FANCL supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner

Abstract

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of selective autophagy, beyond their canonical roles of repairing DNA interstrand cross-links. FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy. Although mutations in FANCL account for only a very small number of cases in FA families, it plays a key role in the FA pathophysiology and might drive carcinogenesis. Here, we demonstrate that FANCL protein is present in mitochondria in the control and oligomycin and antimycin (OA)-treated cells and its ubiquitin ligase activity is not required for its localization to mitochondria. CRISPR/Cas9-mediated knockout of FANCL in HeLa cells overexpressing parkin results in increased sensitivity to mitochondrial stress and defective clearing of damaged mitochondria upon OA treatment. This defect was reversed by the reintroduction of either wild-type FANCL or FANCL(C307A), a mutant lacking ubiquitin ligase activity. To summarize, FANCL protects from mitochondrial stress and supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner.

Fanconi anemia (FA) is a rare autosomal genetic disorder characterized by congenital abnormalities and progressive bone marrow failure (1). FA patients are at higher risk of developing hematological malignancies and solid tumors (2). Loss of function or mutation in any one of the >22 FA genes may lead to development of clinical symptoms of FA (3). The major function of FA proteins is the repair of interstrand cross-links (ICL), where eight FA genes (FANCA, B, C, E, F, G, L and M) act as a FA core complex (FACC) recognizing the ICLs and in turn activate the ID2 complex (FANCD2 and FANCI) through monoubiquitination (4). Monoubiquitylation of FANCD2 and FANCI is mediated by an E3 ubiquitin ligase FANCL/PHF9, present in the core complex (5, 6). Despite the minor proportion of FANCL mutations in FA cases, mutational analysis revealed the existence of pathogenic variants like null mutation (delTAT) and hypomorphic mutation (dupAATT) (7, 8). Further, the crosstalk of FA proteins with other non-FA DNA repair molecules activates homologous recombination, nucleotide excision repair and trans lesion synthesis pathways that aid in ICL repair (4, 9).

In addition to their canonical activities within the nucleus, proteins encoded by FA genes also localize to additional cellular compartments and exert cytoprotective functions independent of DNA Damage Response (DDR) (10–13). A growing body of literature has reported the connection between FA subtypes A, C, D2 and G and mitochondrial functions wherein FA cells experience increased oxidative stress and altered mitochondrial functions (13–15). Mitophagy is a form of selective autophagy in which damaged mitochondria are sequestered inside autophagosomes and delivered to the lysosome for degradation (16). The best understood form of mitophagy utilizes the PINK1-Parkin pathway to label damaged mitochondria with phosphorylated ubiquitin, which recruits autophagy adaptors that in turn initiate autophagosome formation (17). Mitophagy is important for limiting the production of mitochondrial reactive oxygen species from damaged mitochondria and is therefore an important component of the cellular defense against oxidative stress (18). FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy (13). While FANCC interacts directly with Parkin, what role FANCL and its E3 ubiquitin ligase activity play in Parkin-mediated mitophagy remains unknown (13).

E3 ubiquitin ligase activity of FANCL is retained in the ring domain at two major positions, C307 and W341. We focused on the FANCL C307A mutant as the cysteine at 307 position is conserved across species (Figure S1A), and this mutant is defective in binding to E2 ubiquitin-conjugating enzyme and assembling the nuclear core complex (19, 20). To meticulously test the importance of FANCL and its ligase activity, we first generated FANCL KO (Knock Out) Hela cells that mimic the null mutation variants. We then complemented these parkin overexpressed FANCL KO HeLa cells with WT FANCL (Wild Type) or C307A FANCL constructs and characterized FANCL modulated clones (Figure S1B). FANCL knockout cells showed a defect in monoubiquitinating FANCD2 and were sensitive to Mitomycin C (MMC)-induced cell death, which confirms impairment of the canonical nuclear functions of the core FA pathway (Figure S1C & D). Consistent with earlier studies (19), complementing the FANCL KO cells with WT FANCL, but not the C307A FANCL construct, rescued both the defect in monoubiquitination of FANCD2 and the heightened sensitivity to MMC-induced cell death (Figure S1C & D).

As FA cells display mitochondrial dysfunctions, and ubiquitination is a key event in mitochondrial quality control pathways (14, 21), we evaluated if FANCL is localized into mitochondria upon mitochondrial stress induced by combination of two specific inhibitors of mitochondrial respiration, Oligomycin + Anitmycin A, (OA). We observed the colocalization of Flag FANCL and mitochondrial marker TOMM20 by confocal imaging (Figure 1A). We next asked if mitochondrial localization of FANCL relies on its ubiquitin ligase activity using Parkin overexpressing HeLa FANCL KO cells complemented with either FANCLWT or FANCLC307A mutant. We isolated highly purified mitochondria using subcellular fractionation from these cells and confirmed the purity of mitochondria by immunoblotting with cytoplasmic and nuclear markers for contamination (Figure 1B). The mitochondrial lysates were immunoblotted for Flag, and we observed that FANCL-Flag is present in the mitochondrial extracts of both FANCLWT and FANCLC307A mutant HeLa cells. Intriguingly, unlike nuclear localization of FANCL, which is dependent on its ubiquitin ligase activity (22), FANCL is constitutively present in mitochondria and its localization is not initiated by mitochondrial stress nor altered by its ligase activity (Figure 1B). Together, these results suggest that FANCL ligase activity is not required for its mitochondrial localization. We further tested viability of FANCL modulated clones in response to mitochondrial stress and observed that FANCL KO cells are sensitive to OA at all concentrations used, which is rescued by complementing with FANCL WT. FANCL KO cells complemented with FANCL C307A mutant were resistant at lower doses of OA but displayed similar sensitivity to FANCL KO cells at the highest dose tested (Figure 1C). Our observation that FANCL C307A largely rescues OA sensitivity but fails to rescue MMC sensitivity of FANCL knockout cells indicates that the mitochondrial function of FANCL is molecularly distinct from its canonical function in the nuclear DNA damage response (Figure 1C, Figure S1D).

Figure 1: FANCL localizes to mitochondria in a ligase independent manner and protects against mitochondrial stress.

(A) Confocal images of HeLa FANCLWT Parkin cells stained for TOMM20 and Flag with OA for 4 hr, Scale bar, 20 μm. (B) Western blot of indicated proteins in mitochondrial extracts from HeLa FANCLWT/ HeLa FANCL C307A Parkin cells +/− OA for 4 hr. Whole cell lysates from FANCL WT & KO are used to assess mitochondrial purity. (C) Cell viability (resazurin-based assay) was measured after treatment of cells over a range of indicated concentrations of Oligomycin & Antimycin (O, A) for 6hrs. Each data point represents the mean of three independent biological replicates ± SD. Cell viability is expressed as the percentage of the viable cells in the treated group relative to the untreated control group. Differences in cell viability are significant between FANCL KO and other groups at all indicated concentrations (p<0.05). (D) Representative histograms of MitoSOX Red flow cytometry data at 8 hrs of OA treatment. Y-axes represent the number of counts of cells that emit fluorescence. X-axes represent the average fluorescence intensity of cells. (E) Quantified MFI (Mean Fluorescence Intensity) of cells. Error bars are mean ± SEM *p < 0.05 (n = 4). (F) Oxygen Consumption Rate (OCR) in FANCL modulated clones using seahorse analyzer (n = 3). (G & H) Basal and Maximal Respiration rates.

Mitochondrial stress often leads to the production of mitochondrial Reactive Oxygen Species (mtROS), and FA patients display elevated ROS levels which in turn induce detrimental effects (23). Hence, we analyzed mtROS levels using MitoSOX stain by flow cytometry which revealed significantly elevated levels of mtROS in FANCL KO cells compared to FANCL WT or FANCL C307A (Figure 1D & E). As oxidative stress is known to impair mitochondrial bioenergetics, we asked if the loss of FANCL function impacted the mitochondrial oxidative phosphorylation (OXPHOS) capacity by measurement of the oxygen consumption rate (OCR). Addition of the protonophore uncoupler FCCP stimulated the OCR to its maximal activity in FANCL modulated clones but to a significantly lower extent in FANCL KO (Fig 1F, 1G & 1H) when compared to FANCL WT or FANCL C307A mutant. Altogether, these results show increased mtROS levels and reduced mitochondrial OXPHOS capacity in FANCL KO cells, which can be rescued by wild-type FANCL as well as FANCL C307A mutant lacking E3 ligase activity. This indicates that FANCL limits mtROS and supports mitochondrial function in a ubiquitin ligase-independent manner.

It is reasonable to assume that these defective mitochondria could be efficiently cleared by autophagic machinery by a process of selective autophagy (mitophagy) thereby protecting the cell from adverse effects (24). Strikingly, previous studies have identified FANCL as a potential selective autophagy factor in a genome-wide siRNA screen and further revealed a functional link between FANCL and mitophagy, wherein FANCL is required for clearance of damaged mitochondria upon cellular stress (13, 25). However, it is not known if the ligase activity of FANCL is required for mitophagy. In an effort to identify whether the E3 ubiquitin ligase activity of FANCL is required for Parkin-mediated mitophagy, we used FANCL modulated clones of Parkin-overexpressing HeLa cells, a well-studied model of Parkin-mediated mitophagy (24). HeLa FANCL KO cells were deficient in mitophagy after treatment with OA, as measured by quantitation of dsDNA (mitochondrial DNA) puncta, and this deficiency was rescued by re-introduction of WT FANCL or FANCL C307A mutant (Figure 2A & 2B). In corroboration with the immunofluorescence studies, western blot detection of mitochondrial markers TOMM20, HSP60, Parkin and COXIV from whole cell lysates displayed a defect in mitophagy in FANCL KO cells (Figure 2C & 2D). Complementing the FANCL KO cells with either WT FANCL expressing plasmid or FANCL C307A mutant restored the OA-induced mitophagy. Comparably, confocal imaging showed accumulation of COXIV in OA- or CCCP-treated FANCL KO cells that was reversed by complementing with FANCL WT or FANCL C307A mutant (Supplementary Figure 2A & 2B). Despite accelerated mitochondrial biogenesis induced upon OA treatment, there is no significant difference between the clones as evident from the mRNA levels of PGC1α, a critical regulator of mitochondrial biogenesis (Figure 2E) (26). Further, the afore-mentioned elevated mtROS levels observed in FANCL KO cells could be an indirect or direct consequence or a cause of defective mitophagy.

Figure 2: FANCL functions in a ligase-independent manner to support selective mitophagy but is not required for starvation induced autophagy.

(A) Confocal images of HeLa Parental/FANCLKO/ FANCLWT/ FANCLC307A Parkin cells stained for dsDNA after treatment with OA for 8 hr. Cells were treated with OA (Oligomycin, 2.5 μM; Antimycin A, 250 nM) for 8 hr prior to imaging and automated image analysis. Representative images of immunofluorescence staining. Scale bars, 20 μm. (B) Quantitative image analysis. Shown are box plots of at least 50 cells analyzed per condition. Similar results were observed in three independent experiments. **, P<0.05 ***, P<0.005, Mann-Whitney U-test. (C) Mitophagy analysis in HeLa Parental/FANCLKO/ FANCLWT/ FANCLC307A Parkin cells assessed by western blot of the indicated mitochondrial proteins in cells +/− OA for 8hrs. (D) Densitometry graph showing quantification of TOMM20 (outer mitochondrial membrane), COXIV (Matrix) and HSP60 (Intermembrane space) proteins normalized to actin (n=4). (E) PPARGC1A or PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) mRNA levels as assessed by qPCR (n = 3). (F) Autophagy flux analysis using Cyto-ID Autophagy Detection Kit in HeLa FANCL modulated clones in normal (starvation −) or EBSS (starvation +) medium for 4 hr +/− 100 nM bafilomycin A1 (Baf A1) by flow cytometry and the data is presented as a histogram overlay showing mean fluorescence intensity (MFI). (G) Quantified MFI (n = 3). (H) Western blot of p62 and LC3-b in HeLa FANCL modulated clones in normal (starvation −) or EBSS (starvation +) medium for 4 hr +/− 100 nM bafilomycin A1 (Baf A1). (I) LC3B-II/Actin ratio (n=3).

We next asked if FANCL is required for non-selective autophagy, in which starvation triggers cytoplasmic material to be engulfed by the autophagosome in a non-specific manner and the contents are all degraded by the lysosome (27). We used FANCL modulated clones of HeLa cells and evaluated whether FANCL is required for starvation-induced autophagy. Cells under starvation and treated with bafilomycin A1 resulted in a stronger increase in Cyto-ID fluorescence signal. However, FANCL modulated clones did not differ in Cyto-ID fluorescence signal (Fig. 2F & 2G). Western blot analysis of FANCL modulated clones did not differ in starvation-induced degradation of the autophagy substrate p62 or LC3-b in the presence and absence of the lysosomal inhibitor bafilomycin A1 (Figure 2H & 2I) confirming that FANCL is not required for starvation-induced autophagy. This demonstrates that FANCL, like FANCC, is required for selective mitophagy but dispensable for starvation-induced, non-selective autophagy (13).

Altogether our results indicate that FANCL supports mitophagy in a ligase-independent manner. This corroborates previous findings that multiple FA genes are required for mitophagy and that mitochondrial FANCD2 is not ubiquitinated (13). Like FANCC, whose role in mitophagy can be genetically separated from its role in nuclear DNA damage repair (13), our results indicate that FANCL also plays a molecularly distinct and ligase-independent role in mitophagy as opposed to its ligase-dependent role in DNA damage repair. Future investigations will aim to understand in more detail the mechanisms through which FANCL and other FA proteins cooperate to degrade damaged mitochondria and thereby defend against oxidative stress.

Supplementary Material

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Supplementary Figure 1: (A) Multiple sequence alignment of FANCL isoform 2 from different species (supplementary Table 1) showing the conserved amino acid Cysteine at 307 positions. (B) Western blotting of FANCL modulated clones confirming the expression of Parkin and Flag FANCL. (C) Western blotting from whole cell lysates from the cells treated with MMC (1 μM, 24 hours), showing the defect in monoubiquitination of FANCD2 in FANCL modulated clones. (D) MMC sensitivity assay to assess the FA core complex activity. Cells were treated with indicated MMC concentration for 24 hours and the cell viability was analyzed using alamar blue assay (n=3).

Supplementary Figure 2: Confocal images of HeLa Parental/FANCLKO/ FANCLWT/ FANCLC307A Parkin cells stained for COXIV upon treated with (A) OA (Oligomycin & Antimycin) and (B) CCCP (Carbonyl cyanide 3- chlorophenylhydrazone) for 4 hrs. Scale bars, 20 μm.

Supplementary Figure 3: (A) gRNA validation of FANCL using NGS and (B) the final clone’s confirmation. (C) Western blot showing the knockout of endogenous FANCL