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At least 19 recordsLinked to original sources
PubMed2001

The Drosophila Geminin homolog: roles for Geminin in limiting DNA replication, in anaphase and in neurogenesis.

We have identified a Drosophila homolog of the DNA replication initiation inhibitor Geminin (Dm geminin) and show that it has all of the properties of Xenopus and human Geminin. During Drosophila development, Dm Geminin is present in cycling cells; protein accumulates during S phase and is degraded at the metaphase to anaphase transition. Overexpression of Dm geminin in embryos inhibits DNA replication, but cells enter mitosis arresting in metaphase, as in dup (cdt1) mutants, and undergo apoptosis. Overexpression of Dm Geminin also induces ectopic neural differentiation. Dm geminin mutant embryos exhibit anaphase defects at cycle 16 and increased numbers of S phase cells later in embryogenesis. In a partially female-sterile Dm geminin mutant, excessive DNA amplification in the ovarian follicle cells is observed. Our data suggest roles for Dm Geminin in limiting DNA replication, in anaphase and in neural differentiation.

Amino Acid Sequence↗
PubMed2006

Geminin is bound to chromatin in G2/M phase to promote proper cytokinesis.

Previous studies suggested that geminin plays a vital role in both origin assembly and DNA re-replication during S-phase; however, no data to support a role for geminin in G2/M cells have been described. Here it is shown that in G2/M-phase, geminin participates in the promotion of proper cytokinesis. This claim can be supported through a series of observations. First, geminin in G2/M is loaded onto chromatin after it is tyrosine phosphorylated. It is unlike S-phase geminin that resides in the nuclear soluble fraction, where it is exclusively S/T phosphorylated. Secondly, on chromatin, geminin gets S/T phosphorylated in late G1; this modification causes the release of geminin from the chromatin. Cyclins bind and phosphorylate geminin in a sequential, cell cycle-dependent manner. These modifications correlated well with geminin departure from the chromatin. This suggests that cyclin functions to either release geminin from chromatin or at least keep it at bay until late S-phase. Thirdly, depletion of geminin from a diploid mammary epithelial cell line (HME) causes cells to arrest in late G2/M-phase. Massive serine-10 phosphorylated histone H3 staining and survivin localization to mid-body were observed; this suggests that they could be arrested in either mitosis or at cytokinesis. Finally, while in the absence of geminin, cyclin B1, chk1 and cdc7 are all over expressed. This paper will demonstrate that only cdc7 is important in maintaining the cytokinesis arrest in the absence of geminin. Only double depletion of geminin and cdc7 induce apoptosis. Our results taken together show, for the first time, that phosphorylation-induction activates oscillation of geminin between both nuclear soluble and chromatin compartments. Chromatin-bound geminin species functions to initiate or maintain proper cytokineses. In the absence of geminin, cells arrest in cytokinesis; this defines a novel checkpoint, monitored by cdc7, rather than cyclin B1 or chk1.

Cell Cycle↗
PubMed2004

Geminin is targeted for repression by the retinoblastoma tumor suppressor pathway through intragenic E2F sites.

The geminin protein is a critical regulator of DNA replication. It functions to control replication fidelity by blocking the assembly of prereplication complexes in the S and G(2) phases of the cell cycle. Geminin protein levels, which are low in G(0)/G(1) and increase at the G(1)/S transition, are controlled through coordinate transcriptional and proteolytic regulation. Here we show that geminin is regulated transcriptionally by the retinoblastoma tumor suppressor (RB)/E2F pathway. Initially, we observed that the activation of RB led to the repression of geminin transcription. Conversely, Rb-null mouse embryonic fibroblasts have enhanced the expression of geminin relative to wild type mouse embryonic fibroblasts. Similarly, an acute loss of Rb in mouse adult fibroblasts deregulated geminin RNA and protein levels. To delineate the responsible regulatory motifs, luciferase reporter constructs containing fragments of the geminin promoter were generated. An analysis of the critical regulatory cis-acting elements in the geminin promoter indicated that intragenic E2F sites down-stream of the first exon were responsible for RB-mediated repression of geminin. The direct analysis of the endogenous geminin promoter revealed that these intragenic E2F sites are occupied by E2F proteins, and the mutation of these sites eliminates responsiveness to RB. Together, these data link the expression of geminin to the RB/E2F pathway and represent the first promoter analysis of this important regulator of DNA replication.

Animals↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2006

Geminin prevents rereplication during xenopus development.

To maintain a stable genome, it is essential that replication origins fire only once per cell cycle. The protein Geminin is thought to prevent a second round of DNA replication by inhibiting the essential replication factor Cdt1. Geminin also affects the development of several different organs by binding and inhibiting transcription factors and chromatin-remodeling proteins. It is not known if the defects in Geminin-deficient organisms are due to overreplication or to effects on cell differentiation. We previously reported that Geminin depletion in Xenopus causes early embryonic lethality due to a Chk1-dependent G(2) cell cycle arrest just after the midblastula transition. Here we report that expressing a non-Geminin-binding Cdt1 mutant in Xenopus embryos exactly reproduces the phenotype of geminin depletion. Expressing the same mutant in replication extracts induces a partial second round of DNA replication within a single S phase. We conclude that Geminin is required to suppress a second round of DNA replication in vivo and that the phenotype of Geminin-depleted Xenopus embryos is caused by abnormal Cdt1 regulation. Expressing a nondegradable Cdt1 mutant in embryos also reproduces the Geminin-deficient phenotype. In cell extracts, the nondegradable mutant has no effect by itself but augments the amount of rereplication observed when Geminin is depleted. We conclude that Cdt1 is regulated both by Geminin binding and by degradation.

Animals↗
PubMed2004

Cdt1 and geminin are down-regulated upon cell cycle exit and are over-expressed in cancer-derived cell lines.

Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 was shown to predispose cells for malignant transformation. We show here that Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3-6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. Our data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. We show that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and that the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. Our data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells.

Animals↗
PubMed2002

Control of DNA replication and chromosome ploidy by geminin and cyclin A.

Alteration of the control of DNA replication and mitosis is considered to be a major cause of genome instability. To investigate the mechanism that controls DNA replication and genome stability, we used the RNA silencing-interference technique (RNAi) to eliminate the Drosophila geminin homologue from Schneider D2 (SD2) cells. Silencing of geminin by RNAi in SD2 cells leads to the cessation of mitosis and asynchronous overreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content. The effect of geminin deficiency is completely suppressed by cosilencing of Double parked (Dup), the Drosophila homologue of Cdt1, a replication factor to which geminin binds. The geminin deficiency-induced phenotype is also partially suppressed by coablation of Chk1/Grapes, indicating the involvement of Chk1/Grapes in the checkpoint control in response to overreplication. We found that the silencing of cyclin A, but not of cyclin B, also promotes the formation of a giant nucleus and overreplication. However, in contrast to the effect of geminin knockout, cyclin A deficiency leads to the complete duplication of the genome from 4N to 8N. We observed that the silencing of geminin causes rapid downregulation of Cdt1/Dup, which may contribute to the observed partial overreplication in geminin-deficient cells. Analysis of cyclin A and geminin double knockout suggests that the effect of cyclin A deficiency is dominant over that of geminin deficiency for cell cycle arrest and overreplication. Together, our studies indicate that both cyclin A and geminin are required for the suppression of overreplication and for genome stability in Drosophila cells.

Animals↗
PubMed2004

Geminin predicts adverse clinical outcome in breast cancer by reflecting cell-cycle progression.

Geminin inhibits DNA replication by preventing Cdt1 from loading minichromosome maintenance (MCM) proteins onto DNA. The present study has investigated whether the frequency of geminin expression predicts clinical outcome in breast cancer. Immunohistochemistry was used first to examine geminin expression in normal and malignant breast tissue (n = 67). Correlations with cell-cycle parameters, pathological features, and clinical outcome were then determined using an invasive breast carcinoma tissue microarray (n = 165). Breast carcinomas were scanned for mutations (n = 61) and copy number imbalances (n = 241) of the geminin gene. Finally, the cell cycle distribution of geminin in breast cancer cells was investigated in vivo and in vitro. Despite a putative tumour suppressor function, it was found that increased geminin expression is a powerful independent indicator of adverse prognosis in invasive breast cancer. Both poor overall survival (p = 0.0002) and the development of distant metastases (p = 0.005) are predicted by high geminin expression, which performs better in this patient cohort than traditional factors currently used to determine prognosis and appropriate therapy. No mutations or deletions of the geminin gene and no evidence that a high frequency of protein expression is related to gene amplification were found. It is shown that geminin is expressed from S to M phase in breast carcinoma tissue and cell lines, disappearing at the metaphase--anaphase transition. While MCM proteins identify all non-quiescent cells, geminin identifies the sub-fraction that have entered S phase, but not exited mitosis, thereby indicating the rate of cell-cycle progression. It is suggested that this explains its unexpected value as a prognostic marker in breast cancer.

Adult↗
PubMed2006

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.

Animals↗
PubMed1998

Geminin, a neuralizing molecule that demarcates the future neural plate at the onset of gastrulation.

In an expression cloning screen in Xenopus embryos, we identified a gene that when overexpressed expanded the neural plate at the expense of adjacent neural crest and epidermis. This gene, which we named geminin, had no sequence similarity to known gene families. We later discovered that geminin's neuralizing domain was part of a bifunctional protein whose C-terminal coiled-coil domain may play a role in regulating DNA replication. We report here on the neuralizing function of geminin. The localization, effect of misexpression and activity of a dominant negative geminin suggest that the product of this gene has an essential early role in specifying neural cell fate in vertebrates. Maternal geminin mRNA is found throughout the animal hemisphere from oocyte through late blastula. At the early gastrula, however, expression is restricted to a dorsal ectodermal territory that prefigures the neural plate. Misexpression of geminin in gastrula ectoderm suppresses BMP4 expression and converts prospective epidermis into neural tissue. In ectodermal explants, geminin induces expression of the early proneural gene neurogenin-related 1 although not itself being induced by that gene. Later, embryos expressing geminin have an expanded dorsal neural territory and ventral ectoderm is converted to neurons. A putative dominant negative geminin lacking the neuralizing domain suppresses neural differentiation and, when misexpressed dorsally, produces islands of epidermal gene expression within the neurectodermal territory, effects that are rescued by coexpression of the full-length molecule. Taken together, these data indicate that geminin plays an early role in establishing a neural domain during gastrulation.

Amino Acid Sequence↗
PubMed2005

Increased expression of geminin stimulates the growth of mammary epithelial cells and is a frequent event in human tumors.

Geminin is a potent inhibitor of origin assembly and re-replication in multicellular eukaryotes and is a negative regulator of DNA replication during the cell cycle. Thus, it was proposed as an inhibitor of cell proliferation and as a potential tumor suppressor gene. However, the protein was found specifically expressed in proliferating lymphocytes and epithelial cells and up-regulated in several malignancies. Therefore, geminin is now regarded as an oncogene but its role in tumor development remains unknown. In this study, we evaluated by Western blot analysis the expression of geminin in a series of human cancer cell lines of various histogenetic origin and in a series of human primary colon, rectal, and breast cancers. Expression of geminin was variable in different cell lines and not related to the expression level of the corresponding mRNA. Moreover, geminin was expressed at higher level in 56% and 58% of colon and rectal cancers, respectively, compared with the corresponding adjacent normal mucosa. A high expression of geminin was also detected by immunohistochemistry in 60% of human primary breast cancers. We also transfected a full-length geminin cDNA in a human non-tumorigenic and a cancer breast cell lines and obtained derivatives expressing high levels of the protein. Geminin overexpression stimulated cell cycle progression and proliferation in both normal and cancer cells and increased the anchorage--independent growth of breast cancer cells. These results demonstrate that expression of geminin is frequently deregulated in tumor cells and might play an important role in the regulation of cell growth in both normal and malignant cells.

Biomarkers, Tumor↗
PubMed2002

Geminin deficiency causes a Chk1-dependent G2 arrest in Xenopus.

Geminin is an unstable inhibitor of DNA replication that gets destroyed at the metaphase/anaphase transition. The biological function of geminin has been difficult to determine because it is not homologous to a characterized protein and has pleiotropic effects when overexpressed. Geminin is thought to prevent a second round of initiation during S or G2 phase. In some assays, geminin induces uncommitted embryonic cells to differentiate as neurons. In this study, geminin was eliminated from developing Xenopus embryos by using antisense techniques. Geminin-deficient embryos show a novel and unusual phenotype: they complete the early cleavage divisions normally but arrest in G2 phase immediately after the midblastula transition. The arrest requires Chk1, the effector kinase of the DNA replication/DNA damage checkpoint pathway. The results indicate that geminin has an essential function and that loss of this function prevents entry into mitosis by a Chk1-dependent mechanism. Geminin may be required to maintain the structural integrity of the genome or it may directly down-regulate Chk1 activity. The data also show that during the embryonic cell cycles, rereplication is almost entirely prevented by geminin-independent mechanisms.

Animals↗
PubMed2005

Repression of DNA replication licensing in quiescence is independent of geminin and may define the cell cycle state of progenitor cells.

The DNA replication (or origin) licensing machinery ensures precise duplication of the genome and contributes to the regulation of proliferative capacity in metazoa. Using an in vitro fibroblast model system coupled to a cell-free DNA replication assay, we have studied regulation of the origin licensing pathway during exit from and re-entry into the mitotic cell cycle. We show that in the quiescent state (G0) loss of proliferative capacity is achieved in part through down-regulation of the replication licensing factors Cdc6 and Mcm2-7. The origin licensing repressor geminin is absent in quiescent fibroblasts, suggesting that this powerful inhibitor of the licensing machinery is not required to suppress proliferative capacity in G0. Geminin expression is induced at a late stage in the G0-S transition post pre-RC assembly. Ectopic geminin can block re-acquisition of DNA replication competence during re-entry into the cell cycle, indicating that geminin levels must be tightly down-regulated for escape from G0. Analysis of geminin levels in thyroid shows that geminin expression is suppressed in anatomical compartments/tissues harbouring quiescent cells, confirming our in vitro data. Spatio-temporal control of geminin expression may therefore be of particular relevance for multi-potential stem cells which cycle infrequently. In support of this hypothesis, we have identified a unique population of cells in the putative stem cell niche of intestinal epithelium that are unlicensed and lack geminin expression, a prerequisite for successful re-entry into cycle. Our data argue that the prolonged cell cycle times observed for intestinal stem cells could be due to exit of progenitor cells from cycle into an unlicensed "out-of-cycle" state, a powerful mechanism by which rapidly proliferating tissues may resist genotoxic insult.

3T3 Cells↗
PubMed2004

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.

Adenosine Triphosphate↗
PubMed2002

Expression of geminin as a marker of cell proliferation in normal tissues and malignancies.

Geminin interacts with a DNA replication initiation factor, Cdt1p, to suppress initiation of DNA replication in a Xenopus egg extract based cell-free system, leading to the expectation that the protein acts as an inhibitor of cell proliferation. Immunohistochemistry and immunoblotting for geminin, however, reveals that the protein is expressed specifically in proliferating lymphocytes and epithelial cells. This pattern is in contrast to the expression of a bona fide cell cycle inhibitor like p21/WAF1 that is specifically expressed in quiescent cells. Geminin is widely expressed in several malignancies and the number of geminin-expressing cells is directly proportional to the cell proliferation index as measured by Ki-67 expression. Therefore, instead of being a suppressor of cell proliferation, geminin expression is positively correlated with cell proliferation. Consistent with this observation, transient overexpression of wild-type geminin in cancer cells in culture did not produce a cell cycle block. A point mutation in the destruction box of geminin, however, results in a protein that is stabilized in G(1) and capable of arresting cells at the G(1)-S transition.

Biomarkers↗
PubMed2002

Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import.

During late mitosis and early interphase, origins of replication become "licensed" for DNA replication by loading Mcm2-7 complexes. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase. Here, we show that recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Since the initiation of replication at licensed origins depends on nuclear assembly, our results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle.

Active Transport, Cell Nucleus↗
PubMed2004

The cell-cycle regulator geminin inhibits Hox function through direct and polycomb-mediated interactions.

Embryonic development is tightly controlled. The clustered genes of the Hox family of homeobox proteins play an important part in regulating this development and also proliferation. They specify embryonic structures along the body axis, and are associated with normal and malignant cell growth. The cell-cycle regulator geminin controls replication by binding to the licensing factor Cdt1, and is involved in neural differentiation. Here, we show that murine geminin associates transiently with members of the Hox-repressing polycomb complex, with the chromatin of Hox regulatory DNA elements and with Hox proteins. Gain- and loss-of-function experiments in the chick neural tube demonstrate that geminin modulates the anterior boundary of Hoxb9 transcription, which suggests a polycomb-like activity for geminin. The interaction between geminin and Hox proteins prevents Hox proteins from binding to DNA, inhibits Hox-dependent transcriptional activation of reporter and endogenous downstream target genes, and displaces Cdt1 from its complex with geminin. By establishing competitive regulation, geminin functions as a coordinator of developmental and proliferative control.

Amino Acid Sequence↗
PubMed2004

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.

Animals↗