Evidence for subcomplexes in the Fanconi anemia pathway by Annette L. Medhurst, El Houari Laghmani, Jurgen Steltenpool, Miriam Ferrer, Chantal Fontaine, Jan de Groot, Martin A. Rooimans, Rik J. Scheper, Amom Ruhikanta Meetei, Weidong Wang, Hans Joenje, and Johan P. de Winter Blood Volume 108(6):2072-2080 September 15, 2006 ©2006 by American Society of Hematology

FANCL coimmunoprecipitates with FLAG-tagged FA proteins and is required for the assembly of the FA core complex. 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,12 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 FANCC6-105, FANCF1-245, and FANCG480-622. 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. Annette L. Medhurst et al. Blood 2006;108:2072-2080 ©2006 by American Society of Hematology

Direct interactions between FANCL and FANCB in the mammalian 2-hybrid assay. 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. Annette L. Medhurst et al. Blood 2006;108:2072-2080 ©2006 by American Society of Hematology

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. 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. Annette L. Medhurst et al. Blood 2006;108:2072-2080 ©2006 by American Society of Hematology

The interaction between FANCA and FANCL is dependent on other FA proteins and detected in the nucleus. 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 antibody12 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,3 α-FANCG antibody (Rb43), and α-FANCL antibody12 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,13 α-FANCA antibody (Rb89), and α-FANCL antibody12 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. Annette L. Medhurst et al. Blood 2006;108:2072-2080 ©2006 by American Society of Hematology

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 agai... 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.12 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. Annette L. Medhurst et al. Blood 2006;108:2072-2080 ©2006 by American Society of Hematology

Cytoplasmic expression of FLAG-tagged FANCA mutant proteins in transiently transfected MCF-7 cells by indirect immunofluorescence. 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. Annette L. Medhurst et al. Blood 2006;108:2072-2080 ©2006 by American Society of Hematology

A model for the sequential assembly of the nuclear FA core complex. A model for the sequential assembly of the nuclear FA core complex. The model is explained in “Discussion.” Annette L. Medhurst et al. Blood 2006;108:2072-2080 ©2006 by American Society of Hematology