment (Fig. 6A). Similar results were observed after silencing of C/EBPδ (Fig. 6 and Fig. S5). Although these data confirm the role of endogenous C/EBPδ in nuclear translocation of FANCD2, they also identify an important role of IPO4 in nuclear import of both of these proteins.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 6.

IPO4 augments nuclear localization of FANCD2 and cell survival in response to MMC. MDA-MB-468 cells were transiently transfected with siRNA against IPO4 or C/EBPδ or with scrambled oligos as control. MMC (500 ng/mL) was added 24 h later, and cells were incubated for another 20 h before preparation of (A) cytoplasmic (CE) or (B) nuclear (NE) cell extracts, followed by Western blot analysis of protein expression as indicated. The purity of cytoplasmic versus nuclear fractions also is shown in Fig. S7. (C) MDA-MB-468 cells were transfected as above and were placed in 96-well dishes at 5,000 cells/well 8 h later. The next day, MMC (500 ng/mL) was added, and cell viability was assessed 48 h later. Data are mean ± SEM of three experiments, each performed in triplicate, and relative to cells before treatment (set at 100%). **P < 0.01 relative to control siRNA.

Last, we addressed the functional relevance of IPO4 and C/EBPδ in this system by assessing cell survival in response to MMC. Indeed, silencing of either IPO4 or C/EBPδ significantly enhanced the cytotoxicity of MMC on MDA-MB-468 cells (Fig. 6C), demonstrating that, like C/EBPδ, IPO4 promotes cell survival in response to DNA damage. Because numerous reports have documented the role of nuclear FANCD2 in cellular survival in response to MMC (7–9), we suggest that the reduced cell survival after silencing of C/EBPδ or IPO4 is the result, at least in part, of their role in augmenting nuclear import of FANCD2.