Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs upon exposure to DNA damaging agents and during S-phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a CDK regulatory phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during S-phase. Furthermore, FA-D2 ( FANCD2 -/- ) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity.

Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital abnormalities and increased risk for bone marrow failure and cancer. FA is caused by mutation of any one of 23 genes, the protein products of which function primarily in the maintenance of genome stability. An important role for the FA proteins in the repair of DNA interstrand crosslinks (ICLs) has been established in vitro. While the endogenous sources of ICLs relevant to the pathophysiology of FA have yet to be clearly determined, a role for the FA proteins in a two-tier system for the detoxification of reactive metabolic aldehydes has been established. To discover new metabolic pathways linked to FA, we performed RNA-seq analysis on non-transformed FA-D2 (FANCD2-/-) and FANCD2-complemented patient cells. Multiple genes associated with retinoic acid metabolism and signaling were differentially expressed in FA-D2 (FANCD2-/-) patient cells, including ALDH1A1 and RDH10, which encode for retinaldehyde and retinol dehydrogenases, respectively. Increased levels of the ALDH1A1 and RDH10 proteins was confirmed by immunoblotting. FA-D2 (FANCD2-/-) patient cells displayed increased aldehyde dehydrogenase activity compared to the FANCD2-complemented cells. Upon exposure to retinaldehyde, FA-D2 (FANCD2-/-) cells exhibited increased DNA double-strand breaks and checkpoint activation indicative of a defect in the repair of retinaldehyde-induced DNA damage. Our findings describe a novel link between retinoic acid metabolism and FA and identify retinaldehyde as an additional reactive metabolic aldehyde relevant to the pathophysiology of FA.

Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital defects, and increased risk for early‐onset bone marrow failure and cancer. Biallelic mutations in any one of 23 genes leads to FA. In vitro experiments have established a coordinated role for the FA gene network in the repair of DNA interstrand crosslinks (ICLs). However, the endogenous source(s) of DNA damage and chromosome instability in FA patients remain largely unknown. Recent studies have suggested that the FA proteins may play an important role in the repair of DNA damage caused by genotoxic reactive aldehydes produced during cellular metabolism, e.g., acetaldehyde and formaldehyde. To discover novel metabolic pathways linked to FA, we recently performed RNA‐seq analysis on telomerase‐immortalized mutant and functionally complemented FA‐D2 (FANCD2‐/‐) patient cells. Gene ontology enrichment analysis uncovered dysregulation of retinoid metabolism and retinoic acid signaling in mutant FA‐D2 (FANCD2‐/‐) cells. For example, we observed increased expression of the retinoid metabolism genes RDH10, ALDH1A1, and CYP26B1, in the absence of FANCD2. RDH10 converts retinol to retinaldehyde; ALDH1A1 converts retinaldehyde to retinoic acid; CYP26B1 degrades excess retinoic acid. In addition, we observed altered expression of multiple retinoic acid regulated genes including RARA, EGR1, and TGM2, and several members of the HOX, PAX, and SOX gene families. Using immunoblotting and qPCR, we have validated differential expression of many of these genes. We have also observed altered retinoid metabolism in Fancd2‐/‐ mouse cells. Furthermore, in preliminary flow cytometry experiments, we have determined that FA‐D2 (FANCD2‐/‐) patient cells exhibit increased aldehyde dehydrogenase activity. These findings are potentially highly significant and suggest that dysregulated retinoid metabolism and retinoic acid signaling ‐ as well as retinaldehyde‐associated genotoxicity ‐ might be key molecular features of FA. Further characterization of this newly discovered link could pave the way for the discovery of new therapeutic targets for FA patients.

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins under conditions of cellular stress and during S-phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a CDK regulatory phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during S-phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity.Author SummaryFanconi anemia (FA) is a rare genetic disease characterized by high risk for bone marrow failure and cancer. FA has strong genetic and biochemical links to hereditary breast and ovarian cancer. The FA proteins function to repair DNA damage and to maintain genome stability. The FANCD2 protein functions at a critical stage of the FA pathway and its posttranslational modification is defective in >90% of FA patients. However, the function, and regulation of FANCD2, particularly under unperturbed cellular conditions, remains remarkably poorly characterized. In this study, we describe a novel mechanism of regulation of the FANCD2 protein during S-phase of the cell cycle. CDK-mediated phosphorylation of FANCD2 on S592 promotes the ubiquitination of FANCD2 during S-phase. Disruption of this phospho-regulatory mechanism results in compromised mitotic fidelity and an increase in mitotic chromosome instability.