Mouse <i>emi1</i>Has an Essential Function in Mitotic Progression during Early Embryogenesis

Lee, Ho; Lee, Jun Young; Oh, Sang Phil; Park, Hee Dong; Nam, Hyun Hee; Kim, Jin Man; Lim, Dae–Sik

doi:10.1128/mcb.00043-06

Cited by 45 publications

(33 citation statements)

References 39 publications

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“…Moreover, we found no obvious phenotype in salivary gland cells mutant for rca1. The result that Rca1 activity is required in mitotic but not in endoreplicating cells is consistent with the phenotype of Emi1-deficient mice, in which the mitotic cells of the inner cell mass cells die, whereas the endoreplicating trophoblast giant cells appear normal (Lee et al 2006). Therefore, CycE-Cdk2 is the only known APC/C-Fzr inhibitor in larval salivary glands.…”

Section: Cyce-cdk2 Regulates Apc/c-fzr Activity In Salivary Glandssupporting

confidence: 80%

The anaphase-promoting complex/cyclosome (APC/C) is required for rereplication control in endoreplication cycles

Zielke¹,

Querings²,

Rottig

et al. 2008

Genes Dev.

111

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Endoreplicating cells undergo multiple rounds of DNA replication leading to polyploidy or polyteny. Oscillation of Cyclin E (CycE)-dependent kinase activity is the main driving force in Drosophila endocycles. High levels of CycE-Cdk2 activity trigger S phase, while down-regulation of CycE-Cdk2 activity is crucial to allow licensing of replication origins. In mitotic cells relicensing in S phase is prevented by Geminin. Here we show that Geminin protein oscillates in endoreplicating salivary glands of Drosophila. Geminin levels are high in S phase, but drop once DNA replication has been completed. DNA licensing is coupled to mitosis through the action of the anaphase-promoting complex/cyclosome (APC/C). We demonstrate that, even though endoreplicating cells never enter mitosis, APC/C activity is required in endoreplicating cells to mediate Geminin oscillation. Down-regulation of APC/C activity results in stabilization of Geminin protein and blocks endocycle progression. Geminin is only abundant in cells with high CycE-Cdk2 activity, suggesting that APC/C-Fzr activity is periodically inhibited by CycE-Cdk2, to prevent relicensing in S-phase cells.[Keywords: Anaphase-promoting complex; cell cycle; DNA licensing; Drosophila; endoreplication] Supplemental material is available at http://www.genesdev.org. The precise duplication of the genome is crucial for the survival of any organism. In multicellular organisms genome instability potentially gives rise to cancer and thus compromises the life of the whole organism. To maintain the integrity of the genome, DNA replication, and mitosis must be coordinated during cell division cycles so that DNA replication occurs only once per cycle and mitosis only after complete duplication of the genome. To avoid rereplication events, a network of proteins ensures that cells acquire the license for DNA replication only in a specific phase of the cell cycle. DNA licensing involves the formation of prereplication complexes (pre-RC), which can only assemble during late mitosis and G1 (for review, see Bell and Dutta 2002;Blow and Dutta 2005). Pre-RC assembly on replication origins involves a variety of conserved components including the ORC1-6 complex, Cdc6, Cdt1/Double-parked, and the MCM2-7 helicase. Once MCM2-7 proteins are loaded onto the DNA, licensing is completed and the replication origins are ready for firing (for review, see Bell and Dutta 2002;Blow and Dutta 2005).Several mechanisms ensure that replication origins are fired only once during a cell cycle (for review, see Machida et al. 2005). Of particular importance is the temporal separation of pre-RC formation and origin firing. Pre-RC assembly occurs only in G1 because it depends on low Cdk activity while high Cdk activity is required for origin firing. The increase in Cdk activity at the G1-S transition does not only trigger origin firing, but at the same time also results in phosphorylation of several pre-RC components (ORC, Cdc6, Cdt1,. These modifications are thought to prevent reassembly of these components into pre-R...

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Section: Cyce-cdk2 Regulates Apc/c-fzr Activity In Salivary Glandssupporting

confidence: 80%

The anaphase-promoting complex/cyclosome (APC/C) is required for rereplication control in endoreplication cycles

Zielke¹,

Querings²,

Rottig

et al. 2008

Genes Dev.

111

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“…EMI1 depletion in human cells and zebrafish leads to increased cell size, while FACS analysis of human cell lines, as well as zebrafish cells, in the present study indicated that this factor is essential for inhibiting rereplication (6,21,39,42). It is likely that our observation of rereplicated and unsegregated chromosomes or polyploid karyotypes has not been reported before because knockdown of EMI1 in human cell lines induces senescence and the murine Emi1 knockout has a very early lethal phenotype (17,39). Nonetheless, we have shown that emi1 depletion is a mechanism for the genesis of near-tetraploid cells, suggesting that emi1 m/m zebrafish may be a useful model for examining the molecular mechanism regulating this process and the ensuing cellular consequences, such as the derivation of aneuploid cells, as well as identifying additional mechanisms through which emi1 may contribute to leukemogenesis.…”

Section: Discussionmentioning

confidence: 41%

“…Knockout of murine Emi1 results in an embryonic-lethal phenotype prior to implantation, while a deficiency of Emi1 in cultured pronuclear zygotes leads to multipolar and tangled spindle structures, orphan chromosomes, large nuclei, and apoptosis by the 16-cell stage (17). Otherwise, the dynamic influence of EMI1 on early vertebrate development remains undefined.…”

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confidence: 99%

Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression

Rhodes

Amsterdam

Sanda

et al. 2009

Molecular and Cellular Biology

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A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase II␣-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.Successful cell division requires faithful replication of the genome, and defects in this process can contribute to genomic instability and subsequent malignant transformation (23). A key regulator of the normal cell cycle is the early mitotic inhibitor 1 (EMI1/FBXO5), a zinc finger protein expressed by a variety of adult tissues and especially in proliferating Ki-67-positive cells (39). Studies of the mammalian and Xenopus homologues of EMI1 have shown that it inhibits the anaphasepromoting complex/cyclosome (APC/C), a ubiquitin ligase complex that targets cell cycle-regulated proteins, such as the S-and M-phase cyclins (A and B), securin, and geminin (13,25,31). Depletion of EMI1 by small interfering RNA (siRNA) knockdown in cell lines or immunodepletion in cycling Xenopus extracts results in the untimely degradation of APC/C substrates, delaying G 1 /S-and M-phase progression and inducing rereplication (6,21,25,31). Such rereplication is a consequence of decreased levels of the APC/C substrates cyclin A and geminin, which are regulators of replication licensing (6, 21). The result of EMI1 depletion in some cell lines is senescence (39).Despite these insights into the molecular underpinnings of EMI1 function, little is known about the role of this protein in development. Knockout of murine Emi1 results in an embryonic-lethal phenotype prior to implantation, while a deficiency of Emi1 in cultured pronuclear zygotes leads to multipolar and tangled spindle structures, orphan chromosomes, large nuclei, and apoptosis by the 16-cell stage (17). Otherwise, the dynamic influence of EMI1 on early vertebrate development remains undefined. We sought to close this gap by taking advantage of the zebrafish model system. Zebrafish embryos harboring homozygous mutations of emi1 (emi1 m/m ) develop beyond the onset of circulation, providing a unique opportunity to examine the developmental roles of Emi1 in vivo. The zebrafish emi1 mutant (hi2648) line was originally identified by a proviral insertional mutage...

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“…In addition, early embryogenesis was examined using mice deficient in Emi1, which is known to be an APC/C inhibitor (Reimann et al, 2001). Although Emi1 −/− embryos form polyploid TGCs, the inner cell mass does not develop properly, presumably because of the high degree of polyploidy and the abnormal cell-cycle progression (Lee et al, 2006). By contrast, Skp2 appears to be less crucial than Cdh1.…”

Section: Cdh1mentioning

confidence: 99%

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

Sakaue-Sawano

Hoshida

et al. 2013

Development

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The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in ‘licensing’ of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APCCdh1 and SCFSkp2, which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, we used transgenic mice expressing Fucci fluorescent cell-cycle probes that report the activity of APCCdh1 and SCFSkp2. By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APCCdh1 and SCFSkp2 in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. We also obtained three-dimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation.

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Mouse emi1Has an Essential Function in Mitotic Progression during Early Embryogenesis

Cited by 45 publications

References 39 publications

The anaphase-promoting complex/cyclosome (APC/C) is required for rereplication control in endoreplication cycles

The anaphase-promoting complex/cyclosome (APC/C) is required for rereplication control in endoreplication cycles

Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

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