Plasmids

Mammalian expression vectors pCLMSCVhyg-T7-Cdt1, pcDNA3.1–3HA-Cdt1, and pcDNA3.1-zeo-FLAG-ORC1 and bacterial expression vector pGEX-6P1-Cdt1 were described previously (5, 12). The pEGFP-C1 expression vector was purchased from Clontech. pCMV-FLAG-HBO1 was described previously (38). The pCMV6-XL4-SNF2H expression vector, which expresses human SNF2H, was purchased from OriGene Technologies (Rockville, MD). To construct the T7 promoter-driven SNF2H expression vector, pCMV6-XL4-SNF2H was digested with NotI, and the SNF2H fragment was subcloned into pCMV6-XL5. The SNF2H protein was then synthesized by in vitro transcription-translation with rabbit reticulocyte lysate (TnT T7 quick coupled transcription/translation system, Promega, Madison, WI) according to the manufacturer's instructions.

Immunoprecipitation

Nuclear extracts were prepared from HEK293T cells with modified cytoskeleton buffer containing 500 mm NaCl, 0.1% Triton X-100, 0.1 mm ATP, 1 mm DTT, and multiple protease inhibitors as described previously (12, 39). Aliquots of the extracts were then immunoprecipitated using anti-Cdt1 or anti-SNF2H antibody bound to protein G-Sepharose beads (Amersham Biosciences). The beads were washed four times with NET gel buffer (12) containing 200 mm NaCl. The immunoprecipitates were eluted with 1× SDS sample buffer (62.5 mm Tris-HCl (pH 6.8), 2% SDS, 5% β-mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue) and subjected to immunoblotting.

GST-Cdt1 Pulldown Assay

For Fig. 1E, GST-Cdt1 (0.6 μg) was mixed with SNF2H produced by in vitro translation in 50 μl of reaction mixture, diluted with binding buffer A (25 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.01% Nonidet P-40, 1 mm DTT, 0.5 mm PMSF, and 10% glycerol) containing 10 mm glycerophosphate and 5 mm NaF, and incubated at 4 °C for 3 h. GST-Cdt1 and associated SNF2H proteins were collected on glutathione beads and washed with buffer A. The bound proteins were eluted and analyzed by immunoblotting or Coomassie Brilliant Blue staining.

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FIGURE 1.

Cdt1 interacts with SNF2H. A, nuclear extracts were prepared from HEK293T cells and immunoprecipitated with anti-Cdt1 antibody (Cdt1 IP) or control rabbit IgG (cont IP). Immunoprecipitates were subjected to immunoblotting with the indicated antibodies. Three percent of the input was also loaded. B, HEK293T cells were transfected with T7-Cdt1 and SNF2H expression vectors, and nuclear extracts were prepared. After immunoprecipitation with anti-SNF2H antibody, the precipitates were blotted with the indicated antibodies. One percent of the input for SNF2H or 0.3% of the input for Cdt1 was loaded. C, HEK293T cells were transfected with T7-Cdt1 and SNF2H expression vectors, and nuclear extracts were prepared. After immunoprecipitation with anti-Cdt1 antibody, the precipitates were immunoblotted with the indicated antibodies. One percent of the input was loaded. D, HEK293T cells were transfected with the indicated expression vectors, and nuclear extracts were prepared. After immunoprecipitation with anti-Cdt1 antibody or control rabbit IgG, the precipitates were immunoblotted with the indicated antibodies. Three percent of the input was loaded. E, GST-Cdt1 or GST was incubated with SNF2H protein synthesized by in vitro transcription-translation with rabbit reticulocyte lysate. GST-Cdt1 and the associated SNF2H protein were then collected on glutathione beads and subjected either to immunoblotting with anti-SNF2H antibody (upper panel) or to Coomassie Brilliant Blue staining (lower panel). Twenty percent of the input sample was analyzed. F, GST-Cdt1 or GST was incubated with HeLa nuclear extracts, and bound proteins were analyzed by immunoblotting with the indicated antibodies. Fifteen percent of the input was loaded.

For Fig. 1F, GST-Cdt1 was bound to glutathione beads, and the beads were mixed with diluted nuclear extracts prepared from HeLa cells. After washing three times with buffer A, the bound proteins were eluted and analyzed by immunoblotting.

Transfection

For Fig. 1 (B–D), expression plasmids (total ~6 μg) were transiently transfected into 3 × 10⁶ HEK293T cells in 100-mm culture dishes using TransIT-293 (Mirus, Madison, WI) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were harvested and subjected to immunoprecipitation analysis.

For Fig. 3 (D–F), expression plasmids (total ~12 μg) and 1.8 μg of pMSCVpuro (Clontech) were transiently transfected into 6 × 10⁶ HEK293T cells in 150-mm culture dishes using TransIT-293. Twenty-four hours after transfection, cells were selected with 2 μg/ml puromycin for 2 days. Cells were then harvested and subjected to ChIP assay.

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FIGURE 3.

SNF2H binding to origins is inhibited by Cdt1 silencing and enhanced by Cdt1 overexpression. A–C, HeLa cells transfected with control (DS scrambled-Neg; siCont) or Cdt1 (siCdt1) siRNAs for 48 h were subjected to immunoblotting (A), FACS analysis (B), or ChIP assay (C). D--F, HEK293T cells transfected with the indicated expression vectors were analyzed by immunoblotting (D), FACS analysis (E), or ChIP assay (F). Coomassie Brilliant Blue (CBB) staining served as the loading control. For ChIP data, the means ± S.D. are shown (n = 3). *, p < 0.05.

For Fig. 5, expression plasmids (total ~1.6 μg) and siRNA (40 pmol) were transiently transfected into 2 × 10⁵ HEK293T cells in 12-well culture plates using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were lysed in 1× SDS sample buffer containing multiple protease and phosphatase inhibitors and then subjected to immunoblotting.

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FIGURE 5.

Cdt1 overexpression-induced ATM activation is decreased by SNF2H silencing. HEK293T cells were transfected with the T7-Cdt1 Cy+D1m expression vector and SNF2H siRNAs using Lipofectamine 2000 along with a plasmid expressing GFP to allow monitoring of the transfection efficiency. GL2 siRNAs were used as the control. Whole cell lysates were prepared 48 h after transfection and immunoblotted with the indicated antibodies. The signal intensity of each band was quantified, with the intensity of the band corresponding to cells transfected with T7-Cdt1 and treated with control siRNAs set at 100%. The means ± S.D. are shown (n = 3).

siRNA Experiments

HeLa and T98G cells were transfected with siRNA duplexes using HiPerFect transfection reagent (Qiagen) according to the manufacturer's instructions. siRNA oligonucleotides were synthesized (IDT, Coralville, IA, or Dharmacon, Lafayette, Co) with the following sequences (sense strands): Cdt1, 5′-GCUGUUGUACUAUCAUGAGCCCUGGdAdC-3′; SNF2H, 5′-GAGGAGGAUGAAGAGCUAUdTdT-3′; CDC6-1, 5′-AGACUAUAACUCUACAGAUUGUGdAdA-3′; CDC6-3, 5′-GGAGGACACUGGUUAAAGAAUUUdAdT-3′; ORC1, 5′-CUGCACUACCAAACCUAUAdTdT-3′; HBO1, 5′-GGGAUAAGCAGAUAGAAGAAAGGAT-3′; control DS scrambled-Neg, 5′-CUUCCUCUCUUUCUCUCCCUUGUdGdA-3′; and control GL2, 5′-CGUACGCGGAAUACUUCGAdTdT-3′.

FACS

Cells were treated with a Cycletest^TM Plus DNA reagent kit (BD Biosciences) for propidium iodide staining and then analyzed with a BD FACSCalibur.

ChIP

ChIP assays were carried out essentially as described previously (40). Briefly, 6 × 10⁶ cells were fixed with 1% formaldehyde and lysed in SDS lysis buffer (1% SDS, 10 mm EDTA, and 50 mm Tris-HCl (pH 8.0)). Chromatin was sonicated to yield an average fragment size of 0.5 kb. Each sample was incubated overnight at 4 °C with 5 μg of the indicated antibodies or control IgGs and then collected using Dynabeads (Invitrogen). After formaldehyde reversal, phenol/chloroform extraction, and ethanol precipitation, the DNA was dissolved in Tris/EDTA buffer.

Quantitative Real-time PCR Analysis

SYBR® Premix Ex Taq^TM II (Takara, Kyoto, Japan) was used according to the manufacturer's instructions. All PCRs were carried out using an iCycler iQ real-time PCR detection system (Bio-Rad). The annealing temperature was optimized for each primer (supplemental Table S1). For each reaction, the cycling parameters were set as follows: 1 min at 95 °C; five cycles at 95 °C for 30 s, annealing temperature plus 4 °C for 30 s, and 72 °C for 30 s; five cycles at 95 °C for 30 s, annealing temperature plus 2 °C for 30 s, and 72 °C for 30 s; and 50 cycles at 95 °C for 30 s, annealing temperature for 30 s, and 72 °C for 30 s.

Chromatin Binding Assay

The chromatin binding assay was performed as described previously (5, 39).

Immunoblotting and Antibodies

Immunoblotting and quantification of band signals were performed as described previously (12). Preparation of rabbit polyclonal antibodies against human Cdt1, ORC1, CDC6, MCM7, and MCM3 was described previously (10, 13, 41). Anti-human Brg1 antibody was obtained by immunizing rabbits with a peptide corresponding to the 50-amino acid C-terminal region of Brg1.

Other antibodies were purchased from various companies: GFP (catalog no. 46-0092, Invitrogen), Thr⁶⁸-phosphorylated Chk2 (catalog no. 2661) and WSTF (catalog no. 2152) (Cell Signaling Technology, Danvers, MA), ATM (clone 2C1, GeneTex, San Antonio, TX), Ser¹⁹⁸¹-phosphorylated ATM (catalog no. 200-301-400, Rockland Biosciences, Gilbertsville, PA), SNF2H (clone 1B9/D12, Upstate Biotechnology, Lake Placid, NY; or clone H-300, catalog no. sc-13054, Santa Cruz Biotechnology, Santa Cruz, CA), ACF1 (catalog no. A301–319A, Bethyl Laboratories, Montgomery, TX), cyclin E (catalog no. sc-247) and HBO1 (catalog no. sc-13284) (Santa Cruz Biotechnology), and Ser⁷⁸⁰-phosphorylated Rb (catalog no. 555S, MBL International Corp.).

SNF2H Binds to Replication Origins in Human Cells

Cdt1 is a key component of pre-RC that has been shown to be indispensable during the initiation steps of DNA replication. The interaction between Cdt1 and SNF2H observed in this work may thus indicate a functional link between chromatin remodeling and pre-RC formation. To assess this possibility directly, the presence of SNF2H at regions of known replication origins was first examined by ChIP assay. Chromatin prepared from asynchronously growing cells was precipitated with the indicated antibodies, and the presence of the well characterized lamin B2 and MCM4 origin sequences (46–50) was assessed by subsequent quantitative real-time PCR analysis using specific sets of primers (Fig. 2A). In two different human cell lines, HeLa and T98G, Cdt1-, MCM7-, or MCM3-specific antibodies efficiently immunoprecipitated DNA sequences that included origin regions of both loci (Fig. 2, B and C). Although some enrichment for the regions distal to the origins was also observed, it was less efficient compared with the enrichment of the origin regions. In human cells, replication origins are not determined as strictly as those in budding yeast (51). Thus, “origin regions” may in fact represent “preferential origins.” In other words, pre-RCs may be formed at the origin-distal regions with lower frequency than the origins. Overall, these data are in good agreement with previous reports (49, 50, 52, 53).

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FIGURE 2.

SNF2H interacts with cellular replication origins during the G₁ phase. ChIP assay was performed as described under “Experimental Procedures.” Quantitative real-time PCR was performed with DNA templates extracted from chromatin precipitated with control IgGs or antibody against SNF2H, MCM7, MCM3, or Cdt1. A, schematic diagram of the human MCM4 and lamin B2 origin loci. Positions of primer pairs to detect the origins and distal sequences are shown above (black and gray boxes, respectively). Coordinates flanking the origin are given in base pairs. B, results with asynchronously growing HeLa cells are shown as the percent of input DNA. The means ± S.D. are shown (n = 8). C, results with asynchronously growing T98G cells. The means ± S.D. are shown (n = 6). *, p < 0.05. D and E, T98G cells synchronized at the G₀ (serum starvation for 48 h), G₁/S (10 h after serum addition), and late S/G₂/M (24 h in the presence of 50 ng/ml nocodazole after serum addition) phases were examined by FACS analysis (D) or ChIP assay (E). For ChIP data, the means ± S.D. are shown (n = 3).

On the other hand, the anti-SNF2H antibodies efficiently enriched both the origin and distal sequences of lamin B2 and MCM4 loci (Fig. 2, B and C). The binding of SNF2H to the origin-distal regions may be due to its multifunctionality, i.e. SNF2H binds to chromatin not only for the purpose of regulating replication but also to mediate transcription and other functions. Nevertheless, it seems clear that SNF2H does indeed bind to replication origins. Furthermore, SNF2H silencing with siRNAs decreased the origin enrichment in ChIP assay (see Fig. 4), confirming the validity of the assay. Therefore, the origin binding of SNF2H was the focus of further analysis.

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FIGURE 4.

Silencing of SNF2H suppresses MCM, but not Cdt1, loading at origins. A–C, HeLa cells transfected with control (GL2; siCont) or SNF2H (siSNF2H) siRNAs for 48 h were subjected to immunoblotting (A), ChIP assay (B), or FACS analysis (C). Coomassie Brilliant Blue (CBB) staining served as the loading control. For ChIP data, the means ± S.D. are shown (n = 3). *, p < 0.05. D–F, T98G cells were transfected with the siRNAs for 24 h, rendered quiescent by serum starvation for 48 h, and then stimulated to reenter the cell cycle by serum addition. Cells were harvested at the indicated times and subjected to chromatin binding assay (D and E) or FACS analysis (F). D, whole cell extracts and chromatin/matrix-binding fractions were immunoblotted with the indicated antibodies. E, the signal intensities of the pre-RC proteins were quantified, normalized to the signals of Coomassie Brilliant Blue staining, and are shown with the maximal values set at 100%. The means ± S.D. are shown (n = 2).

SNF2H Associates with Replication Origins Specifically during the G₁ Phase

Chromatin association of licensing factors reaches its maximum in the G₁ phase when DNA licensing is complete. Levels are reduced upon progression into the S phase or cell cycle exit. To monitor the cell cycle dependence of SNF2H localization to replication origins, ChIP assays were performed using T98G cells synchronized in the G₁/S phase, and the data were compared with those from cells synchronized in the G₀ or late S/G₂/M phase (Fig. 2D). The specific enrichment of MCM7, MCM3, or Cdt1 at the lamin B2 and MCM4 origins was maximal in the G₁/S phase (Fig. 2E), as reported previously (49, 50, 52, 53). Interestingly, SNF2H levels at replication origins were low in chromatin derived from cells synchronized in the G₀ or late S/G₂/M phase but were significantly elevated in chromatin derived from cells synchronized in the G₁/S phase (Fig. 2E). The association of SNF2H at the origin-distal regions was not significantly altered during the cell cycle progression (Fig. 2E). The pattern of SNF2H binding was similar to that of licensing factors, a finding that is similar to previous results obtained with oriP of Epstein-Barr virus (37). These results suggest that SNF2H is associated with cellular replication origins in the G₁ phase in a cell cycle-dependent manner.

Origin Binding of SNF2H Is Dependent on Cdt1

Given the potential role of a Cdt1-SNF2H interaction in SNF2H-origin binding, the dependence of SNF2H loading at the origins on Cdt1 was examined. Chromatin was prepared from HeLa cells treated with control siRNAs or siRNAs targeting Cdt1 and subjected to ChIP assay. Down-regulation of Cdt1 with the Cdt1 siRNA was confirmed by immunoblotting (Fig. 3A). The cell cycle distribution pattern was not remarkably affected by Cdt1 silencing at this level (Fig. 3B). The specific enrichment of the origin sequences with the anti-Cdt1 antibody was also reduced by siRNA treatment (Fig. 3C and supplemental Fig. S1), demonstrating the validity of ChIP assay.

Cdt1 is essential for the loading of hexameric MCM2–7 complexes onto chromatin (1–3). As expected, MCM7 binding at replication origins was decreased by Cdt1 silencing (Fig. 3C). Importantly, SNF2H binging at origins was also significantly decreased by Cdt1 silencing (Fig. 3C and supplemental Fig. S1). The levels of Cdt1 and MCM7 at the origin-distal regions were also lowered by Cdt1 silencing (Fig. 3C). However, SNF2H binding at the origin-distal regions was not remarkably changed by Cdt1 silencing (Fig. 3C). Similar results were obtained with other siRNAs targeting Cdt1 (data not shown).

The hypothesis that Cdt1 overexpression affects origin binding of SNF2H was also investigated. For this purpose, Cdt1 was overexpressed in HEK293T cells (Fig. 3D). The cell cycle distribution pattern was not remarkably affected by Cdt1 overexpression (Fig. 3E), as we reported previously (5). SNF2H binding at the MCM4 origin was enhanced upon Cdt1 overexpression (Fig. 3F). However, SNF2H binding at the distal region was not significantly affected (Fig. 3F).

Taken together, these results suggest that the origin binding of SNF2H is, at least partly, dependent on Cdt1. On the other hand, recruitment of SNF2H to the origin-distal regions may be mediated mostly by other factors.

Silencing SNF2H Suppresses MCM but Not Cdt1 Loading at Origins

The possibility that origin recruitment of SNF2H might affect MCM loading was next investigated. For this purpose, chromatin was prepared from HeLa cells treated with siRNA targeting SNF2H and then subjected to ChIP assay with the indicated antibodies. The efficient depletion of SNF2H was confirmed by both immunoblotting and ChIP assays (Fig. 4, A and B). Remarkable change in cell cycle distribution pattern was not observed in SNF2H-silenced asynchronously growing HeLa cells (Fig. 4C). In line with the previous observation on oriP of Epstein-Barr virus (37), the binding of MCM7 and MCM3 to the cellular origins was suppressed by SNF2H silencing (Fig. 4B). On the other hand, Cdt1 binding at the origins was not affected by SNF2H silencing. These results suggest that SNF2H association with cellular replication origins occurs after the loading of Cdt1 but prior to the loading of the MCM complex and that both Cdt1 and SNF2H are required for the efficient loading of MCM complexes.

The effect of SNF2H silencing on MCM loading was also investigated using a chromatin binding assay. Control and SNF2H-silenced T98G cells were synchronized in the G₀ phase by serum deprivation and then reentered into the cell cycle by serum stimulation. Cells were harvested sequentially at the indicated times and subjected to chromatin loading assay to estimate the binding of pre-RC components to intact chromatin (Fig. 4, D and E). In control siRNA-treated cells, Cdt1 and CDC6 were absent after serum starvation. The levels of both proteins increased ~10 h after serum addition (Fig. 4, D and E, siCont whole cell extracts), coincident with the loading of MCM7 and MCM3 onto chromatin (Fig. 4, D and E, chromatin/nuclear matrix-binding fractions). This result is in agreement with previous findings (12, 54).

Silencing of SNF2H modestly but significantly reduced the recruitment of MCM7 and MCM3 onto chromatin, whereas CDC6 and Cdt1 loading were not affected (Fig. 4, D and E, siSNF2H chromatin/nuclear matrix-binding fractions). Although SNF2H is also involved in transcription regulation (31–35), its silencing did not affect the expression levels of CDC6, Cdt1, MCM7, MCM3, or cyclin E proteins under these experimental conditions (Fig. 4, D and E, whole cell extracts). In addition, Rb phosphorylation at Ser⁷⁸⁰ by cyclin D-Cdk4/Cdk6 was also investigated. Silencing of SNF2H had no notable effect on the kinetics of the Rb phosphorylation (Fig. 4D). These data give further support for the role of SNF2H in MCM loading.

In SNF2H-silenced cells, a slight delay of the S phase progression was observed (Fig. 4F), which could result from the partial reduction of MCM loading by SNF2H silencing. However, it has been reported that cells with depleted MCM replicate at normal rates (25). SNF2H is also reportedly recruited onto replication foci via a physical interaction between WSTF and PCNA, where it stimulates replication fork progression (42, 55). In the absence of further experimental evidence, it is difficult to distinguish between these two possibilities.