Defective FANCI Binding by a Fanconi Anemia-Related FANCD2 Mutant

<div><p>FANCD2 is a product of one of the genes associated with Fanconi anemia (FA), a rare recessive disease characterized by bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition. FANCD2 forms a complex with FANCI (ID complex) and is monoubiquitinated, which facilitates the downstream interstrand crosslink (ICL) repair steps, such as ICL unhooking and nucleolytic end resection. In the present study, we focused on the chicken FANCD2 (cFANCD2) mutant harboring the Leu234 to Arg (L234R) substitution. cFANCD2 L234R corresponds to the human FANCD2 L231R mutation identified in an FA patient. We found that cFANCD2 L234R did not complement the defective ICL repair in FANCD2^−/− DT40 cells. Purified cFANCD2 L234R did not bind to chicken FANCI, and its monoubiquitination was significantly deficient, probably due to the abnormal ID complex formation. In addition, the histone chaperone activity of cFANCD2 L234R was also defective. These findings may explain some aspects of Fanconi anemia pathogenesis by a FANCD2 missense mutation.</p></div

Chicken FANCD2 L234R is defective in monoubiquitination <i>in vitro</i>.

<p>(A) Purified cFANCD2 and cFANCI proteins used for <i>in vitro</i> assays were analyzed by 15% SDS-PAGE with Coomassie Brilliant Blue staining. Lane 1 indicates the molecular mass markers. Lanes 2–5 indicate purified wild type (WT) cFANCD2, cFANCD2 K563R, cFANCD2 L234R, and cFANCD2 L234R/K563R, respectively. Lane 6 indicates purified WT cFANCI. (B) Schematic diagram of the monoubiquitination assay. The monoubiquitination assay was performed by incubating cFANCD2 (D2) or cFANCI (I)-cFANCD2 with HA-tagged ubiquitin (Ub), human E1, human UBE2T (2T) and GST-cFANCL (L) in the presence of ATP. The high-energy thioester bond between ubiquitin and the cysteine residue of the substrate is indicated as a wavy line. The stable covalent bond between ubiquitin and the lysine residue of the substrate is indicated as a linear line. (C) The monoubiquitination assay was performed in the presence of 100 µM dsDNA. Ubiquitinated proteins were analyzed by 7% SDS-PAGE, and were detected by anti-HA antibody (α-HA) and Coomassie Brilliant Blue staining. Lane 1 indicates a control experiment without WT cFANCD2 and cFANCI. Lane 2 indicates an experiment with WT cFANCD2 in the absence of cFANCI. Lanes 3 and 4 indicate experiments with WT cFANCD2 and cFANCD2 K563R in the presence of WT cFANCI, respectively. Lanes 5–8 indicate experiments with cFANCD2 L234R and cFANCD2 L234R/K563R, instead of WT cFANCD2 and cFANCD2 K563R. (D) Graphical representation of the experiments shown in C, lanes 2–4 and lanes 6–8. The mount of monoubiuqitinated FANCD2 was quantitated. Means of three independent experiments are shown with s.d.’s.</p

Chicken FANCD2 L234R is defective in FANCI binding.

<p>(A, B) Gel filtration analysis of the ID complex formation. cFANCD2, cFANCI, and the mixture of cFANCD2 and cFANCI were fractionated on the Superdex 200 gel filtration column. The SDS-PAGE analysis of the mixture of the cFANCD2 and FANCI fractions is shown below the gel filtration profiles. The void volume of the Superdex200 column is indicated as ‘Vo’ on the gel filtration profiles. Experiments with wild type (WT) cFANCD2 and WT cFANCI (A), cFANCD2 L234R and WT cFANCI (B). (C) Immunoprecipitation analysis of the cFANCD2 and cFANCI interaction. The cell extracts from chicken DT40 cells expressing GFP-WT cFANCD2 or cFANCD2 L234R were immunoprecipitated with an anti-GFP antibody. The immunoprecipitates were separated by 6% SDS-PAGE, and cFANCD2 and cFANCI were detected by western blotting using anti-chicken FANCD2 and FANCI antibodies, respectively. (D) Graphic representation of the relative intensity of the bands corresponding to GFP-cFANCD2 (upper) and endogenous cFANCI (lower) shown in panel (C). The band intensity was normalized relative to that of cFANCD2 or cFANCI.</p

Chicken FANCD2 L234R exhibits impaired histone chaperone activity.

<p>(A) Schematic diagram of the supercoiling assay for nucleosome formation. Core histones were assembled on the relaxed plasmid DNA by a histone chaperone in the presence of wheat germ topoisomerase I. After deproteinization, the topoisomers were separated by agarose gel electrophoresis. (B) Supercoiling assays for nucleosome formation were performed with wild type (WT) cFANCD2 (lanes 4–7) and cFANCD2 L234R (lanes 9–12). Protein concentrations were 0 (lanes 3 and 8), 0.6 (lanes 4 and 9), 1.2 (lanes 5 and 10), and 1.8 µM (lanes 6, 7, 11, and 12). Highly supercoiled DNA is denoted as ‘sc DNA’. (C) Supercoiling assays for nucleosome formation were performed with WT cFANCD2 or cFANCD2 L234R, in the presence of WT cFANCI. The protein concentrations used in the assay are indicated at the top of the panel.</p

The location of the FANCD2 Leu229 residue in the mouse ID complex structure (3S4W).

<p>(A) Amino acid sequences of <i>Homo sapiens</i> FANCD2 surrounding the FA-related missense mutations. Alignment of the FANCD2 sequences of <i>Homo sapiens, Mus musculus</i>, <i>Gallus gallus</i>, <i>Xenopus laevis</i>, <i>Danio rerio</i>, <i>Arabidopsis thaliana</i>, and <i>Drosophila melanogaster</i>. The residues corresponding to human FANCD2 R302, V427, L456, L457, Q815, and W1268, which are mutated in the FA patients, are colored red. (B) The FANCD2 and FANCI proteins are colored grey and green, respectively. The mouse FANCD2 solenoid 1 and the FANCD2 Leu229 residue, corresponding to the cFANCD2 L234 and human FANCD2 L231 residues, are colored black and red, respectively. The FA-associated residues are colored magenta. Ile210 and Phe243 of mouse FANCD2, which are located adjacent to mouse FANCD2 Leu229, are also colored light blue. The van der Waals surfaces of the FANCD2 Ile210, Leu229 and Phe243 side chain atoms are represented.</p

FANCD2^−/− DT40 cells expressing the chicken FANCD2 L234R mutant are defective in ICL repair.

<p>(A) Schematic representation of human FANCD2. S1, HD1, S2, HD2, S3, and S4 denoted on the bar indicate the FANCD2 subdomains, solenoid 1, helical domain 1, solenoid 2, helical domain 2, solenoid 3, and solenoid 4, respectively. The FANCD2 C-terminal acidic region is colored red. The primary structure of the N-terminal regions of <i>Homo sapiens</i>, <i>Mus musculus</i>, <i>Gallus gallus</i>, <i>Xenopus laevis</i>, <i>Danio rerio</i>, <i>Arabidopsis thaliana</i>, and <i>Drosophila melanogaster</i> FANCD2 are aligned. The residues corresponding to human FANCD2 L231 are colored red. (B) ID complex monoubiquitination in cFANCD2^−/− DT40 cells expressing the GFP-wild-type (WT) cFANCD2 or cFANCD2 L234R. Cells were treated with or without mitomycin C (MMC), and whole-cell extracts were analyzed by western blotting using anti-cFANCD2 (α-D2) and anti-cFANCI (α-I) antibodies. S-forms and L-forms indicate non-ubiquitinated and monoubiquitinated forms of cFANCD2 and cFANCI, respectively. Non-specific bands are marked by asterisks. (C) ID complex monoubiquitination in the chromatin fractions from cFANCD2^−/− DT40 cells expressing GFP-WT cFANCD2 or cFANCD2 L234R. Cells were treated with or without MMC, and chromatin fractions were analyzed as in panel (B). (D) Cisplatin sensitivity assay of the cFANCD2^−/− DT40 cells expressing H2B-GFP fusions with the WT cFANCD2, cFANCD2 K563R, or cFANCD2 L234R. The mean values are shown with s.d. from triplicate measurements.</p

FA pathway-related proteins involved in removing DNA lesions induced by metformin.

<p>(A) Histograms of the IC₅₀ values of metformin in wild-type cells and cell lines deficient in various FANC-related proteins. Cells were treated with metformin under glucose-depleted conditions for 24 h and colonies formed on complete media. All data represent IC₅₀ values ± 95% confidence intervals; (B) The toxicity of metformin to cells deficient in the FANCC or FANCL protein and deficient cell lines stably expressing the indicated transgenes. Data represent the mean ± S.D.; (C) The toxicity of metformin to cells deficient in the TDP1 or PARP1 protein and cells simultaneously deficient in both TDP1 and PARP1 proteins. Data represent the means ±S.D.</p

Selective cytotoxicity of the anti-diabetic drug, metformin, in glucose-deprived chicken DT40 cells

<div><p>Metformin is a biguanide drug that is widely used in the treatment of diabetes. Epidemiological studies have indicated that metformin exhibits anti-cancer activity. However, the molecular mechanisms underlying this activity currently remain unclear. We hypothesized that metformin is cytotoxic in a tumor-specific environment such as glucose deprivation and/or low oxygen (O₂) tension. We herein demonstrated that metformin was highly cytotoxic under glucose-depleted, but not hypoxic (2% O₂) conditions. In order to elucidate the underlying mechanisms of this selective cytotoxicity, we treated exposed DNA repair-deficient chicken DT40 cells with metformin under glucose-depleted conditions and measured cellular sensitivity. Under glucose-depleted conditions, metformin specifically killed <i>fancc</i> and <i>fancl</i> cells that were deficient in FANCC and FANCL proteins, respectively, which are involved in DNA interstrand cross-link repair. An analysis of chromosomal aberrations in mitotic chromosome spreads revealed that a clinically relevant concentration of metformin induced DNA double-strand breaks (DSBs) in <i>fancc</i> and <i>fancl</i> cells under glucose-depleted conditions. In summary, metformin induced DNA damage under glucose-depleted conditions and selectively killed cells. This metformin-mediated selective toxicity may suppress the growth of malignant tumors that are intrinsically deprived of glucose.</p></div

Toxicity of metformin and comparison of cellular sensitivities to metformin among various DNA repair-deficient DT40 cell lines under glucose-depleted conditions.

<p>(A) Wild-type cells were treated with the indicated doses of metformin for 24 h in complete medium, no glucose medium, or the 2% O₂ hypoxic condition with complete medium, and colonies formed on methylcellulose-containing complete media under normal conditions for 7 days. All data represent the mean ± S.D. normalized to cells not treated with metformin from three independent experiments. In each experiment, relative viabilities were measured as N/N₀ where N is the mean number of colonies at each dose of metformin in treated cells and N₀ is the mean number of colonies in untreated controls; (B) Histograms of the IC₅₀ values of metformin in the wild-type and various DNA repair-deficient cell lines. Cells were treated with metformin under glucose-depleted conditions for 24 h and colonies formed on complete media. All data represent IC₅₀ values ± 95% confidence intervals normalized to cells not treated with metformin from three independent experiments. In each experiment, relative viabilities were measured as N/N₀ where N is the mean number of colonies at each dose in metformin-treated cells and N₀ is the mean number of colonies in untreated controls. Abbreviations: Wt, wild type; NER, nucleotide excision repair; BER, base excision repair; Topo-DNA, repair of DNA-topoisomerase (Topo) crosslinks; TLS, translation DNA synthesis; NHEJ, non-homologous end joining; HR, homologous recombination repair; FA, FA pathway (ICL repair).</p

Induction of chromosomal breakages by metformin under glucose-depleted conditions.

<p>(A) Wild-type cells, cell lines deficient in FANCC or FANCL, and reconstituted cells were incubated with or without 13 μM metformin for 24 h under glucose-depleted conditions; (B) Wild-type cells and cell lines deficient in FANCC or FANCL were incubated with the indicated doses of metformin for 24 h in glucose-depleted or -containing medium. We analyzed 50 metaphase nuclei, and quantified the number of chromosomal aberrations per cell (Y-axis). Data represent the mean ± S.E. Asterisks (*) indicate <i>p</i> < 0.05 by a multiple comparison one-way ANOVA (Tukey’s test). N.S.: not significant (<i>p</i> ≥ 0.05).</p