Oncogene (2008) 27, 3641–3652 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3 JB Wilson1, K Yamamoto2,9, AS Marriott1, S Hussain3, P Sung4, ME Hoatlin5, CG Mathew6, M Takata2,10, LH Thompson7, GM Kupfer8 and NJ Jones1 1Molecular Oncology and Stem Cell Research Group, School of Biological Sciences, University of Liverpool, Liverpool, UK; 2Department of Immunology and Medical Genetics, Kawasaki Medical School, Kurashiki, Okayama, Japan; 3Department of Biochemistry, University of Cambridge, Cambridge, UK; 4Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA; 5Division of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR, USA; 6Department of Medical and Molecular Genetics, King’s College London School of Medicine, Guy’s Hospital, London, UK; 7Biosciences and Biotechnology Division, L441, Lawrence Livermore National Laboratory, Livermore, CA, USA and 8Department of Pediatrics, Division of Hematology-Oncology, Yale University School of Medicine, New Haven, CT, USA Fanconi anemia (FA) is a human disorder characterized intricate interface between FANC and HRR proteins in by cancer susceptibility and cellular sensitivity to DNA maintaining chromosome stability. crosslinks and other damages. Thirteen complementation Oncogene (2008) 27, 3641–3652; doi:10.1038/sj.onc.1211034; groups and genes are identified, including BRCA2, which published online 21 January 2008 is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex Keywords: Fanconi anemia; ATR; interstrand cross- that is required for the monoubiquitylation of FANCD2 links; DNA repair; RAD51 paralog; replication restart; and FANCI. FANCD2, like FANCD1/BRCA2, is not epistasis part of the core complex, and we previously showed direct BRCA2–FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of Introduction BRCA2and FANCD2.We also show that phosphoryla- tion of FANCG serine 7 is required for its co-precipitation Fanconi anemia (FA) is characterized clinically by with BRCA2, XRCC3 and FANCD2, as well as the direct progressive aplastic anemia, multiple congenital ab- interaction of BRCA2–FANCD2. These results argue normalities and a predisposition to malignancy includ- that FANCG has a role independent of the FA core ing acute myeloid leukaemia and squamous carcinomas complex, and we propose that phosphorylation of serine 7 of the head and neck (Alter, 1996). FA cells universally, is the signalling event required for forming a discrete but not exclusively, display hypersensitivity to DNA complex comprising FANCD1/BRCA2-FANCD2- interstrand crosslinking agents (Carreau et al., 1999; FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to Kennedy and D’Andrea, 2005). As we discussed express either phospho-Ser7-FANCG, or full length previously (Thompson et al., 2005), this phenotype is BRCA2protein, lack the interactions amongst the four likely due to the absolute dual requirement for RAD51- component proteins. A role for D1-D2-G-X3 in homo- mediated homologous recombination repair (HRR) and logous recombination repair (HRR) is supported by our translesion synthesis (TLS) during crosslink repair at finding that FANCG and the RAD51-paralog XRCC3 are broken replication forks (Hinz et al., 2007; Patel and epistatic for sensitivity to DNA crosslinking compounds Joenje, 2007). As many fanc mutants show a suppression in DT40 chicken cells. Our findings further define the of base-substitution mutagenesis (reviewed by Hinz et al., 2006), it is clear that the ‘FA pathway’ has a much broader role than promoting just the repair of crosslink damage. Correspondence: Dr NJ Jones, Molecular Oncology and Stem Cell Thirteen FA genes are now identified (Levitus et al., ResearchGroup, Schoolof Biological Sciences, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK. 2006; Smogorzewska et al., 2007), but the exact E-mail: [email protected] mechanistic function of many FA proteins remains 9Current address: Department of Hematology, Okayama Citizen’s unclear. FANCD1 is identified as the breast cancer Hospital, Okayama City, Okayama 700-8557, Japan. susceptibility protein BRCA2 (Howlett et al., 2002), 10Current address: Radiation Biology Center, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan. whose role lies in regulating RAD51 in HRR (Davies Received 20 September 2007; revised 20 November 2007; accepted 11 et al., 2001; Gudmundsdottir and Ashworth, 2006). December 2007; published online 21 January 2008 BRCA2 function requires a binding partner PALB2 FANCG required for BRCA2–FANCD2–XRCC3 interactions JB Wilson et al 3642 (Xia et al., 2006), recently identified as FANCN (Reid et al., 2007; Xia et al., 2007). The FANCA/B/C/E/F/G/ L/M proteins, together with FAAP24/100, are subunits of a nuclear core complex (Mathew, 2006; Ciccia et al., HeLa (WT)BD220 (A)PD20 (D2)BD215 (C)EUFA409EUFA121 (E) EUFA143 (F) CRL-1583 (G) (WT) 2007; Ling et al., 2007) required for the interdepen- BRCA2 dent monoubiquitylation of FANCD2 and FANCI IP: Anti-FANCD2 (Smogorzewska et al., 2007). IgG Increasing evidence implicates FANCD2 and other 1 2 3 4 5 6 7 8 FANC proteins, including FANCG, being coupled to HRR (Niedzwiedz et al., 2004; Yang et al., or ctor t e tor FANCG 2005; Taniguchi and D’Andrea, 2006). For example, FANCG we previously showed direct interactions between the vec FANCG following pairs of proteins: FANCG–BRCA2; FANCD2– 40BP6 KO40 NM3+ NM3+vec143+ 143+ 326SV+v326SV+ BRCA2; FANCG–XRCC3 (Hussain et al., 2003, 2004, BRCA2 2006). XRCC3 is one of five RAD51 paralogs, which have IP: Anti-FANCD2 nonredundant roles in HRR (Thacker, 2005). A reduced efficiency of HRR, measured after the induction of IgG I-SceI-mediated double-strand breaks, was reported in human and chicken fancg mutants (Yamamoto et al., FANCG 2003; Nakanishi et al., 2005). FANCG protein has at least WB seven tetratricopeptide repeats (TPRs), which are critical ß-Actin for the function of FANCG and its interaction with FANCA, FANCF and the HRR proteins BRCA2 and 1234 5678 XRCC3 (Blom et al., 2004; Hussain et al., 2006). These Figure 1 FANCG is required for the interaction of FANCD2 and TPRs indicate that FANCG likely acts as a scaffold for BRCA2 in mammalian cells. Cell lines were treated with50 n M mitomycin C for 18 hprior to preparation of lysates (for thisfigure the assembly or stabilization of protein complexes (Lamb and subsequent figures unless stated). Cell lysates were immuno- et al., 1995; Groves and Barford, 1999). precipitated withantibody to FANCD2, samples loaded for FANCA/D1/D2/G/M (Yamashita et al., 1998; electrophoresis on a SDS–polyacrylamide gel electrophoresis Taniguchi et al., 2002; Collins and Kupfer, 2005; Esashi (PAGE) gel and blots probed withan anti-BRCA2 C-terminal et al., 2005; Meetei et al., 2005) are all phosphoproteins. antibody (amino acids 3245–3418). (a) FANCD2 and BRCA2 co- precipitate in wild-type (HeLa, CRL-1583) and FA-A (BD220), FANCG phosphorylation occurs at serines 7, 383 and FA-C (BD215), FA-E (EUFA409) and FA-F (EUFA121) human 387, and these modifications are functionally important cell lines, but not in the FA-G cell line EUFA143. (b) FANCD2 for cellular resistance to crosslinking damage (Mi et al., and BRCA2 interaction is restored in fancg mutant cell lines 2004; Qiao et al., 2004). The mutant FANCG-S7A transduced withwild-type human FANCG cDNA, but not withan empty pMMP vector (EUFA143, 326SV and NM3). It is also protein expressed in FA-G EUFA143 lymphoblasts was restored in a knockout hamster cell line (KO40) transfected with able to bind and stabilize FANCA and FANCC, the genomic hamster gene (40BP). Western blot with anti-FANCG although FANCD2 monoubiquitylation was slightly shows FANCG expression in the same cell lines. reduced (Qiao et al., 2004). While it is well established that FANCG is critical for the assembly of a functional FA nuclear core complex (Garcia-Higuera et al., 1999; FA-G lymphoblast cell lines (Hussain et al., 2004). The Waisfisz et al., 1999; Gordon and Buchwald, 2003), here interaction was further investigated by examining other we define an essential role for FANCG in mediating FA complementation groups. BRCA2 co-immunopre- the key interaction between BRCA2 and FANCD2 cipitated using FANCD2 antisera in bothwild-type cells (Hussain et al., 2004; Wang et al., 2004). Moreover, we (HeLa and CRL-1583) and cells from groups FA-A/C/ present evidence that phosphorylation of FANCG at E/F (Figure 1a), while it was absent in FA-D2 cells serine 7 is essential, not only for its own direct (PD20). Western blotting confirmed that each FA cell interactions withBRCA2 and XRCC3, but also the line utilized failed to express detectable levels of the pair-wise interactions among BRCA2, FANCD2 respective FA protein (data not shown). Co-precipita- and XRCC3. We propose that this new complex tion between FANCD2–BRCA2 failed to occur in CHO consisting of at least these four FANC and HRR FancG mutants NM3 and KO40, and in human FA-G proteins (D1-D2-G-X3) is responsible for promoting the fibroblast (326SV) and lymphoblast (EUFA143) cells HRR component of crosslink repair. (Figure 1b). Introduction of wild-type FANCG (either human cDNA or genomic hamster