RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway (original) (raw)
FANCAI939S mutation disrupts FAAP20 binding. We identified a patient with FA with an atypical clinical phenotype (Figure 1). The patient was a 33-year-old woman who developed triple-negative breast cancer (estrogen receptor–negative, progesterone-negative, HER2-negative breast cancer). She was noted to have short stature and mild bone marrow dysfunction, suggesting a diagnosis of FA. Primary peripheral blood lymphocytes exhibited an increased level of DNA damage–inducible quadriradial chromosomes (Figure 1A), consistent with the diagnosis of FA (23, 24). Since subtype A is the most prevalent subtype of FA, accounting for 60% to 70% of patients with FA (25), we next performed genomic sequencing of FANCA from the patient’s primary peripheral blood lymphocytes and her primary skin fibroblast cells (Figure 1B and data not shown). Two mutant FANCA alleles were identified. One mutant allele contained a previously known germline mutation in FANCA, the S947Stop mutation (26–28). The other mutant allele contained a thymine-to-guanine change at nucleotide 2816 (variant of unknown significance), predicted to encode a full-length mutant FANCA protein with one amino acid change (FANCAI939S). The mother of the patient with FA carries the FANCAI939S mutation (data not shown), and the son of the patient with FA is an obligate heterozygous carrier of one of these mutant FANCA alleles (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI79325DS1).
Atypical clinical presentation of a patient with FA reveals a unique mutation in the FANCA gene that disrupts FAAP20 binding. (A) Image and quantification of chromosomal aberrations and radial chromosomes (indicated by red arrows) of peripheral blood lymphocytes and primary skin fibroblast (DF2231) with a positive (+) and a negative (–) control cultures exposed to 20 ng/ml MMC or 0.1 μg/ml diepoxybutane (DEB). Original magnification, ×100. (B) The chromatograms indicate 2 mutant FANCA alleles, a T-to-G point mutation at nucleotide 2816 and a C-to-G point mutation at nucleotide 2840. (C) Relative survival of GM6914 (FANCA-deficient) cells complemented with vector, FANCAWT, or FANCAI939S; exposed to increasing doses of MMC; and plated for 5 days. Three independent experiments were performed. (D) Immunoblot of FANCA, FANCG, and FAAP20 proteins derived from cell lysates of the complemented GM6914 cells. (E) Schematic representation of FANCAI939S and immunoblot of anti-Flag IP immunocomplexes isolated from 293T cell lysates transfected with the Myc-FANCA and Flag-FAAP20 cDNAs. (F) Immunoblot of GST pull-downs, using bacterially expressed GST-FANCA mixed with 293T cell lysates expressing HA-FAAP20.
To test the possible functional significance of the FANCAI939S mutation, we compared the ability of wild-type FANCA (FANCAWT) and the FANCAI939S mutant to complement a protein-null human FA-A fibroblast line, GM6914 (Figure 1C). Unlike the wild-type protein, the mutant protein only partially rescued the mitomycin C (MMC) hypersensitivity of these cells, indicating that the I939S mutation is hypomorphic. FANCA associates directly with FANCG and FAAP20, and this interaction is critical for the stability of all 3 proteins (12, 29, 30). The I939S mutant protein rescued the expression of FANCG but failed to rescue the expression of FAAP20 (Figure 1D), suggesting that the I939S mutation falls in the FAAP20-binding site of FANCA. Indeed, the I939S missense mutation is located in exon 28 of the FANCA gene, a region implicated previously in FAAP20 binding, and our experiments also demonstrated the binding domain to be between amino acids 913 and 1095 (refs. 12, 30–32, and Supplemental Figure 1B). Interestingly, other patients with FA have been identified with FANCA alleles harboring point mutations in this same region of FANCA (33).
To test whether the FANCAI939S mutation blocks FAAP20 binding, we coexpressed Myc-tagged FANCA with Flag-tagged FAAP20 and tested for coimmunoprecipitation. As predicted, unlike FANCAWT, the FANCAI939S protein failed to bind to FAAP20 (Figure 1E). Similarly, a GST-FANCAI939S failed to pull down HA-tagged FAAP20 in vitro (Figure 1F). Taken together, the patient-derived point mutation in FANCA disrupts FAAP20 binding, accounting, at least in part, for its dysfunction (Supplemental Figure 1C) and the patient’s clinical phenotype.
Loss of FAAP20 binding disrupts the TLS function of the FA core complex. The interaction of FANCA and FAAP20 is critical for stabilization of the FA core complex, thereby allowing FANCD2 monoubiquitination and recruitment of the TLS for ICL repair (12). We next determined whether FANCAI939S could restore these functions in the FA pathway. Interestingly, FANCAI939S was able to complement FANCD2 monoubiquitination, albeit at a lower level than FANCAWT (Figure 2A). Consistent with the rescue of FANCD2 monoubiquitination, the FANCA-deficient GM6914 cells expressing FANCAI939S also exhibited DNA damage–inducible FANCD2 foci (Figure 2B and Supplemental Figure 2). This result confirms that the FANCAI939S mutant can support FA core complex assembly and FANCD2 monoubiquitination despite its failure to bind FAAP20.
The FANCAI939S mutant polypeptide promotes monoubiquitination of FANCD2 but fails to activate REV1 recruitment and TLS-mediated mutagenesis. (A) Immunoblot of FANCD2 and FANCA derived from cell lysates of the indicated complemented GM6914 FANCA-deficient cells treated with HU (1 mM). (B) Quantification of immunostaining of FANCD2 in GM6914 cells complemented with FANCAWT or FANCAI939S exposed to HU (1 mM). Three independent experiments were performed. (C) Representative image of GFP fluorescence microscopy of REV1 foci in complemented GM6914 cells 4 hours after psoralen and UVA treatment. Original magnification, ×60. Quantification of cells displaying more than 5 REV1 foci. Data shown are mean ± SEM from 3 independent experiments. *P < 0.05, compared with psoralen plus UVA–treated FANCAWT complemented GM6914 cells, 2-tailed t test (assuming unequal variance). (D) The mutation frequency in damaged (1,000 J/m2 UVC) _Sup_F plasmid recovered from GM6914 cells, complemented with vector, FANCAWT, or FANCAI939S. Data shown are mean ± SEM from 3 independent experiments. *P < 0.05, compared with UVC-treated FANCAWT complemented GM6914 cells, 2-tailed t test (assuming unequal variance).
We demonstrated previously that FAAP20 contains a ubiquitin-binding zinc finger 4 (UBZ4) required for recruiting the TLS scaffold protein, REV1, to sites of DNA cross-link repair (12). REV1 recruitment correlates with cellular TLS repair activity. We next compared the ability of the FANCAI939S protein to correct the DNA cross-linker–inducible assembly of REV1 nuclear foci with that of the FANCAWT protein. Complemented FANCA-deficient GM6914 cells were exposed to psoralen plus UVA, a mechanism known to induce DNA ICLs. Interestingly, while FANCAWT supported the assembly of psoralen-UV–inducible REV1 foci, FANCAI939S failed to rescue REV1 foci (Figure 2C). Previous studies have indicated that DNA damage–inducible REV1 foci assembly correlates with enhanced point mutagenesis, as measured by the _Sup_F assay (34). Consistent with this defect, the FANCAI939S protein, unlike FANCAWT, failed to rescue UVC-inducible point mutagenesis, as measured by the _Sup_F assay (Figure 2D).
Taken together, these results indicate that FANCAI939S is a separation-of-function mutant. The FANCAI939S protein functions efficiently in the assembly of the FA core complex and the DNA damage–inducible monoubiquitination of FANCD2 protein, which is required for FANCD2 foci assembly and recruitment of the downstream endonuclease complex (11). However, the FANCAI939S protein fails to bind to FAAP20, fails to recruit REV1, and fails to restore TLS activity.
Loss of FAAP20 binding results in increased SUMOylation, polyubiquitination, and proteasome-mediated degradation of FANCA. A primary skin fibroblast culture established from the patient with FA was immortalized with SV40 T antigen and tested for MMC-induced cytotoxicity. These transformed fibroblasts (DF2231) were hypersensitive to MMC, comparable to the hypersensitivity observed for a control FANCI-deficient fibroblast culture (Figure 3A). Immunoblotting and immunostaining revealed a detectable but decreased expression level of the full-length mutant FANCAI939S polypeptide (Figure 3B, lanes 5–8, and Supplemental Figure 3A) in DF2231 cells compared with the higher expression level of full-length FANCAWT in wild-type cells. Moreover, decreased monoubiquitination of the FANCD2 protein was observed, consistent with a defect in the FA core complex function. The DF2231 cells also had reduced FANCD2 nuclear foci, consistent with an upstream defect in the pathway (Figure 3B, lanes 5–8, and Supplemental Figure 3B). The MMC hypersensitivity of the DF2231 cells was functionally complemented with the cDNA-encoding FANCAWT protein, further confirming the assignment of this patient with FA to subtype A (Supplemental Figure 3C). Complementation of the cells also resulted in restoration of the stable FA core complex, including FANCA, FANCG, and FAAP20 (Supplemental Figure 3D and refs. 12, 30–32).
The full-length mutant FANCA protein in DF2231 cells is unstable and has enhanced SUMO and ubiquitin conjugation. (A) Relative survival of wild-type human GM0637 cells, FA-I cells, or DF2231 cells derived from a patient with FA-A treated with increasing doses of MMC and plated for 5 days. Three independent experiments were performed. (B) Immunoblot analysis of wild-type (GM0637) or DF2231 fibroblasts treated with various DNA damage-inducing agents. (C) Immunoblot of FANCA, FANCG, and FAAP20 in lysates of DF2231 cells untreated or pretreated with 20 μM MG132 and GM0637 cells treated with siRNAs to control, FAAP20, or FANCG. (D) Endogenous FANCA was immunoprecipitated under denaturing conditions from cell lysates of FA-A (GM6914), wild-type (GM0637), or DF2231 cells, followed by anti-FANCA, -SUMO, or -ubiquitin immunoblot. (E) Endogenous FANCA was immunoprecipitated under denaturing conditions from cell lysates of wild-type (GM0637) or DF2231 cells transfected with siRNA oligos against control or UBC9, followed by anti-SUMO immunoblot. The thin black line indicates that noncontiguous lanes from the same blot are shown. (F) HeLa cells were transfected with HA-tagged SUMO3, and the cells were treated with UV, HU, or MMC for the indicated times. Endogenous FANCA SUMOylation was determined under denaturing conditions by anti-FANCA immunoprecipitation, followed by anti-FANCA or anti-HA immunoblot.
We hypothesized that the reduced expression of FANCAI939S in DF2231 cells results from decreased protein stability. Previous studies suggest that failure of FAAP20 binding might account for the increased degradation of FANCA via the ubiquitin-proteasome system (12, 30, 32). Consistent with this hypothesis, the proteasome inhibitor MG132 stabilized the mutant FANCA protein and its interaction with FANCG and FAAP20 (Figure 3C, lanes 4–6). Cellular exposure to MG132 also resulted in accumulation of a high-molecular-weight FANCA protein ladder, suggesting that the mutant FANCA protein (FANCAI939S) may undergo polyubiquitination and proteasome-dependent degradation.
Many cellular proteins are known to undergo UBC9-mediated SUMOylation prior to their polyubiquitination and degradation by STUbL enzymes (35, 36). STUbL enzymes are ubiquitin E3 ligases, which contain SIMs, and are directed to their substrates by SUMO-SIM interactions. Furthermore, recent studies indicate that some FA proteins are SUMOylated following cellular heat shock (37). To test the hypothesis that FANCA is degraded through this mechanism, we immunoprecipitated FANCA under denaturing conditions from various SV40-transformed fibroblast lines and immunoblotted for SUMO or ubiquitin (Figure 3D). Immunoprecipitation of FANCA from patient-derived DF2231 cells, followed by immunoblotting with anti-SUMO or anti-ubiquitin, indicated that FANCA is modified with SUMO and ubiquitin (lanes 6 and 9) and that it may coprecipitate with other conjugated proteins. In contrast, the full-length FANCAWT immunoprecipitated from wild-type GM0637 cells (lanes 5 and 8), with reduced coprecipitation of SUMO or ubiquitin conjugates. Moreover, an siRNA that silences the expression of UBC9, the SUMO-conjugating enzyme, blocked the SUMOylation of FANCA (Figure 3E). As further evidence of FANCA SUMOylation, we immunoprecipitated endogenous FANCA under denaturing conditions from HeLa cells transfected with HA-tagged SUMO3. In response to DNA damage, FANCA showed enhanced SUMO conjugation, especially 24 hours after hydroxyurea (HU) and MMC exposure (lanes 4 and 6) (Figure 3F). Taken together, these results support the hypothesis that the FANCAI939S protein is degraded by UBC9-dependent SUMOylation followed by polyubiquitination by a STUbL enzyme.
Loss of FAAP20 binding leads to increased UBC9/PIAS1-dependent SUMOylation of FANCA at K921. Protein SUMOylation occurs on a lysine residue within the context of a local consensus sequence, Ψ-K-X-D/E (Ψ is a hydrophobic acid). Using the SUMO prediction program, we identified a potential SUMOylation site on FANCA at K921. Interestingly, K921 is located near the I939S mutation and falls within the putative FAAP20-binding domain of FANCA (Figure 4A). We next demonstrated that the FANCAWT protein is SUMOylated and that the mutant protein, FANCAK921R, has reduced SUMOylation (Figure 4, B and C, and Supplemental Figure 4, A–C). Moreover, FANCAK921R exhibited reduced polyubiquitination (Figure 4D), indicating that SUMOylation of FANCA is required for optimal polyubiquitination. The FANCA mutant protein (K921R) still interacted with FAAP20, suggesting that the lysine residue or its SUMOylation is not required for this interaction (Supplemental Figure 4D).
The SUMO E3 ligase PIAS1 mediates FANCA SUMOylation at K921. (A) Alignment of FANCA from various species. The conserved K921 and I939 residues are indicated with asterisks. (B) 293T cells were transfected with HA-tagged SUMO or cotransfected with HA-tagged SUMO with Myc-tagged FANCAWT. FANCA SUMOylation was determined under denaturing conditions by anti-HA immunoprecipitation, followed by anti-Myc immunoblot. (C) 293T cells were transfected with Myc-tagged FANCAWT or FANCAK921R with HA-SUMO3 as indicated. FANCA SUMOylation was determined under denaturing conditions by anti-HA immunoprecipitation followed by anti-Myc immunoblot. (D) 293T cells were transfected with Myc-tagged FANCAWT or FANCAK921R. Cells were treated with 20 μM MG132 (lanes 2 and 3), and FANCA ubiquitination was examined under denaturing conditions by anti-myc immunoprecipitation, followed by anti-Myc immunoblot. (E) 293T cells were transfected with Myc-tagged FANCAWT alone or with HA-SUMO3 as indicated. Subsequently, cells were exposed to siRNAs against control, UBC9, PIAS1, or PIAS4. Protein expression was determined by immunoblot, and FANCA SUMOylation was examined under denaturing conditions by anti-HA immunoprecipitation, followed by anti-Myc immunoblot. (F) Immunoblot of FANCA derived from cell lysates of the indicated complemented GM6914 cells treated with cycloheximide (CHX) (20 μg/ml). (G) Complemented GM6914 cells were treated with increasing doses of MMC and plated for 4 days. Three independent experiments were performed.
The mammalian SUMO conjugation pathway has 1 known E2 SUMO-conjugating enzyme (UBC9) and 6 known E3 SUMO ligases (PIAS1–PIAS4, ZMIZ1, and NSE2) (17). PIAS1 and PIAS4 are critical for DNA double-strand break response, and suppression of either enzyme results in cisplatin sensitivity (38, 39). Furthermore, PIAS1 interacts with SNM1A, and this association is critical for ICL repair (40). In contrast to that of PIAS4, suppression of PIAS1 with siRNA decreased FANCA SUMOylation to a similar extent as UBC9 suppression (Figure 4E). Taken together, we hypothesized that the absence of FAAP20 binding, resulting from the FANCAI939S point mutation derived from a patient with FA, results in increased exposure of the K921 residue. Exposure of K921 results in increased SUMOylation by UBC9, increased polyubiquitination of the mutant protein, and decreased protein stability. Consistent with this hypothesis, the FANCAI939S mutant protein has a decreased half-life, and its enhanced degradation is rescued by the concomitant mutation of K921R in the same polypeptide (Figure 4F and Supplemental Figure 4E). FANCAI939S, FANCAK921R, and FANCAK921R/I939S complemented cells all exhibited similar MMC sensitivity (Figure 4G).
SUMOylation results in RNF4-mediated polyubiquitination and proteasome-dependent degradation of FANCA. STUbL E3 ligases, such as RNF4, promote the polyubiquitination and degradation of polySUMOylated proteins (35, 36). RNF4 is a strong regulator of DNA damage response (19–22, 41–43), suggesting that it polyubiquitinates DNA repair proteins, which are themselves polySUMOylated. We therefore hypothesized that RNF4, which contains a tandem repeat of SIMs (44), might be the STUbL that promotes the degradation of polySUMOylated FANCA (Figure 5). To test this hypothesis, HeLa cells were transfected with siRNA oligos specific for RNF4, RNF111, and/or FAAP20, and the stability of endogenous FANCA was measured (Figure 5A). Knockdown of FAAP20 resulted in a decreased cellular level of FANCA (Figure 5A, lane 2). Interestingly, concurrent knockdown of RNF4 and FAAP20, but not RNF111 and FAAP20, partially rescued the stability of FANCA, demonstrating that RNF4 promotes the degradation of FANCA protein when FAAP20 is unbound (Figure 5A, lanes 3–5). RNF4 is known to interact with SUMOylated MDC1 in response to DNA double-strand breaks (20). To test the interaction between RNF4 and FANCA, we performed coimmunoprecipitation of Myc-FANCA and Flag-RNF4. FANCA, similar to MDC1, physically associated with RNF4 under these conditions. The interaction was increased after HU treatment (Supplemental Figure 5, A and B), and it was dependent in part on SUMO-SIM interactions (Supplemental Figure 5C). RNF4 suppression also partially rescued the expression of mutant FANCAI939S in DF2231 cells (Figure 5B). Furthermore, RNF4 suppression increased the MMC sensitivity of GM6914 cells expressing FANCAWT but had little effect on the MMC sensitivity of GM6914 cells expressing the mutant FANCAI939S protein (Figure 5C). Interestingly, RNF4 suppression did not further increase the MMC sensitivity of GM6914 cells expressing FANCAK921R (Supplemental Figure 5D). Taken together, these results support the hypothesis that RNF4 functions as the STUbL for FANCA.
RNF4 polyubiquitinates SUMOylated FANCA and regulates its cellular expression level. (A) Immunoblot of FANCA derived from lysates of HeLa cells transfected with the indicated siRNA oligos and relative FANCA level quantified by ImageJ. (B) Immunoblot of FANCA from wild-type (GM0637) or DF2231 cells exposed to siRNAs against control or RNF4 and relative FANCA level quantified by ImageJ. The thin black line indicates that noncontiguous lanes from the same blot are shown. (C) GM6914 (FA-A) fibroblasts were stably transfected with the cDNA encoding either FANCAWT or the FANCAI939S mutant. Cells were treated with the indicated siRNA and subjected to MMC cytotoxicity testing. Three independent experiments were performed. The efficient knockdown of RNF4 expression by the siRNA-RNF4 is indicated by immunoblot.
A normal function of RNF4 in the FA/BRCA pathway. We next hypothesized that RNF4 may promote the degradation of FANCAWT in normal (non-FA) human cells, albeit at a slower rate than the degradation of the mutant FANCAI939S protein in the cells from patients with FA. Indeed, RNF4-mediated degradation of FANCAWT may be required to clear the FA core complex from the site of damaged chromatin as part of the DNA repair process (17). Failure to clear FANCA and the FA core complex may result in disruption of the pathway and persistence of collapsed replication forks. To test this hypothesis, HeLa cells were transfected with a siRNA to RNF4 and treated with HU or MMC. Interestingly, the reduction of RNF4 resulted in hypersensitivity to DNA-damaging agents and increased chromosome aberrations and radials, consistent with a FA phenotype (Supplemental Figure 6, A–C). Furthermore, RNF4 suppression in HeLa cells resulted in increased levels of prolonged retention of FANCD2-Ub in the chromatin fraction, suggesting persistence of the active FA core complex (Supplemental Figure 6D).
We next examined the phenotype of avian DT40 cells, which have a stable knockout of FANCC or RNF4 (Figure 6 and refs. 45, 46). Similar to the observation in HeLa cells, the decreased expression of either protein resulted in MMC and cisplatin hypersensitivity and a FA phenotype. In order to examine the possible epistatic relationship between RNF4 and other FA genes, we generated double knockouts of RNF4 and FANCC in avian DT40 cells (Figure 6). We compared the MMC or cisplatin sensitivity of DT40 cells containing a knockout of FANCC, RNF4, or both FANCC and RNF4. Interestingly, the double-knockout cells had MMC (or cisplatin) sensitivity comparable to that of the single FANCC knockout cells. Taken together, these data demonstrate that RNF4 is a component of the FA/BRCA pathway.
RNF4 functions in the FA/BRCA pathway. (A) Relative survival of wild-type, FANCC–/–, RNF4–/–, or FANCC–/–/RNF4–/– DT40 cells treated with increasing doses of MMC or cisplatin and plated for 3 days. Three independent experiments were performed. (B) Quantification of chromosomal aberrations of wild-type, FANCC–/–, RNF4–/–, or FANCC–/–/RNF4–/– DT40 cells exposed to 50 ng/ml MMC for 16 hours.