Genetic inactivation of FAAP100 causes Fanconi anemia due to disruption of the monoubiquitin ligase core complex
Julia Kuehl, Yutong Xue, Fenghua Yuan, Ramanagouda Ramanagoudr-Bhojappa, Simone Pickel, Reinhard Kalb, Settara C. Chandrasekharappa, Weidong Wang, Yanbin Zhang, Detlev Schindler
Julia Kuehl, Yutong Xue, Fenghua Yuan, Ramanagouda Ramanagoudr-Bhojappa, Simone Pickel, Reinhard Kalb, Settara C. Chandrasekharappa, Weidong Wang, Yanbin Zhang, Detlev Schindler
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Research Article Cell biology Genetics

Genetic inactivation of FAAP100 causes Fanconi anemia due to disruption of the monoubiquitin ligase core complex

Abstract

The Fanconi anemia/breast cancer (FA/BRCA) DNA repair network promotes the removal of DNA interstrand crosslinks (ICLs) to counteract their devastating consequences, including oncogenesis. Network signaling is initiated by the FA core complex, which consists of 7 authentic FA proteins and an FA-associated protein, FAAP100, with incompletely characterized roles and unknown disease associations. Upon activation, the FA core complex functions as a multiprotein E3 ubiquitin ligase centered on its catalytic module, the FANCB-FANCL-FAAP100 (BLP100) subcomplex, for FANCD2 and FANCI monoubiquitylation. Here, we identified a homozygous variant in FAAP100, c.1642A>C, predicting p.(T542P), in a fetus with malformations suggestive of FA. The mutation caused sensitivity to ICL-inducing agents in cells from the affected individual and genetically engineered, FAAP100-inactivated human, avian, zebrafish, and mouse cells. All FAAP100-deficient cell types were rescued by ectopic expression of WT FAAP100, but not FAAP100T542P. In a confirmatory animal model, customized Faap100–/– mice exhibited embryonic lethality, microsomia, malformations, and gonadal atrophy resembling mice with established FA subtypes. Mechanistically, FAAP100T542P impaired ligase activity by preventing BLP100 subcomplex formation, resulting in defective FAAP100T542P nuclear translocation and chromatin recruitment. FAAP100 dysfunction that disrupted the FA pathway and impaired genomic maintenance, together with FA-consistent human manifestations, recommends FAAP100 as a legitimate FA gene, alias FANCX.

Authors

Julia Kuehl, Yutong Xue, Fenghua Yuan, Ramanagouda Ramanagoudr-Bhojappa, Simone Pickel, Reinhard Kalb, Settara C. Chandrasekharappa, Weidong Wang, Yanbin Zhang, Detlev Schindler

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Figure 3

FANCD2 monoubiquitylation capacity of FAAP100-inactivated human and avian cell lines.

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FANCD2 monoubiquitylation capacity of FAAP100-inactivated human and avia...
(A) FAAP100T542P mutant 1176 cells (parental) lacked FANCD2 monoubiquitylation (◄D2) on immunoblots. FANCD2 monoubiquitylation was restored only in 1176+FAAP100WT transduced cells (◄D2-Ub), but not in 1176+FAAP100T542P or mock transduced cells. FA-L was a monoubiquitylation-deficient FA control. (B) FAAP100T542-mutant 1176 cells showed reduced levels of FAAP100 protein (see ratio to vinculin in parental and +[empty] vectorpLVX lanes) and lacked FANCD2 monoubiquitylation (◄D2) on immunoblots. 1176+FAAP100WT- and 1176+FAAP100T542P-transduced cells both overexpressed FAAP100, but FANCD2 monoubiquitylation was restored only in 1176+FAAP100WT cells (◄D2-Ub). FA-D2 was a control for absent FANCD2, and non-FA was a monoubiquitylation-proficient normal control. (C) FANCD2 monoubiquitylation was functional in HEK293T cells (not shown) and HEK293T cells mock transfected with the Cas9-containing vector PX459 (◄D2-Ub), but not in the FAAP100-inactivated HEK293T CRISPR/Cas9 clone Cr10 (parental) (◄D2), where it was rescued by transduction with FAAP100WT, resulting in overexpression of FAAP100 (◄D2-Ub), as shown by FANCD2 and FAAP100 immunoblots. (D) FANCD2 monoubiquitylation was functional in parental HAP1 cells (◄D2-Ub), but not in FAAP100-inactivated HAP1 FAAP100R149Vfs*2 cells (◄D2), where it was restored by transduction with FAAP100WT, resulting in overexpression of FAAP100 (◄D2-Ub), as shown by FANCD2 and FAAP100 immunoblots. (E) FANCD2 monoubiquitylation was functional in parental DT40 cells (◄D2-Ub), but absent in ΔFAAP100-DT40 cells (◄D2). In the latter, it was rescued by the expression of EGFP-FAAP100WT (◄D2-Ub), but not by EGFP-FAAP100T547P (◄D2), which contained the chicken equivalent T547P of human T542P, as shown by FANCD2 and EGFP immunoblots. Note that the strong overexpression of the FAAP100 protein by lentiviral transduction in B–D, where low- and high-exposure blots are shown, was in marked contrast to the transfection in E. Vinculin or RAD50 or tubulin was used as a loading control on all blots. The FAAP100/vinculin ratios were taken from the high-exposure FAAP100 blot. The FANCD2-Ub/FANCD2 ratios semiquantitatively estimated monoubiquitylation.