Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA topoisomerase 1 inhibition

CPT activates the FA pathway.

Several reports implicate the FA pathway in tolerance and repair of DNA damage due to CPT and other Top1 inhibitors.^39,41 However, mechanisms of FA pathway activation in response to CPT have not been studied. To determine whether the FA pathway is activated in response to CPT, we treated H1299 lung carcinoma cells with 100 nM CPT for 1 hour and then examined the sub-cellular distribution and chromatin-binding of FANCD2 using immunofluorescence (IF) microscopy and immunoblotting (IB) respectively. As shown in Figure 1A (40x magnification) and 1B (100x magnification), CPT treatment induced an increase in the number of FANCD2 nuclear foci (likely corresponding to sites of DNA repair) in H1299 cells. Camptothecins are well established S phase-specific chemotherapeutic agents, and most of their cytotoxicity at sub-micromolar concentrations is dependent on DNA replication.⁹ Since we have used relatively low doses of CPT in this study (100 nM), the FANCD2 nuclear foci we observed in CPT-treated cells likely correspond to stalled/collapsed replication forks. Indeed, the FANCD2 foci co-localized with PCNA (Fig. 1C) and therefore represent sites of DNA replication. Since CPT induces DSB at sites of DNA replication, we conclude that the FANCD2 foci represent replication-induced DSB. CPT treatment also induced an increase in the levels of chromatin-bound FANCD2, the monoubiquitinated species of FANCD2⁴² in H1299 cells (Fig. 1D) and in FANCD2-complemented PD-20 cells (Fig. 1E). Supplemental Figure 1 also demonstrates that the CPT-induced chromatin-associated FANCD2 corresponds to the electrophoretically retarded (monoubiquitinated) species.

To determine the role of the FA core complex in FA pathway activation by CPT, we compared CPT-induced FANCD2 ubiquitination in FANCA (−) GM6914 cells and an isogenic FANCA (+) cell line complemented with a functional FANCA cDNA. As shown in Figure 1F, CPT treatment induced chromatin-binding of monoubiquitinated FANCD2 in FANCA (+) cells. However, even though levels of soluble FANCD2 were comparable in FANCA (+) and FANCA (−) cells, CPT did not induce chromatin association of FANCD2 in the absence of FANCA. In siRNA experiments using H1299 cells, we achieved partial FANCA-depletion, which resulted in attenuation of CPT-induced FANCD2 ubiquitination (Fig. 1G). We infer that CPT-induced FANCD2 ubiquitination is FA core complex-dependent. Consistent with previous reports using other cell lines,³⁹ FANCD2-depleted H1299 cells exhibited increased sensitivity to CPT when compared with control siRNA-transfected cells (Fig. 1H). Therefore, CPT induces FA core complex-mediated FANCD2 monoubiquitination. Moreover, FANCD2 is necessary for tolerance of CPT-induced DNA damage.

Discussion

Top1 poisons such as CPT target S-phase cells,⁵⁵ causing DNA replication-associated DNA damage. Thus, Top1ccs are converted to DSB via a “replication run-off” mechanism, whereby the leading strand is replicated only up to the last nucleotide flanking the 5′ end of the TOP1cc and terminates in a DSB.⁵⁶ Genetic alterations in DSB-inducible checkpoint signaling pathways (e.g., ATM, CHK2) and DSB-specific DNA repair mechanisms (e.g., NBS1, Rad51C) sensitize mammalian cells to Top1 poisons,⁹ further consistent with a DSB-mediated mechanism for killing of cells in response to Top1 inhibitors.

While the FA pathway has been studied most extensively in the context of DNA interstrand crosslink repair, FANCs have also been implicated in processing and repair of DSB. In eukaryotic cells, two main pathways exist for repair of DSB: Non-Homologous End Joining (NHEJ) and Homology-Directed Repair (HDR). Studies with cells from mutant Fancd2^−/− mice suggested an NHEJ-independent role for Fancd2 in repair of IR-induced DSB.⁵⁷ Jasin and colleagues showed that monoubiquitinated FANCD2 promotes DSB repair via homologous recombination.⁴⁷ More recent studies suggest that the FA pathway plays a role in resecting dsDNA ends, thereby directing repair away from abortive NHEJ and toward HR.⁴⁸

Consistent with a role for the FA pathway in promoting HR, FA cells show sensitivity to CPT (Nomura et al. and this study) and studies using replication-competent Xenopus egg extracts have suggested a requirement for FANCD2 in replication restart following CPT-induced fork collapse.⁴¹ However, mechanisms of FA pathway activation and integration of the FA pathway with other DNA replication and repair processes have not been studied.

We show here that efficient CPT-induced FANCD2 activation requires the Rad18 E3 ubiquitin ligase. Consistent with a role for Rad18 in CPT-induced FA pathway activation, we show that both FANCD2 and Rad18 are required for survival of CPT-treated cells. We and others have previously shown that FA pathway activation in response to bulky DNA lesions is Rad18-dependent.^27,43 The mechanism of FANCD2 ubiquitination in response to bulky DNA lesions (such as BPDE adducts and CPD) is indirect and occurs secondary to Rad18-induced PCNA monoubiquitination.²⁷ However, in contrast with bulky DNA lesions, CPT does not induce detectable PCNA monoubiquitination as shown in this study and Niimi et al. Moreover, while PCNA monoubiquitination at K164 is important for tolerance of UV- or MMS-induced DNA damage, this PCNA modification does not contribute to CPT-tolerance.⁴⁴ Thus, Rad18-mediated pathways for repair of Top1cc-induced DSB and bulky DNA lesions are separable based on their differential requirements for PCNA monoubiquitination.

Previous work by the Chen lab also implicated Rad18 in tolerance of CPT- and IR-induced DSB.²³ Those elegant studies identified a novel E3 ligase-independent mechanism for Rad18-dependent stimulation of HR, most likely facilitated via direct interactions between Rad18 and a Rad51 paralog, Rad51C.²³ Our report identifies an additional mechanism for Rad18-dependent FANCD2 ubiquitination and tolerance of CPT-induced lesions that is dissociable from the pathway described by the Chen lab²³ based on its requirement for Rad18 E3 ligase activity.

Collectively, these studies indicate that Rad18 plays E3 ligase-dependent as well as E3 ligase-independent roles in tolerance of both bulky DNA lesions (including BPDE adducts, CPD and cis-platin) and DSB (Fig. 8). Our study reveals an interesting analogy between responses to bulky adducts and DSB, whereby Rad18 contributes to tolerance of both forms of damage via separable E3 ligase-dependent and -independent mechanisms: during TLS Rad18 functions as a molecular chaperone that guides Polη to sites of replication fork stalling independently of its E3 ligase activity^21,58 and subsequently monoubiquitinates PCNA to promote stable association of TLS polymerases with the replication machinery.¹⁹ In response to CPT-induced DSB, Rad18 associates with Rad51C and facilitates its chromatin loading in an E3 ligase-independent manner²³ yet also promotes FANCD2 ubiquitination, DNA repair and survival in an E3 ligase-dependent manner (this study).

Our work prompts further questions regarding the mechanism of Rad18 activation at Top1ccs-induced DSB and cellular targets of Rad18 E3 ligase activity that mediate FA pathway activation. Huang et al. showed that RNF8, an E3 ubiquitin ligase involved in Histone H2A ubiquitination is required for efficient recruitment of Rad18 to DSB.²³ In our study, Rnf8-depletion reduced the magnitude of CPT-induced FANCD2 ubiquitination, potentially consistent with a linear activation pathway involving Rnf8, Rad18 and FANCD2. We typically observed a slight reduction of Rnf8 chromatin loading in Rad18-depleted cells (for example see Fig. 5). Chen and colleagues have reported that Rad18 is downstream of RNF8 under certain conditions.²³ Our experiments may indicate that Rnf8 and Rad18 do not always operate in a simple linear pathway. This is by no means unprecedented, and participants of DSB signaling pathways do not always have simple upstream/downstream relationships. For example, p95 and ATM have a complex relationship, whereby p95 has roles both upstream and downstream of ATM.

Interestingly, Rad18 deficiency had a greater inhibitory effect on FANCD2 ubiquitination than on chromatin loading of Rad51, whereas Rnf8 depletion was more inhibitory for Rad51 chromatin loading than for FANCD2 monoubiquitination. It is possible, therefore, that additional Rnf8-independent mechanisms contribute to Rad18-mediated FANCD2 ubiquitination. In responses to bulky DNA lesions, recruitment of Rad18 to stalled replication forks and the ensuing PCNA ubiquitination is dependent on RPA-coated ssDNA.^53,59 Top1ccs likely generate RPA-coated ssDNA via resection of replication-dependent DSB and at sites of discontinuous DNA replication on the lagging strand.⁹ Indeed, CPT rapidly induces Chk1 phosphorylation, a signal that is thought to be initiated by recruitment of the ATRIP/ATR complex to RPA-coated ssDNA.⁶⁰ Thus, it is possible that RPA-coated ssDNA contributes to Rad18 activation at Top1cc-induced DSB.

Potentially, Rad18 might promote CPT-induced FANCD2 monoubiquitination activation via inhibition of FANCD2 deubiquitination or by stimulation of FANCD2-directed E3 Ligase activity. USP1 is a deubiquitinating enzyme for FANCD2, and DNA damage-induced USP1 cleavage has been proposed as a major mechanism for increasing levels of monoubiquitinated FANCD2 following genotoxin treatments.^54,61

We show here that Rad18 status does not affect levels of USP1 basally or in CPT-treated cells (Fig. 7). Thus, Rad18 is unlikely to contribute to CPT-induced FA pathway activation via changes in USP1 expression levels. We cannot exclude the possibility that Rad18 affects USP1 activity or that Rad18 influences alternative (putative) FANCD2-directed DUBs. However, since Rad18-depletion reduces the chromatin loading of FA core complex proteins, we consider it most likely that Rad18 contributes to FA pathway activation by promoting FA core complex activation. Precisely how Rad18 E3 ligase activity regulates core complex activation remains unclear. The most direct potential mechanism for Rad18-dependent FA core complex activation involves ubiquitination of one or more members of the core complex. There is a precedent for monoubiquitination of core complex members: Dutta and colleagues demonstrated that UBE2T, an E2 ubiquitin-conjugating enzyme that mediates FANCD2 ubiquitination, is itself monoubiquitinated, although this modification is inhibitory for E2 ligase activity.⁶² In preliminary experiments, we have not noticed any effects of Rad18 status on electrophoretic mobility of UBE2T or FA core complex members that we have examined. Nevertheless, it is conceivable that reduced ubiquitination of FA core complex proteins in Rad18-deficient cells compromises core complex function. An alternative possibility is that Rad18-mediated modification of other replication fork or DNA damage signaling components in CPT-treated cells contributes to recruitment of the FA core complex. While Rad18 is most commonly studied in the context of PCNA ubiquitination, alternative Rad18 substrates have been described, including RFC2 and 53BP1.^25,63 Therefore, it possible that ubiquitinated forms of RFC2, 53BP1 or other unidentified Rad18 targets mediates activation of the FA core complex in response to CPT. Regardless of the precise mechanism(s) by which Rad18 promotes FA pathway activation, this report expands the repertoire of DNA repair mechanisms that are known to be regulated by Rad18 and reveals a new pathway that protects cells from Top1-inhibiting anticancer drugs. Further studies are underway to identify additional substrates of Rad18 that mediate FA pathway activation in response to CPT.

Cell culture.

H1299, A549 and HDF cells were maintained as described previously in reference ²⁷. FANCD2-deficient PD20 cells, FANCA-deficient GM6914 and isogenic cell lines stably expressing reconstituted wild-type FANCs were obtained from Dr. Alan D'Andrea, Dana Farber Cancer Institute and were validated for FANC status by immunoblotting as described in recent publications.^45,64,65 All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, streptomycin sulfate (100 µg/ml) and penicillin (100 U/ml), 5 mM L-glutamine, and cultures were incubated at 37°C in humidified chambers with 5% CO₂.

Antibodies and drugs.

Top1 antibody (Abcam); PCNA; Rad51; FANCD2; FANCL; Rnf8; β-actin (Santa Cruz) Rad18; (Bethyle); Rad6; γH2AX; p-Chk1; p-Chk2; p-ATM (Cell Signaling). Camptothecin (Sigma, St. Louis, MO) was dissolved in anhydrous DMSO at 10 mM concentration and stored at −20°C. The 10 mM CPT stock solution was diluted further in to anhydrous DMSO and added to the cells. All other reagents used in this study were analytical grade and obtained from certified vendors.

RNA interference (RNAi).

All siRNAs used in this study were purchased from Dharmacon (Lafayette, CO) and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols as described previously in reference ²⁷ and ⁵⁸. The siRNAs used in this study are as follows: siRad18-3, 5′-GAG CAU GGA UUA UCU AUU CAA UU-3′, previously described by Hicks et al. siRad18-3′UTR, 5′-UUA UAA AUG CCC AAG GAA AUU-3′; siFANCD2, 5′-GCA CCG UAU UCA AGU ACA AUU-3′; siFANCA, 5′-CAG CGT TGA GAT ATC AAA GAT-3′; siFANCI, 5′-CTG GCT AAT CAC CAA GCT TAA-3′; siRNF8, 5′-GAG AAG CUU ACA GAU GUU U-3′; non-targeting siControl, 5′-UAG CGA CUA AAC ACA UCA AUU-3′. For all knockdowns, cells were transfected twice (8–10 hr for each transfection) using 100–150 nM siRNA with a 24 hour recovery period between the first and second transfections. Cells were typically subjected to genotoxin treatments 24 hr after the second transfection.

SDS-PAGE and immunoblotting.

Cells were harvested by aspirating the medium and washing twice with cold phosphate buffer saline (PBS). Chromatin (essentially nucleated cytoskeletons) and soluble fractions were prepared according to standard protocols essentially as described by Izumi and colleagues.⁶⁷ Briefly, cells were resuspended in cytoskeleton (CSK) buffer (10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM ATP, 1 mM Na₃VO₄, 10 mM NaF and 0.1% Triton X-100, freshly supplemented with protease inhibitor and phos-stop cocktail tablets from Roche), scraped into microfuge tubes and allowed to lyse for 10–20 min on ice. Detergent-insoluble nuclei and soluble fractions were separated by centrifugation at 3,000 g for 5 min. The soluble fractions were removed, and the remaining insoluble nuclear pellets were washed twice with 0.5 ml of lysis buffer. The resulting washed nuclei were resuspended in an approximately equal volume of CSK. Protein concentrations of soluble and nuclear fractions were determined immediately. Extracts were normalized for protein content, dissolved in 4x SDS-PAGE denaturing buffer and heated to 100°C for 10 min. Denatured samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were typically stained with Ponceau to verify transfer and equivalent loading protein loading. Used as Ponceau stain was removed by extensive rinsing with PBS and the resulting membranes were probed with appropriate antibodies. Bound antibodies were detected using chemiluminiscence. Actin (present in soluble and chromatin fractions prepared by this method) was generally used as a loading control. Films were scanned, and relative intensities of protein bands were determined densitometry using Image J software.

In vivo complex of enzyme (ICE) assay.

To assess the formation of Top1ccs in vivo, exponentially growing cells in 60 mm dishes were treated with 1 µM CPT for 30 min and lysed in TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) containing 1% sarkosyl. The cell lysates were fractionated by centrifugation in a CsCl gradient for 22 hr as described previously in reference ⁶⁸ and ⁶⁹. For each sample equal volumes of DNA-containing fractions were diluted in 25 mM sodium phosphate buffer and loaded on to a pre-soaked nitrocellulose membrane using a dot-blot apparatus (Bio-Rad). The membranes containing immobilized genomic DNA were immunoblotted with Top1 antibody.

Fluorescence microscopy.

Exponentially growing cells were transfected with indicated siRNAs and plasmid vectors. Transfected cells were trypsinized and seeded in two- or four-well LabTek chamber slides to give a confluence of 40–60% 16–24 hours after replating. The resulting cells were treated with CPT (or DMSO for controls). At various times after CPT treatment, cells were washed with cold PBS and permeabilized with PBS containing 0.2% Triton X-100 (PBS-T) for ∼1 min and then washed twice with PBS. Permeabilized cells were fixed in 3% formaldehyde for 10 min and then in 100% methanol (−20°C) for 10 min at room temperature. Fixed cells were blocked in 10% FBS for 30 min. After three washes with PBS-T, cells were incubated overnight at 4°C with primary antibodies in PBS containing 5% bovine serum albumin (BSA) and 0.1% Triton X-100 (PBST). The slides were washed three times with PBST containing 1% BSA then incubated with anti-mouse IgG-Cy3 antibodies (Sigma-Aldrich) for 2 hr at RT. After washing extensively with PBST to remove unbound secondary antibody, the slides were mounted with DAPI-containing VECTASHIELD^® Mounting Medium (Vector Laboratories) and imaged and analyzed with an Olympus BX61 upright fluorescence microscope using a U Plan Fluorite Apo 40x objective lens.

Survival assays.

H1299 or A549 cells were transfected with indicated siRNAs and plasmid vectors. 48 hours post-transfection, 300–500 cells were plated in triplicate in six-well tissue culture dishes. Cells were allowed to attach for 16 hours and treated with indicated concentrations of CPT for 16–20 h. Following CPT treatment, cells were washed three times with PBS, once with (CPT-free) growth medium and placed in fresh complete medium. After 7–10 days, surviving colonies were fixed with methanol for 10 min and stained with crystal violet. Colonies containing at least 50 cells were enumerated.

Reproducibility.

All data shown are representative of experiments that were performed at least three times with qualitatively similar results on each occasion.