A patient-derived mutant form of the Fanconi anemia protein, FANCA, is defective in nuclear accumulation

Experimental Hematology 27 (1999) 587–593

A patient-derived mutant form of the Fanconi anemia protein, FANCA, is defective in nuclear accumulation Gary Kupfera, Dieter Naf a, Irene Garcia-Higueraa, Jennifer Wasika, Andrew Chenga, Takayuki Yamashitab, Alex Tippingc, Neil Morganc, Christopher G. Mathewc, Alan D. D’Andreaa a Division of Pediatric Oncology, Dana-Farber Cancer Institute, and Department of Pediatrics, Children’s Hospital, Harvard Medical School, Boston, MA; bThe Institute of Medical Science, The University of Tokyo, Tokyo, Japan; c Division of Medical and Molecular Genetics, UMDS Guy’s Hospital, London, United Kingdom

(Received 6 January 1999; revised 3 February 1999; accepted 5 February 1999)

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A–H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but the function of the encoded FA proteins remains unknown. We recently demonstrated that the FANCA and FANCC proteins bind and form a nuclear complex. In the current study, we identified a homozygous mutation in the FANCA gene (3329A.C) in an Egyptian FA patient from a consanguineous family. This mutant FANCA allele is predicted to encode a mutant FANCA protein, FANCA(H1110P), in which histidine 1110 is changed to proline. Initially, we characterized the FANCA(H1110P) protein, expressed in an Epstein Barr virus (EBV)-immortalized lymphoblast line derived from the patient. Unlike wild-type FANCA protein expressed in normal lymphoblasts, FANCA(H1110P) was not phosphorylated and failed to bind to FANCC. To test directly the effect of this mutation on FANCA function, we used retroviral-mediated transduction to express either wildtype FANCA or FANCA(H1110P) protein in the FA-A fibroblast line, GM6914. Unlike wild-type FANCA, the mutant protein failed to complement the mitomycin C sensitivity of these cells. In addition, the FANCA(H1110P) protein was defective in nuclear accumulation in the transduced cells. The characteristics of this mutant protein underscore the importance of FANCA phosphorylation, FANCA/FANCC binding, and nuclear accumulation in the function of the FA pathway. © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Fanconi anemia—Mitomycin C—Leukemia—Cancer susceptibility

Introduction Fanconi anemia (FA) is an autosomal recessive disease characterized by chromosomal instability, cancer susceptiOffprint requests to: Alan D. D’Andrea, Dana-Farber Cancer Institute, Harvard Medical School, Division of Pediatric Oncology, 44 Binney Street, Boston MA 02115; E-mail: [email protected]

bility, and progressive bone marrow failure [1–3]. Somatic cell fusion studies have defined at least eight genetic complementation groups (FA-A through FA-H) [4–6]. The genes corresponding to groups A, C, and G have been cloned [7–10], and mutations in these genes appear to account for more than 80% of FA patients [6,11]. The FANCG cDNA only recently was cloned [10] and shown to be identical to the XRCC9 cDNA [12]. The FANCA, FANCC, and FANCG proteins have no sequence similarity to each other or to other proteins in GenBank, and their biochemical functions remain unknown. We recently determined that the FANCA and FANCC proteins bind and form a complex in the nucleus [13,14]. The complex is not observed in cells from several other FA complementation groups, including groups B, E, F, and H, suggesting that other FA gene products are required for its assembly [15]. The FA protein complex presumably mediates a nuclear function such as DNA repair, transcription, or chromosome segregation. Cells derived from FA patients display multiple phenotypic abnormalities. FA cells are hypersensitive to bifunctional alkylating agents, such as diepoxybutane and mitomycin C (MMC), suggesting a defect in DNA repair. FA cells also exhibit abnormal cell cycle progression [16–18] and reduced cell survival [19–23]. Many of these abnormalities are observed in primary cells derived from mice homozygous for a disrupted FancC gene [20,24]. How the absence of the FANCA/FANCC protein complex leads to these cellular abnormalities remains unknown. To date, little is known regarding the functional domains of FANCA. FANCA contains a nuclear localization signal (NLS) at its N-terminus [14] and a partial leucine zipper motif between amino acids 1069 and 1090. The importance of this leucine zipper region remains unclear. Some, but not all, mutations in this region of FANCA result in loss of FANCA function [25]. Although the primary amino acid sequence of FANCA provides little direct insight into its biochemical function, missense mutations derived from FA-A

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patients have identified critical functional domains of the FANCA protein. Based on mutational screens published to date [26–28], a region of the FANCA protein from amino acids 1046–1320, encoded by exons 32–39 [29], appears to be critical for FANCA function. Multiple patient-derived missense mutations have been identified in this region, including R1055W (exon 32), S1088F (exon 33) [27], R1117G, Q1128E, and T1131A (exon 34), and W1302R (exon 39) [26]. One mutation, FANCA1263delF (exon 34), encoded by the mutant FANCA allele, 3788–3790del, was found in 30 FA-A patients and appears to account for 5% of known FANCA mutations [26]. In many cases, it is difficult to distinguish between pathogenic point mutations and simple polymorphisms. In the current study, we identified an FA-A patient with a novel missense mutation of the FANCA gene, which resides in this critical region. The mutation is found in exon 33, downstream from the partial leucine zipper motif, and is predicted to disrupt an a-helical region of the FANCA protein. Lymphoblasts derived from this FA patient expressed the mutant FANCA protein, suggesting that the mutation has little effect on protein stability but instead ablates a functional domain of FANCA. When expressed in an FA-A fibroblast line (GM6914), the mutant protein failed to complement MMC sensitivity. Interestingly, the protein was defective in phosphorylation, FANCC binding, and nuclear accumulation. Our results suggest that the wild-type FANCA protein contains a domain in its carboxy-terminal region that is required for its functional activity and which is disrupted by this point mutation.

Materials and methods Characterization of cell lines GM6914 fibroblasts (ATCC) were maintained in Dulbecco’s modified Eagle medium (DMEM) medium containing 15% (v/v) fetal bovine serum (DMEM/15% FCS). Cells were grown at 378C in a humidified atmosphere containing 5% CO2. 293GPG producer cells [30] were cultivated in DMEM/10% FCS, supplemented with tetracycline (1 mg/mL; Sigma), Geneticin (0.3 mg/mL; Gibco), and puromycin (2 mg/mL; Sigma). Maintenance of lymphoblast lines was described previously [7,31,32]. Subtyping of FA lymphoblasts by retroviral transduction FA patient-derived lymphoblast lines were analyzed by retroviral gene transfer, as described previously [32]. We used modified forms of the retroviral vectors, pMMP-FANCA and pMMPFANCC, constructed by ligation of a cDNA sequence encoding the eight amino acid Flag peptide (DYKDDDDK), upstream and inframe of the expressed protein. In addition, the vectors were modified by ligation with the puromycin resistance cDNA cassette [33] into the BspMII site of pMMP. Concentrated retroviral supernatants were prepared as described previously [30,32]. Lymphoblasts (5 3 105 cells/mL) (1 mL) in RPMI/15% FCS were incubated with an equal volume (1 mL) of concentrated pMMP-Flag-FANCA-

(puro) or pMMP-Flag-FANCC-(puro) retroviral supernatant for 4 hours, in the presence of 8 mg/mL polybrene. Following infection, cells were washed and grown in RPMI/15% FCS for 24 hours and selected in puromycin (Sigma) (1 mg/mL) for 7 days. Cellular expression of the Flag-FANCA or Flag-FANCC proteins was confirmed by Western blotting with an anti-Flag antibody (Kodak, Rochester, NY). Selected lymphoblasts were analyzed for MMC sensitivity, as described previously [13,31]. EC50 values (mean values 6 SD, from three separate MMC assays) were calculated. Genotype analysis of the BD32 cell line An Epstein Barr virus (EBV)-transformed lymphoblast cell line was established from an Egyptian patient with FA. For this patient, onset of aplastic anemia was at age 8 years, and congenital anomalies included cafe au lait spots, an abnormal aorta, micropthalmia, and growth retardation. The clinical diagnosis of FA was confirmed by the diepoxybutane test [34], and an assignment of complementation group A was established by somatic cell fusion studies [27]. Mutation screening of the FANCA gene was carried out by reverse transcriptase–polymerase chain reaction (RT-PCR) of the coding sequence, followed by automated sequencing on an ABI377 sequencer as described previously [27]. A homozygous mutation, FANCA (3329A.C), predicted to encode a mutant protein, FANCA(H1110P), was detected in exon 33 and confirmed by sequencing from genomic DNA. The mutation was confirmed further by restriction enzyme digest, because the mutation creates a new StyI site. Amplification of exon 33 with the primers F-GACACAGGCCAAGGCTCTG and R-GGCATTCCAGACACTGTTCC and digestion of the amplified product with Sty1 yielded fragments of 314, 67, and 9 bp for wild-type DNA and 165, 149, 67, and 9 bp for mutant DNA. The mutation was not found in 42 Middle Eastern controls or in a series of European and North American FA patients [26–28]. Production of pMMP-FANCA and pMMP-FANCA(H1110P) retroviral supernatants and infection of GM6914 cells The patient-derived point mutation in FANCA (3329A.C) was generated by PCR with Pfu polymerase (Stratagene, San Diego, CA). The full-length mutant cDNA insert was subcloned into the retroviral vector, pMMP, and verified by DNA sequencing. The pMMP-FANCA(wt) or pMMP-FANCA(H1110P) retroviral vector was transfected into 293GPG producer cells, and retroviral supernatants were collected [30,32]. GM6914 fibroblasts were infected with retroviral supernatants, and the MMC sensitivity of the fibroblasts was determined as described previously [14,32]. Western blotting and immunoprecipitation Western blotting and immunoprecipitation of FANCA and FANCC were performed as described previously, using affinity-purified polyclonal rabbit antisera [13,14,31]. In vivo labeling of lymphoblasts with 32P-orthophosphate Lymphoblasts were labeled with 32P-orthophosphate, as described previously [15]. Immunofluorescence microscopy Immunofluorescence of retrovirally infected GM6914 fibroblasts was performed as described previously [14].

G. Kupfer et al./Experimental Hematology 27 (1999) 587–593

Results Expression of a mutant FANCA protein, FANCA(H1110P), in a patient-derived cell line Initially, we performed a genotype analysis of an EBVtransformed lymphoblast line, BD32, derived from this patient. We amplified the coding sequence of FANCA by RTPCR and found a single homozygous mutation, 3329A.C, in exon 33. This mutation was confirmed by automated sequencing and restriction digestion of genomic DNA (data not shown). We next confirmed the subtype classification of BD32 cells, using retroviral gene transfer [32] (Table 1). BD32 cells were sensitive to MMC, with an EC50 of 19 nM. Infection of these cells with a retrovirus encoding a Flag-tagged FANCA protein (Flag-FANCA) resulted in functional complementation (EC50 5 132 nM), thereby confirming the subtype of BD32 as FA-A. As controls, Flag-tagged FANCA selectively complemented the FA-A cell line, HSC72, whereas Flag-FANCC selectively complemented the FA-C cell line, PD4. Several FA lymphoblast lines, including BD32, were analyzed by immunoblotting for FANCA and FANCC (Fig. 1). The FA-A cell line, HSC72, did not express FANCA protein (lane 1), whereas HSC72 cells infected with pMMPFANCA overexpressed the FANCA protein (lane 2). The BD32 cell line expressed a full-length mutant FANCA protein (lane 4). The FA-C cell line, PD4 (lane 5), expressed the FANCA protein but did not express FANCC, as described previously [31].

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immunoprecipitate with the anti-FANCA antiserum. These FA-A cell lines (HSC72, PD9, and PD113) were described previously [32]. Interestingly, for BD32 cells, the mutant FANCA(H111OP) protein was expressed but did not coimmunoprecipitate with FANCC (lane 5). These FA-A cell lines (lanes 2–5) expressed comparable levels of FANCC protein (Fig. 2A, whole-cell extract, anti-FANCC immunoblot). We next compared the binding of FANCA and FANCC proteins in uncomplemented vs Flag-FANCA complemented cell lines (Fig. 2B). HSC72 cells and BD32 cells, transduced with pMMP-FlagFANCA and selected in puromycin, expressed the Flag-FANCA(wt) protein (lanes 3 and 4) and normally were resistant to MMC (Table 1). Endogenous FANCC protein expressed in these complemented cell lines coimmunoprecipitated with Flag-FANCA (anti-FANCC immunoblot, lanes 3 and 4). Therefore, the presence of the mutant FANCA(H1110P) protein did not inhibit the interaction of Flag-FANCA(wt) and FANCC (lane 4). Recent studies demonstrated that the phosphorylation of FANCA correlates with its functional activity [15]. We therefore analyzed the phosphorylation of the FANCA protein in the BD32 cells (Fig. 3). The mutant FANCA(H111OP) pro-

The FANCA(H1110P) protein is defective in FANCC binding and phosphorylation Several FA lymphoblast lines, including BD32, were analyzed for FANCA/FANCC protein binding (Fig. 2). For normal lymphoblasts (PD7), the FANCA protein coimmunoprecipitated with the FANCC protein (Fig. 2A, lane 1), as described previously [13]. Three FA-A lymphoblast lines expressed no FANCA protein (lanes 2–4), and FANCC did not

Table 1. Assays for cellular MMC sensitivity

Lymphoblast cell line/plasmid

FA group

EC50 (concentration MMC in nM)

PD7 HSC72 HSC72/Flag-FANCA HSC72/Flag-FANCC PD4 PD4/Flag-FANCA PD4/Flag-FANCC BD32 BD32/Flag-FANCA BD32/Flag-FANCC

Wild type A A A C C C A A A

138 6 26 13 6 5 118 6 16 17 6 4 21 6 8 18 6 11 127 6 24 19 6 10 132 6 14 15 6 8

MMC 5 mitomycin C.

Figure 1. Lymphoblasts derived from FA patients express mutant forms of the FANCA polypeptide. Protein extracts from the indicated EBVimmortalized lymphoblasts were screened by immunoblot with either antiFANCA antiserum (upper blot) or anti-FANCC antiserum (lower blot). Cell lines tested were HSC72 (FA-A) (lane 1), HSC72 1 pMMP-FANCA (lane 2), PD7 (normal adult control) (lane 3), BD32 (FA-A) (lane 4), and PD4 (FA-C) (lane 5).

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Figure 2. The FANCA(H1110P) mutant protein is defective in FANCC binding. (A) EBV-transformed lymphoblast lines, derived from a normal adult control or from five FA patients, were analyzed for FANCA/FANCC binding. Protein from the indicated cell lines was immunoprecipitated with an anti-FANCA (carboxyl-terminal) antiserum. Protein was electrophoresed and transferred to nitrocellulose and immunoblotted with either anti-FANCA (amino-terminal) or anti-FANCC antiserum. Alternatively, whole-cell extracts (WCE) were analyzed directly by anti-FANCC immunoblot (bottom panel). Cell lines analyzed included PD7 (normal control) (lane 1), HSC72 (lane 2), PD9 (lane 3), PD113 (lane 4), BD32 (lane 5), and PD4 (FA-C) cells (lane 6). The HSC72, PD9, and PD113 cells are FA-A cells previously shown to lack the FANCA protein [32]. (B) Protein from the indicated EBV-transformed lymphoblast lines was analyzed, as described in (A). Cell lines included HSC72, BD-32, or HSC72 and BD32 cells transduced with the pMMP-Flag-FANCA retroviral supernatant. MMC sensitivity of these cells lines is shown in Table 1.

tein was not phosphorylated in BD32 cells (lane 1), whereas wild-type FANCA was phosphorylated in wild-type (PD7) cells (lane 3). Taken together, these results demonstrate that the FANCA(H111OP) mutation disrupts FANCA phosphorylation, and that FANCA phosphorylation correlates with FANCC binding and functional activity for this mutant form of FANCA. The FANCA(H1110P) mutant protein fails to complement an FA-A cell line To assess directly the effect of the FANCA(H111OP) mutation on function, we used site-directed mutagenesis to generate a cDNA encoding FANCA(H111OP). Using retrovi-

Figure 3. The FANCA(H1110P) mutant protein is not phosphorylated in vivo. The indicated human lymphoblast lines were radiolabeled in vivo by incubation with 32P-orthophosphate, as described previously [15]. Cell extracts were immunoprecipitated with an anti-FANCA (carboxy terminal) antiserum and separated by denaturing polyacylamide gel electrophoresis. Gels were dried, and phosphorylated proteins were visualized by autoradiography for 12 to 24 hours at 2708deg;C. Cell lines examined were BD32 (lane 1), HSC72 (lane 2), PD7(wt) (lane 3).

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ral-mediated transduction, we expressed either wild-type FANCA or FANCA(H111OP) protein in the MMC-sensitive FA-A fibroblast line, GM6914, which expresses no endogenous FANCA protein [14]. Retrovirally transduced cells were analyzed for MMC sensitivity (Fig. 4). Consistent with previous reports, GM6914 cells infected with pMMP-FANCA(wt) were resistant to MMC. Cells infected with pMMP-FANCA(H111OP) or pMMP-nlsLacZ [14,30] remained sensitive to MMC. Taken together, these results indicate that the FANCA(H111OP) mutation disrupts the activity of the FANCA protein. The FANCA(H1110P) protein is not translocated to the nucleus We next analyzed the subcellular localization of the various FANCA polypeptides expressed in GM6914 fibroblasts by immunofluorescence microscopy (Fig. 5). Cells expressing wild-type FANCA protein displayed FANCA-specific staining predominately in the nucleus, with a faint diffuse staining of the cytoplasm, consistent with previous results [14]. In contrast, predominantly cytoplasmic staining was observed in GM6914 cells expressing the FANCA(H111OP) mutant.

Discussion Based on recent studies, the FA proteins appear to cooperate in a novel pathway, leading to the maintenance of chromosome stability. This pathway requires several biochemical events, including the phosphorylation of the FANCA protein [15], the binding of FANCA and FANCC [13], and the nuclear accumulation of the FANCA/FANCC complex

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[14]. Although the precise nuclear function of the FANCA/ FANCC complex remains unknown, the complex may play a role in DNA repair or chromosome segregation. The FA pathway is disrupted not only in FA-A and FA-C cell lines, but also in other FA complementation groups, suggesting that products of other FA genes participate in the pathway [15]. In the current study, we characterized a patient-derived mutant form of the FANCA protein, FANCA(H111OP). We analyzed the mutant protein in FA patient-derived lymphoblasts (BD32) and in retrovirally infected FA-A fibroblasts (GM6914). Interestingly, the mutant FANCA protein was defective in phosphorylation, FANCC binding, and nuclear accumulation, further demonstrating the importance of these events in the FA pathway. The FANCA(H111OP) mutation may disrupt a domain of the protein required for its functional activity [26,27]. The direct molecular function of such a domain remains to be determined. The domain may be required for phosphorylation, FANCC binding, or binding to other protein subunits of the FA complex. Alternatively, this domain may be directly required for nuclear transport or stabilization of the FANCA/FANCC protein complex in the nucleus. Finally, the domain may be required for activation of some nuclear function, such as DNA binding or DNA repair. It remains unknown whether the normal FANCA/FANCC protein complex has DNA binding or repair activity. The absence of nuclear accumulation of the FANCA(H111OP) protein has many possible explanations. First, although FANCA(H111OP) has an NLS at its amino terminus [14], an intact carboxy-terminal functional domain may be required for efficient nuclear transport. Second, the FANCA-

Figure 4. The FANCA(H1110P) mutant protein fails to complement the mitomycin C (MMC) sensitivity of an FA-A fibroblast line. Following retroviral transduction, the indicated FANCA polypeptides were expressed in the FA-A fibroblast line, GM6914. Infected cells were grown in the presence of various concentrations of MMC, and cell survival was assayed by crystal violet staining of viable colonies. MMC sensitivity of an SV40-transformed (normal adult) control cell line, GM0637, also is shown. The values shown are representative of three separate experiments.

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Figure 5. The FANCA(H1110P) mutant protein fails to accumulate in the nucleus. The parental FA-A fibroblast line, GM6914, does not express detectable FANCA protein [14]. GM6914 cells were infected with the indicated retroviral supernatants, including pMMP-FANCA(wt), pMMP-FANCA(H1110P), or pMMP-nlsLacZ (encoding b-galactosidase). Pools of infected cells were stained with anti-FANCA(C) and the DNA-specific dye, DAPI (49, 6-diamidino-2phenylindole) and analyzed by immunofluorescence, as described in the text.

(H111OP) protein may be transported to the nucleus but may be degraded rapidly in the nucleus, due to the absence of FANCC binding. Third, the FANCA(H111OP) mutation may disrupt a nuclear retention signal of the carboxy terminus, resulting in diffusion or export of the protein back to the cytoplasm. Because immunofluorescence only measures the steady-state level of nuclear localization, our studies cannot distinguish among these possibilities. Studies regarding the interaction and cellular localization of the FA proteins have been largely conflicting. First, some studies show that the FANCA and FANCC protein bind in a functional complex [13,15], whereas other studies fail to detect the complex [35]. Second, some studies show that the FANCC protein is localized to the cytoplasm and nucleus [13,14], whereas other studies show that FANCC is primarily cytoplasmic [36]. The current study helps to resolve some of the controversy in the field. The new data reported herein further demonstrate the critical functional importance of FANCA phosphorylation, FANCA/FANCC binding, and nuclear accumulation of the complex. Increasing evidence suggests that FANCA and FANCC are part of a larger protein complex. For instance, the nuclear FANCA/FANCC protein complex has a high molecular mass (.400 kDa), consistent with the presence of addi-

tional (unknown) protein subunits of the complex (G. Kupfer, unpublished observation). Recent studies suggest that FANCC binds to additional proteins, such as GRP94 [37] and cdc2 [18]. Interestingly, the recently cloned FANCG protein [10,12] also is a component of the FANCA/FANCC protein complex (I. Garcia Higuera, unpublished observation). The identification of additional components of the FANCA/FANCC complex may help to define the biochemical functions of FANCA and FANCC. It will be interesting to determine whether other FA gene products also are assembled in the protein complex. Acknowledgments Supported by NIH grants R01-HL52725 and PO1HL54785. G.M.K. is supported by NIH grant K08-H103420. A.D.D. is a Scholar of the Leukemia Society of America (LSA). We thank members of the D’Andrea lab for helpful discussions. We thank Markus Grompe and Petra Jakobs for FA cell lines. We thank Dr. Irene Roberts for referring the FA-A patient.

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