Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication

Abstract

Cdc10-dependent transcript 1 (Cdt1) is an essential DNA replication protein whose accumulation at the end of the cell cycle promotes the formation of pre-replicative complexes and replication in the next cell cycle. Geminin is thought to be involved in licensing replication by promoting the accumulation of Cdt1 in mitosis, because decreasing the Geminin levels prevents Cdt1 accumulation and impairs DNA replication. Geminin is known to inhibit Cdt1 function; its depletion during G2 leads to DNA rereplication and checkpoint activation. Here we show that, despite rapid Cdt1 protein turnover in G2 phase, Geminin promotes Cdt1 accumulation by increasing its RNA and protein levels in the unperturbed cell cycle. Therefore, Geminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents its rereplication.

Fig. 1.

Cdt1 is abundant in G2 phase but is highly unstable. (A) Schematic of the experiment. (B) U2OS cells were synchronized by double thymidine treatment and then were released for the indicated times. At 8 h after release (i.e., at the end of S phase) cells were left untreated or were treated with either cycloheximide (CHX) or carbobenzoxy-Leu-Leu-leucinal (MG132). Western blotting analysis for the indicated proteins is shown. APC2 protein was used as loading control. As, nonsynchronized cells; Thy, cells arrested in S phase. (C) Schematic of the experiment. (D) U2OS cells were synchronized by double thymidine treatment and then were released for the indicated times. At 11 h after release (i.e., in early G2 phase) cells were left untreated or were treated with either cycloheximide or MG132. Western blotting analysis for the indicated proteins is shown. APC2 protein was used as loading control.

Fig. 2.

Geminin depletion causes decrease of Cdt1 protein in the presence of DNA synthesis inhibitors. (A) U2OS cells were synchronized by double thymidine treatment and then were released from S-phase block for 2 h. Cells then were transfected with Geminin siRNA or mock-transfected before they were treated with nocodazole. Aphidicolin was added at the S/G2 transition where indicated. Cells then were incubated for another 9 h before the adherent cells (end-G2 cells) or the shake-off cells (mitosis) were harvested (see also Fig. S1A). Western blotting analysis for the indicated proteins is shown. APC2 protein was used as a loading control. The slower mobility of Cdc27 in mitosis is shown as a marker of good separation between G2 and mitotic cells (25). Cdt1 mobility decreases in mitosis, as previously reported (4). Spaces are intentionally left between pairs of bands to indicate that the images shown are taken from distant parts of the same gel. (B) FACS analyses were performed with the samples shown in A to control for good synchronization in G2 phase after depletion of Geminin protein. (C) U2OS cells were synchronized by double thymidine treatment and then were released from S-phase block. Ten hours later (at S/G2 transition), cells were treated with control siRNA or Geminin siRNA. Nocodazole and optional third thymidine were also added at this time. Cells then were incubated for an additional14 h before the mitotic cells were harvested by manual shake-off. Western blotting analysis for the indicated proteins is shown. APC2 protein was used as a loading control.

Fig. 3.

Geminin positively regulates the RNA levels of Cdt1. (A) Geminin depletion decreases Cdt1 mRNA. U2OS cells were transfected with siRNA for Geminin or Cdt1 or were mock-transfected (Contr.) and incubated for 48 h. After cell lysis, mRNA for Cdt1 was quantified by quantitative RT-PCR, and quantifications were represented in a bar chart. Samples were normalized to β-actin. The control (mock-transfected) sample was arbitrarily given a value of 1. SDs are shown. (B) Geminin depletion decreases Cdt1 mRNA in G2 phase and mitosis. U2OS cells were treated with thymidine and released for 5 h. Cells then were transfected with siRNA for Geminin or Cdt1 or were mock-transfected, as previously reported (4) (see also Fig. S2A). Nocodazole was added 4 h after transfections, and cells were incubated for another 16 h before the adherent cells (end-G2 cells) or the shake-off cells (mitotic cells) were harvested. Cdt1 mRNA levels were evaluated by quantitative RT-PCR, and quantification is shown as bar charts. Control (mock-transfected) samples for G2 and mitotic cells were arbitrarily assigned a value of 1. SDs are shown. Samples were normalized to β-actin. (C) Western blotting analysis for the samples in B is shown to evaluate protein levels of Cdt1 and Geminin. (D) Geminin regulates Cdt1 RNA levels. Normal U2OS cells or U2OS cells stably overexpressing full-length WT Geminin or a form of Geminin deleted of amino acids 112–118 (4) were used for this experiment. Cells were synchronized by thymidine treatment and were released for 5 h before transfection with Geminin siRNA or mock-transfection as indicated. Nocodazole was added 4 h after transfections, and cells were incubated for another 12 h before the adherent cells (end-G2 cells) or the shake-off cells (mitotic cells) were harvested. Cdt1 mRNA levels were evaluated by quantitative RT-PCR, and quantification is shown as bar charts. Control (mock-transfected) samples for G2 and mitotic cells are arbitrarily assigned a value of 1. SDs are shown. Samples are normalized to β-actin. (E) Cdt1 mRNA decreases in G2 phase. U2OS cells were synchronized as described in Fig. S2B. Time points were taken at early S phase, mid S phase, and G2 phase. Cdt1, Cdc6, and Mcm6 mRNA levels were evaluated by quantitative RT-PCR, and quantifications are shown as bar charts. The first time points (early S phase) are arbitrarily assigned a value of 1. SDs are shown. Samples are normalized to β-actin. (F) Geminin depletion decreases Cdt1 mRNA in S and G2 phases. Some plates of U2OS cells were treated in parallel with siRNA for Geminin in the experiment shown in E (see also Fig. S2B). Cells were taken at early S phase and G2 phase, and Cdt1, Cdc6, and Mcm6 mRNA levels then were evaluated by quantitative RT-PCR. The first time points (in early S phase) are arbitrarily assigned a value of 1. SDs are shown. Samples are normalized to β-actin. (G) Geminin depletion has a little effect on stability of Cdt1 RNA. Some plates of U2OS cells also were treated in parallel with Actinomycin D in early S phase in the experiment shown in E and F (see also Fig. S2B). The treatment with the transcription inhibitor Actinomycin D was as long as 10 h. Cells were taken at early S phase, mid S phase, and G2 phase. Cdt1 mRNA levels then were evaluated by quantitative RT-PCR. The first time points (in early S phase) are arbitrarily assigned a value of 1. SDs are shown. Samples are normalized to β-actin.

Fig. 4.

Geminin positively regulates the stability of Cdt1 protein. (A) Geminin increase amplifies Cdt1 protein levels. Western blotting analysis for samples of cells treated as in Fig. 3D is shown to evaluate a positive correlation between Geminin and Cdt1 protein levels. Note that both the WT and mutant Geminin are Myc-tagged and mutated in the target sequence for siRNA, and for this reason the exogenous proteins are not affected by siRNA. (B) Geminin increase amplifies Cdt1 protein levels. HeLa cells were transfected with a vector expressing a stable version of Geminin mutated in the destruction box (4) or control. After synchronization in mitosis, cells were released in G1 phase for 4 h before treatment with cycloheximide (CHX) for the indicated times. Western blotting analysis for the indicated proteins is shown. (C) Geminin depletion decreases the stability of Cdt1 protein. U2OS cells treated with siRNA for Geminin or control were synchronized in mitosis and treated with cycloheximide for the indicated times. Western blotting analyses for Cdt1, Geminin, and Mcm6 are shown. To facilitate the comparison of Cdt1 half-lives in control cells and cells treated with siRNA for Geminin, the different amounts of total protein loaded on gel are represented by asterisk size; that is, the total protein loaded on gel in lanes marked by large asterisks is 3.5-fold the total protein in the lanes marked by small asterisks. (D) Geminin depletion decreases the stability of Cdt1 protein. U2OS cells treated with siRNA for Geminin and control cells were synchronized in mitosis or end-G2 phase and treated with puromycin (a protein-synthesis inhibitor) for the indicated times. Western blotting analyses for the indicated proteins are shown. To facilitate the comparison of Cdt1 half-lives in control cells and cells treated with siRNA for Geminin, the different amounts of total protein loaded on gel are represented by asterisk size; that is, total protein loaded on gel in lanes marked by large asterisks is 3.5-fold the total protein in the lanes marked by small asterisks. Ponceau staining of the nitrocellulose membrane used for the Western blot analysis is shown to control for total protein loaded in each lane of the gel.

Fig. 5.

Dual regulation of Cdt1 in G2 phase. Geminin inhibits and amplifies Cdt1 protein during the G2 phase of the cell cycle. In this way Geminin prevents DNA rereplication during the same G2 phase and promotes the timely onset of DNA replication in the S phase of the next cell cycle.