Evidence for subcomplexes in the Fanconi anemia pathway

Abstract

Fanconi anemia (FA) is a genomic instability disorder, clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. Cells derived from patients with FA display a marked sensitivity to DNA cross-linking agents, such as mitomycin C (MMC). This observation has led to the hypothesis that the proteins defective in FA are involved in the sensing or repair of interstrand cross-link lesions of the DNA. A nuclear complex consisting of a majority of the FA proteins plays a crucial role in this process and is required for the monoubiquitination of a downstream target, FANCD2. Two new FA genes, FANCB and FANCL, have recently been identified, and their discovery has allowed a more detailed study into the molecular architecture of the FA pathway. We demonstrate a direct interaction between FANCB and FANCL and that a complex of these proteins binds FANCA. The interaction between FANCA and FANCL is dependent on FANCB, FANCG, and FANCM, but independent of FANCC, FANCE, and FANCF. These findings provide a framework for the protein interactions that occur "upstream" in the FA pathway and suggest that besides the FA core complex different subcomplexes exist that may have specific functions other than the monoubiquitination of FANCD2.

Figure 1.

FANCL coimmunoprecipitates with FLAG-tagged FA proteins and is required for the assembly of the FA core complex. Lymphoblastoid cell lines stably expressing functionally active FLAG-tagged FANCA (A), FANCF (B), and FANCC (C) were generated. Whole-cell extracts of 10 million cells were immunoprecipitated with an α-FLAG affinity gel and precipitated proteins eluted by competition with a FLAG peptide. Untransfected cells (lane 1) were included as negative controls. Precipitated proteins were detected with α-FANCA (Rb89), α-FANCL, α-FANCF (Rb7), and α-FANCC (Rb39). In panel B, the bottom part of the blot was first probed for FANCL and then subsequently probed for FANCF, thus allowing detection of both proteins that run at a very similar molecular weight, 45 and 42 kDa, respectively. The asterisk in panels B and C mark a nonspecific polypeptide that cross-reacts with the FANCL antiserum. (D) Whole-cell extracts of 10 million WT, FA-L, and respective patient control lymphoblasts were immunoprecipitated using guinea pig antibodies against FANCC_6-105, FANCF_1-245, and FANCG_480-622. Precipitated proteins were detected using rabbit antibodies against FANCA (Rb89), FANCG (Rb43), and FANCF (Rb7). The FANCG protein is indicated with an asterisk. (E) Direct FANCC Western blot on whole-cell extracts of 500 000 lymphoblastoid cells to show the total cellular FANCC levels.

Figure 2.

Direct interactions between FANCL and FANCB in the mammalian 2-hybrid assay. Protein pairs were cotransfected in 293 cells in the presence of a luciferase reporter construct to test for direct interactions. All experiments were performed in triplicate. (A) AD-FANCL was cotransfected with BD-FA proteins. Fold induction of luciferase expression is relative to FANCL alone. (B) AD-FANCB was cotransfected with BD-FA proteins. Fold induction of luciferase expression is relative to FANCB alone.

Figure 3.

Characterizing the regions that are required for direct interaction between FANCB and FANCL. (A) Mammalian 2-hybrid assays coexpressing mutant fragments of BD-FANCL with full-length AD-FANCB. Fold induction of luciferase expression was measured relative to AD-FANCB alone. +++ indicates a strong activation of the luciferase reporter gene; –, no activation of the reporter gene. (B) FANCL constructs that failed to activate the luciferase reporter in the mammalian 2-hybrid experiments were transfected in 293 cells. Whole-cell extracts were immunoblotted with anti–GAL4-BD to show expression of the constructs. The fragments 219-375 and 275-375 are probably difficult to detect because of the presence of background bands. (C) BD-FANCL–containing point mutations of the RING domain were coexpressed with full-length AD-FANCB and fold induction was measured relative to AD-FANCB alone. +++ indicates a strong activation of the luciferase reporter gene; –, no activation of the reporter gene. (D) FANCL missense mutants were transfected in FA-L cell line EUFA868 and tested for their ability to restore the MMC hypersensitive phenotype of this cell line in an MMC growth inhibition test.

Figure 4.

The interaction between FANCA and FANCL is dependent on other FA proteins and detected in the nucleus. (A) Whole-cell extracts of 10 million lymphoblastoid cells were immunoprecipitated with an antiserum against the N-terminus of FANCA (amino acid 1-271). After blotting, membranes were probed with α-FANCA antibody (Rb89) and α-FANCL antibody to show the interaction between FANCA and FANCL. A direct FANCL Western on whole-cell extracts (WCE) of 500 000 lymphoblastoid cells was performed to show the total cellular FANCL levels. (B) Whole-cell extracts of 10 million lymphoblastoid cells were immunoprecipitated with an antiserum against the C-terminus of FANCG (amino acid 480-622). After blotting, membranes were probed with α-FANCA antibody (Rb89), α-FANCB antibody, α-FANCG antibody (Rb43), and α-FANCL antibody to show the interaction between FANCA, FANCB, FANCG, and FANCL. (C) Whole-cell extracts of 10 million lymphoblastoid cells were immunoprecipitated with an antiserum against the N-terminus of FANCM (amino acid 1-70). After blotting, membranes were probed with α-FANCM antibody, α-FANCA antibody (Rb89), and α-FANCL antibody to show the interaction between FANCA, FANCL, and FANCM. (D) Mammalian 3-hybrid assay in 293 cells expressing FLAG-FANCB. Cells were cotransfected with the indicated protein pairs and assayed for luciferase activation. Fold induction relative to reporter gene activation when full-length FANCL is expressed alone. Error bars represent SD of 3 experiments. (E) Nuclear and cytoplasmic fractions of lymphoblasts cell lines were immunoprecipitated with an anti-FANCA antibody (as described for panel A). In addition, aliquots of protein lysate were probed with anti–beta tubulin and anti–TOPO I to check integrity of fractionation procedure.

Figure 5.

Interaction of FANCA mutants with the FANCB/FANCL complex and with FANCG. Stable cell lines were generated expressing FANCA protein containing patient-derived missense mutations and immunoprecipitation with an α-FLAG affinity gel (A-C), or an antiserum against the N-terminus of FANCA (D) was performed. Western blots were probed for FANCA (Rb89), FANCG (Rb43), and FANCL. The FANCG protein is indicated with an asterisk. (E-G) Mammalian 3-hybrid assay in 293 cells expressing FLAG-FANCB. Cells were cotransfected with the indicated protein pairs and assayed for luciferase activation. Fold induction relative to reporter gene activation when full-length FANCL is expressed alone. (E-G) Error bars represent SD of 3 experiments.

Figure 6.

Cytoplasmic expression of FLAG-tagged FANCA mutant proteins in transiently transfected MCF-7 cells by indirect immunofluorescence. The different FLAG-tagged FANCA mutant proteins are shown in green. The counterstaining with Hoechst is shown in red to visualize the nuclei.

Figure 7.

A model for the sequential assembly of the nuclear FA core complex. The model is explained in “Discussion.”