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Human Molecular Genetics Advance Access originally published online on November 20, 2007
Human Molecular Genetics 2008 17(5):679-689; doi:10.1093/hmg/ddm340
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Loss of CHK1 function impedes DNA damage-induced FANCD2 monoubiquitination but normalizes the abnormal G2 arrest in Fanconi anemia

Jean-Hugues Guervilly{dagger}, Gaëtane Macé-Aimé{dagger} and Filippo Rosselli*

Equipe voie FANC/BRCA et Cancer, CNRS FRE2939, CEA LRC43V, Univ Paris-Sud, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France

* To whom correspondence should be addressed: Tel: +33 142115116; Fax: +33 142115008; Email: rosselli{at}igr.fr

Received November 8, 2007; Revised November 8, 2007; Accepted November 17, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Fanconi anemia (FA) is a cancer-prone hereditary disease resulting from mutations in one of the 13 genes defining the FANC/BRCA pathway. This pathway is involved in the cellular resistance to DNA-cross-linking agents. How the FANC/BRCA pathway is activated and why its deficiency leads to the accumulation of FA cells with a 4N DNA content are still poorly answered questions. We investigated the involvement of ATR pathway members in these processes. We show here that RAD9 and RAD17 are required for DNA interstrand cross-link (ICL) resistance and for the optimal activation of FANCD2. Moreover, we demonstrate that CHK1 and its interacting partner CLASPIN that act downstream in the ATR pathway are required for both FANCD2 monoubiquitination and assembling in subnuclear foci in response to DNA damage. Paradoxically, in the absence of any genotoxic stress, CHK1 or CLASPIN depletion results in an increased basal level of FANCD2 monoubiquitination and focalization. We also demonstrate that the ICL-induced accumulation of FA cells in late S/G2 phase is dependent on ATR and CHK1. In agreement with this, CHK1 phosphorylation is enhanced in FA cells, and chemical inhibition of the ATR/CHK1 axis in FA lymphoblasts decreases their sensitivity to mitomycin C. In conclusion, this work describes a complex crosstalk between CHK1 and the FANC/BRCA pathway: CHK1 activates this pathway through FANCD2 monoubiquitination, whereas FA deficiency leads to a CHK1-dependent G2 accumulation, raising the possibility that the FANC/BRCA pathway downregulates CHK1 activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The cellular response to DNA damage or stalled replication forks is crucial for the maintenance of the genetic stability and is a fundamental mechanism to prevent cancer. As a consequence of DNA damage, stalled or delayed replication forks activate an intricate network of proteins under the control of ATR, a nuclear kinase belonging to the phosphatidylinositol 3-kinase-like family (1). The ATR-dependent DNA damage signaling requires several proteins to achieve the proper cellular response through checkpoint activation and DNA repair followed by replication forks and cell cycle restart and/or apoptosis (1,2). Notably, ATR forms a complex with its interacting protein ATRIP that mediates their interaction with replication protein A (RPA)-coated single-stranded (ss) DNA regions, generated as a consequence of stalled replication forks (3). Independently from ATR, the 9-1-1 complex, a PCNA-like trimeric clamp formed by RAD9, RAD1 and HUS1 proteins, is recruited onto damaged chromatin by the clamp loader complex RAD17:RFC2-5 in a RPA-dependent manner (4,5). Once loaded onto DNA, the 9-1-1 complex participates to ATR kinase activation and helps ATR to phosphorylate its substrates, including RAD9 itself, RAD17 and RPA32 (6–8). Therefore, the replication stress-induced assembling and activation of the ‘molecular module’ constituted by RPA:ATR:ATRIP:RAD17:RFC2-5:RAD9:RAD1:HUS1 initiates the signaling pathway(s) leading to the intra-S and G2/M checkpoints (via phosphorylation of several targets including CHK1, NBS1 and SMC1), DNA repair [mainly via homologous recombination (HR)] and, eventually, apoptosis (1,9).

The downstream kinase CHK1 is one of the major ATR targets and is essential for cell survival (1). In response to DNA damage, ATR phosphorylates CHK1 in a CLASPIN-mediated manner on S317 and S345, inducing its subcellular relocalization and checkpoint initiation (10). The adaptator protein CLASPIN is also phosphorylated by CHK1, promoting the formation of a heterodimeric complex between the two proteins (11). Whether mammalian CHK1 phosphorylation modifies its intrinsic kinase activity is still controversial (10,12). Activated CHK1 participates to HR by phosphorylating RAD51 and to intra-S and G2/M checkpoint via the phosphorylation of CDC25A and CDC25C, which promotes their degradation and/or inactivation (13,14).

DNA interstrand cross-links (ICLs) are highly cytotoxic lesions induced by several antiproliferative agents such as mitomycin C (MMC), cisplatin or photoactivated psoralens [e.g. 8-methoxypsoralen (8-MOP) plus UVA]. The cytotoxicity of ICL-inducing agents is related to their ability to block DNA synthesis. For this reason they are widely used to target more specifically highly proliferating tumoral cells. The mechanisms leading to ICLs removal from DNA and to the reconstitution of the damaged template remain poorly understood. However, it is generally accepted that ICL repair needs S-phase transition and could involve proteins of the nucleotide excision repair, translesion synthesis, HR as well as mismatch repair systems (15). Among the multiple actors required to efficiently cope with ICLs, the proteins defining the so-called FANC-BRCA pathway constitute key players in mammalian cells.

The FANC-BRCA pathway is encoded by genes mutated in Fanconi anemia (FA) and/or in breast and ovarian cancer predisposition syndromes. FA is a rare recessive syndrome characterized by aplastic anemia, developmental birth defects, reduced fertility and predisposition to acute myeloid leukemia as well as head and neck cancers. FA cells exhibit cellular and chromosomal hypersensitivity to ICL-inducing agents as well as late S/G2-phase arrest induced by such genotoxic stress. During replication or in response to DNA damage, the FANCcore complex (FANCA, B, C, E, F, G, L and M) monoubiquitinates FANCD2 allowing its assembly in subnuclear foci (16,17). The DNA damage-induced FANCD2 monoubiquitination is also dependent on ATR and RPA (18). The recently identified FANCI is monoubiquitinated in a FANCcore complex and FANCD2-dependent manner while FANCD1/BRCA2, FANCN/PALB2 and FANCJ/BRIP1 work downstream of or in parallel to the FANCcore complex/FANCD2/FANCI pathway (16,19,20). Moreover, the FANC/BRCA pathway interacts biochemically and/or functionally with several other players of the DNA damage response, including {gamma}H2AX, BLM and the MRE11 complex (21–25).

Despite the progress in deciphering the mechanisms leading to ICLs resistance and the role of the FANC/BRCA pathway in the response to ICLs, it is unclear how ICLs contribute to checkpoint activation and whether checkpoint players modulate FANC/BRCA pathway activation. Here we show that RAD17 and the 9-1-1 complex are involved in ICL resistance through their participation to the optimal activation of FANCD2. We demonstrate that FANCD2 monoubiquitination and subnuclear assembling in response to DNA damage also require the CHK1/CLASPIN complex. Paradoxically, in the absence of genotoxic stress, CHK1 or CLASPIN depletion induces spontaneous FANCD2 monoubiquitination and focalization. Finally, we show that the aberrant G2 arrest in DNA-damaged FA cells is dependent on CHK1 activation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Rad17 and 9-1-1 complex are involved in the response to ICL and required for optimal FANCD2 monoubiquitination
It is thought that ATR and RPA are activated by ICLs as a consequence of the stalling of replication forks (18,25). We asked whether RAD17 and the 9-1-1 complex, actors of the ATR pathway, also participate to the cellular response induced by ICLs. HeLa cells were treated with photoactivated 8-MOP, a potent ICL-inducer, or exposed to UVC irradiation, a treatment known to activate the ATR pathway. In response to both genotoxic stresses, the RAD1 and HUS1 components of the 9-1-1 complex accumulated on chromatin, similarly to RPA and FANCD2-L, the monoubiquitinated form of FANCD2 (Fig. 1A). Moreover, in response to 8-MOP+UVA or UVC, RAD17 was phosphorylated on S645, and the 32 kDa RPA subunit became hyper-phosphorylated (Fig. 1B). Similar results were obtained in MRC5 human fibroblasts (data not shown). Having shown that the ‘ATR module’ proteins RAD17 and 9-1-1 were activated by cross-linked DNA, we asked for the cellular significance of their response. Since RAD17 and 9-1-1 are essential for long-term cell survival, we used siRNAs to transiently knock-down their expression in HeLa cells. Depletion of RAD17 or RAD9 sensitized HeLa cells treated with 8-MOP+UVA (Fig. 1C). Therefore, RAD17 and the 9-1-1 complex are activated by ICL and are necessary for the cellular response towards this genotoxic stress.


Figure 1
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  		Figure 1. RAD17 and 9-1-1 complex are activated by ICLs. (A) HeLa cells were treated with 10 µM 8-MOP+10 kJ/m2 UVA irradiation (8-MOP), irradiated with 30 J/m2 UVC or left untreated (Untr). Nuclear cell extracts isolated 6 h after treatment were fractionated as previously described (8) and the presence of the indicated protein within the chromatin-enriched fraction was assessed by western blot. Numbers indicate the fold enrichment of the protein within the chromatin fraction after treatment normalized to the loading control ORC2. (B) Whole-cell extracts from HeLa cells treated as in (A) were separated by SDS–PAGE, and RAD17 and RPA32 phosphorylations were analyzed by western blot using a phospho-specific antibody or revealed by a mobility shift of the protein (bands marked by asterisks represent hyperphosphorylated RPA). (C) HeLa cells were transfected with siRNA targeting RAD9, RAD17 or Luciferase (Luc) as indicated and treated with 8-MOP+UVA as described in (A). Cell viability was assessed by XTT assay. The figure shows the means and standard deviations of three independent experiments, each one done in triplicate. Data are expressed as the percentage of living cells in untreated matched siRNAs.

 
The so-called FANC/BRCA pathway is one of the major determinants of the cellular resistance to cross-linking agent exposure (16,17). Consequently, we sought to determine whether the ICLs’ hypersensitivity resulting from RAD17 or RAD9 depletion was associated with a disruption of the FANC/BRCA pathway. Hence, we examined the status of FANCD2 monoubiquitination as a readout for FANC/BRCA pathway activation in RAD17- or RAD9-depleted cells treated with ionizing radiations (IRs), replication inhibitor hydroxyurea (HU), photoactivated psoralen or MMC. The level of induced monoubiquitinated FANCD2 was lower in RAD9- or RAD17-deficient cells than in wild-type cells irrespective of the kind of DNA damage and the recovery time (Fig. 2A and B, and Supplementary Material, Fig. S1). This effect was specific because transfection of a FLAG-tagged RAD17 largely complemented the decreased induction of monoubiquitinated FANCD2 due to a siRNA targeting only the endogenous form of RAD17 (Supplementary Material, Fig. S2).


Figure 2
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  		Figure 2. RAD17 is required for optimal DNA damage-induced FANCD2 monoubiquitination. HeLa cells were transfected with siRNA targeting RAD17 (siRAD17) or Luciferase (siLuc), treated 72 h later with 10 Gy of ionizing radiation (IR), 2 mM hydroxyurea (HU) or 10 µM 8-MOP+10 kJ/m2 UVA (8-MOP) and harvested at the indicated times. Monoubiquitination of FANCD2 [(A) and (B)] as well as the phosphorylation of ATR targets (B) were analyzed by western blot using specific antibodies. L/S indicates in both (A) and (B) the ratio of monoubiquitinated (L) to non-monoubiquitinated (S) FANCD2. FI indicates the fold induction of the L/S ratio of the treated versus the non-treated samples for each siRNA.

 
It has been previously reported that RPA and ATR contribute to both cellular resistance to ICLs and activation of the FANC/BRCA pathway, as demonstrated by their requirement for the DNA damage-inducible phosphorylation and monoubiquitination of FANCD2 (18,25,26). In agreement with the role of RAD17 and the 9-1-1 complex in the activation and/or maintenance of the RPA/ATR-signaling pathway, following UVC or HU exposure (6,8,9), RAD17- and RAD9-depleted cells presented a defect in the phosphorylation of the ATR targets CHK1, SMC1 and NBS1 after treatment with photoactivated 8-MOP (Fig. 2B and Supplementary Material, Fig. S1A). Taken together, our data show that the clamp loader RAD17 and the loaded clamp 9-1-1 are necessary for the optimal activation of both ATR and FANCD2.

CHK1 inhibition affects both basal and DNA damage-induced monoubiquitination of FANCD2
We showed that RAD17 depletion reduces both ATR phosphorylation events and FANCD2 monoubiquitination. Consequently, the decreased FANCD2 monoubiquitination in the absence of RAD17 could be the consequence of an impaired activation of one or several ATR targets. NBS1 phosphorylation is dispensable for FANCD2 monoubiquitination (22,25). On the contrary, the involvement of CHK1, whose phosphorylation is strongly impaired in the absence of RAD17, in the activation of the FANC/BRCA pathway is not clearly established. To investigate the putative role for CHK1 in the FANC/BRCA pathway, we first used the siRNA approach to transiently deplete CHK1 in HeLa cells. As previously reported (27), CHK1-depleted cells exhibited phosphorylation of many ATR substrates, namely SMC1, NBS1, H2AX and RPA32, in the absence of any exogenous genotoxic stress (Fig. 3A and B, compare the untreated lines in siLuc- versus siCHK1-transfected cells). Depletion of CHK1 also induced a significant increase in the basal level of monoubiquitinated FANCD2 (Fig. 3B).


Figure 3
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  		Figure 3. CHK1 inhibition affects both the basal and the DNA damage-induced FANCD2 monoubiquitination. (A) HeLa cells transfected with siRNA targeting CHK1 or Luciferase (Luc) were treated with 20 J/m2 UVC or 10 µM 8-MOP+10 kJ/m2 UVA 72 h after transfection and harvested at the indicated times. Phosphorylation of several ATR targets was analyzed by western blotting with the indicated antibodies. (B) HeLa cells were transfected with siRNA targeting ATR, CHK1, both or Luciferase. Cells were lysed 72 h after transfection, and FANCD2 monoubiquitination and NBS1 phosphorylation were assessed by western blot. Asterisk indicates the remaining signal of CHK1 just below β-tubulin signal. (C) FANCD2 monoubiquitination was monitored by western blot in extracts from HeLa cells transfected and treated as in (A). (D) HeLa cells were pretreated with either 5 µM CHK1 inhibitor sb-218078 or solvent (DMSO) for 1 h and then exposed to 5 mM HU or 10 µM 8-MOP+10 kJ/m2 UVA (8M). Cells were cultivated with or without CHK1 inhibitor until lysis 5 h later. FANCD2 ubiquitination was monitored by western blot. (E) HeLa cells were pretreated with either 300 nM CHK1 inhibitor UCN-01 or solvent (DMSO) for 1 h and then exposed to 2 mM HU or 20 J/m2 UVC. Cells were cultivated with or without CHK1 inhibitor until lysis 6 h later. FANCD2 ubiquitination and NBS1 phosphorylation were analyzed by western blot. Where indicated, L/S represents the ratio of monoubiquitinated (L) to non-monoubiquitinated (S) FANCD2. Fold induction (FI) of the L/S ratio of the treated samples relative to the non-treated for each siRNA or drug is also indicated.

 
We wondered if the increased basal level of FANCD2 monoubiquitination in the absence of CHK1 resulted from the basal activation of the ATR pathway. To check this, we compared cells transfected with siRNA targeting only CHK1 to cells depleted in both CHK1 and ATR. The ATR/CHK1-depleted cells presented less ATR activity, as judged by the level of NBS1 phosphorylation, and less monoubiquitinated FANCD2 than CHK1-only-depleted cells (Fig. 3B). In agreement with this result, exposure to the CHK1 inhibitors sb-218078 or UCN-01 during 18 h enhanced basal FANCD2 monoubiquitination in wild-type but not in Seckel syndrome lymphoblasts that display diminished ATR activity and protein level (Supplementary Material, Fig. S3) (28). Taken together, these data suggest that enhanced ATR signaling in cells lacking CHK1 activity is responsible for the induction of monoubiquitinated FANCD2.

Interestingly, CHK1-depleted cells were still able to increase SMC1, H2AX and RPA32 phosphorylation after exposure to UV or 8-MOP+UVA, reflecting a normal ability to activate ATR in response to DNA damage (Fig. 3A). In contrast, FANCD2 monoubiquitination could not be further induced after various genotoxic treatments (Fig. 3C). Thus, it appears that CHK1 is specifically required for FANCD2 monoubiquitination in response to exogenously induced DNA damage.

We then asked whether CHK1 kinase activity was required for the DNA damage-dependent activation of the FA pathway. A short treatment with the CHK1 kinase inhibitor sb-218078, without affecting the basal level of FANCD2 monoubiquitination, significantly impaired the induction of monoubiquitinated FANCD2 after HU or 8-MOP+UVA treatments (Fig. 3D). Similar results were obtained with the CHK1 inhibitor UCN-01 in HU- or UV- treated cells (Fig. 3E). These results support a specific requirement for CHK1 kinase activity in the activation of FANCD2. As expected, a longer exposure of HeLa cells to the CHK1 inhibitor sb-218078 could activate ATR, thereby inducing phosphorylation of CHK1 itself and monoubiquitination of FANCD2 at the basal level. However, CHK1 inhibition drastically reduced HU-induced FANCD2 monoubiquitination (Supplementary Material, Fig. S4). Collectively, these results indicate that the DNA damage-induced FANCD2 monoubiquitination is linked to CHK1 kinase activity.

CLASPIN is also required for the inducible FANCD2 monoubiquitination
Following DNA damage, optimal CHK1 activation requires the adaptator protein CLASPIN (29,30), which is also phosphorylated by CHK1, promoting the formation of a CHK1/CLASPIN complex (11). Hence, we examined whether CLASPIN was also involved in the regulation of FANCD2 monoubiquitination. As shown in Figure 4A, transient inhibition of CLASPIN expression by siRNA in HeLa cells mimicked the inhibition of CHK1. Indeed, CLASPIN-depleted cells displayed an induction of ATR signaling in the absence of exogenous DNA damage as judged by NBS1, SMC1 and RPA32 phosphorylation. Moreover, ATR signaling could be further increased by HU treatment. Again, as in the absence of CHK1 expression, CLASPIN deficiency led to an increase in FANCD2 monoubiquitination in non-exogenously stressed cells but monoubiquitinated FANCD2 could not be further enhanced by HU exposure. CLASPIN was also required or the UV-induced monoubiquitination of FANCD2 (Supplementary Material, Fig. S5). Therefore, both CHK1 and CLASPIN regulate FANCD2 monoubiquitination.


Figure 4
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  		Figure 4. Both CLASPIN and CHK1 regulate FANCD2 monoubiquitination and foci formation. (A) CLASPIN or CHK1 expression was downregulated by siRNA in HeLa cells, and cells were treated 72 h later with 2 mM HU for 6 h. Two different siRNAs were used in the case of CHK1: the one shown in Figure 3 (termed siCHK1) and the other less efficient one directed against the 3'-UTR of CHK1 mRNA (termed si3'CHK1). Protein expression, phosphorylation or monoubiquitination was monitored by western blot. L/S indicates the ratio of monoubiquitinated (L) to non-monoubiquitinated (S) FANCD2, and FI represents the fold induction of the L/S ratio after HU treatment. (B) Forty-eight hours after transfection with siRNA against Luciferase, CHK1 or CLASPIN, HeLa cells were treated with 10 µM 8-MOP+10 kJ/m2 UVA. Cells were fixed 8 h later, permeabilized with 0.5% Triton X-100 and immunofluorescence was performed using anti-FANCD2 and anti-{gamma}H2AX antibodies. Magnification, x630. (C) Quantification of FANCD2 foci-positive cells and {gamma}H2AX-positive cells. The values are the means of three independent experiments. Error bars represent standard deviations.

 
It is interesting to note that, in response to DNA damage, CLASPIN-depleted cells showed a reduced but substantial level of CHK1 phosphorylation. The same level of phosphorylated CHK1 could be observed in cells expressing roughly 50% of the basal CHK1 level (Fig. 4A, si3'CHK1 lines), a condition that did not affect either the basal or the induced FANCD2 monoubiquitination. Moreover, this partial CHK1 depletion did not destabilize CLASPIN level, although efficient CHK1 depletion did (Fig. 4A, compare siCHK1 versus si3'CHK1 lines), as previously described (31). This suggests that the DNA damage-induced FANCD2 monoubiquitination requires a fully functional CHK1/CLASPIN complex but possibly not CHK1 phosphorylation.

As FANCD2 monoubiquitination is a prerequisite for its relocalization in nuclear foci, we also looked at FANCD2 foci formation in CHK1- or CLASPIN-depleted cells. In agreement with the effect on monoubiquitination of FANCD2, silencing of CHK1 or CLASPIN increased the number of cells exhibiting FANCD2 foci in the absence of exogenous DNA damage. However, FANCD2 foci-positive cells were not increased after exposure to photoactivated 8-MOP (Fig 4B and C). In contrast, siRNA against CHK1 or CLASPIN enhanced the number of {gamma}H2AX-positive cells compared with control cells both without and with 8-MOP+UVA. Taken together, these data strongly support the requirement of the CHK1/CLASPIN complex to activate the FA pathway in the presence of DNA damage through FANCD2 monoubiquitination and foci formation.

CHK1 is required for the MMC-induced G2 arrest of FA cells
CHK1 is involved in S and G2 checkpoints and it has been shown that CHK1 is responsible for an excessive IR-induced G2 accumulation in cells with mutations in DNA repair genes such as PARP, Ku80, or ATM (13,32–34). Since FA cells accumulate in late S/G2-phase after treatment with ICL inducers, we tested whether CHK1 is involved in this cell cycle abnormality (35). In contrast to wild-type cells, FA cells with an apparent 4N DNA content accumulated 24 h after exposure to a pulse treatment with MMC, reflecting their late S/G2 arrest/accumulation (now simply called G2 arrest). However, CHK1-depleted FA cells did not accumulate in G2 upon MMC treatment, showing that CHK1 was required to block and accumulate FA cells in the G2-phase (Fig 5A). In a similar way, the MMC-induced accumulation of FA-C lymphoblasts with a 4N DNA content was abrogated by treating these cells with the CHK1 inhibitor sb-218078 (Fig 5B). Finally, siRNA-mediated depletion of FANCD2 in HeLa cells induced an abnormal DNA damage-dependent G2 accumulation that was normalized by the co-depletion of CHK1 and FANCD2 (Supplementary Material, Fig. S6). Taken together, our results demonstrate that CHK1 is required for the ICL-induced late S/G2-phase accumulation of FA cells.


Figure 5
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  		Figure 5. The ATR/CHK1 axis regulates MMC-induced G2 arrest in FANC-pathway-deficient cells. (A) MRC5 (WT) and FA-A PD220 fibroblasts (FA-A) were transfected in six-well plates with siRNA targeting Luciferase (Luc) or CHK1. Forty-eight hours later, cells were detached, re-suspended in fresh medium and seeded in 60 mm dishes. The day after, cells were treated with 100 ng/ml mitomycin C (MMC) for 2 h and then extensively washed with PBS. Cell cycle profiles were analyzed 24 h after MMC treatment by FACS using propidium iodide staining to visualized DNA content. The percentage of cells with a 4N DNA content (G2) is reported. Right panel, western blot showing the knock-down of CHK1 and subsequent ATR activation as revealed by NBS1 phosphorylation in MRC5 and FA-A fibroblasts. (B) Exponentially growing HSC536N (FA-C) and HSC536N-corrected (FA-C+FANCC) lymphoblasts were treated with 50 ng/ml MMC for 2 h, washed twice in PBS and incubated with 1 µM CHK1 inhibitor sb-218078 (CHK1i) or an equivalent volume of solvent (DMSO). Twenty-four hours later, cells were fixed, and cell cycle was analyzed as in (A). Right part, western blotting revealing CHK1 phosphorylation on S345 in FA-C or FA-C+FANCC lymphoblasts 24 h after exposure to 50 ng/ml MMC for 2 h. (C) FA-A PD220 fibroblasts were transfected with siRNA against Luciferase, ATR, CHK1, CLASPIN or RAD17 as indicated, treated with 100 ng/ml MMC for 2 h and analyzed as in (A). Right panel, western blot revealing the levels of depleted proteins as well as CHK1 phosphorylation on S345. (D) Growth inhibition test on HSC536N (FA-C) and HSC536N-corrected (FA-C+FANCC) lymphoblasts treated with 5 ng/ml MMC, 1 mM caffeine and 500 nM sb-218078 alone or in combination for 4 days of culture as described in Material and Methods. Values represent the means ± standard variations from three independent experiments.

 
It has been previously reported that the ATR/ATM inhibitor caffeine abrogates G2 accumulation in FA cells, suggesting that ATR could also be required to arrest FA cells in G2-phase (36). Indeed, we found that transfection of FA cells with siRNA targeting ATR but not CLASPIN or RAD17 could efficiently suppress the MMC-induced accumulation of these cells in G2 (Fig 5C). These results suggest that ATR-mediated activation of CHK1 in FA cells is necessary for their late S/G2 arrest upon ICL induction. In agreement with this, we found that MMC-induced CHK1 phosphorylation was enhanced and/or prolonged in FA cells compared with corrected or wild-type cells (Fig 5B and Supplementary Material, Fig. S7). Moreover CHK1 inhibition in FA lymphoblasts partially restored their growth in the presence of MMC, suggesting that FA cells escaping from G2 arrest were viable and still able to divide (Fig 5D). Caffeine also suppressed in part the cytostatic effect of MMC in FA cells (Fig 5D). Taken together, these data suggest that inhibition of the ATR/CHK1 axis abrogates the late S/G2 arrest of FA cells, thereby alleviating the growth inhibition due to the ICL-inducing agent exposure.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
We showed in this work that RAD17 and RAD9, two early actors of the ATR pathway, play a role in the cellular response to cross-linking agents and are required for the optimal monoubiquitination of FANCD2 after DNA damage. CHK1 and CLASPIN, later players of the ATR pathway, also appear to regulate FANCD2 monoubiquitination in an apparently paradoxical manner. Their absence induces FANCD2 monoubiquitination in unperturbed cells but impairs the exogenous DNA damage-induced FANCD2 activation. Finally, we showed that the ATR-CHK1 axis is responsible for the characteristic accumulation of late S/G2-blocked FA cells, following exposure to ICL-inducing agents (Fig 6).


Figure 6
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  		Figure 6. Model depicting the connections of the ATR-CHK1 axis and the FANC/BRCA pathway. (A) Pathway(s) leading to FANCD2 monoubiquitination. The protein analyzed in this paper are represented in red. (B) G2 checkpoint in normal and FANC/BRCA pathway-deficient cells. See Discussion for details.

 
Our findings that human RAD17 and the 9-1-1 complex are activated by ICLs and required to deal with these lesions are consistent with results showing that mouse Rad17–/– ES cells are hypersensitive to MMC (37). The disruption of the FANC pathway through a decreased FANCD2 monoubiquitination could be the molecular link between the loss of RAD17 or RAD9 expression and the acquired cellular hypersensitivity to cross-linking agent exposure. RAD17 or RAD9 depletion did not affect the percentage of cells in S-phase (roughly 20% of control cells, RAD17- or RAD9-depleted cells are replicating), thereby excluding an indirect effect of the cell cycle on the decreased FANCD2 monoubiquitination in these cells. It has been suggested that phosphorylation of FANCD2 itself at two conserved ATR/ATM sites (S/TQ) promotes its monoubiquitination (26). RAD17 and the 9-1-1 complex are involved in amplifying ATR signaling (6) when a replication fork meets a bulky DNA lesion (similar to an ICL) and stalls (1). Therefore, it is conceivable that FANCD2 might need the presence of RAD17 and RAD9 to be fully phosphorylated by ATR and subsequently monoubiquitinated. In an alternative but not exclusive manner, RAD17 and RAD9 loss could also impact on the activation of other ATR targets involved in turn in FANC pathway activation. Notably, RAD17 depletion strongly reduces NBS1 and CHK1 phosphorylation. Since NBS1 phosphorylation has been already reported as dispensable for FANCD2 monoubiquitination (22,25), we looked for a role of CHK1 in FANC pathway activation. We downregulated CHK1 activity and/or expression by siRNA, by pharmacological approach as well as by knocking-down its partner CLASPIN, and demonstrated that loss of CHK1 function affects both basal and DNA damage-induced FANCD2 monoubiquitination. Indeed, unperturbed CHK1- or CLASPIN-depleted cells display high levels of monoubiquitinated and focalized FANCD2 as well as phosphorylation of many ATR targets. This last phenomenon was already reported in CHK1-deficient cells (27). Two other groups have recently reported that CHK1 silencing by siRNA increases the basal level of monoubiquitinated and focalized FANCD2, concluding that CHK1 is not required for FANCD2 monoubiquitination and that it may even have a role in downregulating the FA pathway (38,39). However, we demonstrated here that compared with cells depleted for CHK1 only, co-depletion of ATR and CHK1 significantly reduces the level of FANCD2 monoubiquitination. Similarly, CHK1 inhibition induces FANCD2 monoubiquitination in wild-type but not in Seckel syndrome cells. In agreement with the proposed role of the ATR-mediated FANCD2 phosphorylation for its own monoubiquitination (26), our data suggest that increased FANCD2 monoubiquitination in the absence of a functional CHK1/CLASPIN complex is more likely a consequence of the ATR activation rather than a direct role of this heterodimer in shutting off the FA pathway.

Despite the fact that the CHK1/CLASPIN complex is not absolutely required to mediate FANCD2 monoubiquitination per se, CHK1- and CLASPIN-depleted cells fail to upregulate FANCD2 monoubiquitination after exposure to various genotoxic stresses. In contrast, these cells can further increase ATR kinase activity towards numerous substrates. Moreover, we demonstrated that CHK1 kinase activity is required for the DNA damage-induced FANCD2 monoubiquitination. All together, these observations suggest that CHK1/CLASPIN heterodimer is specifically required for the DNA damage-induced monoubiquitination of FANCD2. Is the ATR-dependent CHK1 phosphorylation involved in the induced FANCD2 monoubiquitination? Indeed, the requirement of CHK1 phosphorylation for its own kinase activity is still controversial (10,12). Our data suggest that CHK1 phosphorylation per se is not sufficient to mediate FANCD2 monoubiquitination. Indeed, despite a similar level of CHK1 phosphorylation in both cases, CLASPIN-depleted cells are not able to induce FANCD2 monoubiquitination, whereas cells expressing only ~50% of CHK1 are. Interestingly, CLASPIN could still associate after DNA damage with a non-phosphorylable S317A/S345A CHK1 mutant, demonstrating that DNA damage could induce the CHK1/CLASPIN complex independently of the ATR-mediated CHK1 phosphorylation (30) (data not shown). Moreover, an ATR- and RAD17-independent role for the CHK1/CLASPIN complex in the DNA replication checkpoint was already reported (40). Collectively, our data and already published observations could be summarized in a model in which DNA damage-induced FANCD2 monoubiquitination requires both its ATR/RAD17/RAD9-dependent phosphorylation and a CHK1/CLASPIN-mediated but ATR-independent event (Fig 6A). Unfortunately, the ectopic overexpression of non-phosphorylable or kinase-dead CHK1-mutants failed to show any dominant-negative effect (41) (data not shown), precluding the possibility to unambiguously determine the requirement of the ATR-mediated phosphorylation of CHK1/CLASPIN to mediate or enhance DNA damage-dependent FANCD2 monoubiquitination.

Whether the quantity of monoubiquitinated FANCD2 in the absence of either CHK1 or CLASPIN is sufficient to perform its function in response to DNA damage is an important question. Because the precise function(s) and consequence(s) of FANCD2 monoubiquitination are not well known, one way to address this question is to look at FANCD2 foci formation. We reported that CHK1- and CLASPIN-depleted cells are impaired in forming FANCD2 foci in response to photoactivated 8-MOP. Similar data were reported by D’Andrea and co-workers (39) in response to UV irradiation, but they are in contrast to the observations of Boulton and co-workers (38), who showed that HU- or MMC-treated CHK1-deprived cells are able to normally assemble FANCD2 foci while RAD51 focal activity is highly compromised in these cells. Besides the different kinds of DNA-damaging agents used in these studies, these apparent discrepancies could arise from the time of recovery. Indeed, we and D’Andrea’s group analyzed FANCD2 foci formation at ‘early’ time points (from 3 to 8 h recovery), whereas Boulton’s colleagues analyzed it at a later time point (18 h recovery). Unfortunately, in our hands, the quality of immunofluorescence in CHK1-depleted cells exposed to HU or MMC for 18 h was not good enough to allow us to draw definitive conclusions. In conclusion, it is possible that the level of monoubiquitinated FANCD2 in CHK1- or CLASPIN-depleted cells is sufficient to relocalize into nuclear foci at a late time after DNA damage but is unable to quickly react and redistribute into foci. Alternatively, this efficient FANCD2 foci formation at a later time could simply reflect accumulation of HU- or MMC-treated cells in S-phase in which FANCD2 monoubiquitination and foci formation normally occur.

D’Andrea and colleagues convincingly showed that CHK1 phosphorylates the core complex member FANCE after DNA damage. They demonstrated that FANCE phosphorylation at T346 and S374 is required for MMC resistance (39). Intriguingly, transfection of a non-phosphorylable FANCE mutant is sufficient to restore spontaneous FANCD2 monoubiquitination and foci formation in unperturbed FA-E cells but these cells cannot increase FANCD2 monoubiquitination and foci assembling in response to induced DNA damage (39). Interestingly, it has also been recently shown that CHK1 could efficiently phosphorylate peptides derived from FANCA or FANCG in vitro (42). From these data, we propose that DNA damage, but not S-phase, specifically requires CHK1/CLASPIN-mediated phosphorylation of FANCcore complex members to increase the ubiquitin ligase activity of the FANCcore complex towards FANCD2 or to stabilize the interaction between some FANCcore complex members and FANCD2 (Fig 6A).

CHK1 participates to both intra-S-phase and G2/M checkpoint (13). We previously reported that CHK1 and FANCD2 work in parallel pathways to regulate the ICL-induced intra-S-phase checkpoint (25). At first glance, this can seem in contradiction with our finding that CHK1 is required to monoubiquitinate FANCD2 after DNA damage. However, the role of FANCD2 in the intra-S-phase checkpoint is related to its CHK1-independent but ATR/ATM-dependent phosphorylation and not to its CHK1- and ATR-mediated monoubiquitination (25,43). In contrast, FANCD2 monoubiquitination seems mainly involved in DNA repair and HR (44).

Finally, we showed that CHK1 is not only involved in the DNA-damage-induced FANCD2 monoubiquitination but is also implicated in the typical ICL-induced accumulation of FA cells with a 4N DNA content (35) (Fig 6B). Indeed, the prolonged G2 arrest induced by cross-linking agent treatment can be abrogated by CHK1 loss of function. Moreover, MMC induces a higher level of CHK1 phosphorylation in FA cells compared with corrected or wild-type cells. These data indicate that in FA the G2 accumulation is actively determined by a cell cycle checkpoint dependent on CHK1. Moreover, we observed that CHK1 inhibition as well as caffeine treatment alleviates the MMC hypersensitivity of FA-C lymphoblasts. This observation is in agreement with previous data reporting that treatment with caffeine or adenine, which also inhibits ATR kinase activity (45), abrogates G2 arrest in FA-C lymphoblasts (36) and increases the survival of FA cells treated with MMC in a clonogenic assay (46). Taken together, these observations support the model in which an overactivated ATR/CHK1 axis in MMC-treated FA cells impairs cell proliferation and survival by blocking cells in late S/G2. Two hypotheses for the overactivation of ATR/CHK1 are conceivable. First, ICL induction could lead to more DNA damage in FA cells, and this higher level of DNA lesions would be expected to trigger more activation of ATR and CHK1 in these cells. Alternatively, a more direct role of the FANC/BRCA pathway in suppressing the ATR/CHK1 pathway could be proposed. Hence, FANC/BRCA pathway disruption induces a higher ATR-dependent CHK1 activation or a decreased checkpoint recovery. In this scenario, DNA damage in normal cells induces the CHK1-dependent activation of the FANC/BRCA pathway which, in turn, will shut off the checkpoint signal (Fig 6B).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Cell lines and culture conditions
HeLa cells, SV40-immortalized MRC5 and FA complementation group A (FA-A: PD220) fibroblasts were routinely grown in DMEM (Gibco) supplemented with 10% fetal calf serum (FCS) (Dutcher), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 µg/ml streptomycin (all from Gibco) at 37°C in 5% CO2. Lymphoblastoid cell lines AHH1, FA-C (HSC536N), ectopically corrected FA-C (HSC536N+FANCC) and Seckel cells (GM18367) were grown in RPMI medium (Gibco) supplemented with 12–15% FCS, glutamine and antibiotics as mentioned earlier.

Transfection
ATR, RAD9, RAD17 and RPA70 smart pool siRNAs were obtained from Dharmacon. The following siRNAs were also used: 3'-UTR RAD17 (5'-CCUACUCUUCUGUCAUCUUdTdT-3') from Invitrogen, FANCD2 (5'-GGAGAUUGAUGGUCUACUAdTdT-3'), Luciferase (5'-CGUACGCGGAAUACUUCGAdTdT-3'), CHK1 (5'-GCGUGCCGUAGACUGUCCAdTdT-3'), 3'-UTR CHK1 (5'-GCUGCUAUGUUGACAUUAUdTdT-3'), CLASPIN 1 (5'-GCACAUACAUGAUAAAGAAdTdT) and CLASPIN 2 (5'-CGCGAAGCAUCUUCAAAUAdTdT-3'), all from Eurogentec.

For transfection, cells were plated in six-well plates at the concentration of 180 000 cells/well and 24 h later were transfected with 100 nM of siRNA using Oligofectamine (Invitrogen) according to the manufacturer’s protocol.

Genotoxic treatments and drugs
ICL induction was performed using either MMC (in pulse or chronic treatment as indicated) (Sigma) or 8-methoxypsoralen (8-MOP, Sigma) photoactivated by exposure to UVA light, as previously described (25). Alternatively, cells were exposed to HU (Sigma), UVC light or to {gamma}-rays from a 60Co source at indicated doses. CHK1 inhibitors sb-218078 (Calbiochem) and UCN-01 (Sigma) diluted in DMSO or caffeine (Sigma) freshly diluted in culture medium were used at indicated doses.

Chromatin fractionation, cell extracts, immunoblotting and antibodies
Chromatin fractionation was performed as previously described (8). For whole-cell extracts, cells were washed in ice-cold PBS and lysed in Laemmli loading buffer (Biorad) containing β-2-mercaptoethanol. Cell lysates were sonicated twice for 10 s each. Alternatively, cells were disrupted in lysis buffer [150 mM NaCl, 50 mM Tris, pH 7.9, 1% NP-40 supplemented with protease inhibitors (Roche), 20 mM sodium fluoride and 2 mM sodium orthovanadate] and the lysates were incubated 10 min on ice and cleared by centrifugation at 25 000g for 10 min at 4°C. For immunoblotting, lysates were separated by SDS–PAGE and transferred onto nitrocellulose membranes (Schleicher & Schull). The membranes were saturated in 5% non-fat milk in PBS-Tween20 (PBS-T) (0.1%) for at least 1 h at room temperature (RT). The following primary antibodies were used and incubated in 5% non-fat milk PBS-T (or 5% BSA in TBS-T for antibodies from Cell Signaling) overnight at 4°C: FANCD2, ATR, RAD1, HUS1, RAD17, CHK1, ACTIN, NBS1 (Santa Cruz Biotechnology), FANCD2, RAD9, RPA32, p343NBS1, SMC1, p966SMC1, H2AX (Abcam), RAD9 (Stratagene), p645RAD17, p345CHK1 (Cell Signaling), RPA70 (Oncogene), CLASPIN (Bethyl), NBS1 (Calbiochem), ORC2 (BD Biosciences) and {gamma}H2AX (Upstate). Binding of the primary antibodies was detected with horseradish peroxydase-coupled specie-specific secondary antibodies (Santa Cruz Biotechnology), followed by enhanced chemiluminescence detection (Amersham or Pierce). Signals were detected by Gene Gnome apparatus and their intensities quantified by the Genetools software (Syngene Bio Imaging). Alternatively, Image J software was also used.

Cell sensitivity assays
Seventy-two hours after siRNA transfection, HeLa cells were seeded into 96-well plates at the concentration of 104 cells/well in complete medium. Genotoxic treatments were performed and cells were cultivated at 37°C in complete medium for 72 h. Cell viability was assessed using Cell Proliferation Kit II (XTT) (Roche) according to the manufacturer’s instructions. Briefly, 50 µl of XTT labeling mixture was added to the cells and incubated for 4 h at 37°C. Absorbance was measured at 450 nm with a microplate ELISA reader (Dynex Technologies).

For the growth inhibition test, lymphoblasts were seeded in 12-well plates at 1 x 105 cells/ml in 15% FCS RPMI medium and eventually treated with 5 ng/ml MMC, 1 mM caffeine and 500 nM sb-218078 alone or in combination for 4 days of culture (3–4 cell divisions in untreated samples). After 4 days, cells were counted (Beckman Coulter) and the growth of treated samples was normalized with respect to the growth of untreated cultures.

Immunofluorescence
Forty-eight hours after siRNA transfection, cells cultivated on microscope slides were treated with 8-MOP+UVA and 8 h later fixed for 10 min at RT in a 4% paraformaldehyde buffered solution and permeabilized with 0.5% Triton X-100 in PBS for 10 min at RT. Staining with rabbit anti-FANCD2 (Abcam) and mouse anti-{gamma}H2AX (Upstate) antibodies was performed for 1 h at RT in 1% BSA/PBS. Primary antibody detection was achieved by donkey anti-mouse alexafluor 488 and donkey anti-rabbit alexafluor 594 secondary antibodies (Molecular Probes) for 1 h at RT. Slides were mounted in Vectashield containing DAPI (Vector Laboratories). For each time point, at least 200 nuclei were examined, and FANCD2 and {gamma}H2AX signals were scored blindly by two independent investigators at a magnification of x630 using a fluorescence microscope (Olympus). Only nuclei showing more than five FANCD2 foci were considered FANCD2 foci-positive, whereas in the case of {gamma}H2AX that presents foci or rather pan-nuclear staining, all cells with {gamma}H2AX signal were considered positive.

Flow cytometry analysis
Cells were trypsinized, washed with PBS and fixed with chilled 80% ethanol for at least 3 h at –20°C. The staining was performed at RT for 30 min in PBS containing 20 µg/ml propidium iodide (Sigma) and 50 µg/ml RNAse (Roche). Data were collected with a Becton Dickinson FACS machine and analyzed with Cell Quest software.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by grants from the Ligue Nationale contre le Cancer (Equipe Labellisée 2006), Electricité de France (EDF), Institut National du Cancer (INCa) and by a Marie Curie European Reintegration Grant (513612). J.-H.G. was supported by a fellowship from the Ministère de l’Enseignement supérieur et de la Recherche.


   ACKNOWLEDGEMENTS
 
We thank Y. Lécluse for help with the FACS experiments, as well as all the members of F.R.’s laboratory and C. Guillouf for helpful discussion and comments on the manuscript. We are grateful to Dr B. Xu and Dr K.K. Khanna for providing us the pSG5 plasmid encoding FLAG-RAD17 and the CHK1 constructs, respectively.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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