Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair
2012; Springer Nature; Volume: 31; Issue: 17 Linguagem: Inglês
10.1038/emboj.2012.197
ISSN1460-2075
AutoresKoichi Sato, Masamichi Ishiai, Kazue Toda, Satoshi Furukoshi, Akihisa Osakabe, Hiroaki Tachiwana, Yoshimasa Takizawa, Wataru Kagawa, Hiroyuki Kitao, Naoshi Dohmae, Chikashi Obuse, Hiroshi Kimurâ, Minoru Takata, Hitoshi Kurumizaka,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoArticle24 July 2012free access Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair Koichi Sato Koichi Sato Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Masamichi Ishiai Masamichi Ishiai Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan Search for more papers by this author Kazue Toda Kazue Toda Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Satoshi Furukoshi Satoshi Furukoshi Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Akihisa Osakabe Akihisa Osakabe Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Hiroaki Tachiwana Hiroaki Tachiwana Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Yoshimasa Takizawa Yoshimasa Takizawa Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Wataru Kagawa Wataru Kagawa Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Hiroyuki Kitao Hiroyuki Kitao Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan Graduate School of Medical Sciences, Department of Molecular Oncology, Kyushu University, Fukuoka, Japan Search for more papers by this author Naoshi Dohmae Naoshi Dohmae RIKEN Advanced Science Institute, Saitama, Japan Search for more papers by this author Chikashi Obuse Chikashi Obuse Graduate School of Life Science, Hokkaido University, Hokkaido, Japan Search for more papers by this author Hiroshi Kimura Hiroshi Kimura Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Minoru Takata Corresponding Author Minoru Takata Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan Search for more papers by this author Hitoshi Kurumizaka Corresponding Author Hitoshi Kurumizaka Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Koichi Sato Koichi Sato Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Masamichi Ishiai Masamichi Ishiai Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan Search for more papers by this author Kazue Toda Kazue Toda Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Satoshi Furukoshi Satoshi Furukoshi Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Akihisa Osakabe Akihisa Osakabe Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Hiroaki Tachiwana Hiroaki Tachiwana Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Yoshimasa Takizawa Yoshimasa Takizawa Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Wataru Kagawa Wataru Kagawa Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Hiroyuki Kitao Hiroyuki Kitao Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan Graduate School of Medical Sciences, Department of Molecular Oncology, Kyushu University, Fukuoka, Japan Search for more papers by this author Naoshi Dohmae Naoshi Dohmae RIKEN Advanced Science Institute, Saitama, Japan Search for more papers by this author Chikashi Obuse Chikashi Obuse Graduate School of Life Science, Hokkaido University, Hokkaido, Japan Search for more papers by this author Hiroshi Kimura Hiroshi Kimura Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Minoru Takata Corresponding Author Minoru Takata Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan Search for more papers by this author Hitoshi Kurumizaka Corresponding Author Hitoshi Kurumizaka Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Search for more papers by this author Author Information Koichi Sato1,‡, Masamichi Ishiai2,‡, Kazue Toda1, Satoshi Furukoshi1, Akihisa Osakabe1, Hiroaki Tachiwana1, Yoshimasa Takizawa1, Wataru Kagawa1, Hiroyuki Kitao2,3, Naoshi Dohmae4, Chikashi Obuse5, Hiroshi Kimura6, Minoru Takata 2 and Hitoshi Kurumizaka 1 1Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan 2Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan 3Graduate School of Medical Sciences, Department of Molecular Oncology, Kyushu University, Fukuoka, Japan 4RIKEN Advanced Science Institute, Saitama, Japan 5Graduate School of Life Science, Hokkaido University, Hokkaido, Japan 6Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan ‡These authors contributed equally to this work *Corresponding authors: Laboratory of DNA Damage Signaling, Radiation Biology Center, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan. Tel.:+81 75 753 7563; Fax:+81 75 753 7565; E-mail: [email protected] of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan. Tel.:+81 3 5369 7315; Fax:+81 3 5367 2820; E-mail: [email protected] The EMBO Journal (2012)31:3524-3536https://doi.org/10.1038/emboj.2012.197 There is a Have you seen? (August 2012) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair. Introduction Fanconi anaemia (FA) is a rare hereditary disorder characterized by skeletal abnormalities, progressive bone marrow failure, and genomic instability accompanied by cancer susceptibility (Venkitaraman, 2004; Niedernhofer et al, 2005; Taniguchi and D'Andrea, 2006; Wang, 2007). FA-mutant cells are highly sensitive to interstrand DNA crosslinking reagents, which induce stalled replication forks, suggesting that FA proteins promote the stabilization and restarting of the replisome (Thompson et al, 2005; Wang, 2007). Thirteen genes, FANCA, -B, -C, -D1 (BRCA2), -D2, -E, -F, -G, -I, -J (BRIP1), -L, -M, and -N (PALB2), corresponding to individual FA complementation groups, have been cloned (Thompson et al, 2005; Wang, 2007; Kee and D'Andrea, 2010; Garner and Smogorzewska, 2011; Kitao and Takata, 2011). In addition, homozygous Rad51C mutations have recently been identified in a family with an FA-like disorder, as the FANCO gene (Vaz et al, 2010), and SLX4 has been confirmed as the FANCP gene (Crossan et al, 2011; Kim et al, 2011; Stoepker et al, 2011). These FA gene products constitute a common DNA damage response pathway that is often referred to as the 'FA pathway'. In this pathway, eight proteins, FANCA, -B, -C, -E, -F, -G, -L, and -M, and three FANCA-associated polypeptides (FAAPs) form the FA core E3 ligase complex (Garcia-Higuera et al, 2001; Wang, 2007; Ali et al, 2012; Kim et al, 2012; Leung et al, 2012). On the other hand, FANCD2 and FANCI associate with each other to form a different complex, called the ID complex (Sims et al, 2007; Smogorzewska et al, 2007). Upon DNA damage during S-phase, multiple phosphorylations of FANCI trigger the monoubiquitination of FANCD2 and FANCI by the FA core complex (Ishiai et al, 2008). The monoubiquitinated ID complex is then targeted to the chromatin, where it plays a critical role in DNA-repair pathways, such as homologous recombination and translesion synthesis (Matsushita et al, 2005; Thompson et al, 2005; Yamamoto et al, 2005; Wang, 2007; Kee and D'Andrea, 2010; Garner and Smogorzewska, 2011; Kitao and Takata, 2011). Recent studies indicated that monoubiquitinated FANCD2 (and FANCI) recruit the FAN1 nuclease, which possesses endo- and exonuclease activities, providing a partial explanation for their roles in DNA repair (Kratz et al, 2010; MacKay et al, 2010; Smogorzewska et al, 2010; Yoshikiyo et al, 2010). Monoubiquitinated FANCD2 also reportedly recruits SLX4 (Garner and Smogorzewska, 2011; Yamamoto et al, 2011), which is considered to function as a scaffold that interacts with the other nucleases, SLX1, XPF, and MUS81 (Fekairi et al, 2009; Svendsen et al, 2009; Yamamoto et al, 2011). Furthermore, a recent report found that FANCD2 itself might have exonuclease activity (Pace et al, 2010). However, whether chromatin-bound FANCD2 and FANCI have any additional functions remains to be determined. The nucleosome is the fundamental repeating unit of chromatin (Wolffe, 1998). Four core histones, H2A, H2B, H3, and H4, are the protein components of the nucleosome. H2A forms a specific dimer with H2B (H2A/H2B dimer), and H3 forms a specific dimer with H4 (H3/H4 dimer). During nucleosome assembly, two H3/H4 dimers (H3/H4 tetramer) are first deposited on DNA, forming a tetrasome, in which the DNA is wrapped around the H3/H4 tetramer. Two H2A/H2B dimers are then incorporated into the tetrasome to form the mature nucleosome, in which about 150 base pairs of DNA are wrapped around a histone octamer, containing two each of the H2A/H2B and H3/H4 dimers. In cells, nucleosomes are dynamically assembled and disassembled during the replication, transcription, recombination, and repair processes, and such nucleosome dynamics are accomplished with the aid of histone chaperones and/or ATP-dependent chromatin remodelling factors (Avvakumov et al, 2011). In the present study, we purified the human and chicken FANCD2 proteins, and found that FANCD2 possesses nucleosome-assembly activity in vitro. We also purified FANCI, and showed that it significantly stimulated FANCD2-mediated nucleosome assembly, although FANCI itself lacked nucleosome-assembly activity. A histone-binding domain was mapped in the chicken FANCD2 C-terminal region (residues 1268–1439). The FANCD2 mutants, in which either the histone-binding domain was deleted or the Arg1336 and Lys1346 residues were replaced by Ala, were significantly defective in nucleosome assembly in vitro, and cells bearing these mutants displayed impaired survival upon cisplatin treatment in vivo. Furthermore, a disease-related mutation, human FANCD2(R302W) (Timmers et al, 2001), compromised histone dynamics, and the corresponding chicken FANCD2(R305W) also showed impaired histone chaperone activity. These data suggest that the histone chaperone activity of FANCD2 is crucial for the histone dynamics and the DNA crosslink repair in cells. Results Human FANCD2 promotes nucleosome assembly In a proteome analysis to search for proteins in HeLa cell extracts that bind to the histone H3/H4 complex (Supplementary Figure S1), we unexpectedly detected human FANCD2 (hFANCD2) as a candidate interacting protein. Indeed, hFANCD2 was efficiently captured from a HeLa cell extract, using H3/H4 beads (Figure 1A). We purified hFANCD2 as a recombinant protein expressed in insect cells (Supplementary Figure S2A), and confirmed that purified hFANCD2 also bound to H3/H4 (Figure 1B). The hFANCD2-H3/H4 binding was also detected in the presence of DNaseI (Figure 1B, lane 5), indicating that the interaction is not mediated by DNA contamination. These results indicated that hFANCD2 directly binds to H3/H4, which prompted us to examine its nucleosome-assembly activity. Figure 1.hFANCD2 promotes nucleosome assembly. (A) The H3/H4-conjugated beads were incubated with a HeLa WCE, and the endogenous FANCD2 bound to the beads was detected by western blotting with an anti-FANCD2 monoclonal antibody (α-FANCD2). N and H indicate the control Affi-Gel 10 beads and the H3/H4-conjugated beads, respectively. Input WCE (30 μg of protein) and hFANCD2 (125 ng) were applied in lanes 4 and 5, respectively. (B) The H3/H4 beads were incubated with purified hFANCD2 in the absence or presence of DNaseI, washed with buffer, and mixed with two-fold SDS sample buffer. The proteins bound to the beads were analysed by 15% SDS–PAGE. (C) Topological assay. A schematic diagram of topological assay is shown in the left panel. Nucleosomes were reconstituted on the relaxed plasmid DNA by hFANCD2 (0.2, 0.5, and 0.9 μM), in the presence of wheat germ topoisomerase I. After deproteinization, the topoisomers were separated by agarose gel electrophoresis. Highly supercoiled and relaxed DNAs are denoted as 'sc' and 'relaxed', respectively. (D) Nucleosome-assembly assay. A schematic diagram of the nucleosome-assembly assay is shown in the left panel. Nucleosomes were reconstituted on the linear 195 base-pair DNA by hNap1 (0.4, 0.8, and 1.6 μM) or hFANCD2 (0.2, 0.4, and 0.8 μM). Nucleosomes positioned at the edge and centre of the 195-bp DNA are indicated by cartoons on the right side of the panel. Download figure Download PowerPoint We tested hFANCD2-mediated nucleosome assembly by a topological assay, using relaxed circular DNA in the presence of topoisomerase (Figure 1C). The extent of nucleosome formation was assessed by analysing the superhelicity of circular DNA fractionated through an agarose gel, because negative supercoils are introduced when nucleosomes are formed. As shown in Figure 1C, the number of superhelical turns in the DNA substrate increased with greater amounts of hFANCD2. The faster migration of the DNA substrate was not due to DNA degradation (Supplementary Figure S2B). Therefore, hFANCD2 actually promoted nucleosome assembly in vitro. We next performed the nucleosome-assembly assay with a short DNA fragment, to directly detect the nucleosomes by an electrophoretic mobility shift assay. hFANCD2 stimulated the nucleosome assembly in this assay (Figure 1D, lanes 6–9). The nucleosome-assembly activity of hFANCD2 was slightly lower than that of human Nap1, which is a prominent nucleosome-assembly protein (Figure 1D). These biochemical results suggest that FANCD2 may regulate chromatin reorganization during DNA repair in higher eukaryotes. Chromatin-bound FANCD2 is known to be monoubiquitinated, and the ubiquitin moiety may function to recruit its associated nucleases (Fekairi et al, 2009; Svendsen et al, 2009; Kratz et al, 2010; MacKay et al, 2010; Smogorzewska et al, 2010; Yoshikiyo et al, 2010; Yamamoto et al, 2011). Therefore, we tested whether FANCD2 monoubiquitination affects the nucleosome-assembly activity. For this purpose, we utilized the chicken FANCD2 protein (cFANCD2) (Yamamoto et al, 2005), which was bacterially expressed and purified to homogeneity (Supplementary Figure S2C). We then prepared monoubiquitinated cFANCD2, using purified components for the conjugation (i.e., FANCL, UBE2T, E1, and ubiquitin; Supplementary Figure S2D–F). As we previously reported, the cFANCD2 monoubiquitination was robustly enhanced in the presence of DNA (Sato et al, 2012), and about 40% of cFANCD2 was monoubiquitinated in this study (Supplementary Figure S2F). This monoubiquitinated cFANCD2 fraction was purified, and was subjected to the topological assay. However, we did not find a clear difference in the nucleosome-assembly activities between the fractions containing monoubiquitinated cFANCD2 and the monoubiquitination-deficient cFANCD2(K563R) mutant (Supplementary Figure S2G). Therefore, the monoubiquitination does not affect the activity. However, this could be due to the incomplete monoubiquitination of cFANCD2. Therefore, we prepared the monoubiquitination-mimicking version of FANCD2, by genetically fusing FANCD2(K563R) with ubiquitin to create FANCD2(K563R)-Ub (Supplementary Figure S2H), which is known to complement the DNA-repair-defective phenotype in the FANCD2−/− DT40 cells (Matsushita et al, 2005). We found that purified FANCD2(K563R)-Ub possessed similar histone-binding and nucleosome-assembly activities to those of cFANCD2 (Supplementary Figure S2I and J). These results suggested that the FANCD2 monoubiquitination may not be directly involved in the nucleosome assembly. The C-terminal region of FANCD2 is responsible for interacting with histone H3/H4 and for promoting nucleosome assembly To gain further insights into the molecular mechanism and the functional relevance of the nucleosome-assembly activity of FANCD2, we first searched for the FANCD2 region that interacts with the H3/H4 complex. We subjected cFANCD2 to limited proteolysis, and two fragments, cFANCD2(1-1167) and cFANCD2(1-1389), were identified (Figure 2A). These fragments lacked the acidic region, which is composed of the C-terminal 50 amino-acid residues of FANCD2. In addition, FANCD2(1268X), which also lacks the C-terminal region, is present in FA patients (FA Mutation Database, http://www.rockefeller.edu/fanconi/mutate/), suggesting the functional importance of the FANCD2 C-terminal region. As histone chaperones are generally acidic, we investigated whether this acidic C-terminal region is essential for histone binding. The C-terminal deletion mutants, cFANCD2(1-1167), cFANCD2(1-1267), and cFANCD2(1-1389), were expressed as GFP-tagged forms in HEK293T cells, and their H3/H4-binding activity was examined by a pull-down assay, using the H3/H4 beads. We found that cFANCD2(1-1167) and cFANCD2(1-1267) displayed diminished H3/H4 binding (Figure 2B, lanes 3 and 4). In contrast, cFANCD2(1-1389) retained residual H3/H4-binding activity (Figure 2B, lane 2). Consistently, cFANCD2(1-1167) and cFANCD2(1-1267) showed significant defects in nucleosome assembly (Figure 2C, lanes 13–17 and 18–22, and D; Supplementary Figure S3A). cFANCD2(1-1389) was completely proficient in the nucleosome-assembly activity under low protein concentration conditions (Figure 2C, lanes 8–12, and D; Supplementary Figure S3B). These observations suggest that the C-terminal region of FANCD2 (amino acids 1268–1389) is important for interacting with H3/H4 and for promoting nucleosome assembly. It should be noted that cFANCD2(1-1389) was defective in nucleosome assembly under high protein concentration conditions (Figure 2C, lanes 10–11, and D; Supplementary Figure S3C). This may reflect the biochemical property of cFANCD2(1-1389), which tends to form large protein–DNA aggregates that are unable to enter an agarose gel during electrophoresis (Supplementary Figure S3D). Figure 2.The C-terminal region of FANCD2 is responsible for histone binding and nucleosome assembly. (A) Schematic representations of full-length cFANCD2, and the cFANCD2(1-1389), cFANCD2(1-1267), cFANCD2(1-1167), cFANCD2(669-1439), and cFANCD2(953-1439) deletion mutants. The cFANCD2 domains, solenoid 1, helical domain 1, solenoid 2, helical domain 2, solenoid 3, and solenoid 4, are denoted as S1, HD1, S2, HD2, S3, and S4, respectively (Joo et al, 2011). The FANCD2 C-terminal acidic region is coloured red. The amino-acid sequences of the C-terminal regions of the Homo sapiens, Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio, and Drosophila melanogaster FANCD2 proteins are aligned. The highly conserved residues are coloured red. The mutated residues in cFANCD2(R1336A/K1346A) and cFANCD2(D1350A/E1365A/E1382A) are indicated by orange and purple arrowheads, respectively. (B) The H3/H4 beads were incubated with extracts of HEK293T cells, producing either GFP-tagged cFANCD2, cFANCD2(1-1389), cFANCD2(1-1267), cFANCD2(1-1167), cFANCD2(669-1439), cFANCD2(953-1439), cFANCD2(R305W), cFANCD2(K563R), cFANCD2(D1350A/E1365A/E1382A), or cFANCD2(R1336A/K1346A). Proteins bound to the beads were detected by western blotting with an anti-chFANCD2 (polyclonal) antibody. The bottom panel indicates negative control experiments with beads lacking histones. (C) Nucleosomes were reconstituted on the relaxed plasmid DNA by cFANCD2 (lanes 3–7), cFANCD2(1-1389) (lanes 8–12), cFANCD2(1-1267) (lanes 13–17), cFANCD2(1-1167) (lanes 18–22), and cFANCD2(953-1439) (lanes 23–27) in the presence of wheat germ topoisomerase I. After deproteinization, the topoisomers were separated by agarose gel electrophoresis with ethidium bromide staining. The cFANCD2 concentrations were 0, 0.45, 0.90, and 1.8 μM. Highly supercoiled and relaxed DNAs are denoted as 'sc' and 'relaxed', respectively. (D) Graphic representation of nucleosome-assembly activities of the cFANCD2 mutants shown in C. Representative images are shown in C. The supercoiled DNA fractions were generated by nucleosome assembly in the presence of cFANCD2, and the intensities of the bands indicated by the arrows in C were quantitated by an LAS-4000 Image Analyser (GE Healthcare). Means of three independent experiments are shown with s.d.'s. (E) Nucleosomes were reconstituted on the relaxed plasmid DNA by cFANCD2 (lanes 3–7), cFANCD2(R1336A/K1346A) (lanes 8–12), and cFANCD2(D1350A/E1365A/E1382A) (lanes 13–17) in the presence of wheat germ topoisomerase I. The cFANCD2 concentrations were 0, 0.45, 0.90, and 1.8 μM. Highly supercoiled and relaxed DNAs are denoted as 'sc' and 'relaxed', respectively. (F) Graphical representation of the nucleosome-assembly activities of the cFANCD2 mutants shown in E. Representative images are shown in E. The supercoiled DNA fractions were generated by nucleosome assembly in the presence of cFANCD2, and the intensities of the bands indicated by the arrows in E were quantitated by an LAS-4000 Image Analyser (GE Healthcare). Means of three independent experiments are shown with s.d.'s. Download figure Download PowerPoint Since the cFANCD2 C-terminal deletion may induce the improper folding of the cFANCD2 structure, we next performed the H3/H4-binding and nucleosome-assembly experiments with cFANCD2 point mutants. Based on the amino-acid conservation among the human, mouse, chicken, frog, fish, and fly FANCD2 proteins, we mutated the conserved amino-acid residues, which might be functionally important (Figure 2A). We found that the FANCD2(R1336A/K1346A) mutant, in which the Arg1336 and Lys1346 residues are replaced by Ala, was significantly defective in nucleosome assembly in vitro, while another mutant, cFANCD2(D1350A/E1365A/E1382A), in which the Asp1350, Glu1365, and Glu1382 residues are replaced by Ala, did not affect the nucleosome-assembly activity (Figure 2A, E; Supplementary Figure S3E). Consistently, the histone-binding activity was substantially reduced in cFANCD2(R1336A/K1346A) (Figure 2B lane 11), but not in cFANCD2(D1350A/E1365A/E1382A) (Figure 2B, lane 10). These results strongly support the conclusion that FANCD2 promotes nucleosome assembly through its C-terminal histone-binding domain. Finally, we tested the histone binding of the C-terminal cFANCD2 fragments, cFANCD2(669-1439) and cFANCD2(953-1439), which contain the C-terminal amino-acid residues 669-1439 and 953-1439, respectively (Figure 2A). These cFANCD2 fragments were identified by a protease mapping experiment. As expected, the cFANCD2(669-1439) and cFANCD2(953-1439) fragments both efficiently bound to histones (Figure 2B, lanes 5 and 6). Surprisingly, cFANCD2(953-1439), which contained only one-third of cFANCD2, promoted nucleosome assembly (Figure 2C, lanes 23–27, and D). Therefore, we concluded that the histone-binding domain is located in the C-terminal region of FANCD2. FANCD2 mediates histone mobilization in living cells in a DNA damage-dependent manner To determine whether FANCD2 plays a role in histone dynamics in living cells during DNA repair, we knocked down hFANCD2 in HeLa cells expressing histone H3-GFP (Kimura and Cook, 2001), using small inhibitory RNA (siRNA). Three days after the transfection of the specific siRNA, the level of hFANCD2 had decreased substantially, to <10% of the normal level (Supplementary Figure S4A and B). Using these cells, the mobility of H3 was analysed by fluorescence recovery after photobleaching (FRAP) (Kimura et al, 2006). The recovery kinetics (the curve shapes) of the exchanging fractions were similar in the hFANCD2-knockdown and control cells (Figure 3A), suggesting that hFANCD2 does not play a major role in H3 assembly or exchange under normal conditions. As FA-mutant cells display significant sensitivity to interstrand DNA crosslinking reagents, such as mitomycin C (MMC) (Niedernhofer et al, 2005; Thompson et al, 2005; Wang, 2007; Kee and D'Andrea, 2010; Garner and Smogorzewska, 2011; Kitao and Takata, 2011), we next tested the effect of MMC on the H3 mobility in the hFANCD2-knockdown HeLa cells. Interestingly, the recovery of H3-GFP in the hFANCD2-knockdown cells was clearly slower in the presence of MMC (Figure 3B). Similar results were obtained with a different FANCD2-specific siRNA (Supplementary Figure S4C). The slower H3-GFP exchange observed in the MMC-treated FANCD2-knockdown cells could be due to a different cell cycle distribution, since the FA-deficient cells may be arrested at S and/or G2 due to the deficiency of DNA crosslink repair. We therefore performed FRAP experiments in cells stably expressing both H3-GFP and mCherry-tagged PCNA, which shows characteristic patterns representing replication and repair foci (Leonhardt et al, 2000). We first examined the mobility of H3-GFP in different cell cycle stages under the normal growth conditions. The H3-GFP mobility in PCNA foci-positive (S-phase) cells did not differ from that in PCNA foci-negative cells (Figure 3C). In the presence of MMC, the PCNA foci-positive cells were indeed enriched by the hFANCD2 knockdown, but the mobility of H3-GFP was still slower than that in the MMC-treated PCNA foci-positive control cells (Figure 3D; Supplementary Figure S4D). Therefore, the reduced histone H3 mobility in the MMC-treated hFANCD2-knockdown cells does not appear to be attributable to a difference in the cell cycle phase. Furthermore, the slower H3-GFP recovery in the presence of MMC was also detected in the hFANCA-knockdown cells, in which the damage-dependent focus formation of hFANCD2 on chromosomes was significantly inhibited (Supplementary Figure S4E–H). These data suggest that hFANCD2 may mediate nucleosome assembly and/or histone exchange in human cells, in a damage-dependent manner. Figure 3.Histone H3 mobility is decreased in FANCD2-knockdown cells in the presence of a DNA crosslinking reagent. (A, B) FRAP with HeLa cells. Three days after the transfection of hFANCD2-siRNA or control RNA, the mobility of histone H3-GFP was analysed by bleaching one-half of a nucleus in the absence (A) or presence (B) of 50 ng/ml MMC for 12–18 h. The mean of the relative fluorescence intensity with the s.d. (n=10–11) and examples are shown. (C) FRAP with HeLa cells stably expressing mCherry-PCNA. Three days after the transfection of control RNA, the mobility of histone H3-GFP was analysed in the absence of MMC. The PCNA foci-positive (S-phase) cells were identified by the characteristic mCherry-PCNA distribution. The mean of the relative fluorescence intensity with the s.d. (n=10–14) and examples are shown. (D) FRAP with MMC-treated HeLa cells expressing H3-GFP and mCherry-PCNA in S-phase. Three days after the transfection of hFANCD2-siRNA or control RNA, the mobility of histone H3-GFP in the PCNA foci-positive (S-phase) cells was analysed in the presence of 50 ng/ml MMC. The mean of the relative fluorescence intensity with the s.d. (n=10–18) and examples are shown. Bars: 10 μm. Download figure Download PowerPoint The C-terminal histone-binding region of FANCD2 is important for the DNA repair mediated by the FA pathway To
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