ATM mediated phosphorylation of CHD4 contributes to genome maintenance
© Urquhart et al; licensee BioMed Central Ltd. 2011
Received: 8 December 2010
Accepted: 10 January 2011
Published: 10 January 2011
In order to maintain cellular viability and genetic integrity cells must respond quickly following the induction of cytotoxic double strand DNA breaks (DSB). This response requires a number of processes including stabilisation of the DSB, signalling of the break and repair. It is becoming increasingly apparent that one key step in this process is chromatin remodelling.
Here we describe the chromodomain helicase DNA-binding protein (CHD4) as a target of ATM kinase. We show that ionising radiation (IR)-induced phosphorylation of CHD4 affects its intranuclear organization resulting in increased chromatin binding/retention. We also show assembly of phosphorylated CHD4 foci at sites of DNA damage, which might be required to fulfil its function in the regulation of DNA repair. Consistent with this, cells overexpressing a phospho-mutant version of CHD4 that cannot be phosphorylated by ATM fail to show enhanced chromatin retention after DSBs and display high rates of spontaneous damage.
These results provide insight into how CHD4 phosphorylation might be required to remodel chromatin around DNA breaks allowing efficient DNA repair to occur.
It is essential that human cells detect, signal and repair DNA damage in order to prevent chromosomal instability or malignant transformation. DNA double strand breaks can be induced by a number of agents including ionising radiation (IR), reactive chemical species and during endogenous DNA processing events such as DNA replication . These breaks must be repaired in order to maintain cellular viability and genomic stability. Once a break has occurred, cells respond by recruiting DNA repair proteins to the DSB sites and initiate a complex DSB response pathway, which includes altered transcriptional and translational regulation, activation of DSB repair and cell cycle checkpoint arrest.
It is clear that chromatin restructuring in response to DNA damage is essential for initiation, propagation and termination of DNA repair and may even precede DNA end resection. This process opens the DNA allowing the recruitment of repair factors and the amplification of the checkpoint and downstream signals . Consistent with this, the DNA damage response can be activated in the absence of exogenous DNA damage by the tethering of DNA damage response proteins to chromatin, demonstrating the importance of chromatin as a scaffold in the activation and amplification of the DNA damage response. In mammalian cells, the accumulation of any one of early response proteins MDC1, Mre11, Nbs1 or ATM is sufficient to achieve checkpoint activation . This work extends the seminal discovery by Bakkenist and Kastan  that changes in chromatin structure can lead to ATM activation . Whether ATM directly senses the disturbance in chromatin structure or requires an unidentified DSB sensor to transmit the signal is thus far unclear. Nonetheless, ATM kinase activity is a primary driving force for chromatin alterations emanating from DSB induction. Emerging evidence suggests that rapid IR-induced phosphorylation of H2AX and MDC1 by ATM serves as a recruiting signal for E3 ubiquitin ligase RNF8 [4–7]. RNF8 acts together with RNF168 to ubiquitinate histone H2AX and other chromatin proteins in the vicinity of the break [8–10]. The combined activity of these ligases is required for productive recruitment of repair proteins including 53BP1 and BRCA1. Notably, histone ubiquitination on chromatin surrounding the DSBs has recently been shown to mediate RNF8 and RNF168 dependent tra!nscription repression, which suggests the existence of cross talk between cellular processes mediated by these post-translational modifications .
Here we describe how CHD4 functions directly in the processes required for the efficient repair of DSBs. Our data demonstrates that CHD4 is phosphorylated rapidly at Ser-1349 by ATM following induction of DSBs and that this phosphoryated CHD4 is specifically located at sites of DSBs. We also demonstrate that the CHD4 Ser-1349 phosphorylation is required for the CHD4 chromatin retention and for the efficient repair of DSBs.
Cell culture, plasmids, siRNA and antibodies
Human cell lines 293T, HeLa, CaCo2, U2OS, C3ABR, L3 and Seckle cells were maintained in RPMI 1640 medium supplemented with 10% foetal calf serum and incubated at 37°C with 5% CO2. Transfection of plasmids and siRNA was performed using Lipofectamine 2000 (Invitrogen) as per manufacturer's instructions. A series of GST-CHD4 constructs were generated, which together spanned the full length of CHD4. These constructs corresponding to F1 (1-1106), F2 (308-1307), F3 (930-1584), F4 (1528-2511), F5 (2109-3111), F6 (2802-3815), F7 (3666-4521), F8 (4000-5037) and F9 (4431-5037) were PCR amplified and then cloned into the GST-5x-3 vector (GE Healthcare) for expression. GST-CHD4 fusion proteins were purified as described previously . Full length Flag-CHD4 plasmid was a kind gift from Yi Zhang. The Flag-CHD4 S1349A mutant was prepared by site-directed mutagenesis using the Quikchange mutagenesis kit (Stratagene) according to manufacturers instructions. RNA interference was performed using 5'-CAGUUACCAAGAAGACUUAdTdT-3' siRNA specific to CHD4 or control 5'UUCUCCGAACGUGUCACGUdTdT-3' siRNA. The siRNA sequences were synthesized as duplexes (Invitrogen).
Antibodies used in this study were supplied by: Cell Signaling (53BP1, Chk1 S317, p53 S15, ATM S1981), Oncogene (Brca2), Upstate (Acetyl-Lys-H4), Millipore (H2AX), Sigma (FLAG), Santa Cruz (p53, Chk1) GeneTex (ATM), anti-ATM AT9 for immunoprecipitation analysis was prepared in house . The CHD4 antibody was a kind gift from Wei Dong. Rabbit polyclonal anti-phosphopeptide antisera against CHD4 (Serine 1349) was raised using the synthetic peptide NYNDG phospho-SQEDRDW-NH2 conjugated to keyhole limpet hemocyanin (KLH) at the Institute of Medical and Veterinary Sciences, Adelaide, Australia.
Immunoblotting and Immunoprecipitation
Cells were harvested in PBS and cell extracts were prepared by lysis in universal immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 25 mM sodium fluoride, 25 mM β-glycerophsphate, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethysulfonyl fluoride, 0.2% Triton-X 100, 0.3% Igepal CA-630, 0.5 mM dithiothreitol, protease inhibitor cocktail (Sigma)) and incubated on ice for 30 min and then sonicated with a Barnson Sonifier 450. Supernatants were collected following centrifugation at 14,000 × g for 15 mins. 25 μg of protein sample was then analysed by SDS- polyacrylamide gel electrophoresis and immunoblotting with appropriate antibodies. For co-immunoprecipitations protein samples were pre-cleared with protein A beads for 1 hr at 4°C. The supernatants were then incubated with the appropriate antibody for 2 hr. The immune complexes were collected with protein A and protein G beads. The complexes were washed twice with lysis buffer and then fractionated by SDS-polyacrylamide gel electrophoresis for immunoblot analysis. For protein pull-down assays, protein samples were pre-cleared with Glutathione Sepharose beads for 1hr at 4°C. The supernatants were then incubated with purified GST fusion protein fragments linked to Glutathione Sepharose beads for 2 hr. The complexes were washed twice with lysis buffer and then fractionated by SDS-polyacrylamide gel electrophoresis for immunoblot analysis.
Chromatin Fractionation was undertaken using a Sub-cellular Protein Fractionation Kit (Thermo Scientific Cat# 78840) as per manufacturer's instructions.
Cells were harvested in PBS and cell extracts were prepared by lysis in TGN buffer (50 mM Tris-HCl, pH 7.5, 50 mM β-glycerophsphate, 150 mM NaCl, 10% glycerol, 1% Tween 20, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethysulfonyl fluoride, 2 μg/ml pepstatin, 10 μg/ml aprotinin, 5 μg/ml leupeptin and 1 mM dithiothreitol) as described previously . The immunoprecipitation was carried out as described above using an anti-ATM antibody. The immunoprecipitates were washed twice with TGN buffer, once with 100 mM Tris-HCl (pH 7.5) and 0.5 M LiCl, and twice with kinase buffer (10 mM Hepes, pH 7.5, 50 mM β-glycerophosphate, 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 5 mM ATP, and 1 mM dithiothreitol). Kinase reactions were prepared by resuspending washed beads in 30 μl of kinase buffer containing 10 μCi of [γ-32P] ATP and 1 μg of GST-CHD4 fusion protein. Immune complex reactions were incubated at 30°C for 30 min and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.
Immunofluorescence was performed as described previously . Briefly, cells grown on glass coverslips or on Ibidi 8 well μ-slides were washed with PBS, treated for 5 min with Triton buffer (0.5% Triton X-100 in 20 mM Hepes, pH 7.4, 50 mM NaCl, 3 mM MgCl2, and 300 mM sucrose) on ice to remove soluble proteins. Cells with the remaining chromatin-bound proteins were fixed with PBS-buffered 4% paraformaldehyde in 2% sucrose solution at room temperature for 10 min. Fixed cells were washed 3 times with TBS-T (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Tween 20) and blocked in 3% BSA in TBS-T buffer for 30 min. Cells were then incubated with an appropriate primary antibody in blocking buffer at 4°C overnight. After washing three times in blocking buffer cells were incubated with the appropriate secondary antibody (Alexa 488 or Alexa 546, Invitrogen). Images were taken on a Deltavision PDV microscope or GE Healthcare In Cell Analyzer 2000.
These assays were performed as described previously , using the GE Healthcare In Cell Analyzer 2000 and the data analysed using In Cell Investigator software. Assays were performed using GE Healthcare 96 well Matriplates. Immunofluorescence was performed as described above. The In Cell Analyser automatically counted 300 cells from each well prior to measuring the mean nuclear intensity for each signal (Alexa 488).
CHD4 interacts with the ATM kinase
We originally identified CHD4 as an ATM interacting protein during co-precipitation (co-IP) experiments of GFP-tagged ATM and Flag-tagged CRM1. We consistently noticed an additional cross-reacting (Flag-antibody) band of ~200KDa in a GFP-ATM IP from irradiated cells. Bioinformatic analysis of proteins of similar size, in database, that show sequence similarity to Flag-epitope, identified CHD4 as a possible candidate. To confirm this initial observation we precipitated ATM from HeLa cellular extract before and after the induction of DSBs by IR (6 Gy) and blotted the immunoprecipitates with anti-CHD4 antibody. This confirmed that IR stimulated the interaction between ATM and CHD4 (Figure 1b). We also performed the reciprocal co-immunoprecipitation using CHD4 antibody followed by immunoblotting with anti-ATM and this also demonstrated that both proteins interacted (Figure 1c).
CHD4 is phosphorylated by the ATM kinase
ATM phosphorylated CHD4 (Ser1349) colocalises with γH2AX
The loss of chromatin remodeling has been shown to perturb the normal kinetics of the DSB repair process . To examine if CHD4 regulates the normal processing of DSBs we determined the kinetics of loading of two well-established markers of the DSB repair, γH2AX and 53BP1 on to chromatin. Using in-nuclear-westerns , to measure chromatin bound 53BP1 and γH2AX, we observed that depletion of CHD4 (siRNA) resulted in a more pronounced recruitment of 53BP1 to chromatin (Figure 3de). The level of chromatin bound 53BP1 remained higher in the CHD4 depleted cells during the course of the experiment. γH2AX also showed a more rapid activation following IR; however, this was less marked than was observed for 53BP1 (Figure 3de, f). Interestingly, although ATM autophosphorylation (S1981) and SMC1 phosphorylation (S957) occurs in a timely manner in CHD4 depleted cells, these cells show a more rapid loss of signal (Figure 3g). These results are interesting as they demonstrate an abnormality in the repair kinetics of CHD4-deficient cells. It is possible that the loss of CHD4 function has a global effect on chromatin structure, effecting overall DNA repair protein accessibility of these damage sites and also exposing more DNA to damage. This could potentially result in higher rates of spontaneous damage as more DNA is exposed to the solute environment. However we failed to observe an increase in markers for spontaneous damage e.g. spontaneous γH2AX foci formation or spontaneous activation of ATM/ATR signaling events in CHD4-depleted cells. This may be due to technical limitation in achieving a complete knockout of CHD4 expression or that areas of DNA damage are rapidly repaired in these cells.
CHD4 phosphorylation by ATM is required for its recruitment to chromatin and the timely repair of DSBs following IR
We have now established that CHD4 responds to DSB induction by IR. To determine if CHD4 is required for DNA damage repair we next analysed the appearance of spontaneous DNA damage in cells ectopically expressing Flag-CHD4 and the non-phosphorylatable Flag-CHD4-A. The ectopic expression of Flag-CHD4 had no effect on the number of γH2AX positive cells in the population, interestingly neither did the depletion of CHD4 (siRNA) which support the enhanced DNA repair phenotype observed earlier in this study. However, the ectopic expression of Flag-CHD4-A significantly increased the percentage of cells showing spontaneous (undamaged cells) γH2AX and 53BP1 foci (Figure 5de) suggesting that these cells had impaired ability to cope with endogenous DNA damage.
To further explore this we next treated HeLa cells with low dose IR (2Gy) to determine the repair kinetics of DSBs. To do this, we analysed the ability of HeLa cells expressing wt and phospho-mutant CHD4 to resolve γH2AX foci. Maximal γH2AX foci were observed in both wt-Flag CHD4 and Flag-CHD41349A mutant expressing cells at 1 h after irradiation. In wt-CHD4 transfected cells, γH2AX foci were present in ~ 7% of cells by 6 h after damage (Figure 5f). In contrast, Flag-CHD41349A mutant expressing cells were slower at resolving the γH2AX foci and retained γH2AX foci in ~20% of cells by 6 h which returned to predamage level 24 h after IR. These data indicate the phosphorylation of CHD4 at Ser 1349 is required for the efficient repair of double strand DNA breaks.
It is important to understand how cells respond to and attempt to repair double strand DNA breaks. Much is already known in regard to the recruitment of repair factors and the checkpoint activation pathways, however, it is becoming increasingly clear that chromatin remodeling must take place to allow efficient repair. CHD4 has previously been characterized as a component of the NuRD transcriptional repression complex [15, 30] that exists as a part of a complex with ATM-related kinase ATR .
Here we identified CHD4, as an ATM interacting protein and subsequently showed that it is a direct target of ATM kinase. CHD4 is phosphorylated on Ser1349 in vivo in cells in an ATM dependent manner in response to IR. During the course of our study, another study reported CHD4 as a phosphorylation target of ATM kinase that supports our findings . This study reported that association of CHD4 with DNA lesions occurs independently of ATM instead the authors found that it is dependent on poly (ADP-ribose) specific pathway. Another study reported significant enrichment in chromatin binding of CHD4 in response to IR, which could be explained in part by its retention directly at the sites of DNA damage . Consistent with this report, we also observed ATM-dependent increase in chromatin retention of CHD4 after IR. Moreover, we found that Ser1349 phosphorylated CHD4 accumulated into IR-induced foci which colocalize with γH2AX, while staining with antibody that detects the total pool of CHD4 remained unchanged in response to IR suggesting that phosphorylated CHD4 might play a role in the regulation of DNA repair processes. Although CHD4 is not an essential component of the repair process, our data demonstrates that CHD4 functions to allow the timely repair of DNA damage, which is essential to maintain cellular viability. Consistent with this, CHD4-depleted cells show an elevated level of recruitment of DNA repair factors to chromatin following DNA damage. This however may be due to the general opening of the chromatin structure in cells depleted of CHD4, which would allow more rapid recruitment and would also result in elevated levels of DNA damage. Indeed, we demonstrate that markers of euchromatin increase significantly in cells depleted of CHD4 and there are large cellular changes in the transcriptome. This supports a general role of CHD4 and the NuRD complex in regulating global chromatin structure and expla!ins the global impact of CHD4 depletion.
To explore the possible role of CHD4 in DSB repair further we decided to study the role of the ATM mediated phosphorylation of CHD4 on Ser1349. Following IR treatment, CHD4 becomes more tightly chromatin associated in an ATM dependent manner, which might help localize CHD4 in close proximity to DNA lesions. It is likely that ATM modifies the cellular function of CHD4 specifically at sites of DSBs since DNA damage is known to cause retention of active ATM kinase in detergent-resistant nuclear fractions . Cells ectopically expressing Flag-CHD4-A, which cannot be phosphorylated by ATM, fail to show enhanced chromatin retention after IR suggesting that ATM dependent phosphorylation might result in CHD4 becoming more tightly associated with the chromatin and this allows for the opening of the chromatin structure and repair. Consistent with this, cells ectopically expressing ATM-phosphosite mutant CHD4 (Flag-CHD4-A) displayed higher rates of spontaneous DNA damage and showed a defect in their ability to resolve IR induced γH2AX foci in a timely manner.
Collectively, our data demonstrates how phosphorylated CHD4 functions to facilitate the repair of DSBs. Understanding the cellular processes that control chromosomal stability are of vital importance. Our data provides evidence that CHD4, a component of NuRD complex is required to allow the timely repair of breaks and provide further support to the idea that there is an intimate link between ATM kinase and chromatin remodelling complexes involved in different aspects of chromatin dynamics in mammalian cells. Therefore, dissecting the interplay of ATM kinase and chromatin modifying activities has become a vital step towards understanding of DNA damage response. This data gives us interesting insight into the function chromatin remodelers may play in modulating the effect of chemotherapeutic drugs . Furthermore, therapies that target the chromatin modifying enzymes are being investigated for the treatment of cancer [34, 35].
K.K.K. is a NHMRC Senior Principal Research Fellow and this work is supported in part by Program grants from the National Health and Medical Research Council of Australia to K.K.K. We wish to thank Weidong Wang for providing anti-CHD4 antibodies.
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