Disease relevance of FANCG

• Carboxy terminal region of the Fanconi anemia protein, FANCG/XRCC9, is required for functional activity [1].
• Unlike the lymphoblasts, this myeloid leukemia cell line (UoC-M1) was hypersensitive to mitomycin-C (MMC) and diepoxybutane (DEB) and exhibited a marked decrease in nuclear FANCA, FANCG, and FANCD2-L [2].
• Recently van der Heijden et al. identified FANCC and FANCG gene mutations in patients with young-onset pancreatic cancer [3].
• Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer [4].
• METHODS: We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance [4].

High impact information on FANCG

• We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control [5].
• On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation [4].
• RESULTS: FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function [4].
• We demonstrate that the FA group G (FANCG) protein is found in mitochondria [6].
• A physical complex of the Fanconi anemia proteins FANCG/XRCC9 and FANCA [7].

Chemical compound and disease context of FANCG

• Phosphorylation of fanconi anemia (FA) complementation group G protein, FANCG, at serine 7 is important for function of the FA pathway [8].
• We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and gamma-radiation [9].

Biological context of FANCG

• Additional amino acid sequences at the carboxy terminus of FANCG are required for the binding of FANCC in the complex [1].
• Patient-derived mutant forms of FANCA, containing an intact NLS sequence but point mutations in the carboxy-terminal leucine zipper region, bound FANCG in the cytoplasm [10].
• We used a strategy involving amplification of FANCG/XRCC9 exons and direct sequencing to identify novel FANCG mutations in cell lines derived from these FA-G patients [11].
• RESULTS: Our results demonstrate a wide range of mutations in the FANCG gene (splice, nonsense, and missense mutations) [11].
• We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA [12].

Anatomical context of FANCG

• Similar results were obtained upon correction of an FA-A cell line, with a reciprocal increase in the half-life of FANCG [10].
• Four human FANCG polymorphic variants show normal biological function in hamster CHO cells [13].
• To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40 [14].
• The recent identification of FANCC and FANCG mutations in resected pancreatic tumors selected for loss of heterozygosity on chromosome 9, some of which were present in the germ line DNA, suggests that inactivation of these and other Fanconi complementation group genes may contribute to pancreatic cancer [15].
• Conversely, stable expression of FANCA mutants encoding intact FANCG interaction domains induced hypersensitivity to MMC in HeLa cells [16].

Associations of FANCG with chemical compounds

• After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, the accumulation of epitope-tagged FANCA in the nucleus increased, whereas FANCC was affected to a lesser extent and FANCG showed no response [17].
• The phosphorylation of FANCG at serine 7 by using mass spectrometry was previously mapped [18].
• By alanine mutagenesis, Arg(1), Arg(2), and Leu(8) but not Arg(3), Trp(5), and Glu(7) appeared to be critical for binding to FANCG [16].
• N-Ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro [19].
• Using DNA affinity chromatography, we now show that alphaSpIISigma, present in HeLa cell nuclei, specifically binds to DNA containing psoralen interstrand cross-links and that the FANCA, FANCC, and FANCG proteins are bound to this damaged DNA as well [20].

Physical interactions of FANCG

• A FANCG mutant truncated at the carboxyl-terminus retained the ability to interact with FANCA [21].
• We found that FANCG was capable of binding to two separate sites in the BRCA2 protein, located either side of the BRC repeats [22].
• We found that the C terminus of FANCF interacts directly with FANCG and allows the assembly of other FA proteins into a stable complex [23].
• The FANCG Fanconi anemia protein interacts with CYP2E1: possible role in protection against oxidative DNA damage [24].
• FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P [25].

Regulatory relationships of FANCG

• However, a high level of activation was found when FANCA was co-expressed with FANCG, indicating strong, direct interaction between these proteins [26].
• We found that treatment with TNF-alpha induced FANCG protein expression [27].

Other interactions of FANCG

• To further examine complex assembly, the yeast 3-hybrid system was used to investigate the ability of FANCG to act as a molecular bridge in mediating interaction between other FA proteins [21].
• Direct interaction of the Fanconi anaemia protein FANCG with BRCA2/FANCD1 [22].
• The interaction between FANCG and FANCF was ablated by a Leu71Pro mutant of FANCG [21].
• Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity [12].
• The amino-terminal region of the FANCA protein is required for FANCG binding, FANCC binding, nuclear localization, and functional activity of the complex [28].

Analytical, diagnostic and therapeutic context of FANCG

• Sequence analysis of 38 individuals with familial pancreatic cancer enrolled in the National Familial Pancreatic Tumor Registry (NFPTR) revealed previously identified polymorphisms within two exons and one intron of FANCC, and in three introns of FANCG [3].
• To address to what extent FANCG variants contribute to sporadic childhood AML, we determined the spectrum of FANCG sequence variants in 107 children diagnosed with sporadic AML, using polymerase chain reaction (PCR), fluorescent single-strand conformational polymorphism (SSCP) and sequencing methodologies [29].
• We used the yeast two-hybrid assay to test for interactions of the FANCA, FANCC, and FANCG proteins [26].
• DNA single-strand conformation polymorphism of each exon of the FANCA and FANCG genes was followed by sequence analysis of the aberrantly migrating fragments and by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the splice-site mutations identified [30].

References