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. 2014 Jun 26;7(6):1849-57.
doi: 10.1016/j.celrep.2014.04.029. Epub 2014 Jun 5.

Modularized functions of the Fanconi anemia core complex

Yaling Huang  1 , Justin W C Leung  1 , Megan Lowery  2 , Nobuko Matsushita  3 , Yucai Wang  1 , Xi Shen  1 , Do Huong  1 , Minoru Takata  4 , Junjie Chen  1 , Lei Li  5
Affiliations

Modularized functions of the Fanconi anemia core complex

Yaling Huang et al. Cell Rep. .

Erratum in

  • Cell Rep. 2016 Mar 22;14(11):2761-3

Abstract

The Fanconi anemia (FA) core complex provides the essential E3 ligase function for spatially defined FANCD2 ubiquitination and FA pathway activation. Of the seven FA gene products forming the core complex, FANCL possesses a RING domain with demonstrated E3 ligase activity. The other six components do not have clearly defined roles. Through epistasis analyses, we identify three functional modules in the FA core complex: a catalytic module consisting of FANCL, FANCB, and FAAP100 is absolutely required for the E3 ligase function, and the FANCA-FANCG-FAAP20 and the FANCC-FANCE-FANCF modules provide nonredundant and ancillary functions that help the catalytic module bind chromatin or sites of DNA damage. Disruption of the catalytic module causes complete loss of the core complex function, whereas loss of any ancillary module component does not. Our work reveals the roles of several FA gene products with previously undefined functions and a modularized assembly of the FA core complex.

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Figures

Fig. 1
Fig. 1. Various sensitivities of FA knockout mutants identify the FA core complex catalytic module
(A) Depiction of three protein interaction modules within the FA core complex and the FANCM-FAAP24 complex. See also Fig. S1A-C. (B) Clonogenic survival of wild-type HCT116 (WT), FANCB−, FANCG−/−, and FANCB− G−/− mutants treated with mitomycin C. See also Fig. S1D-E and S2. (C) Clonogenic survival of wild-type HCT116 (WT), FANCB−, FAAP20−/−, and FANCB− FAAP20−/− mutants treated with mitomycin C. See also Fig. S2. (D) Clonogenic survival of wild-type HCT116 (WT), FAAP20−/−, FANCG−/−, FANCB−, and FANCL−/− mutants treated with mitomycin C. (E) Immunoblots detecting MMC- or cisplatin-induced monoubiquitination of chromatin-bound FANCD2 and FANCI in wild-type HCT116 cells (WT) and the indicated knockout mutants. See also Fig. S3A-B. (F) Immunoblot detecting MMC- or cisplatin-induced monoubiquitination of chromatin-bound FANCD2 in wild-type chicken DT40 (WT) and indicated knockout mutants. *FANCG gene localizes in the single Z chromosome and only one allele exists in DT40 cells. See also Fig. S3C-D. (G) Immunoblots detecting FANCL auto ubiquitination in protein extracts prepared from 293T cells stably expressing SPB-FAAP100 and/or SFB-FANCL with or without MMC treatment.. Protein extracts from indicated cell lines were subjected to S beads pull-down and immunoblotted by anti-ubiquitin or Flag antibodies. (H) Superose 6 gel filtration profiling of the FA core complex in HCT116 WT, FANCG−/−, and FANCL−/− mutants. Nuclear extracts were fractionated by Superose 6 gel filtration and the indicated fractions were immunoblotted (IB:) with FANCL or FAAP100 antibodies. The arrow indicates the elution position of the FA core complex (670kD). Fraction 21 marks the void. Error bars in survival curves were derived from SDs from 4–6 independent experiments with triplicates.
Fig. 2
Fig. 2. The A-G-20 module is required for FANCL association with DNA damage
(A) Chromatin binding of FANCG and FANCL in wild-type HCT116 cells (WT) and the indicated mutants exposed to mitomycin. Histone H3 served as a loading control for chromatin-bound protein fractions. (B) Quantification of chromatin-bound FANCG in FANCB−, G−/−, L−/−, and FAAP20−/− knockout mutants. (C) Quantification of chromatin-bound FANCL in FANCB−, G−/−, L−/−, and FAAP20−/− knockout mutants. (D) eChIP analysis of FANCG enrichment to a defined psoralen lesion in wild-type HCT116 cells (WT)and the FANCL−/− mutant. (E) eChIP analysis of FANCG enrichment to a cisplatin-adducted plasmid substrate in wild-type HCT116 cells (WT) and the FANCL−/− mutant. (F) eChIP analysis of FANCL enrichment to a defined psoralen lesion in wild-type HCT116 cells (WT) and the FANCG−/− mutant. (G) eChIP analysis of FANCL enrichment to a cisplatin-adducted plasmid substrate in wild-type HCT116 cells (WT) and the FANCG−/− mutant. (H) Immunoblotting of FANCA in cytoplasmic (C), nuclear (N), and whole cell protein extracts from FANCB−, FANCL−/−, and FANCG−/− mutant cells. Tubulin, Histone H3, and MCM4 are controls for loading and extraction. Error bars for chromatin fraction and eChIP analyses represent SDs derived from three independent experiments.
Fig. 3
Fig. 3. The A-G-20 and C-E-F modules exhibit non-redundant functions in the chromatin recruitment of the FA core complex and resistance to crosslink damage
(A) Immunoblot detecting monoubiquitination of FANCD2 and chromatin-bound FANCL in cells exposed to mitomycin C (left panel) or cisplatin (right panel) in wild-type HCT116 (WT) and the indicated knockout/knockdown mutant cells. See also Fig. S4. (B) Quantification of chromatin-bound FANCL (left panel of A) in wild-type HCT116 (WT), FANCG−/− + Ctrl shRNA, WT + shFANCF alone (shFANCF), FANCG−/− + shFANCF, and FANCL−/− cells exposed to mitomycin C. See also Fig. S4. (C) Clonogenic survival of parental HCT116 (WT), FANCG−/− + Ctrl shRNA, FANCG−/− + shFANCF, and FANCL−/− cells treated with mitomycin C. See also Fig. S4. (D) Clonogenic survival of parental DT40 (WT), FANCE−/−, FANCG−, and FANCE−/− FANCG− cells treated with cisplatin. See also Fig. S2C. Error bars for chromatin-bound FANCL quantification (B) and clonogenic survivals (C and D) were derived from SDs from three or more independent tests.
Fig. 4
Fig. 4. FANCM functions epistatically with the C-E-F module in FA core complex chromatin association
(A) Immunoblot detecting mitomycin C-induced monoubiquitination of FANCD2 and FANCI in wild-type HCT116 cells (WT) and the indicated knockout/knockdown mutant cells. See also Fig. S4. (B) Immunoblot detecting cisplatin-induced monoubiquitination of FANCD2 in wild-type HCT116 cells (WT) and the indicated knockout/knockdown mutant cells. See also Fig. S4. (C) Clonogenic survival of HCT116 FANCF knockdown alone (shFANCF), FANCM−/− +shFANCF, and FANCM−/− +Ctrl shRNA mutants treated with mitomycin C. See also Fig. S4. (D) Immunoblot detecting MMC-induced monoubiquitination of FANCD2 and chromatin binding of FANCL in wild-type HCT116 cells (WT) and the indicated mutant cells exposed to mitomycin C. Histone H3 serves as a loading control for chromatin-bound protein fractions. See also Fig. S2F. (E) Quantification of (C) - chromatin-bound FANCL in FANCG−/−, FANCM−/−, and FANCG−/− M−/− double mutants. See also Fig. S2F. (F) Clonogenic survival of wild-type HCT116 cells (WT), FANCM−/−, FANCG−/−, FANCL−/−, and FANCG−/− M−/− mutants treated with mitomycin C. See also Fig. S2F. Error bars for chromatin fraction represent SDs derived from three independent experiments. Error bars for clonogenic survival are derived from SDs from four ndependent tests done in triplications.
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References

    1. Ali AM, Pradhan A, Singh TR, Du C, Li J, Wahengbam K, Grassman E, Auerbach AD, Pang Q, Meetei AR. FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway. Blood. 2012;119:3285–3294. - PMC - PubMed
    1. Alpi AF, Pace PE, Babu MM, Patel KJ. Mechanistic Insight into Site-Restricted Monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI. Molecular Cell. 2008;32:767–777. - PubMed
    1. Blom E, van de Vrugt HJ, de Vries Y, de Winter JP, Arwert F, Joenje H. Multiple TPR motifs characterize the Fanconi anemia FANCG protein. DNA repair. 2004;3:77–84. - PubMed
    1. Bogliolo M, Schuster B, Stoepker C, Derkunt B, Su Y, Raams A, Trujillo Juan P, Minguillón J, Ramírez María J, Pujol R, et al. Mutations in ERCC4, Encoding the DNA-Repair Endonuclease XPF, Cause Fanconi Anemia. The American Journal of Human Genetics. 2013;92:800–806. - PMC - PubMed
    1. D'Andrea AD. Susceptibility pathways in Fanconi's anemia and breast cancer. The New England journal of medicine. 2010;362:1909–1919. - PMC - PubMed

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