The Fanconi anemia core complex promotes CtIP-dependent end-resection to drive homologous recombination at DNA double-strand breaks

Abstract

Homologous Recombination (HR) is a high-fidelity repair mechanism of DNA Double-Strand Breaks (DSBs), which are induced by irradiation, genotoxic chemicals or physiological DNA damaging processes. DSBs are also generated as intermediates during the repair of interstrand crosslinks (ICLs). In this context, the Fanconi anemia (FA) core complex, which is effectively recruited to ICLs, promotes HR-mediated DSB-repair. However, whether the FA core complex also promotes HR at ICL-independent DSBs remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen with cells carrying the DSB-repair reporter DSB-Spectrum. Using isogenic cell-line models, we validated the HR-function of FANCL and Ube2T, and demonstrated a similar function for their ubiquitination-substrate FANCD2. We further show that FANCL and Ube2T are directly recruited to DSBs and are required for the accumulation of FANCD2 at these break sites. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of the nuclease CtIP at DSBs, and consequently for optimal end-resection and Rad51 loading. CtIP overexpression rescues HR in FANCL-deficient cells, validating that FANCL primarily regulates HR by promoting CtIP recruitment. Together, these data demonstrate that the FA core complex and FANCD2 have a dual genome maintenance function by promoting repair of DSBs as well as the repair of ICLs.

Figure 1.. A targeted CRISPR screen in DSB-Spectrum reporter cells identifies FANCM, Ube2T and FANCL as HR-promoting factors

(A) Schematic of the DSB-Spectrum_V2 reporter. Adpated from van de Kooij et al., 2022. (B) HEK 293T+Cas9+DSB-Spectrum_V2 cells were lentivirally infected to express mCherry and the BFPsg targeting the DSB-Spectrum_V2 reporter locus. Next, at indicated time points, BFP and GFP expression was analyzed by flow cytometry. Depicted is the mean±SEM of a technical triplicate. (C) Schematic displaying the CRISPR screen lay-out in HEK 293T+Cas9+DSB-Spectrum_V2 cells. (D) Volcano plot showing the gene targets of sgRNAs that were either enriched or depleted from the GFP⁺ HR population as compared to the reference population. BRCA1/BARD1 and Ube2T/FANCM/FANCL are indicated in green as HR-promoting factors. BRE/BABAM1/BRCC3, all members of the BRCA1-A complex, and PRKDC are indicated in red as HR-inhibiting factors.

Figure 2.. Ube2T and FANCL promote HR-mediated repair of Cas9-induced DSBs.

(A) Ube2T expression was analyzed by western blot in HEK 293T+DSB-Spectrum_V3 Ube2T^KO clones 3.3 and 4.1, and in the parental control. (B) Indicated HEK 293T+DSB-Spectrum_V3 cell-lines were transfected to express Cas9 and either a control sgRNA, or the sgRNA targeting the BFP gene in the reporter locus. Next, cells were analyzed by flow cytometry to determine the frequency of repair by each of the three indicated pathways (n=7; mean±SEM; One-way ANOVA+Dunnett’s multiple comparison). (C) DNA sequence alignment of the FANCL sgRNA target site in unedited parental control cells and the HEK 293T+DSB-Spectrum_V3 FANCL^KO clones. Depicted are representative sequence chromatograms, red shaded boxes indicate deviations in the DNA sequence of the FANCL^KO clone compared to control. (D) As in panel B, now analyzing FANCL^KO cells. Note that for four biological repeats the same control was shared between panel B and this panel (n=7; mean±SEM; One-way ANOVA+Dunnett’s multiple comparison). (E) HEK 293T+DSB-Spectrum_V3 FANCL^KO clones were transduced with an empty vector (EV), FANCL wild-type cDNA (WT), or FANCL Ligase-Dead cDNA (LD), and treated with Mitomycin C (MMC, 500μM) for 24h. Next, FANCD2 ubiquitination was analyzed by western blot. (F) As in panel B, now analyzing the HEK 293T+DSB-Spectrum_V3 FANCL^KO cells described in panel E (n=3; mean±SEM; One-way ANOVA+Dunnett’s multiple comparison).

Figure 3.. Loss of Ube2T, FANCL and FANCD2 sensitizes to PARP inhibitor-induced toxicity.

(A) Indicated U-2 OS Ube2T^KO clones, as well as the parental control, were treated with Mitomycin C (MMC, 500 μM) for 24h. Next, FANCD2 ubiquitination status and protein levels of Ube2T were analyzed by western blot. (B) As in panel A, but now for U-2 OS FANCL^KO clones. (C and D) U-2 OS cells, either wild-type control (Con.) or knock-out for Ube2T (panel C) or FANCL (panel D), were treated with olaparib for 14 days. Cell viability was assed by clonogenic survival. Note that the 0 μM value was added manually to the X-axis. Inset shows mean IC50 and p-value (n=3, expect for the 0.1 μM and 10 μM concentration in panel C for which n=2; mean±SEM; Ratio paired t-test). (E) As in panel B, now for FANCL^KO cells that were transduced with an empty vector (EV), FANCL wild-type cDNA (WT), or FANCL ligase-dead cDNA (LD). (F and G) Indicated cell-lines were treated with 0.1 μM olaparib, or left untreated, for 14 days. Cell viability was assessed by clonogenic survival. Panel F shows a representative picture of Methylene Blue-stained colonies, panel G shows the quantification (n=3; mean±SEM; One-way ANOVA+Dunnett’s multiple comparison). (H) As in panel B, but now for untreated FANCD2^KO clones. (I) As in panels C and D, but for FANCD2^KO cells (n=3; mean±SEM; Ratio paired t-test).

Figure 4.. FANCL, Ube2T and FANCD2 are recruited to DNA Double-Strand Breaks.

(A, B) U-2 OS cells expressing either GFP-NLS as a control, or GFP-tagged FANCL or Ube2T were exposed to UV-A laser micro-irradiation. Next, cells were fixed and analyzed by immunofluorescence microscopy. Laser-induced DNA damage tracks were identified by α-γH2AX staining. Panel A shows representative images, panel B shows the quantification of a representative experiment. Dotted line is set at 1 (i.e. no recruitment to the track), red lines indicate median (n=2; one-way ANOVA with post-hoc Kruskal-Wallis). (C, D). As in panels A, B, but now analyzing recruitment of endogenous FANCD2 to UV-A laser induced DNA damage tracks in U-2 OS FANCL^KO cell-lines (EV=empty vector). DNA damage tracks were identified by α-MDC1 staining. Panel C shows representative images, panel D shows the quantification of a representative experiment. Red lines indicate the median (n=2; one-way ANOVA with post-hoc Kruskal-Wallis). (E) Cartoon schematic of a DSB recruiment assay in U2OS 2-6-3 cells. In short, LacI-fused mCherry-tagged FokI is tethered to an array of LacO repeats where it generates a large number of DSBs. DSB-repair factors accumulate at these DSBs and form microscopically discernable foci. Adapted from Singh et al., 2021 (F, G) Accumulation of GFP-NLS or GFP-FANCL at γH2AX-marked FokI-generated DSBs in U-2 OS 2-6-3 cells was assessed by immunofluorescence microscopy. Panel F shows representative images, panel G shows the quantification of a representative experiment. Red lines indicate the median (n=2; Mann-Whitney test). (H,I) As in panels F and G but now analyzing endogenous FANCD2 recruitment to MDC1-marked FokI-induced DSBs (n=2; Mann-Whitney test).

Figure 5.. FANCL promotes end-resection at DNA double-strand breaks.

(A, B) U-2 OS FANCL^KO and wild-type control cells were exposed to 10 Gy ionizing radiation (IR), followed by IF microscopy to detect foci containing S4/8 phosphorylated RPA (pRPA) in S-phase (EdU+) nuclei. Panel A shows representative images, panel B shows the quantification (n=4; mean±SEM; one-way ANOVA with post-hoc Dunnett’s). (C) As in panel B, but now plotting total pRPA intensity per nucleus in FANCL^KO cells reconstituted with FANCL WT or LD (n=3; mean±SEM; one-way ANOVA with post-hoc Dunnett’s). (D) Schematic of the qPCR-based quantification of end-resection in AsiSI cells. (E) Western blot of MMC-treated (500 μM, 24h) U-2 OS AsiSI cells. (F) Quantification by qPCR of single-strand DNA (ssDNA) at 335 bp or 1618 bp distance from a defined AsiSI-induced DSB (n≥3; one-way ANOVA with post-hoc Dunnett’s). (G) As in panel F, but now including treatment with the DNA-PKcs inhibitor NU7441 (PKi; 2 μM; n=5; mean±SEM; paired t-test). (H, I) As in panels A and B, respectively, but now analyzing total Rad51 intensity per S-phase nucleus (n=4; mean±SEM; one-way ANOVA with post-hoc Dunnett’s). (J, K) IF-micropscopy of UV-A laser micro-irradiated cells. Panel J shows representative images, panel K shows the quantification. Plotted are the data from all biological repeats. Each grey or green dot represents an individual track, black dots are the median for each biological repeat (n=4; mean±SEM; ratio paired t-test).

Figure 6.. FANCL promotes CtIP recruitment to DNA double-strand breaks.

(A, B) U-2 OS FANCL^KO and wild-type control cells were exposed to 10 Gy ionizing radiation (IR), followed by IF microscopy to detect CtIP foci in S-phase (EdU+) nuclei. Panel A shows representative images, panel B shows the quantification. Plotted are the data from all biological repeats. Each grey or green dot represents an individual nucleus, black dots are the median for each biological repeat (n=3; mean±SEM; one-way ANOVA with post-hoc Dunnett’s). (C, D) IF-microscopy of UV-A laser micro-irradiated cells. Panel C shows representative images, panel D shows the quantification. Plotted are the data from all biological repeats. Each grey or green dot represents an individual track, black dots are the median for each biological repeat (n=3; mean±SEM; ratio paired t-test). (E) Western blot analysis of CtIP overexpression in the cells decribed in panel F. (F) Indicated HEK 293T+DSB-Spectrum_V3 cell-lines were transfected to express either CtIP or an empty vector control, together with Cas9 and the sgRNA targeting the BFP gene in the reporter locus. Next, cells were analyzed by flow cytometry to determine the frequency of repair by each of the three indicated pathways. Data were normalized to the Con.+EV (n=4; mean±SEM; One-way ANOVA+Dunnett’s multiple comparison). (G) Model depicting how FANCL/Ube2T promotes repair of DSBs by homologous recombination. See main text for details.