Human Molecular Genetics, 2002, Vol. 11, No. 25 3125-3134
© 2002 Oxford University Press
Heterogeneous activation of the Fanconi anemia pathway by patient-derived FANCA mutants
1Division of Genetic Diagnosis, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 2Department of Molecular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan and 3Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA
Received July 22, 2002; Revised September 26, 2002; Accepted October 4, 2002
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ABSTRACT |
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Fanconi anemia (FA) is an autosomal recessive disorder of hematopoiesis characterized by hypersensitivity to DNA crosslinkers such as mitomycin C (MMC). There is growing evidence for a model of the FA pathway, wherein a nuclear multiprotein complex of five FA proteins (FANCA, C, E, F and G) regulates activation of FANCD2 into a monoubiquitinated form, which, collaborating with the BRCA1 machinery, affects cellular response to DNA damage. However, the role of the FA pathway in defective DNA damage response caused by various mutant forms of FA proteins has not been fully assessed. In the present study, 21 patient-derived FANCA mutants with a missense or a small in-frame deletion were expressed in FANCA-deficient fibroblasts and examined for complementation of MMC sensitivity and for reconstitution of the FA pathway: FANCA phosphorylation, interaction with FANCC, FANCF and FANCG and nuclear localization and FANCD2 monoubiquitination. The altered FANCA proteins complemented MMC sensitivity at different grades: five proteins (group I) behaved like wild-type FANCA, whereas the other proteins were either mildly (group II, n=4) or severely (group III, n=12) impaired. Group I proteins showed an apparently normal reconstitution of the FA pathway, thus they may be pathogenic by reducing endogenous expression or possibly benign polymorphisms. Reconstitution of the FA pathway by group II and III mutants closely correlated with cellular sensitivity to MMC. The different activation of the FA pathway may partly account for the phenotypic variation seen in FA patients.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, progressive bone marrow failure and leukemia susceptibility (for reviews, see 1–3). A characteristic cellular phenotype of FA is chromosomal instability and hypersensitivity to DNA crosslinkers such as mitomycin C (MMC) (1–3). FA has at least eight different complementation groups (FA–A to FA–G) (4–8). Growing evidence indicates that protein products of six FA genes (FANCA, C, D2, E, F and G) which have been cloned to date (8–14) function in a common pathway, termed the FA pathway (15–27).
In a current model of the FA pathway, FANCA, C, E, F and G assemble into a nuclear multiprotein complex, which converts FANCD2 into a monoubiquitinated form (15–27). This active isoform is likely to affect DNA damage response in collaboration with the BRCA1 machinery, through homology-directed repair (26). Furthermore, a recent study showed that biallelic inactivating mutations of BRCA2, a component involved in homologous recombination, cause a clinical phenotype of FA (28). The model of the FA pathway has been largely based on studies using FA protein-deficient cells. However, it has not been verified that various mutant forms of FA proteins similarly affect the FA pathway. It is also conceivable that some mutants cause aberrant DNA damage response or hematopoiesis through impairment of distinct pathways. This notion is consistent with recent findings that anti-apoptotic functions of FANCC and the ATM-dependent cell cycle checkpoint function of FANCD2, the defects of which may partly account for FA phenotypes, are separated from activation of the FA pathway by mutational analyses (29,30).
FANCA is a relatively large protein with 1455 amino acids (Mr=163 kDa) and contains a bipartite nuclear localization signal (NLS) at its N-terminus and a leucine zipper-like motif between amino acids 1069 and 1090 (10,11). Subsequent studies revealed several features of this protein: (i) FANCA is predominantly localized in the nuclei (15–23,32); (ii) FANCA indirectly interacts with FANCC and FANCF (15–19,23–25) and directly interacts with FANCG and possibly FANCE (19–22,24,27); (iii) a serine kinase binds and phosphorylates FANCA (17,31). However, little is known of functional domains of FANCA, except that its N-terminal portion, including NLS, is required for nuclear localization and for direct interaction with FANCG (16,19–22,32). The FANCA gene abnormalities are seen in the majority (60–70%) of FA patients and more than 100 types of mutations throughout the gene have been found to date (10,11,33–38). While most of these mutations are either small insertion/deletion mutations resulting in premature termination or intragenic large deletions and presumably lack protein expression (null-mutations), 30 or more mutations are predicted to produce altered proteins with a single amino acid substitution or a small in-frame deletion (33–38). Although a few mutants, such as H1110P, R1117G and delF1263, were shown to be defective in complementation of MMC sensitivity and reconstitution of the FA pathway (17–20), other mutants have not been thoroughly characterized.
The goal of the present study was to assess the role of the FA pathway in defective DNA damage response caused by various mutants of FANCA proteins. For this, 19 uncharacterized or poorly characterized mutants were expressed in FANCA-deficient cells and systematically analysed for complementation of MMC sensitivity and for reconstitution of the FA pathway, in comparison with the well-characterized mutants H1110P and delF1263 (17–20). We found that various mutants show different complementation of MMC sensitivities. FANCA interactions with FANCC and FANCF, FANCA phosphorylation and FANCD2 monoubiquitination were closely associated with MMC sensitivity, whereas the nuclear localization of FANCA was dissociated from these events. Our present findings provide fundamental information which will serve to better understand mechanisms and the pathophysiological role of the FA pathway.
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RESULTS |
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Table 1 summarizes data on the 21 patient-derived FANCA mutations examined in the present study. Sixteen mutations were predicted to result in substitution of a single amino acid, and five were presumed to result in in-frame small deletions. Many of the mutations were detected in a single patient, i.e. ‘private mutation’ and reported without identification of a mutation in a second allele. Figure 1 shows a schematic presentation of the FANCA protein structure and locations of the patient-derived mutations.
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To characterize these mutants, we introduced into GM6914 FANCA-null fibroblasts wild-type (wt) and mutant FANCA proteins carrying a Flag epitope at their N-termini. These proteins were stably expressed at similar levels (Fig. 2A). MMC hypersensitivity of GM6914 cells was corrected by introduction of Flag-wt-FANCA (Fig. 2B). Different mutants corrected MMC sensitivities of GM6914 cells, to various degrees (Fig. 2B and C). Five mutants (D598N, Q1128E, T1131A, F1262L and H1417D: group I) behaved like wt-FANCA, whereas 12 mutants (R435C, H492R, L845P, FQ868–869del, R1055L, R1055W, H1110P, F1135del, W1174del, 1239–43del, F1263del and W1302R: group III) failed to correct MMC sensitivities. Four mutants (L817P, P1324L, D1359Y and M1360I: group II) exhibited partial correction.
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In a subsequent series of experiments, we assessed effects of mutations on reconstitution of the FA pathway. For this, we first examined the interaction of the FANCA mutants with FANCC, FANCF and FANCG. Flag-FANCA immunoprecipitates from GM6914 cell lysates were analysed by immunoblotting with anti-FANCC (17), anti-FANCF (25) and anti-FANCG (19) antibodies. Group I mutants interacted with FANCC as strongly as did wt-FANCA, whereas group III mutants interacted to a much lesser extent with FANCC (Fig. 3A). Unlike FANCC, FANCG constantly interacted with all group I and III mutants (Fig. 3A). The interaction of group II mutants with FANCC was intermediate between group I and group III mutants (Fig. 3B and C). The interaction of group II and III mutants with FANCF was impaired in parallel with their interaction with FANCC (Fig. 3C). D1359Y interacted with FANCC and FANCF more weakly than other group II mutants, whereas R1055L interacted with FANCC and FANCF more strongly than other group III mutants. The deviation of these two mutants was observed in repeated experiments. The FANCA/FANCG interaction was not affected by group II mutations. Some data are not shown (see Table 2).
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Group I mutants were phosphorylated much like wt-FANCA, whereas phosphorylation of group III mutants was markedly reduced (Fig. 4A). Again, group II mutants were phosphorylated intermediately between group I and group III mutants (Fig. 4B).
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To analyse the subcellular distribution of the mutants, we did immunofluorescence studies, using an anti-FANCA antibody. Group I mutants, like wt-FANCA, were predominant in the nuclei in more than 90% of cells, whereas most group II and group III mutants were primarily cytoplasmic in more than 95% of cells (Fig. 5). However, there were two exceptional mutants, L817P (group II) and R1055L (group III), which showed homogenous staining mixed with nuclear and cytoplasmic predominance: homogenous, nuclear and cytoplasmic staining patterns were observed in 65, 20 and 15% of L817P transformants and 63, 2 and 35% of R1055L transformants, respectively (Fig. 5). Cytoplasmic and nuclear levels of wt-FANCA and representative mutants of groups I, II and III were determined using immunoblotting (Fig. 6). Group II and III proteins in the nuclear fraction were significantly reduced in comparison with wt and group I proteins, whereas no difference was noted between group II and group III mutants.
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Finally, we examined effects of FANCA mutations on FANCD2 monoubiquitination. Group I transformants express a monoubiquitinated form of FANCD2 (D2-Ub) to some extent, like GM6914/wt-FANCA, whereas D2-Ub was not detected in group III transformants (Fig. 7A, top). The difference between these two groups was much more pronounced when these cells were treated with MMC (Fig. 7A, middle). The same blot shows that D2-Ub was slightly evident in group III cells, but not in mock cells (Fig. 7A, bottom). Although the difference in D2-Ub levels between group II and group III cells was not clear under basal conditions, MMC treatment revealed a higher induction of D2-Ub in group II cells than in group III cells (Fig. 7B).
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DISCUSSION |
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The present study revealed that various patient-derived mutants have a different extent of influence, but not an all-or-none type impact, on functions of FANCA. In the complementation of MMC sensitivity, the mutants showed nearly wt activities (group I, n=5), partial activities (group II, n=4) or no activities (group III, n=12). Reconstitution of the FA pathway by these mutants is summarized in Table 2. FANCA phosphorylation, interactions with FANCC and FANCF and MMC-induced FANCD2 monoubiquitination were closely associated with correction of MMC sensitivities. wt-FANCA and group I mutants were predominantly localized to the nuclei, whereas most of group II and III dysfunctional mutants were predominantly cytoplasmic. Interestingly, L817P (group II) and R1055L (group III) showed a mixed staining pattern. Thus, various mutant forms of FANCA affect DNA damage response through different activation of the FA pathway.
We analysed 16 missense variants and five mutants with small in-frame deletions (Table 1, Fig. 1). All of the deletion mutants were classified into group III. Eleven (seven of group III and four of group II proteins) of 16 missense variants proved to be disease-associated, whereas five missense variants of group I behaved like wt-FANCA. There are possible interpretations for wt activities of group I proteins. Firstly, these proteins may be pathogenic by increased degradation of proteins resulting in reduced endogenous expression. Although group I proteins did not show significantly reduced levels or increased degradation rates (not shown) in the present system, we cannot exclude the possibility that enforced overexpression masked the increased degradation. A similar problem is true for other mutants. Although group II mutants showed partial activities in the present system, these mutations may result in more severe effects at endogenous expression levels. Secondly, when only genomic DNA is sequenced, the possibility should be considered that a nucleotide change in coding regions, predicted to be a missense or nonsense mutation, may result in defective pre-mRNA splicing (39). Finally, group I variants may be benign polymorphisms, although they have not been detected in the normal population. To distinguish these possibilities, analyses of mRNAs and proteins and complementation tests, using patient cells, will be required.
FANCA interacts with other FA proteins (C, E, F and G) and a protein kinase phosphorylating FANCA in a multiprotein complex (15–25,27,31), but little is known regarding the structural basis of these protein interactions, except for the direct and constitutive interaction between FANCA and FANCG (19–22,24). Previous analyses using several mutants led to the notion that specific regions of FANCA may be involved in interactions with FANCC and FANCA-protein kinase (17–19,31). However, the present results showed that various point-mutations, regardless of their locations, similarly impaired FANCA phosphorylation and association with FANCC and FANCF in a correlative manner. Thus, it is likely that these mutations prevent FANCA from proper interaction with other proteins by affecting tertiary, but not local, structure of the molecule.
Monoubiquitination of FANCD2 was shown to be essential for normal cellular response to MMC (26). However, it has not been determined if FANCD2 is the only downstream transducer of the FA pathway signaling. It can be hypothesized that the FA–protein complex activates distinct signaling pathways, required for normal DNA damage response. However, dysfunction of this hypothetical pathway is, if present, unlikely to play a major role in MMC hypersensitivity in FA–A cells, since effects of diverse FANCA mutations on MMC-triggered FANCD2 monoubiquitination were tightly linked with those for MMC-induced cell death.
Previous studies showed that not only an N-terminal region including NLS but also a C-terminal region plays a pivotal role in the nuclear localization of FANCA (16,19,32), yet little is known of regulatory mechanisms of its subcellular localization. Diverse mutations had similar effects on nuclear localization of FANCA, thereby suggesting that alteration of its tertiary structure, rather than local structures, prevents FANCA from proper interaction with nuclear import machinery (40). Earlier observations led to the notion that interaction with other FA proteins and phosphorylation may regulate the nuclear import of FANCA (15–19,22). While this notion is consistent with results of most mutants examined in the present study, the results of L817P (group II) and R1055L (group III) showeda dissociation between nuclear localization and phosphorylation or interactions with other FA proteins. Unlike R1055L, R1055W (group III) was predominantly cytoplasmic, which suggests that a side chain of an amino acid at this position specifically affects nuclear import of FANCA.
FA is characterized by variability of clinical phenotypes (1,2,41), which are likely to be affected not only by genotypes but also by ethnic and individual genetic backgrounds (modifier genes) and environmental factors (42–45). A recent study revealed a significant genotype–phenotype correlation in FA–A: patients homozygous for FANCA null-mutations have an earlier onset of anemia and a higher incidence of leukemia than did those with mutations to produce an altered protein (46). The present study indicates that altered FANCA proteins can activate the FA pathway to various degrees, in response to DNA damage, as assessed by FANCD2 monoubiquitination, while no activation of this pathway was detected in FANCA-null cells. The different activation of the FA pathway may, in part, account for the genotype–phenotype correlation in FA–A patients. Measurement of FANCD2 monoubiquitination levels in cells treated with DNA crosslinkers provides a sensitive and specific assay for function of the FA pathway. Determination of the relationship between function of the FA pathway in patient cells and clinical phenotypes will aid in a better understanding of the molecular basis of the genotype–phenotype correlation concerning FA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture
SV40-immortalized fibroblasts GM6914 were maintained in DMEM containing 10% fetal calf serum (FCS), as described previously (16,19). 293T cells were maintained in DMEM containing 10% FCS.
Generation of FANCA mutant constructs and retroviral infection of GM6914 cells
cDNAs of FANCA mutants with a Flag epitope at its N-terminus were generated using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, CA) and synthetic oligonucleotide primers containing patient-derived mutations. cDNA inserts of wt and mutant FANCA, verified by DNA sequencing, were subcloned into a retroviral expression vector, pMMP, as described previously (16). The indicated pMMP constructs were co-transfected by lipofection using Fugene (Roche Diagnostics, IN, USA) into 293T producer cells with cDNAs of VSV-G envelope protein. Retroviral supernatants were collected on day 2 following the lipofection. Infection of GM6914 cells was carried out as described previously (16). After 2 days, selection with 0.5 µg/ml of puromycin was begun.
Cell survival assay
Cell survival assay was carried out as described previously (16).
Immunoprecipitation and immunoblotting
Cell extracts of GM6914 cells (
107 cells/sample) were prepared in lysis buffer supplemented with protease inhibitors and subjected to immunoprecipitation using an anti-Flag monoclonal antibody (M2, Sigma), as described previously (19). Immunoprecipitates or lysates were separated on SDS–polyacrylamide gels, transferred to PVDF membranes and immunoblotted with the indicated antibodies. Protein bands were visualized using enhanced chemiluminescence detection reagents (DuPont).
In vivo phosphorylation of FANCA
In vivo phosphorylation of FANCA was examined as described previously (17). Briefly, cells (107 cells/sample) were incubated in medium containing [32P]orthphosphate (1 mCi/ml) for 2 h and lysed in 1 ml of lysis buffer. Immunoprecipitates with affinity-purified anti-FANCA antibody (2 µg) raised against the N terminus (3) were separated on SDS–polyacrylamide gels and blotted onto a PVDF membrane. Phosphorylation of proteins on the membrane was visualized autoradiographically, and the same membrane was probed using the indicated antibodies, as described previously (19).
Immunofluorescence microscopy
Cells were fixed with 2% paraformaldehyde in PBS (pH, 7.4) for 20 min followed by permeabilization with 0.3% Triton X-100 in PBS for 10 min. Next, the cells were incubated for 1 h in blocking buffer [PBS containing 10% normal goat serum and 0.1% Nonidet P40 (NP40)], followed by incubation in blocking buffer containing anti-FANCA antibody (3) for 2 h at room temperature. Cells were washed with washing buffer (PBS containing 0.1% NP40) and incubated for 1 h in blocking buffer containing goat anti-rabbit secondary antibody conjugated to fluorescein isothiocyanate. Cells were again washed with washing buffer. Cell nuclei were stained with a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, CA). Fluorescence microscopy was carried out as described previously (47). To determine percentages of cells with different staining patterns, 100 cells were scored in two independent experiments.
Mono-ubiquitination of FANCD2
A monoubiquitinated form of FANCD2 was detected as a protein band with a slower mobility on immunoblotting using an anti-FANCD2 monoclonal antibody (26).
Cell fractionation
Fractionation of GM6914 cells was carried out as described previously (19). Each fraction was analysed on SDS–polyacrylamide gels. Proteins were analysed by immunoblotting with the indicated antibodies.
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ACKNOWLEDGEMENTS |
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We thank T. Nakahata for support and M. Hoatlin for antibodies. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports and Culture of Japan and grants from the Ministry of Health, Labor and Welfare of Japan. The Division of Genetic Diagnosis is supported in part by Otsuka Pharmaceutical Co. Ltd.
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FOOTNOTES |
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* To whom correspondence should be addressed at: The Institute of Medical Science, The University of Tokyo, Division of Genetic Diagnosis, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel: +81 354495765; Fax: +81 354495764; Email: y-taka{at}ims.u-tokyo.ac.jp
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