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1 Department of Immunology and Molecular Genetics, and 2 Department of Rehabilitation Medicine, and 3 Department of Nephrology, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan
4 Department of Hematology, Oncology and Respiratory Medicine, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Okayama 700-8558, Japan
5 Laboratory of Molecular Genetics, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, Japan
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Introduction |
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Recent studies have indicated that the FA pathway promotes DNA repair function through both HR and TLS pathways (Yamamoto et al. 2003; Niedzwiedz et al. 2004; Hirano et al. 2005; Nakanishi et al. 2005). However, how FA proteins exert their function is still unclear. Eight FA proteins constitute the nuclear FA core complex (FancA/B/C/E/F/G/L/M). Only two of them, FancL and FancM, have conserved sequence motifs that suggest an obvious catalytic function. FancL contains a PHD-type zinc-finger (PHD finger) domain (Meetei et al. 2003a), while helicase and nuclease domains are found in the FancM protein (Meetei et al. 2005; Mosedale et al. 2005), which is homologous to Archea Hef (Nishino et al. 2005). Upon DNA damage or replication fork stalling, the FancD2 protein is modified by monoubiquitination on a specific Lys residue (Lys 561 in human (h) protein). Following monoubiquitination, which is likely mediated by FancL (Meetei et al. 2003a), FancD2 is targeted to chromatin and accumulates at or close to sites of damaged DNA, forming nuclear foci that can be detected by immunofluorescence. Consistent with the functional studies (Nakanishi et al. 2005; Yamamoto et al. 2005), FancD2 foci co-localize with HR proteins, such as Rad51, BRCA1 (Taniguchi et al. 2002a) and BRCA2 (Wang et al. 2004), or with the TLS factor Rev1 (Niedzwiedz et al. 2004) following ICL induction. This monoubiquitination seems both necessary and sufficient for FancD2 activation as shown by ectopic expression of either mutated FancD2 (Garcia-Higuera et al. 2001; Taniguchi et al. 2002b) or a FancD2-ubiquitin fusion protein (Matsushita et al. 2005). Any mutation that has been identified in the core complex components appears to compromise the E3 ligase activity as well as structural integrity of the complex, resulting in common down-stream defects in monoubiquitination and focus formation of FancD2. In addition, the disrupted core complex may lose its own effector function, which is still undefined but might be carried out by components such as FancM (Matsushita et al. 2005; Meetei et al. 2005; Mosedale et al. 2005).
Identification of the other two FA genes, FancD1/BRCA2 (Howlett et al. 2002) and FancJ/BRIP1 (Bridge et al. 2005; Levitus et al. 2005; Levran et al. 2005; Litman et al. 2005), has forged a strong link between FA and familial breast cancer. While mutations in a single allele of BRCA2 predispose carriers to breast or ovarian cancer, biallelic hypomorphic mutations are found in a subset of FA patients (Howlett et al. 2002). Such patients are clinically rather distinct, for they often develop early onset leukaemia and brain tumors, which are rarely seen in individuals from the other complementation groups (Hirsch et al. 2003; Wagner et al. 2004). FancJ/BRIP1 was originally identified through interaction with another breast cancer suppressor, BRCA1 (Cantor et al. 2001). The role of BRCA2 is well characterized as a major regulator of Rad51, a central molecule in HR that mediates homology searching and strand transfer (Venkitaraman 2002). On the other hand, BRIP1 protein has a helicase activity, which preferentially unwinds a forked duplex structure that may mimic a stalled replication fork (Gupta et al. 2005). Of importance, in both fancd1 and fancj cell lines, monoubiquitination of FancD2 occurs normally, placing these molecules down-stream of, or parallel to, the FA core complex–FancD2 pathway (Howlett et al. 2002; Levitus et al. 2004).
In this study, we have identified chicken FancL as a FancD2-interacting protein by yeast two-hybrid (Y2H) screening. We have established and characterized a FANCL-deficient mutant DT40 cell line, and confirmed a role for FancL in HR. To rigorously test whether the physiological function of FancD2 totally relies on its monoubiquitination, we generated DT40 cells carrying a monoubiquitination site "knock-in" mutation (Lys 563 changed to Arg) at the endogenous FANCD2 locus, and compared phenotypes among FANCD2-null mutant (designated fancd2-null), the "knock-in" K563R mutant (fancd2-K563R) and FANCL-deficient (fancl) cells. Our results establish a requirement for FancL and FancD2 monoubiquitination in the repair of DSBs mediated by HR.
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Results |
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Previous studies showed that FancD2 plays crucial roles in the DNA damage response; however, the precise molecular mechanism by which FancD2 is activated is largely unknown. To analyze this, we employed the Y2H screening to search for proteins that interact with FancD2, using an N-terminal chFancD2 fragment (chFancD2 2-722) (Fig. 2A). Full length chFancD2 was not suitable for screening because of autoactivation of the reporters (data not shown). We isolated 15 positive clones. DNA sequencing of the interacting clones revealed that one of them encoded full-length chPHF9, later identified as FancL (Meetei et al. 2003a). Chicken PHF9/FancL encodes a protein with 373 amino acids, and the percentage amino acid sequence identity with hFancL is 68%. Human FancL has three potential WD40 repeats and a PHD finger motif (Meetei et al. 2003a), and these were all well conserved in chFancL (NCBI/EMBL/DDBJ accession number: AB214907 for chFancL). A part of the amino acid sequence has been described in Matsushita et al. (2005) as supplement Fig. S4A.
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Next, we tried to narrow down the interacting portion in chFancL. To test whether the PHD domain is important for FancL–FancD2 interaction, we changed the two conserved amino acid residues to Ala (chFancL C305A, which was described in Matsushita et al. (2005), and W339A). In addition, the Pro 330 and the Gln 348 residues, which are conserved from human to fish, were each mutated to Lys and Thr (P330K and Q348T), the corresponding residues found in the Drosophila FancL. The C305A mutation reduced the chFancD2–chFancL interaction in both Y2H (Fig. 2C) and co-immunoprecipitation (Fig. 1) assays. This could be due to destruction of the whole protein structure, since the equivalent C307A mutation in hFancL impaired the interaction with a few other FA proteins (Table 1, see below). However, the other PHD domain mutations (P330K and Q348T), but not W339A, also affected the interaction (Fig. 2B,C) indicating that the PHD finger domain is required for interaction with chFancD2. We also divided the full length chFancL cDNA into four overlapping fragments as shown in Fig. 2B, and tested Y2H interaction. None of them interacted significantly with the chFancD2 2-722 bait, indicating that both PHD domain (305-374 region) and N-terminal 1-143 region are required for the interaction. The inverse combination of the bait–prey was not feasible, since the bait contained the full-length chFancL autoactivated reporters (data not shown).
Since hFancL was isolated as a FancA-associated protein, it should be a part of the FA core complex (Meetei et al. 2003a). We examined whether FancL directly binds to other components of the FA core complex by Y2H. The bait plasmids were prepared by subcloning a variety of human FA core complex genes, because fewer chicken FA genes were available. Using hFancL as a prey, we found that, besides hFancD2, only hFancA and hFancF displayed significant binding with hFancL (Table 1). These interactions were disrupted by the C307A mutation (equivalent to chFancL C305A). Consistently, a previous report showed that co-immunoprecipitation between hFancA and hFancL was reduced by the C307A mutation (Meetei et al. 2003a).
FANCL-deficient cells show defects in FancD2 activation and repair of DNA damage
To characterize the physiological function of FancL, we generated FANCL gene disruptants in the chicken B cell line DT40. Based on the cDNA sequence, we PCR-amplified genomic DNA fragments of chFANCL and designed the targeting construct (Fig. 3A). The gene targeting was achieved by serial transfections with the construct, and gene disruption was verified by Southern (Fig. 3B) and Northern blot analyzes (Fig. 3C).
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To test whether fancl cells have HR defects, we examined the frequencies of gene targeting events that occur at two independent genomic loci. Wild-type and fancl cells were transfected with gene targeting vectors in parallel, and targeting events were examined by Southern blot analysis. FANCL-deficient cells had a drastically reduced gene targeting efficiency compared to wild-type cells, and this defect was partially complemented by expression of GFP-chFancL (Table 2). These results indicate that fancl cells display phenotypes that are observed in other DT40 FA mutants, and that the severity of the defects is very similar to that seen in fancd2-null mutant cells (Yamamoto et al. 2005).
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In DT40 cells, it is quite straightforward to introduce a specific mutation into an endogenous locus by "knock-in" gene targeting. To examine the physiological significance of monoubiquitination of FancD2, we generated a FANCD2 mutation in which the critical Lys residue was changed to Arg (fancd2-K563R). As summarized in (Fig. 4A,B), we first deleted three exons containing the monoubiquitination site in one allele by the FANCD2-bsr vector. Then the other allele was "knocked-in" with K563R mutation using D2-K563R-his vector. Finally, the bsr and his resistance gene cassettes were excised by GFP-Cre expression as described in Experimental procedures. Genotypes generated in each step in these procedures were verified by Southern blotting (Fig. 4C), and the mutation in fancd2-K563R cells was confirmed by DNA sequencing of both PCR-amplified genomic DNA and transcripts (data not shown). Indeed, Western blotting showed that FancD2 L-form was not detected in fancd2-K563R cells before or after MMC treatment (Fig. 4D).
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We compared the phenotypes of fancl, fancd2-null and fancd2-K563R cells. First, we examined focus formation and chromatin targeting of FancD2 in fancl and fancd2-K563R cells. While wild-type cells displayed robust formation of FancD2 foci following MMC treatment, similarly treated fancl and fancd2-K563R cells could not form any significant levels of FancD2 foci (Fig. 5A). In wild-type cells, the FancD2 L-form was found to be targeted to chromatin after MMC treatment; however, both s- and L-forms in fancl and fancd2-K563R cells were not significantly localized in chromatin fraction (Fig. 5B). These results were consistent with the notion that chFancD2 was monoubiquitinated at K563 site, which was most likely catalyzed by chFancL. We next carried out cell growth analysis in parallel, and found that fancl, fancd2-null and fancd2-K563R cells displayed a similarly reduced growth rate compared to wild-type cells (Fig. 6A). We also found that the plating efficiency of these mutants was comparable (
30%), but was reduced relative to that of wild-type cells (nearly 100%).
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Next, we examined the cross-linking sensitivity of these mutant cells using a colony formation assay in the presence of cisplatin (Fig. 6C) or following brief exposure to MMC (Fig. 6D). Fancd2-null and fancd2-K563R mutants showed almost the same level of sensitivity to these cross-linking agents, indicating that the cross-link repair function of FancD2 depends on its monoubiquitination. In contrast, fancl cells were slightly, but significantly, more sensitive than either fancd2-null or fancd2-K563R cells.
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Discussion |
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We also found that any partial deletion of FancL or some of the tested point mutations in the PHD domain abrogated the interaction (Fig. 2), indicating that at least the PHD domain and the N-terminal 1-143 region are required to interact with chFancD2. hFancL C307A mutation also disrupted interactions with hFancA and hFancF (Table 1). As we have previously described, the equivalent chicken C305A mutation abrogated the capacity of the GFP-chFancL protein to complement cisplatin sensitivity as well as chromatin localization when expressed in fancl cells (Matsushita et al. 2005). Consistent with these results, a previous study reported that the human mutation (C307A) disrupted co-immunoprecipitation with hFancA, the ability to complement fancl patient's cells with FancD2 monoubiquitination and in vitro auto-ubiquitination activity (Meetei et al. 2003a). Thus this mutation likely disrupts multiple aspects of FancL function, including maintenance of the integrity of the core complex as well as ubiquitin E3 ligase activity. It would be interesting to test other mutations in a similar manner to dissect functions of FancL protein.
We have provided evidence of roles for FancL in ICL repair and HR. These functions were not unexpected, given the proposed role of FancL in the monoubiquitination and activation of FancD2. Several studies have implicated the monouibiquitination as a critical modification for activation of FancD2 for DNA repair. For example, mutant FancD2 protein lacking the monoubiquitination site abolished these functions when expressed in FANCD2-deficient PD20 cells (Garcia-Higuera et al. 2001; Taniguchi et al. 2002b). However, such ectopic expression does not necessarily provide definitive proof for a physiological role of the monoubiquitination, since this may lead to overproduction of FancD2. Furthermore, the endogenous cis element could be crucial for regulation of FANCD2, since defective immunoglobulin gene conversion in fancd2 cells could not be complemented by CMV promoter-driven expression of FancD2 (Yamamoto et al. 2005). Similarly, the defects in targeted integration frequencies could not be fully complemented in both fancd2 (Yamamoto et al. 2005) and fancl (this study) by expression of the corresponding expression vectors.
To more rigorously test the role of monoubiquitination (as well as FancL) for FancD2 activation, we generated fancd2-K563R cells and compared these cells with fancd2-null and fancl mutants. We found that fancd2-K563R and fancd2-null mutants displayed essentially the same degree of defects in cell growth, HR-mediated chromosomal DSB repair, and cisplatin and MMC sensitivity, indicating that absence of monoubiquitination functionally equates to absence of the protein. Thus, the monoubiquitination is critically important for FancD2 to function, at least in the assays analyzed here.
In cell growth and HR-directed DSB repair assays, the fancl and fancd2 mutants showed a similar degree of impairment. In contrast, fancl cells showed consistently more severe cross-linking agent sensitivity compared to the other two fancd2 mutants. Since two independently derived fancl clones (data not shown) were both more sensitive to cross-linkers than were the fancd2 mutants, it seems unlikely that these results were due to simple clonal variation. This raises the following two possibilities. First, it is possible that FancL may ubiquitinate another substrate that modulates ICL repair, but not HR. Absence of FancL affects the function of this putative substrate as well as that of FancD2, and therefore the level of cisplatin sensitivity in fancl cells is the combined effects of the failure of two modifications. Alternatively, or additionally, FANCL mutation may disrupt the core complex, which will have the effect of disrupting the DNA repair activity mediated by the core complex. Based on our data using FancD2-fusion proteins, we have proposed that the core complex has its own role in DNA repair (Matsushita et al. 2005). FancM is an obvious candidate for an effector molecule of the core complex because of its potential DNA modifying activity. For example, FANCL mutation may affect mobilization of FancM into sites of cross-link damage. Consistently, fancm mutant DT40 cells do not have a defect in the I-SceI-based HR repair assay (Mosedale et al. 2005).
In summary, we isolated chicken FancL as an interacting partner of FancD2, and confirmed the role of FancL in HR repair. Generation of fancd2-K563R cells, and direct comparison with fancd2-null cells, provided more definitive proof for a crucial role of monoubiquitination in FancD2 activation. Our data also suggest that there is a FancD2-independent role for FancL in ICL repair, but not in HR. The functional relationships between FancD2 and the core complex are still poorly defined and clearly deserve further investigation.
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Experimental procedures |
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The Y2H assay was performed using the Matchmaker LexA two-hybrid system (BD Clontech, Palo Alto, CA, USA) following the manufacturer's protocol (Ishiai et al. 2004). For screening of FancD2-interacting proteins, an N-terminal fragment of chFancD2 (amino acid residues 2-722) was cloned in-frame with the LexA DNA binding domain in the bait plasmid, pEG202. EGY48 yeast cells containing both the bait and the pSH18-34 reporter plasmids were transformed with a pJG4.5-DT40 cDNA library (kindly provided by Dr Ryo Goitsuka, Science University of Tokyo, Chiba, Japan). Using a dual reporter expression system (lacZ and LEU2), double positive clones was selected from 9 x 105 transfectants. Plasmid DNA was recovered, and the cDNA inserts were PCR-amplified and sequenced. To detect the interaction between chFancD2 and chFancL, various chFancD2 and chFancL fragments were cloned into the bait or prey plasmid, respectively. Point mutation was introduced using QuikChange mutagenesis kit (Stratagene, La Jolla, CA, USA). hFANCA, hFANCB, hFANCE and hFANCG cDNAs or the hFANCC cDNA were kindly provided by Dr Hans Joenje (VU University Medical Center, Amsterdam, the Netherlands) or Dr Manuel Buchwald (Hospital for Sick Children, Toronto, Canada), respectively. hFANCL cDNA was obtained from Biological Resource Center, National Institute of Technology and Evaluation (Kisarazu, Chiba, Japan). hFANCF (full length) or hFANCD2 (1-719 amino acids) cDNAs were isolated by RT-PCR and verified by sequencing. Various human FA genes were cloned into the bait plasmids, while hFANCL was cloned into the prey plasmid. The interactions of two proteins were evaluated by growth on synthetic assay media lacking leucine and by blue/white colony coloration on synthetic media containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) (Nacalai Tesque Inc., Kyoto, Japan). For quantitative analysis, the ß-galactosidase activity (Miller unit) was measured by liquid culture assay using o-nitrophenyl-ß-D-galactopyranoside (Nacalai Tesque Inc.) as the substrate.
Cell culture and expression plasmids
Chicken DT40 cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 1% chicken serum, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, penicillin and streptomycin in a 5% CO2 incubator at 39.5 °C. Generation of fancd2 (hereafter designated fancd2-null) DT40 cells were previously described (Yamamoto et al. 2005). Cell growth analysis was done as described (Yamamoto et al. 2003). Construction of GFP-chFancL expression plasmid was previously described (Matsushita et al. 2005). An expression vector for FLAG-chFancD2 was constructed by inserting full-length chFANCD2 cDNA in frame into pFLAG-C1, which was made by replacing the EGFP segment of pEGFP-C1 (Clontech) with synthetic FLAG oligonucleotides. The GFP-Cre expression plasmid pBS598 was kindly provided by Dr Brian Sauer (Stowers Institute for Medical Research, Kansas City, MO, USA) (Gagneten et al. 1997). Stable or transient transfections were done by electroporation as described (Yamamoto et al. 2003).
Co-immunoprecipitation experiments
293T cells were transiently transfected with GFP-chFancL and FLAG-chFancD2 expression vectors, and were harvested 24 h later. Cells were lyzed in lysis buffer (1% NP-40, 10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 10 mM NaF, 1 mM PMSF) supplemented with proteinase inhibitor cocktail (Complete EDTA-free Tablet, Roche Diagnostics, Basel, Switzerland). FLAG-chFancD2 was immunoprecipitated using agarose-beads conjugated with monoclonal anti-FLAG antibody M2 (Sigma, St. Louis, MO, USA). The immunoprecipitates or whole cell extracts were separated with 6% (for FancD2) or 7.5% (for FancL) SDS-PAGE, and then transferred to a membrane and detected with anti-FLAG or anti-GFP (Clontech) antibodies.
Generation of FANCL-deficient or FANCD2-K563R "knock-in" mutant DT40 cells
The genomic fragment of chFANCL or chFANCD2 was isolated by PCR amplification from DT40 genomic DNA. The FANCL-targeting vector was designed by replacing a 20 kb genomic fragment containing six exons that correspond to chFancL amino acids 105-272, with a bsr- or his-resistant gene cassette. For making the fancd2-K563R
"knock-in" vector, K563R mutation was introduced using a QuikChange Kit (Stratagene), and a his-resistance cassette was inserted in the intron (see Fig. 4B). DT40 cells were targeted sequentially by the FancD2-bsr targeting vector (Yamamoto et al. 2005) followed by the fancd2-K563R-his vector. The GFP-Cre expression plasmid was transiently transfected by electroporation to remove the bsr- and his-cassettes, which are flanked by loxP sequences. Twenty-four hours after transfection, GFP-positive cells were clone-sorted using FACSAria (Becton Dickinson). Removal of the cassettes was ensured by Southern blot analysis with chFANCD2 probe.
Measurement of sensitivity of cells to DNA damaging agents
Colony formation was assayed in medium containing 1.4% methylcellulose. Cells were irradiated with X-rays (Linear Accelerator; Mitsubishi Electric, Inc., Tokyo, Japan), or exposed for 1 h to MMC (Kyowa Hakkou, Tokyo, Japan) or continuously to cisplatin (Nihon Kayaku, Tokyo, Japan) as previously described (Yamamoto et al. 2003). After the cells were cultured for 7 (for wild-type) to 14 days (for mutant cells), visible colonies were counted.
Measurement of HR-mediated repair of DSBs by I-SceI expression
Analysis of HR-mediated repair of I-SceI-induced DSBs was done as described (Yamamoto et al. 2003). The recombination substrate, SCneo (kindly provided by Dr Maria Jasin, Sloan-Kettering Institute, NY, USA), was targeted into the OVALBUMIN locus in wild-type and FA mutant cells. I-SceI expression vector, or the control plasmid, pBluescript, was transiently transfected by electroporation, and cells were selected in 96-well culture plates containing 2 mg/mL G418 (Nacalai Tesque Inc.). Surviving colonies were counted after 14 days.
General techniques for DT40 cell analysis
Northern blot analysis, Western blotting, measurement of targeted integration frequencies, subnuclear focus formation assay, preparation of subcellular fractionation into the soluble and chromatin were done as previously described (Yamamoto et al. 2003; Ishiai et al. 2004; Matsushita et al. 2005).
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Acknowledgements |
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Footnotes |
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aThese authors contributed equally to this work.
bPresent address: Department of Human Genetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553 Japan.
*Correspondence: E-mail: minorut{at}hiroshima-u.ac.jp |
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Received: 16 September 2006
Accepted: 30 November 2006
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