CDT-2 functions as a CRL4 complex SRS to target CKI-1 degradation

To test if CDT-2 functions as an SRS, we analyzed the physical interactions of CDT-2 with CRL4 components and the putative substrates CDT-1 and CKI-1 using the yeast two-hybrid assay. The two-hybrid results are consistent with CDT-2 functioning as an SRS: CDT-2 interacted with the adaptor DDB-1 and with the substrates CKI-1 and CDT-1, but did not interact directly with CUL-4 (Fig. 2A [top panel], B). Also consistent with the CRL structure, the adaptor DDB-1 did not interact directly with the substrates CKI-1 or CDT-1 in the two-hybrid assay (Fig. 2A, top; data not shown). We tested whether CDT-2 can act as a bridge between DDB-1 and CKI-1 using the yeast three-hybrid test (Licitra and Liu 1996). CDT-2 was expressed from the GAL1 promoter, which is induced by galactose and repressed by glucose (Giniger et al. 1985). Yeast containing the GAL1 promoter/CDT-2 construct plus a DDB-1/GAL4 DNA-binding domain fusion and CKI-1/GAL4 activation domain fusion were able to grow on histidine(−) selection plates containing galactose but not glucose, indicating that CDT-2 can bridge interaction between DDB-1 and CKI-1 (Fig. 2A, bottom). A similar experiment showed that DDB-1 can mediate interaction between CUL-4 and CDT-2 (Fig. 2A, bottom).

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Figure 2.

CDT-2 interacts with the CRL4 adaptor DDB-1 and the substrates CDT-1 and CKI-1. (A) Yeast two-hybrid (top panel) and three-hybrid (bottom panel) analysis to examine the interactions between CRL4 complex components and substrates. Note that CDT-2 binds to the putative substrates CDT-1 and CKI-1 and to the adaptor DDB-1 in the two-hybrid assay, but not to CUL-4. For the three-hybrid assay, DDB-1 and CDT-2 were expressed under control of the galactose-inducible GAL1 promoter. Note that CUL-4 can interact with CDT-2 only in the presence of DDB-1, and that DDB-1 can interact with CKI-1 only in the presence of CDT-2 (left and right sides of the plate, respectively). The first protein listed was expressed as a fusion with the GAL4 DNA-binding domain, and the second protein was expressed as a fusion with the GAL4 activation domain. (B) Diagram of the predicted CRL4^CDT-2 structure with bound substrate. Components are labeled. (C) C. elegans CDT-2 can physically associate with human TAP-DDB1 when coexpressed in 293T cells. Note that pulldown of TAP-DDB1 coprecipitates C. elegans CDT-2. (D) The in vivo ubiquitylation of C. elegans CKI-1 expressed in HeLa cells is stimulated by C. elegans CDT-2. Flag-CKI-1 and HA-ubiquitin were coexpressed with or without Myc-CDT-2. Flag-CKI-1 was immunoprecipitated under denaturing conditions (0.1% of SDS), and analyzed by Western blot with anti-HA and anti-Flag antibodies. Whole-cell lysate was blotted to assess Myc-CDT-2 expression.

To test if CKI-1 is a ubiquitylation substrate of CDT-2, we utilized a mammalian expression system. We observed that C. elegans CDT-2 can interact with TAP-tagged human DDB1, suggesting that it can interact with human CRL4 components (Fig. 2C). Coexpression of C. elegans CDT-2 with CKI-1 in HeLa cells stimulated the in vivo ubiquitylation of CKI-1, suggesting that CKI-1 is a direct substrate of the CRL4^CDT-2 E3 complex (Fig. 2D). Overall, our data indicate that CDT-2 functions as a CRL4 SRS to catalyze the ubiquitin-mediated degradation of the replication-licensing factor CDT-1 and the Cip/Kip family CDK inhibitor CKI-1 in C. elegans.

Human CRL4^Cdt2 targets p21^Cip1 for degradation

The mammalian Cip/Kip family includes three members (p21^Cip1, p27^Kip1, and p57^Kip2) that are each capable of inhibiting a wide-range of CDK holoenzymes (complexes of CDKs and cyclins) (Besson et al. 2008). To test if Cdt2 negatively regulates the levels of Cip/Kip family CDK inhibitors in human cells, we determined whether Cdt2 siRNA knockdown would lead to increases in the protein levels of the three mammalian CDK inhibitors. We observed that Cdt2 knockdown in HeLa cells produced a 5.6-fold increase in p21^Cip1 protein levels (Fig. 3A). In contrast, Cdt2 knockdown did not increase the levels of the Cip/Kip family members p27^Kip1 or p57^Kip2 (Fig. 3A). It has been reported that the E3 SCF^Skp2 can target p21, p27, and p57 for degradation (Yu et al. 1998; Carrano et al. 1999; Sutterluty et al. 1999; Tsvetkov et al. 1999; Bornstein et al. 2003; Kamura et al. 2003). In our experiments, Skp2 knockdown in HeLa cells increased the levels of p21 and p27, but did not affect p57 levels. Notably, coknockdown of Cdt2 and Skp2 increased p21 levels approximately twofold higher than either single knockdown (an 11-fold increase), indicating that Cdt2 and Skp2 redundantly regulate p21 (Fig. 3A). Unless otherwise noted, siRNA experiments were performed with pools of four siRNAs. Knockdowns with individual siRNAs for Cdt2 and Skp2 also produced increased p21 levels (Fig. 3B).

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Figure 3.

Cdt2 targets p21 for degradation in S phase. (A) Asynchronously growing HeLa cells were transfected with siRNA for GFP (a control), Cdt2, Skp2, or Cdt2 and Skp2 together, and the levels of the indicated proteins in the lysates were assessed by Western blot. Note that p21 protein levels increase upon siRNA knockdown of either Cdt2 or Skp2, and are further increased upon siRNA knockdown of both Cdt2 and Skp2. (B) Comparison of siRNA knockdown of individual siRNAs (numbered) and pooled siRNAs (P) for Cdt2, Skp2, and p21. The pools contain the individual siRNAs #2–#5. Note that knockdowns with individual siRNAs for Cdt2 and Skp2 increase p21 levels similar to the pooled siRNAs. (C) A comparison of p21 levels in asynchronous or S-phase-enriched HU-treated HeLa cells subjected to GFP, Cdt2, or Skp2 siRNA. Cyclin A is a marker of S/G2 phase, and α-tubulin is a loading control. Note that p21 levels are higher in HU-treated than asynchronous Cdt2 siRNA cells. The increased Cyclin A protein level in asynchronous Cdt2 siRNA cells reflects the increased number of cells in S phase (undergoing rereplication with greater than 4C DNA content, see Fig. 6). (D) Analysis of p21 protein turnover in Cdt2 knockdown cells. GFP or Cdt2 siRNA-treated HeLa cells were arrested in S phase with HU and incubated with the proteasome inhibitor MG132. At time 0, HU and MG132 were removed and replaced with media containing the protein synthesis inhibitor cycloheximide (CHX). Cells were harvested at the indicated times and analyzed by Western blot. Dark and light exposures of the p21 blot are shown to facilitate a comparison of p21 turnover in GFP and Cdt2 siRNA cells. A graph of the ratio of p21 to tubulin levels is presented with values standardized to 100% at time 0 for each siRNA cell type. (E) Time course of p21 levels in serum-stimulated Cdt2 and control knockdown cells. HeLa cells were serum starved and treated with the individual Cdt2 siRNA (#1) or GFP siRNA. The quiescent cells were stimulated to enter the cell cycle by the addition of serum, and collected at the times indicated. Cyclin B1 is presented as a cell cycle marker whose expression begins in S phase (Pines and Hunter 1992).

To determine if Cdt2 regulates p21 levels in S phase, we inactivated Cdt2 in asynchronous cells and in cells enriched in S phase by hydroxyurea (HU) treatment. The level of p21 was significantly increased in S-phase Cdt2 siRNA cells relative to asynchronous Cdt2 siRNA cells, suggesting that CRL4^Cdt2 predominantly regulates p21 levels during S phase (Fig. 3C). A similar increase in p21 levels was also observed in aphidicolin-blocked Cdt2 siRNA cells (data not shown). In contrast to the Cdt2 knockdown, Skp2 siRNA treatment produced similar elevated levels of p21 in asynchronous and HU-treated cells, suggesting that SCF^Skp2-mediated p21 degradation is not restricted to S phase (Fig. 3C). To determine if Cdt2 affects the rate of p21 protein turnover, we performed a time-course experiment in which Cdt2 knockdown or control GFP knockdown cells were arrested in S phase with HU in the presence of the proteasome inhibitor MG132. At time 0, HU and MG132 were removed while cycloheximide was added to block protein synthesis. We found that the half-life of endogenous p21 was significantly increased in Cdt2 knockdown cells, indicating that Cdt2 contributes to p21 turnover during S phase (Fig. 3D).

It has been observed that p21 levels decrease during S phase as serum-stimulated quiescent mammalian cells progress through the cell cycle (Dulic et al. 1998; Amador et al. 2007). We asked whether Cdt2 is required for this drop in p21 protein levels. To test this, serum-starved HeLa cells were treated with either Cdt2 siRNA or control GFP siRNA, and then stimulated with serum. As previously reported, p21 levels increased in control cells after serum stimulation and then decreased as cells progressed through S phase (Fig. 3E). Cdt2 protein levels were very low in quiescent control cells, but increased significantly upon serum stimulation, with the highest level observed at 9 h post-serum stimulation coincident with increased expression of Cyclin B1, indicating that the cells had entered S phase (Fig. 3E). The increase in Cdt2 levels correlated with the decrease in p21 levels. In Cdt2 knockdown cells, the induction of Cdt2 protein after serum stimulation was blocked, and p21 was markedly stabilized as cells progressed through S phase (Fig. 3E). Our results indicate that Cdt2 contributes significantly to the degradation of p21 during S phase in mitotically dividing cells.

To determine if Cdt2 directly targets the degradation of p21, we tested for physical interaction between p21 and Cdt2. We observed that when expressed in HEK293T cells, Flag-Cdt2 and Myc-p21 reciprocally coimmunoprecipitate with each other, while Flag-Cdt2 did not show significant interaction with Myc-p27^Kip1 (Fig. 4A). We also asked whether the expression of Cdt2 would allow interaction between p21 and DDB1, as expected for an SRS. We observed that significantly more p21 associated with expressed TAP-tagged DDB1 in the presence of Flag-Cdt2 than in its absence (a 6.7-fold difference) (Fig. 4B). The observed low-level of p21 bound to TAP-DDB1 in the absence of Flag-Cdt2 was potentially mediated by interaction with the endogenous Cdt2.

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Figure 4.

CRL4^Cdt2 can physically interact with p21. (A) Cdt2 can bind p21. Flag-Cdt2 was coexpressed with either Myc-p21 or Myc-p27 in HEK293T cells as noted by plus symbols (+) above the lanes. Anti-Flag immunoprecipitation (IP) or anti-Myc IP were analyzed by Western blot with anti-Flag antibody (top panel) or anti-Myc antibody (bottom panel). (B) DDB1 binds to p21 in a Cdt2-dependent manner. Flag-Cdt2 and nontagged p21 were expressed in HEK293T cells stably expressing TAP–DDB1 as noted above the lanes. Total protein extract and the TAP-DDB1 pulldown were probed with the indicated antibodies.

We next sought to determine if Cdt2 facilitated p21 ubiquitylation. We observed that expression of Flag-Cdt2 stimulated the in vivo conjugation of HA-ubiquitin to endogenous p21 (Fig. 5A). Expression of Flag-Skp2 also stimulated the in vivo ubiquitylation of p21, consistent with the siRNA data that showed that both E3 complexes regulate p21 levels (Fig. 5A). The CRL4^Cdt2 complex can catalyze the in vitro ubiquitylation of p21 (Fig. 5B). Taken together, our results suggest that p21^Cip1 is a direct target for ubiquitylation by the CRL4^Cdt2 E3 ligase.

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Figure 5.

CRL4^Cdt2-mediated ubiquitylation of p21. (A) Overexpressing Cdt2 and Skp2 stimulates the in vivo ubiquitylation of endogenous p21 in HeLa cells. Flag-Cdt2 and Flag-Skp2 were coexpressed with HA-ubiquitin (noted at the top). Endogenous p21 was immunoprecipitated from whole-cell lysate under denaturing conditions (0.1% SDS), followed by anti-HA and anti-p21 immunoblotting. Note that the lysates were probed with anti-Cdt2 and anti-Skp2 antibodies to show total Cdt2 and Skp2 levels, both endogenous and overexpressed. Lane 2 shows a total Cdt2 level that is 1.7-fold that of the control lane and a higher-molecular-weight HA-ubiquitin signal that is 2.8-fold that of the control lane. The level of Flag-Cdt2 and Flag-Skp2 proteins in lanes 2 and 3 were suppressed relative to lane 4 by coexpression with the empty vector pEGFP-N1 (included in the first three transfections). (B) In vitro ubiquitylation of p21 by the CRL4^Cdt2 complex. Flag-Cdt2 and nontagged p21 were coexpressed in HEK293T cells in the presence of MG132. The CRL4^Cdt2 complex (with associated overexpressed p21) was immunoprecipitated with anti-Flag antibody, and used in an in vitro ubiquitylation reaction. p21 was subsequently immunoprecipitated under denaturing conditions (0.1% SDS), followed by immunoblotting for HA-ubiquitin and p21. (C) Coimmunoprecipitation of PCNA withMyc-p21 in HEK293T cells. The transgenes noted were coexpressed and Flag-Cdt2 was immunoprecipitated. Western blots with antibodies to the indicated proteins were performed to assess coimmunoprecipitation. (D) Both wild-type and PIP-box mutant p21 immunoprecipitate with Flag-Cdt2 from a chromatin fraction of HeLa cells. Note that PCNA efficiently coprecipitated with Flag-Cdt2 in the presence of overexpressed wild-type p21 but not PIP-box mutant p21. (E) Wild-type p21 is more efficiently ubiquitylated in vivo than PIP-box mutant p21 upon cotransfection with Flag-Cdt2 in HeLa cells. Transfected p21 was immunoprecipitated from a nuclear fraction under denaturing conditions, followed by anti-HA and anti-p21 immunoblotting. Asterisks mark nonspecific bands.

p21 degradation is PIP-box-dependent

The ubiquitylation of vertebrate Cdt1 by CRL4^Cdt2 is dependent on the interaction of Cdt1 with PCNA, mediated by the PIP-box sequence of Cdt1 (Higa et al. 2006a; Hu and Xiong 2006; Jin et al. 2006; Nishitani et al. 2006; Senga et al. 2006). p21 has a C-terminal PIP-box motif and can bind to PCNA (Waga et al. 1994). We observed that the precipitated Flag-Cdt2 immunocomplex, which was capable of in vitro ubiquitylation of Myc-p21, contains PCNA (Fig. 5C; data not shown). To determine the importance of the PIP-box for the physical interaction between p21 and Cdt2, we converted the PIP-box sequence of p21 (QTSMTDFY) to eight alanine residues. The p21 PIP-box mutant protein coprecipitated with Flag-Cdt2 at levels equivalent to that of wild-type p21 (Fig. 5D). As expected, PCNA was only coprecipitated above background levels with wild-type p21 but not the p21 PIP-box mutant, suggesting that Cdt2 can bind p21 independently of PCNA association (Fig. 5D).

To determine if the PIP-box of p21 is required for its in vivo ubiquitylation, we expressed wild-type p21 or PIP-box mutant p21 along with HA-ubiquitin and Flag-Cdt2. We observed that the in vivo ubiquitylation of wild-type p21 was significantly greater than the ubiquitylation of the p21 PIP-box mutant (Fig. 5E). This suggests that p21 ubiquitylation depends on PCNA binding, providing a mechanistic link between the CRL4^Cdt2-mediated degradation of the two substrates Cdt1 and p21, with both ubiquitylation reactions dependent on interaction with PCNA.

p21 is required for the rereplication induced upon inactivation of CRL4^Cdt2

We observed that Cdt2 siRNA treatment of HeLa cells induces rereplication, producing increased numbers of cells with DNA content greater than 4C (Fig. 6), similar to what has been reported previously (Jin et al. 2006). Notably, Skp2 siRNA did not produce cells with DNA content greater than 4C, and coinactivation of Cdt2 and Skp2 did not increase the percentage of cells with greater than 4C DNA content beyond that of Cdt2 siRNA alone (Fig. 6). Thus, Cdt2 is required to prevent DNA rereplication in HeLa cells, while Skp2 does not significantly contribute to the prevention of overreplication. Strikingly, the rereplication observed with both Cdt2 knockdown and the coknockdown of Cdt2 and Skp2 was abolished upon coinactivation of p21 (Fig. 6). Thus, p21 is essential for the rereplication that occurs when Cdt2 is inactivated.

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Figure 6.

Cdt2 siRNA induces increased DNA content, which is reversed by p21 siRNA. Flow cytometry profiles of DNA content of HeLa cells transfected with the indicated siRNAs. Pools of four siRNA were used with the exception of the third graphs from the left, which used individual siRNAs. The increase in DNA content upon Cdt2 knockdown was observed in siRNA treatments with the Cdt2 siRNA pool and two different individual Cdt2 siRNAs that were tested (#1 and #2), and the block on rereplication by coinactivation of p21 was observed with the p21 siRNA pool and two different individual p21 siRNAs that were tested (#1 and #2) (above; data not shown). The percentages of cells in each cell cycle phase are provided for each graph. (G0/G1) 2C DNA content; (S) 2C < × < 4C; (G2/M) 4C; and >4C. Control GFP siRNA cells had a DNA distribution that was similar to that of p21 siRNA: (G0/G1) 62.4%; (S,) 22.5%; (G2/M) 13.3%; and (>4c) 1.9%. The position of 2C and 4C are noted by triangles at the bottom of each graph.

Yeast two- and three-hybrid assays

For two-hybrid analysis, the full-length cul-4, ddb-1, cdt-2, cki-1, and cdt-1 cDNAs were cloned into the vectors pACT2 (which encodes GAL4 activation domain fusion) and pAS2 (encodes GAL4 DNA-binding domain fusion) (Clontech). Plasmids were transformed into the Saccharomyces cerevisiae strain pJ69-4A (James et al. 1996) using standard procedures (Ausubel et al. 1995). Interaction in the two-hybrid system was assayed on histidine-deficient selective media containing 1 mM 3-amino-1,2,4-triazole (3-AT, Sigma). For three-hybrid tests, cdt-2 and ddb-1 cDNAs were cloned into the pYES2 vector (Invitrogen), in which the gene of interest is under the control of the GAL1 promoter. Two-hybrid bait and prey plasmids as well as the pYES2 construct were cotransformed into pJ69-4A. Interaction in the three-hybrid system was determined on histidine-deficient selective media containing 1 mM 3-AT in the presence of 2% glucose or galactose.

Expression constructs

To express Flag-tagged proteins in mammalian systems, cDNAs of human Cdt2 and C. elegans cki-1 were cloned into pCMV-Tag2 (Stratagene). For Myc-tagged proteins, cDNAs of human p21 and p27 were cloned into pCS3-MT (Hallier et al. 1998), and the C. elegans cdt-2 cDNA was cloned into pcDNA3.1/Myc-His (Invitrogen). The expression constructs pcDNA3/FlagSkp2 (Bashir et al. 2004), pCMV/HA-UB (Furukawa et al. 2005), p3XFlag-CMV10/Hs Cdt2 (Higa et al. 2006b), pCS2/p21 (Sheaff et al. 2000), and pGLUE-hDDB1 (Angers et al. 2006) were gifts from M. Pagano, Y. Xiong, H. Zhang, B. Clurman, and S. Angers, respectively. The PIP-box in p21 was mutated by overlapping PCR mutagenesis (Mikaelian and Sergeant 1992) to convert the PIP-box sequence (QTSMTDFY) to eight alanine residues.

Mammalian cell culture

HeLa and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO-BRL). For expression in HeLa and HEK293T cells, expression constructs were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Flag-Cdt2 and wild-type p21 were expressed in an HEK293T cell line stably expressing TAP-DDB1. To isolate HEK293T cell lines stably expressing TAP-DDB1, cells were transfected with pGLUE-hDDB1, and selected in a medium containing 0.7 μg/mL puromycine (Sigma). For the study of p21 protein turnover, HeLa cells were treated with GFP or Cdt2 siRNA and then incubated with 2.5 mM HU (Sigma) for 19 h, and with the proteasome inhibitor MG132 (Sigma) for the last 7 h of the HU incubation. At time 0, HU and MG132 were removed from the culture media, and 25 μg/mL cycloheximide (Sigma) was added. For the analysis of p21 levels upon cell cycle entry, HeLa cells were serum-starved for 54 h by incubation in DMEM containing 0.02% fetal calf serum (FCS) in order to induce G0 entry. Cells were transfected with GFP or Cdt2 siRNA for the last 18 h of the serum starvation. At time 0, cells were stimulated with 15% FCS plus a second siRNA transfection. At set time points, cells were harvested for Western blot analysis.

Antibodies, immunofluorescence, and immunoprecipitation

For Western blots, whole-cell lysates were prepared by treating with NP-40 buffer: 1% or 0.5% NP-40, 0.15 M NaCl, 10 mM Sodium phosphate (pH 7.5), 10% glycerol, Protease Inhibitor Cocktail (Roche), 2 mM DTT, 1 mM EDTA, and 15 μM MG132. Monoclonal antibodies used were anti-p21 (Upstate Biotechnologies), anti-Cdc6 (DCS-180, Upstate Biotechnologies), anti-Myc (9E10, Covance), anti-Flag (M2, Sigma), anti-Cyclin A (Ab-6, Thermo Scientific), anti-Cyclin B1 (GNS1, Santa Cruz Biotechnologies), and anti-α-tubulin (DM1A, Sigma). Rabbit polyclonal antibodies used were anti-Cdt2 (Bethyl Laboratories), anti-Skp2 (H-435, Santa Cruz Biotechnologies), anti-p27 (Upstate Biotechnologies), anti-p57 (Upstate Biotechnologies), anti-Cyclin A (H-432, Santa Cruz Biotechnologies), anti-CUL4 (H-66, Santa Cruz Biotechnologies), anti-PCNA (PC10, Santa Cruz Biotechnologies), and anti-HA (ab9110, Abcam). For immunofluorescence, HeLa cells were fixed in 4.0% paraformaldehyde in PBS for 10 min at room temperature, washed twice with 0.4% Triton X-100 in PBS, and stained with monoclonal anti-Cdc6 (1:100) and rabbit polyclonal anti-Cyclin A (1:250). Anti-mouse Alexa-Fluor 488 (Molecular Probes) and anti-rabbit Alexa-Fluor 546 (Molecular Probes) were used as secondary antibodies. Alternatively, cells were stained with monoclonal anti-Cdc6 (1:100) and monoclonal anti-Cyclin A (1:40) using the Zenon Mouse Tricolor IgG1 labeling kit (Invitrogen). DNA was stained with 1 μg/mL Hoechst 33258 dye (Sigma). Immunofluorescence images were taken with the same exposure times and the images were processed identically using Adobe Photoshop software. Quantitation of nuclear and total immunofluorescence signal was performed with OpenLab software (version 5.0; Improvision). For the quantitation of Cdc6 localization in Figure 7B, 21–23 cells were analyzed for each condition.

Immunoprecipitation using anti-Myc and anti-Flag antibodies was performed as described (Kim and Kipreos 2007b). The purification of TAP-tagged DDB1 with associated proteins was modified from Angers et al. (2006). Briefly, HEK293T cells stably expressing TAP-DDB1 were cultured in a six-well plate and transfected using Lipofectamine 2000. After an 8-h incubation at 37°C, cells were treated with 2.5 mM HU and 10 μM MG132, and then harvested after an additional 10 h of incubation at 37°C. Whole-cell lysates were prepared with NP-40 buffer. Cell extracts were incubated at 4°C with 20 μL of packed streptavidin resin (Amersham); the resin was washed, and associated proteins were analyzed by Western blot using the ECL Advance chemiluminescence kit (Amersham). The immunoprecipitation of Flag-Cdt2 shown in Figure 5D was performed from a chromatin fraction of HeLa cells, prepared essentially as in Sarcinella et al. (2007), except that micrococcal nuclease (Sigma) treatment of nuclei was for 2 min at 37°C, and the nuclear extract was further prepared by osmotic lysis of nuclei. Determining the intensity of Western blot protein bands was performed with unsaturated images, and involved subtracting the mean background signal and standardizing to the level of α-tubulin in the same sample.

siRNA and cell cycle analysis

The following ON-TARGETplus siRNAs were generated by Dharmacon (overhanging bases are in lowercase): siGFP, GCUGACCCUGAAGUUCAUCUGdTdT; siCdt2(#2), GAAU UAUACUGCUUAUCGAuu; siCdt2(#3), GCGCUUGAAUAGA GGCUUAuu; siCdt2(#4), GUCAAGACCUGGCCUAGUAuu; siCdt2(#5), ACUCCUACGUUCUCUAUUAuu; siSkp2(#1), GC AUGUACAGGUGGCUGUUuu; siSkp2(#2), UGUCAAUACU CUCGCAAAAuu; siSkp2(#3), UCGGUGCUAUGAUAUAA UAuu; siSkp2(#4), GGUAUCGCCUAGCGUCUGAuu; siSkp2(#5), GGAUGUGACUGGUCGGUUGuu; sip21(#1), CUUCGACUU UGUCACCGAGuu; sip21(#2), AGACCAGCAUGACAGAUU Uuu; sip21(#3), CGACUGUGAUGCGCUAAUGuu; sip21(#4), CCUAAUCCGCCCACAGGAAuu; and sip21(#5), CGUCAG AACCCAUGCGGCAuu. The Cdt2(#1) siRNA, CAAUGGACA CCAGAACUCUACCUUUdTdT, was generated by Invitrogen. ON-TARGETplus SMARTpools of siRNAs (comprised of individual siRNAs #2–#5) were used for Cdt2, Skp2, and p21 knockdowns in all experiments except Figure 3E, which used individual Cdt2(#1) siRNA. Both pooled and individual siRNAs were used for the experiments in Figures. 3B, ​,6,6, and ​and7A,7A, for which the individual siRNAs gave results that were similar to the pooled siRNAs. siRNA SMARTpools were transfected once at 100 nM (individual siRNAs in each pool were at 25 nM), and individual (nonpool) siRNAs were transfected two or three times sequentially at 100 nM. siRNAs were transfected with Oligofectamine (Invitrogen) according to the manufacturer’s instructions. HeLa cells were analyzed 60 h after transfection. For flow cytometry to determine DNA content, HeLa cells were fixed with 70% ethanol and stained with 0.5 mg/mL propidium iodide and 20 μg/mL RNAse A (Sigma). Propidium iodide-stained cells were analyzed using a FACSCalibur flow cytometer (Beckton-Dickinson), and the cell cycle phase distributions were determined with ModFit software (Verity).

We are grateful to S. Angers, B.E. Clurman, J. Jin, M. Pagano, Y. Xiong, and H. Zhang for reagents; J.G. Nelson and the UGA Flow Cytometry facility for DNA content analysis; the Caenorhabditis Genetics Center for strains; and members of the Kipreos laboratory for critical reading of the manuscript. This work was supported by grants from the NIH National Institute of General Medical Sciences (NIGMS) (R01GM055297 and R01GM074212) to E.T.K.