Life without Geminin

The interplay of proliferation and differentiation is essential for normal development and organogenesis. Geminin is a cell cycle regulator which controls licensing of origins for DNA replication, safeguarding genomic stability. Geminin has also been shown to regulate cellular decisions of self-renewal versus commitment of neuronal progenitor cells. We discuss here our recent analysis of mice with conditional inactivation of the Geminin gene in the immune system. Our data indicate that Geminin is not indispensable for every cell division: in the absence of Geminin, development of progenitor T-cells appears largely unaffected. In contrast, rapid cell divisions, taking place in vitro upon TCR receptor activation or in vivo during homeostatic proliferation, are defective.Key words: Geminin, Cdt1, stem cells, licensing, self-renewal, differentiation, cell cycle duration     

The Geminin and Idas Coiled Coils Preferentially Form a Heterodimer That Inhibits Geminin Function in DNA Replication Licensing

Geminin is an important regulator of proliferation and differentiation in metazoans, which predominantly inhibits the DNA replication licensing factor Cdt1, preventing genome over-replication. We show that Geminin preferentially forms stable coiled-coil heterodimers with its homologue, Idas. In contrast to Idas-Geminin heterodimers, Idas homodimers are thermodynamically unstable and are unlikely to exist as a stable macromolecule under physiological conditions. The crystal structure of the homology regions of Idas in complex with Geminin showed a tight head-to-head heterodimeric coiled-coil. This Idas-Geminin heterodimer binds Cdt1 less strongly than Geminin-Geminin, still with high affinity (∼30 nm), but with notably different thermodynamic properties. Consistently, in Xenopus egg extracts, Idas-Geminin is less active in licensing inhibition compared with a Geminin-Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression. The properties of the Idas-Geminin complex suggest it as the functional form of Idas and provide a possible mechanism to modulate Geminin activity.     

Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of geminin through its N-terminal region

Licensing of replication origins is carefully regulated in a cell cycle to maintain genome integrity. Using an in vivo ubiquitination assay and temperature-sensitive cell lines we demonstrate that Cdt1 in mammalian cells is degraded through ubiquitin-dependent proteolysis in S-phase. siRNA experiments for Geminin indicate that Cdt1 is degraded in the absence of Geminin. The N terminus of Cdt1 is required for its nuclear localization, associates with cyclin A, but is dispensable for the association of Cdt1 with Geminin in cells. This region is responsible for proteolysis of Cdt1 in S-phase. On the other hand, the N terminus-truncated Cdt1 is stable in S-phase, and associates with the licensing inhibitor, Geminin. High level expression of this form of Cdt1 brings about cells having higher DNA content. Proteasome inhibitors stabilize Cdt1 in S-phase, and the protein is detected in the nucleus in a complex with Geminin. This form of Cdt1 associates with chromatin as tightly as that of G1-cells, when no Geminin is detected. Our data show that proteolysis and Geminin binding independently inactivate Cdt1 after the onset of S-phase to prevent re-replication.     

Protein kinase CK2 phosphorylates the cell cycle regulatory protein Geminin

Geminin contributes to cell cycle regulation by a timely inhibition of Cdt1p, the loading factor required for the assembly of pre-replication complexes. Geminin is expressed during S and G2 phase of the HeLa cell cycle and phosphorylated soon after its synthesis. We show here that Geminin is an excellent substrate for protein kinase CK2 in vitro; and that the highly specific CK2 inhibitor tetrabromobenzotriazole (TBB) blocks the phosphorylation of Geminin in HeLa protein extracts and HeLa cells in vivo. The sites of CK2 phosphorylation are located in the carboxyterminal region of Geminin, which carries several consensus sequence motifs for CK2. We also show that a minor phosphorylating activity in protein extracts can be attributed to glycogen synthase kinase 3 (GSK3), which most likely targets a central peptide in Geminin. Treatment of HeLa cells with TBB does not interfere with the ability of Geminin to interact with the loading factor Cdt1.     

Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin

To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.     

Geminin has dimerization, Cdt1-binding, and destruction domains that are required for biological activity

Geminin is an unstable regulatory protein that affects both cell division and cell differentiation. Geminin inhibits a second round of DNA synthesis during S and G(2) phase by binding the essential replication protein Cdt1. Geminin is also required for entry into mitosis, either by preventing replication abnormalities or by down-regulating the checkpoint kinase Chk1. Geminin overexpression during embryonic development induces ectopic neural tissue, inhibits eye formation, and perturbs the segmental patterning of the embryo. In order to define the structural and functional domains of the geminin protein, we generated over 40 missense and deletion mutations and tested their phenotypes in biological and biochemical assays. We find that geminin self-associates through the coiled-coil domain to form dimers and that dimerization is required for activity. Geminin contains a typical bipartite nuclear localization signal that is also required for its destruction during mitosis. Nondegradable mutants of geminin interfere with DNA replication in succeeding cell cycles. Geminin's Cdt1-binding domain lies immediately adjacent to the dimerization domain and overlaps it. We constructed two nonbinding mutants in this domain and found that they neither inhibited replication nor permitted entry into mitosis, indicating that this domain is necessary for both activities. We identified several missense mutations in geminin's Cdt1 binding domain that were deficient in their ability to inhibit replication yet were still able to allow mitotic entry, suggesting that these are separate functions of geminin.     

Cdt1 Interactions in the Licensing Process: A Model for Dynamic Spatio-temporal Control of Licensing

Within each cell cycle, a cell must ensure that the processes of selection of replication origins (licensing) and initiation of DNA replication are well coordinated to prevent re-initiation of DNA replication from the same DNA segment during the same cell cycle. This is achieved by restricting the licensing process to G1 phase when the prereplicative complexes (preRCs) are assembled onto the origin DNA, while DNA replication is initiated only during S phase when de novo preRC assembly is blocked. Cdt1 is an important member of the preRC complex and its tight regulation through ubiquitin-dependent proteolysis and binding to its inhibitor Geminin ensure that Cdt1 will only be present in G1 phase, preventing relicensing of replication origins. We have recently reported that Cdt1 associates with chromatin in a dynamic way and recruits its inhibitor Geminin onto chromatin in vivo. Here we discuss how these dynamic Cdt1-chromatin interactions and the local recruitment of Geminin onto origins of replication by Cdt1 may provide a tight control of the licensing process in time and in space.     

Depletion of licensing inhibitor geminin causes centrosome overduplication and mitotic defects

Metazoans limit origin firing to once per cell cycle by oscillations in cyclin-dependent kinases and the replication licensing inhibitor geminin. Geminin inhibits pre-replication complex assembly by preventing Cdt1 from recruiting the minichromosome maintenance proteins to chromatin. Geminin depletion results in genomic over-replication in Drosophila and human cell lines. Here, we show that loss of geminin affects other cell cycle-dependent events in addition to DNA replication. Geminin inactivation causes centrosome overduplication without passage through mitosis in human normal and cancer cells. Centrosomes are microtubule-organizing centres that are duplicated during S phase and have an important role in the fidelity of chromosome transmission by nucleating the mitotic spindle. Consistent with this, geminin-depleted cells show multiple mitotic defects, including multipolar spindles, when driven into mitosis by checkpoint abrogation. These results show that the consequences of geminin loss exceed its immediate role in DNA replication and extend to promoting chromosome mis-segregation in mitosis.     

Geminin Stabilizes Cdt1 during Meiosis in Xenopus Oocytes

During the mitotic cell cycle, Geminin can act both as a promoter and inhibitor of initiation of DNA replication. As a promoter, Geminin stabilizes Cdt1 and facilitates its accumulation leading to the assembly of the pre-replication complex on DNA. As an inhibitor, Geminin prevents Cdt1 from loading the mini-chromosome maintenance complex onto pre-replication complexes in late S, G₂, and M phases. Here we show that during meiosis Geminin functions as a stabilizer of Cdt1 promoting its accumulation for the early division cycles of the embryo. Depletion of Geminin in Xenopus immature oocytes leads to a decrease of Cdt1 protein levels during maturation and after activation of these oocytes. Injection of exogenous recombinant Geminin into the depleted oocytes rescues Cdt1 levels demonstrating that Geminin stabilizes Cdt1 during meiosis and after fertilization. Furthermore, Geminin-depleted oocytes did not replicate their DNA after meiosis I indicating that Geminin does not act as an inhibitor of initiation of DNA replication between meiosis I and meiosis II.In eukaryotes, initiation of DNA replication involves the formation and activation of the pre-replication complex (pre-RC)³ at the origins of replication. Pre-RCs are formed by the sequential binding of the origin recognition complex components, Cdc6, Cdt1, and mini-chromosome maintenance complex (MCM 2–7) proteins, to DNA. After loading the MCM complex, the pre-RCs are activated by S phase kinases (Dbf4-dependent kinase and Cdks) to initiate DNA replication (1). Replication of DNA, limited to only once per cell cycle, is critical to maintain genomic stability. Redundant mechanisms exist to ensure that DNA replication is tightly regulated during the cell cycle (1, 2). A small protein named Geminin has been shown to play a significant role in such regulatory mechanisms during mitosis (2–6). Geminin, a multifunctional 25-kDa protein, was first identified in a screen for proteins degraded during mitosis in Xenopus laevis egg extracts (7). Geminin is present in higher eukaryotes, but its presence in yeast has not yet been reported (7–10). Geminin plays a major role in regulating the function of Cdt1, one of the pre-RC components (8, 11–13). Numerous studies suggest that in higher eukaryotes the interaction between Geminin and Cdt1 is pivotal to restrict DNA replication to only once per cell cycle (6, 14–22). Furthermore, in Xenopus egg extracts, the Geminin/Cdt1 ratio seems to control the assembly of pre-RCs at replication origins and to determine whether the origins are licensed or not (23). The positive and negative roles of Geminin in origin licensing and DNA replication are made possible by their temporal separation during the cell cycle. Pre-RC formation occurs during late M and early G₁ phase, whereas pre-RC inhibition occurs from late S to mid M phase.As a positive regulator of DNA replication, Geminin has been shown to stabilize Cdt1. In human osteosarcoma cells, silencing of GEMININ expression limits CDT1 accumulation during mitosis and therefore the formation of pre-RCs in the subsequent cell cycle. This stabilizing effect is the result of a direct interaction between CDT1 and GEMININ preventing CDT1 ubiquitination and degradation (13). Similar findings were also recently observed in normal human cells and various cancer cells (24). However, in both human normal and tumor cells, the low level of CDT1, generated by the absence of GEMININ, did not always prevent cellular proliferation or re-replication of the genome (5, 24, 25). Therefore, one might question the importance of the role of GEMININ in stabilizing CDT1 in human cells. Beyond its role as a stabilizer of Cdt1 levels, Geminin has also been shown to participate directly in the formation of pre-RCs in Xenopus egg extracts. A complex between Cdt1 and Geminin binds to chromatin and supports pre-RC assembly. However, the recruitment of additional Geminin molecules to this complex on the chromatin blocks further pre-RC formation. These results indicate that the stoichiometry of Cdt1 and Geminin in this complex regulates its activity as a promoter or inhibitor of pre-RC assembly and DNA replication (23, 26). Several mechanisms have been shown to modulate the Geminin/Cdt1 balance on the chromatin. In Xenopus the binding of Cdt1 to the MCM9 protein seems to block the recruitment of an excess of Geminin to the chromatin and therefore favors pre-RC assembly (27). Similarly, the inactivation of Geminin by either ubiquitination or degradation also has a positive effect on pre-RC assembly (8, 11, 28–30). On the other hand, the replication-dependent degradation of Cdt1 has the opposite effect and prevents refiring of replication origins during S and G₂ phases of the mitotic cell cycle (18, 20, 31).Although the role of Geminin during mitosis has been extensively studied, not much is known about its function during meiosis. The expression pattern of Geminin during oocyte maturation is unclear. The presence of Geminin in immature stage VI Xenopus oocytes is controversial, but the protein is fully expressed in mature oocytes arrested in metaphase of meiosis II (7, 32). To form haploid gametes, DNA replication has to be inhibited between meiosis I (MI) and meiosis II (MII). In Xenopus oocytes, cyclin B-dependent kinase 1 (Cdk1) also known as maturation-promoting factor (MPF) plays a role in preventing DNA replication between the two meiotic divisions (33–36). Inhibition of Cdk1 activity between MI and MII leads to the formation of interphase nucleus and DNA replication. However, the role of Geminin in preventing DNA replication between meiotic divisions has not been tested so far. Finally, the possibility that Geminin stabilizes Cdt1 during meiosis and ensures its accumulation for the early embryonic divisions has not been formally examined.Here we show that the levels of Geminin and Cdt1 proteins increase significantly during meiosis in Xenopus oocytes and that the primary role of geminin is to promote the accumulation of Cdt1 and not to repress DNA replication between meiosis I and meiosis II. Depletion of Geminin in Xenopus immature oocytes does not lead to DNA replication after the first meiotic division but to a decrease in Cdt1 stability during the maturation and activation of these oocytes. Rescue of Cdt1 levels in these Geminin-depleted oocytes is achieved by injection of exogenous recombinant Geminin protein confirming the role of Geminin as a stabilizer of Cdt1 during meiosis and the early embryonic division cycles. These results provide further support for the idea that Geminin functions universally in stabilizing Cdt1. Although the stabilizing role of Geminin might not be its most important function in somatic cells, we show here that stabilizing Cdt1 is a dominant function for Geminin in Xenopus oocytes undergoing meiosis. This stabilizing role of Geminin is essential for the stockpiling of Cdt1 before fertilization that is required to sustain the rapid divisions of the early embryo.     

Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex

All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2～7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.     

A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus

Initiation of DNA synthesis involves the loading of the MCM2-7 helicase onto chromatin by Cdt1 (origin licensing). Geminin is thought to prevent relicensing by binding and inhibiting Cdt1. Here we show, using Xenopus egg extracts, that geminin binding to Cdt1 is not sufficient to block its activity and that a Cdt1-geminin complex licenses chromatin, but prevents rereplication, working as a molecular switch at replication origins. We demonstrate that geminin is recruited to chromatin already during licensing, while bulk geminin is recruited at the onset of S phase. A recombinant Cdt1-geminin complex binds chromatin, interacts with the MCM2-7 complex and licenses chromatin once per cell cycle. Accordingly, while recombinant Cdt1 induces rereplication in G1 or G2 and activates an ATM/ATR-dependent checkpoint, the Cdt1-geminin complex does not. We further demonstrate that the stoichiometry of the Cdt1-geminin complex regulates its activity. Our results suggest a model in which the MCM2-7 helicase is loaded onto chromatin by a Cdt1-geminin complex, which is inactivated upon origin firing by binding additional geminin. This origin inactivation reaction does not occur if only free Cdt1 is present on chromatin.     

Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis

Geminin is an unstable inhibitor of DNA replication that negatively regulates the licensing factor CDT1 and inhibits pre-replicative complex (pre-RC) formation in Xenopus egg extracts. Here we describe a novel function of Geminin. We demonstrate that human Geminin protects CDT1 from proteasome-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle. Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate that Geminin is both a negative and positive regulator of pre-RC formation in human cells, playing a positive role in allowing CDT1 accumulation in G2-M, and preventing relicensing of origins in S-G2.     

Life without geminin

The interplay of proliferation and differentiation is essential for normal development and organogenesis. Geminin is a cell cycle regulator which controls licensing of origins for DNA replication, safeguarding genomic stability. Geminin has also been shown to regulate cellular decisions of self-renewal versus commitment of neuronal progenitor cells. We discuss here our recent analysis of mice with conditional inactivation of the Geminin gene in the immune system. Our data indicate that Geminin is not indispensable for every cell division: in the absence of Geminin, development of progenitor T cells appears largely unaffected. In contrast, rapid cell divisions, taking place in vitro upon TCR receptor activation or in vivo during homeostatic proliferation, are defective.