Inhibition of SET8 expression results in DSBs

Next, we investigated whether the slower progression through S phase might be related to DNA replication–associated lesions. To address this, we stained U2OS cells using an antibody against phosphorylated H2AX (γ-H2AX), a well-established marker for DNA DSBs (Pilch et al., 2003). As shown in Fig. 3 A, inhibition of SET8 expression led to a dramatic increase in γ-H2AX–positive cells as early as 24 h after siRNA transfection, suggesting that SET8 depletion leads to massive DNA damage. To further corroborate these findings, we analyzed and quantified γ-H2AX levels in a FACS-based assay. Again, SET8 silencing lead to marked DNA damage (Fig. 3 B). Cells with DNA damage were negative for H4-K20 monomethylation, which is consistent with the concomitant loss of the monomethylase function of SET8 in these cells (Figs. 3 C and S1 C). γ-H2AX foci formation after SET8 depletion was also observed in HeLa cells (Fig. S1 D) as well as by direct analysis of DNA strand breaks using pulsed-field gel electrophoresis (Fig. 4 D). Collectively, our data demonstrate that SET8 has a critical role in maintaining correct genomic structure.

Open in a separate window

Figure 3.

SET8 depletion leads to DSBs and checkpoint activation in S-phase cells. (A) SET8 was depleted in U2OS cells for 24 and 48 h. Cells were preextracted and fixed before staining with antibody toward γ-H2AX and Rad51. (B) U2OS cells were treated as in A and were fixed and stained for γ-H2AX and PI followed by FACS analysis. (C) Cells were treated as in A and stained with antibodies recognizing γ-H2AX and monomethylated H4 Lys20. Cells were also collected and processed for Western blotting with the indicated antibodies. Reduction of H4 Lys20 monomethylation upon SET8 depletion is quantified and indicated below the immunoblot. (D) Cells were treated as in A and were stained with 53BP1 and RPA antibodies. Bars, 10 μm.

Open in a separate window

Figure 4.

SET8 depletion leads to replication-dependent DNA damage that activates Chk1. (A) SET8 was depleted in U2OS cells using siRNA for 52 h. Nocodazole was added for the last 16 h as indicated. Cells were collected and processed for immunoblotting. (B) U2OS cells were treated with siRNA against mock, SET8, or Chk1 for 48 h. Nocodazole was added 13 h before harvest. Cells were fixed, stained with PI, and analyzed by FACS. A graph was generated by overlaying the FACS profiles from SET8-depleted cells and cells codepleted for SET8 and Chk1. (C) SET8 was depleted in U2OS cells using siRNA for 52 h. Nocodazole (16 h) and the Chk1 inhibitor Gö6976 (3 h) were added before harvest. Samples were fixed, stained with PI, and analyzed by FACS. (B and C) A fraction of the cells was collected and processed for immunoblotting. (D) 48 h after SET8 siRNA transfection, cells were treated with 5 μM aphidicolin and/or the Chk1 inhibitor CEP-3891 for an additional 24 h. Cells were then processed for pulsed-field gel electrophoresis. (E) U2OS cells were treated with siRNA against mock, SET8, Cdc45, or Rad51 for 48 h. Cells were then fixed and stained for γ-H2AX and PI followed by FACS analysis.

We reasoned that massive DNA damage observed after SET8 depletion could result from the inhibition of vital DNA repair processes because SET8 status could affect the recruitment of 53BP1 as well as other proteins to sites of DNA damage. 53BP1 is a checkpoint mediator involved in the initial sensing and signaling of DNA strand breaks (Ward et al., 2006). The protein has been suggested to be recruited to DNA DSBs via interaction with dimethylated histone H3-K79 and mono- or dimethylated H4-K20, and this interaction has been suggested to be dependent on SET8 (Botuyan et al., 2006). To understand whether the inhibition of SET8 expression affected the recruitment of key DNA repair proteins to sites of DNA damage, we investigated the nuclear accumulation of these proteins by confocal microscopy. As demonstrated in Fig. 3 (C and D), decreased SET8 expression lead to 53BP1 recruitment to sites of DNA damage and a marked increase in Rad51 and replication protein A (RPA) foci. Thus, inhibition of SET8 expression and reduction of H4-K20 monomethylation led to an increased recruitment of 53BP1. To test whether SET8 would be required for 53BP1 recruitment after exogenously induced DSB, we also treated SET8-depleted cells with ionizing irradiation. Yet again, 53BP1 readily relocated to radiation-induced DNA damage foci (Fig. S1 D). This strongly indicates that the presence of SET8 is not essential for the recruitment of 53BP1. 53BP1 was found primarily to bind dimethylated H4-K20, and we noticed that dimethylated H4-K20 persists after SET8 silencing (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200706150/DC1). This indicates that 53BP1 is recruited via persisting dimethylated H4-K20 or via interaction with accessible dimethylated H3-K79 or γ-H2AX (Huyen et al., 2004; Ward et al., 2006).

The S-phase delay in SET8-depleted cells is Chk1 mediated

DNA replication can cease for a variety of reasons before scheduled termination, including progression into areas with DNA damage lesions. Chk1 is a key regulator of the cellular response induced by stalled replication forks, a response that leads to the inhibition of DNA replication initiation at origins of replication (Feijoo et al., 2001). Therefore, we investigated whether Chk1 is activated in SET8-depleted cells. To do this, we transfected cells with SET8 siRNA. Nocodazole was added during the last 16 h, and cells were processed for immunoblotting analysis. The inhibition of SET8 expression led to a dramatic activation of Chk1 as measured by the phosphorylation of Ser317 on Chk1 (Fig. 4 A; Zhao and Piwnica-Worms, 2001). Phosphorylation of RPA was also markedly increased when SET8 was depleted. RPA is required for activation of the ataxia telangiectasia related kinase, which activates Chk1 in the presence of DNA damage (Zou et al., 2003). The activation of Chk1 suggested a role for the checkpoint kinase in mediating the cell cycle delay in SET8-depleted cells. To explore this, Chk1 was specifically long-term depleted using siRNA in combination with SET8 depletion. Inhibition of Chk1 prevented the delay in S phase (Fig. 4 B). Similar data were obtained with the Chk1 inhibitors Gö6976 (Fig. 4 C; Kohn et al., 2003), CEP-3891 (Sorensen et al., 2003), and UCN-01 (Sarkaria et al., 1999; and unpublished data). These cells progress through the cell cycle with markedly damaged DNA as judged by quantitative γ-H2AX FACS analysis (Figs. 4 C and S2 B) and pulsed-field gel electrophoresis (Figs. 4 D and S2 C). This is consistent with a critical role for Chk1 in restraining cell cycle progression after DNA damage.

DNA damage occurring after SET8 depletion requires replication and the functional homologous recombination repair pathway

We next wanted to address whether the lesions generated by SET8 silencing were dependent on DNA replication. Importantly, as shown in Fig. 4 D, the DNA replication inhibitor aphidicolin abrogated the DNA damage induced by SET8 depletion, suggesting that the lesions depend on ongoing DNA replication (Fig. 4 D). To corroborate this, we cosilenced several genes with an important role in DNA replication. Rapid accumulation of γ-H2AX foci was not observed by individual depletion of other replication-associated proteins such as Cdc45 (Syljuasen et al., 2005) and MCM4, which are both essential for the initiation of DNA replication (Fig. 4 E and not depicted; Bell and Dutta, 2002; Pacek and Walter, 2004). When codepleted with SET8, these proteins all reduced the DNA damage. This confirmed that DNA replication is necessary for SET8 silencing to cause DNA damage. The homologous recombination repair pathway plays an important role in the repair of DNA damage occurring during DNA replication (Saintigny et al., 2001; Helleday et al., 2007). Depletion of Rad51, a key component of this repair pathway, did not cause such massive DNA damage at the time points analyzed (Fig. 4 E). At such early time points, cells depleted for Rad51 were still viable and progressed through S phase like control depleted cells (Fig. S3 C, available at http://www.jcb.org/cgi/content/full/jcb.200706150/DC1). Notably, Rad51 depletion blocked DNA damage after SET8 depletion. We suggest that SET8 in unperturbed cells is involved downstream of Rad51 in resolving recombination structures forming spontaneously in cells during DNA replication. When SET8 is depleted, these structures are collapsed into DSBs.

Recent results have suggested that Drosophila PR-Set7 function is required for chromosome condensation and mitosis (Sakaguchi and Steward, 2007). Thus, to investigate whether the S-phase checkpoint observed in mammalian cells is dependent on progression through mitosis, we arrested cells at the G1/S transition by addition of the DNA replication inhibitor thymidine. At the same time, SET8 was depleted using siRNA, and cells were released 30 h after arrest and analyzed by FACS. As seen in Fig. 5 A, cells released from the thymidine arrest were also affected by the depletion of SET8, as these cells progressed slower through S phase compared with mock-treated cells. Therefore, we conclude that the S-phase delay in response to SET8 silencing can occur independently of progression through mitosis.

Open in a separate window

Figure 5.

SET8 interacts with PCNA and is required for replication fork progression. (A) U2OS cells were transfected with siRNA against mock or SET8 and treated with thymidine for 30 h. Cells in the bottom panel were released from G1 arrest by the addition of new media. Nocodazole was added as the cells were released 10 h before harvest. (B) 48 h after SET8 siRNA transfection, cells were pulse labeled with [³H]thymidine for 30 min and were allowed to progress with and without Chk1 inhibitor CEP-3891 for the indicated times. Replication fork elongation from the incorporated [³H]thymidine was then measured as described in Fig. S3 D. 100% labeled single-stranded DNA (ssDNA) is equivalent to no fork progression. (C) Colocalization between HA-FLAG–tagged SET8 and YFP-PCNA transfected in U2OS cells. Cells were preextracted before fixation and processed for immunostaining with HA antibody. (D) Immunoprecipitation of HA-FLAG–tagged SET8 wild type and PIP-box mutant (LTDFY mutated to ATDAA) from HEK293 cells. Cells were transfected with the indicated constructs followed by lysis, immunoprecipitation, and immunoblotting. (E) Endogenous SET8 from HEK293 cells was immunoprecipitated and processed for immunoblotting. Bars, 10 μm.

Our results suggest that depletion of SET8 in the Drosophila and mammalian organisms may have different outcomes. This can partly be explained by the fact that Drosophila PR-Set7 and human SET8 only are moderately homologous. However, the different phenotypes could also be a consequence of the experimental approaches used. In the Drosophila study (Sakaguchi and Steward, 2007), the investigators used cells from PR-Set7 knockout flies. The cells originated from third instar larvae flies and, thus, had progressed through several cell cycles before analysis. In contrast, we used mammalian cells and investigated the defects of SET8 depletion after cells were released from a G1/S block. We cannot eliminate the possibility that cells depleted for SET8 experience defects in progression through M phase; however, such defects were not detected in our study.

FACS

Cells pulse labeled with 10 μM BrdU (Roche) for 5 min were analyzed according to standard protocols. In brief, cells were fixed in 70% ethanol and incubated in 2 M HCl for 30 min. Cells were stained with mouse antibody to BrdU (1:100) for 1 h followed by 1-h incubation with conjugated anti–mouse IgG (AlexaFluor488 at 1:1,000). DNA was then counterstained with 0.1 mg/ml propidium iodide (PI) containing RNase for 30 min at 37°C. γ-H2AX, Cdc45, and Rad51 were stained as described above for BrdU except for the omission of HCl treatment. Analysis was performed on a FACSCalibur (BD Biosciences) using CellQuest software (Becton Dickinson). Quantifications were performed using Modfit LT software (Verity Software House).

Microscopy and immunofluorescence

Cells were grown on coverslips and treated as indicated in the figure legends. The coverslips were then washed briefly in PBS followed by extraction for 10 min on ice with Triton buffer (0.5% Triton X-100 in 20 mM Hepes, pH 7.4, 50 mM NaCl, 3 mM MgCl, and 300 mM sucrose) and fixation for 10 min with 4% PFA. Samples were incubated with primary antibodies in PBS/1% FCS for 1 h at room temperature, washed in PBS/1% FCS three times for 10 min, and incubated with AlexaFluor488– or 594–conjugated secondary antibodies (1:1,000; Invitrogen) for 1 h at room temperature. Cells were washed in PBS three times for 10 min and mounted using Vectashield mounting medium containing DAPI (Vector Laboratories). Pictures were acquired on a microscope (Axiovert 200M LSM 510; Carl Zeiss, Inc.) using a 40× c-Apochromat objective with an NA of 1.2 in H₂O. Pictures were taken at room temperature using LSM 510 META software and LSM image examiner software (Carl Zeiss, Inc.). Brightfield pictures were taken with a microscope (Axiovert 135; Carl Zeiss, Inc.) using a 10× Achroplan objective with an NA of 0.25. Pictures were acquired at room temperature with a camera (CoolSNAP cf²; Photometrics) using MetaMorph software (MDS Analytical Technologies). All pictures were exported in preparation for printing using Photoshop (Adobe).

Immunoprecipitation

Antibodies for immunoprecipitation were coupled to protein A beads for 1 h at 4°C. Extracts for immunoprecipitation were prepared using immunoprecipitation buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween 20, and protease inhibitors leupeptin, aprotinin, and PMSF). The lysis mixture was incubated on ice for 15 min, sonicated three times with a digital sonifier for 3 s (20%; S250; Branson), and microfuged for 10 min at 4°C. Extracts were precleared with 20 μl of protein A–Sepharose and 2 μg of normal IgG. After microcentrifugation, 20 μl of protein A–Sepharose conjugated with 1 μg of antibody was added and allowed to precipitate for 1.5 h at 4°C with rotation. The immune complexes were pelleted by gentle centrifugation and washed four times with 1 ml of immunoprecipitation buffer. After a final wash with immunoprecipitation buffer, the buffer was aspirated completely, and beads were resuspended in laemmli buffer. Immunoblotting was performed as indicated in the next section.

Immunoblotting and antibodies

In brief, cells were lysed on ice in radioimmunoprecipitation assay buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 1% IgePal630, 1% deoxycholic acid [sodium salt], 0.1% SDS, 1 mM PMSF, 5 μg/ml leupeptin, 1% vol/vol aprotinin, 50 mM NaF, and 1 mM DTT). Proteins were separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were incubated in primary antibody diluted in 5% milk as indicated in the figures followed by incubation with secondary antibody (peroxidase-labeled anti–mouse or –rabbit IgG; 1:10,000; Vector Laboratories). Films were developed using an x-ray machine (Valsoe; Ferrania). Phospho-Chk1 antibody (Chk1-pSer317) was purchased from Cell Signaling Technology. Antibodies to MCM7 (DCS-141) and Chk1 (DCS-310) have been described previously (Sorensen et al., 2005). Cdc25A (F-6), cyclin A1 (H-432), Rad51 (H-92), and 53BP1 (H-300) were obtained from Santa Cruz Biotechnology, Inc. Phosphorylated H2AX (JBW103), monomethylated histone H4 Lys20, and phosphorylated histone H3 Ser10 were purchased from Millipore. SET8 (ab3744) and PCNA (PC10) were purchased from Abcam, and RPA (Ab-3) was purchased from EMD.

Pulsed-field gel electrophoresis

48 h after SET8 siRNA transfection, cells were treated with 5 μM aphidicolin (Fluka) and/or 500 nM CEP-3891 (Cephalon) for an additional 24 h. 10⁶ cells were inserted into 10 mg/ml InCert agarose (Cambrex) plugs and incubated for 48 h in 0.5 M EDTA, 1% N-laurylsarcosyl, and 2 mg/ml proteinase K at 20°C in darkness. After washing of plugs four times in Tris-EDTA buffer, separation was performed for 20 h as described previously (Lundin et al., 2005) on a 1% certified megabase agarose (Bio-Rad Laboratories) gel. The gel was then stained overnight with ethidium bromide. Three independent experiments were performed.

Replication fork elongation

Analysis of replication fork elongation was performed as described previously (Johansson et al., 2004). Basically, [³H]thymidine was incorporated in ongoing forks, and, after labeling, the fork progressed from the labeled area. In alkaline solution, unwinding was initiated from the single-stranded ends of the fork; thus, the amount of labeled single-stranded DNA is greater the slower the fork is moving. 24 h after SET8 siRNA transfection, cells were replated in 24-well plates (10⁵ cells/well) and allowed to grow for an additional 24 h. Cells were then pulse labeled with 37 kBq/ml [³H]thymidine (GE Healthcare) in DME at 37°C with 5% CO₂ for 30 min, washed, and incubated with prewarmed media ±500 nM CEP-3891 at 37°C with 5% CO₂ for the indicated times. After incubation, replication was terminated by rinsing the cells with ice-cold 0.15 M NaCl before the addition of 0.5 ml of ice-cold 0.15 M NaCl and 0.03 M NaOH unwinding solution. After 30-min incubation on ice in darkness, unwinding was terminated by the addition of 1 ml of 0.02-M NaH₂PO₄. DNA was fragmented to ~3 kb by sonication (B-12 sonifier with micro-tip; Branson) for 15 s, and SDS was added to a final concentration of 0.25%. After overnight storage at −20°C, single- and double-stranded DNA was separated on hydroxy apatite columns at 60°C. Three independent experiments were performed.

We thank Randi G. Syljuåsen and Adrian P. Bracken for thorough and critical reading of the manuscript. We thank Claudia Lukas for the YFP-PCNA construct and Reidar Albrechtsen for helpful assistance during image acquisition.

We thank the Novo Nordisk Foundation, the Danish Cancer Society, the Danish Medical Research Council, the Danish National Research Foundation, the Swedish Cancer Society, the Swedish Research Council, the Swedish Pain Relief Foundation, and the Medical Research Council for financial support.