Mcrs1 regulates G2/M transition and spindle assembly during mouse oocyte meiosis.

Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.

Propylparaben exposure impairs G2/M and metaphase-anaphase transition during mouse oocyte maturation.

Propylparaben (PrPB) is a known endocrine disrupting chemicals that is widely applied as preservative in pharmaceuticals, food and cosmetics. PrPB has been detected in human urine samples and human serum and has been proven to cause functional decline in reproduction. However, the direct effects of PrPB on mammalian oocyte are still unknown. Here, we demonstrationed that exposure to PrPB disturbed mouse oocyte maturation in vitro, causing meiotic resumption arrest and first polar body extrusion failure. Our results indicated that 600 µM PrPB reduced the rate of oocyte germinal vesicle breakdown (GVBD). Further research revealed that PrPB caused mitochondrial dysfunction and oxidative stress, which led to oocyte DNA damage. This damage further disturbed the activity of the maturation promoting factor (MPF) complex Cyclin B1/ Cyclin-dependent kinase 1 (CDK1) and induced G2/M arrest. Subsequent experiments revealed that PrPB exposure can lead to spindle morphology disorder and chromosome misalignment due to unstable microtubules. In addition, PrPB adversely affected the attachment between microtubules and kinetochore, resulting in persistent activation of BUB3 amd BubR1, which are two spindle-assembly checkpoint (SAC) protein. Taken together, our studies indicated that PrPB damaged mouse oocyte maturation via disrupting MPF related G2/M transition and SAC depended metaphase-anaphase transition.

Mis12 controls cyclin B1 stabilization via Cdc14B-mediated APC/CCdh1 regulation during meiotic G2/M transition in mouse oocytes.

Mammalian oocytes are arrested at G2/prophase of the first meiosis. After a hormone surge, oocytes resume meiosis, undergoing germinal vesicle breakdown (GVBD). This process is regulated by Cdk1/cyclin B1. Here, we report that Mis12 is required for G2/M transition by regulating cyclin B1 accumulation via Cdc14B-mediated APC/CCdh1 regulation, but is not essential for spindle and chromosome dynamics during meiotic maturation. Depletion of Mis12 severely compromised GVBD by impairing cyclin B1 accumulation. Importantly, impaired GVBD after Mis12 depletion was rescued not only by overexpressing cyclin B1 but also by depleting Cdc14B or Cdh1. Notably, oocytes rescued by cyclin B1 overexpression exhibited normal spindle and chromosome organization with intact kinetochore-microtubule attachments. In addition, after being rescued by cyclin B1 overexpression, Mis12-depleted oocytes normally extruded polar bodies. Moreover, Mis12-depleted oocytes formed pronuclear structures after fertilization but failed to develop beyond zygotes. Interestingly, Mis12 was localized in the cytoplasm and spindle poles in oocytes, in contrast to kinetochore localization in somatic cells. Therefore, our results demonstrate that Mis12 is required for meiotic G2/M transition but is dispensable for meiotic progression through meiosis I and II.

MDC1 is essential for G2/M transition and spindle assembly in mouse oocytes.

Mammalian oocytes are particularly susceptible to accumulating DNA damage. However, unlike mitotic cells in which DNA damage induces G2 arrest by activating the ATM-Chk1/2-Cdc25 pathway, oocytes readily enter M-phase immediately following DNA damage. This implies a lack of a robust canonical G2/M DNA damage checkpoint in oocytes. Here we show that MDC1 plays a non-canonical role in controlling G2/M transition by regulating APC/C-Cdh1-mediated cyclin B1 degradation in response to DNA damage in mouse oocytes. Depletion of MDC1 impaired M-phase entry by decreasing cyclin B1 levels via the APC/C-Cdh1 pathway. Notably, the APC/C-Cdh1 regulation mediated by MDC1 was achieved by a direct interaction between MDC1 and APC/C-Cdh1. This interaction was transiently disrupted after DNA damage with a concomitant increase in Cdh1 levels, which, in turn, decreased cyclin B1 levels and delayed M-phase entry. Moreover, MDC1 depletion impaired spindle assembly by decreasing the integrity of microtubule organizing centers (MTOCs). Therefore, our results demonstrate that MDC1 is an essential molecule in regulating G2/M transition in response to DNA damage and in regulating spindle assembly in mouse oocytes. These results provide new insights into the regulation of the G2/M DNA damage checkpoint and cell cycle control in oocytes.

Genome-wide CRISPR/Cas9 knockout screening uncovers ZNF319 as a novel tumor suppressor critical for breast cancer metastasis.

Breast cancer is prone to relapse and metastasize to many vital organs, contributing to most of the breast cancer-related death and accentuating the importance of systematic identification of key factors regulating the metastasis of breast cancer. In this study, we performed a genome-wide CRISPR/Cas9 knock out screen in an orthotopic murine model of breast cancer for essential genes monitoring the progression and metastasis of breast cancer. We found one member of the zinc finger protein (ZNF) family, i.e., ZNF319, was among the top candidate genes. We further confirmed the lower expression of ZNF319 in the tumor tissue of breast cancer patients by analyzing tissue sections with IHC staining and TCGA database. Consistently, higher expression of ZNF319 correlates with better clinical outcome in almost all subtypes of breast cancer. Moreover, knocking down or overexpressing ZNF319 in breast cancer cells dramatically affects the breast cancer growth and metastasis capacity both in vitro and in vivo, suggesting ZNF319 functions as a strong suppressor of breast cancer progression. Lastly, the transcriptome analysis on ZNF319-silenced breast cancer cells shows that ZNF319 is involved in multiple crucial signaling pathways and biological processes, especially in cell cycle and proliferation. GO and KEGG analyses of our RNA-seq results reveal the up-regulation of E2F and G2/M related genes in ZNF319-silenced cells, suggesting that ZNF319 monitors the cell cycle during the breast cancer progression through the regulation of the E2F target genes and G2/M checkpoint. In summary, our study identifies ZNF319 as a novel metastasis suppressor gene arresting tumor cell cycle in breast cancer and thus presents a novel potential therapeutic target for breast cancer treatment.

Prognostic Value of BUB1 for Predicting Non-Muscle-Invasive Bladder Cancer Progression.

Non-muscle-invasive bladder cancer (NMIBC) is a common disease with a high recurrence rate requiring lifetime surveillance. Although NMIBC is not life-threatening, it can progress to muscle-invasive bladder cancer (MIBC), a lethal form of the disease. The management of the two diseases differs, and patients with MIBC require aggressive treatments such as chemotherapy and radical cystectomy. NMIBC patients at a high risk of progression benefit from early immediate cystectomy. Thus, identifying concordant markers for accurate risk stratification is critical to predict the prognosis of NMIBC. Candidate genetic biomarkers associated with NMIBC prognosis were screened by RNA-sequencing of 24 tissue samples, including 16 NMIBC and eight normal controls, and by microarray analysis (GSE13507). Lastly, we selected and investigated a mitotic checkpoint serine/threonine kinase, BUB1, that regulates chromosome segregation during the cell cycle. BUB1 gene expression was tested in 86 NMIBC samples and 15 controls by real-time qPCR. The performance of BUB1 as a prognostic biomarker for NMIBC was validated in the internal Chungbuk cohort (GSE13507) and the external UROMOL cohort (E-MTAB-4321). BUB1 expression was higher in NMIBC patients than in normal controls (p < 0.05), and the overexpression of BUB1 was correlated with NMIBC progression (log-rank test, p = 0.007). In in vitro analyses, BUB1 promoted the proliferation of bladder cancer cells by accelerating the G2/M transition of the cell cycle. Conclusively, BUB1 modulates the G2/M transition to promote the proliferation of bladder cancer cells, suggesting that it could serve as a prognostic marker in NMIBC.

Lack of Cyclin B1 in zebrafish causes lengthening of G2 and M phases.

An essential part of the Mitosis Promoting Factor, Cyclin B1 is indispensable for cells to enter mitosis. We report here that the zebrafish early arrest mutant specter is a loss-of-function mutation in the сyclin B1 gene. cyclin B1 is maternally transcribed in zebrafish, and the zygotic phenotype is apparent by early segmentation. Lack of zygotic Cyclin B1 does not stop cells from dividing, rather it causes an abnormal and elongated progression through the G2 and M phases of the cell cycle. Many mutant cells show signs of chromosomal instability or enter apoptosis. Using CRISPR-mediated gene editing, we produced a more severe gain-of-function mutation confirming that specter is the result of nonfunctional Cyclin B1. Although also a recessive phenotype, this new mutation produces an alternative splice-form of cyclin B1 mRNA, whose product lacks several key components for Cyclin B1, but not the Cdk1-binding domain. This mutant form of Cyclin B1 completely prevents cell division. We conclude that, although Cyclin B1 is critical for cells to enter mitosis, another cell cycle protein may be cooperating with Cdk1 at the G2/M checkpoint to sustain a partly functional Mitosis Promoting Factor.

SNHG16 as the miRNA let-7b-5p sponge facilitates the G2/M and epithelial-mesenchymal transition by regulating CDC25B and HMGA2 expression in hepatocellular carcinoma.

Long noncoding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of small nucleolar RNA host gene 16 (SNHG16) for regulating the cell cycle and epithelial to mesenchymal transition (EMT) remain elusive. In this study, SNHG16 expression profiles of HCC tissues or cell lines were compared with those of normal tissues or hepatocyte cell line. The effect of SNHG16 knockdown in HCC cell lines was investigated by using in vitro loss-of-function experiments and in vivo nude mouse experiments. The potential molecular regulatory mechanism of SNHG16 in HCC progression was investigated by using mechanistic experiments and rescue assays. The results revealed that SNHG16 was highly expressed in HCC tissues and cell lines, which predicted poor prognosis of HCC patients. On one hand, the downregulation of SNHG16 induced G2/M cell cycle arrest, inducing cell apoptosis and suppression of cell proliferation. On the other hand, it inhibited cell metastasis and EMT progression demonstrated by in vitro loss-of-function cell experiments. Besides, knockdown of SNHG16 increased the sensitivity of HCC cells to cisplatin. For the detailed mechanism, SNHG16 was demonstrated to act as a let-7b-5p sponge in HCC. SNHG16 facilitated the G2/M cell cycle transition by directly acting on the let-7b-5p/CDC25B/CDK1 axis, and promoted cell metastasis and EMT progression by regulating the let-7b-5p/HMGA2 axis in HCC. In addition, the mechanism of SNHG16 for regulating HCC cell proliferation and metastasis was further confirmed in vivo by mouse experiments. Furthermore, these results can provide new insights into HCC treatment and its molecular pathogenesis, which may enlighten the further research of the molecular pathogenesis of HCC.

CenpH regulates meiotic G2/M transition by modulating the APC/CCdh1-cyclin B1 pathway in oocytes.

Meiotic resumption (G2/M transition) and progression through meiosis I (MI) are two key stages for producing fertilization-competent eggs. Here, we report that CenpH, a component of the kinetochore inner plate, is responsible for G2/M transition in meiotic mouse oocytes. Depletion of CenpH by morpholino injection decreased cyclin B1 levels, resulting in attenuation of maturation-promoting factor (MPF) activation, and severely compromised meiotic resumption. CenpH protects cyclin B1 from destruction by competing with the action of APC/CCdh1 Impaired G2/M transition after CenpH depletion could be rescued by expression of exogenous cyclin B1. Unexpectedly, blocking CenpH did not affect spindle organization and meiotic cell cycle progression after germinal vesicle breakdown. Our findings reveal a novel role of CenpH in regulating meiotic G2/M transition by acting via the APC/CCdh1-cyclin B1 pathway.

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development.

NEK5, a member of never in mitosis-gene A-related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA-mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin-dependent kinase 1 in oocytes, resulting in a decrease of maturation-promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1-cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.

A Mathematical Model for the Effect of Low-Dose Radiation on the G2/M Transition.

We develop a mathematical model to study the immediate effect of low-dose radiation on the G2 checkpoint and the G2/M transition of the cell cycle via a radiation pathway (the ATM-Chk2 pathway) of an individual mammalian cell. The model consists of a system of nonlinear differential equations describing the dynamics of a network of regulatory proteins that play key roles in the G2/M transition, cell cycle oscillations, and the radiation pathway. We simulate the application of a single pulse of low-dose radiation at different intensities ([Formula: see text] 0-0.4 Gy) and times during the latter part of the G2-phase. We use bifurcation analysis to characterize the effect of radiation on the G2/M transition via the ATM-Chk2 pathway. We show that radiation between 0.1 and 0.3 Gy can delay the G2/M transition, and radiation higher than 0.3 Gy can fully activate the G2 checkpoint. Also, our results show that radiation can be low enough to neither delay the G2/M transition nor activate the G2 checkpoint ([Formula: see text] 0.1 Gy). Our model supports the idea that the cell response to radiation during G2-phase explains hyper-radiosensitivity and increased radioresistance (HRS/IRR) observed at low dose.

Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition.

Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K-Akt pathway through the Gßγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B-Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B-Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gßγ-dependent pathway enhances PI3K-Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gßγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries.

FBW7 suppresses cell proliferation and G2/M cell cycle transition via promoting γ-catenin K63-linked ubiquitylation.

FBW7 is an E3 ubiquitin ligase and frequently mutated in various types of cancer. As a component of SCF ubiquitin ligase complex, FBW7 usually targets the substrates via K11 or K48-linked ubiquitylation and subsequent degradation of target proteins. Nevertheless, the role of FBW7 in mediating non-degradable ubiquitin signaling remains unknown in human cancers. In this study, we identified γ-catenin as a new binding protein of FBW7 by TAP-MS (tandem affinity purification-mass spectrum). Knockdown of FBW7 did not affect the stability of γ-catenin, but significantly reduced the K63-linked ubiquitin of γ-catenin, resulting in decreased expression of γ-catenin downstream gene 14-3-3σ. Rescue experiment revealed that γ-catenin promoted the expression of 14-3-3σ in a K63-linked ubiquitin signaling dependent manner. Furthermore, we showed that FBW7 cooperated with γ-catenin to inhibit G2/M cell cycle transition and cell proliferation. Taken together, our study uncovered a novel mechanism that FBW7 associated with γ-catenin and promoted its K63-linked ubiquitylation, providing new insights in understanding the role of FBW7 in inhibiting G2/M cell cycle transition and tumor cell proliferation.

Ribosomal protein NtRPL17 interacts with kinesin-12 family protein NtKRP and functions in the regulation of embryo/seed size and radicle growth.

We previously reported that a novel motor protein belonging to the kinesin-12 family, NtKRP, displays critical roles in regulating embryo and seed size establishment. However, it remains unknown exactly how NtKRP contributes to this developmental process. Here, we report that a 60S ribosomal protein NtRPL17 directly interacts with NtKRP. The phenotypes of NtRPL17 RNAi lines show notable embryo and seed size reduction. Structural observations of the NtRPL17-silenced embryos/seeds reveal that the embryo size reduction is due to a decrease in cell number. In these embryos, cell division cycle progression is delayed at the G2/M transition. These phenotypes are similar to that in NtKRP-silenced embryos/seeds, indicating that NtKRP and NtRPL17 function as partners in the same regulatory pathway during seed development and specifically regulate cell cycle progression to control embryo/seed size. This work reveals that NtRPL17, as a widely distributed ribosomal protein, plays a critical role in seed development and provides a new clue in the regulation of seed size. Confirmation of the interaction between NtKRP and NtRPL17 and their co-function in the control of the cell cycle also suggests that the mechanism might be conserved in both plants and animals.

Inhibition of casein kinase 2 blocks G2/M transition in early embryo mitosis but not in oocyte meiosis in mouse.

Casein kinase 2 (CK2) is a highly conserved, ubiquitously expressed serine/threonine protein kinase with hundreds of substrates. The role of CK2 in the G2/M transition of oocytes, zygotes, and 2-cell embryos was studied in mouse by enzyme activity inhibition using the specific inhibitor 4, 5, 6, 7-tetrabromobenzotriazole (TBB). Zygotes and 2-cell embryos were arrested at G2 phase by TBB treatment, and DNA damage was increased in the female pronucleus of arrested zygotes. Further developmental ability of arrested zygotes was reduced, but that of arrested 2-cell embryos was not affected after releasing from inhibition. By contrast, the G2/M transition in oocytes was not affected by TBB. These results indicate that CK2 activity is essential for mitotic G2/M transition in early embryos but not for meiotic G2/M transition in oocytes.

Regulation of G2/M Transition by Inhibition of WEE1 and PKMYT1 Kinases.

In the cell cycle, there are two checkpoint arrests that allow cells to repair damaged DNA in order to maintain genomic integrity. Many cancer cells have defective G1 checkpoint mechanisms, thus depending on the G2 checkpoint far more than normal cells. G2 checkpoint abrogation is therefore a promising concept to preferably damage cancerous cells over normal cells. The main factor influencing the decision to enter mitosis is a complex composed of Cdk1 and cyclin B. Cdk1/CycB is regulated by various feedback mechanisms, in particular inhibitory phosphorylations at Thr14 and Tyr15 of Cdk1. In fact, Cdk1/CycB activity is restricted by the balance between WEE family kinases and Cdc25 phosphatases. The WEE kinase family consists of three proteins: WEE1, PKMYT1, and the less important WEE1B. WEE1 exclusively mediates phosphorylation at Tyr15, whereas PKMYT1 is dual-specific for Tyr15 as well as Thr14. Inhibition by a small molecule inhibitor is therefore proposed to be a promising option since WEE kinases bind Cdk1, altering equilibria and thus affecting G2/M transition.

A humanized anti-CD26 monoclonal antibody inhibits cell growth of malignant mesothelioma via retarded G2/M cell cycle transition.

BACKGROUND: Malignant Mesothelioma (MM) is a highly aggressive tumor with poor prognosis. Multimodal treatments and novel molecular targeted therapies against MM are in high demand in order treat this disease effectively. We have developed a humanized monoclonal antibody YS110 against CD26 expressed in 85 % of MM cases. CD26 is thought to be involved in tumor growth and invasion by interacting with collagen and fibronectin, or affecting signal transduction processes. METHODS: We evaluated the direct anti-tumor effect of YS110 against MM cell lines, NCI-H2452 and JMN, and investigated its effects on cell cycle and on the cell cycle regulator molecules. In addition, we investigated synergistic effects of YS110 and anti-tumor agent pemetrexed (PMX) against MM cell line both in vitro and in vivo. RESULTS: YS110 suppressed the proliferation of NCI-H2452 cells by approximately 20 % in 48 h. Based on cell cycle analysis, percentage of cells in G2/M phase increased 8.0 % on the average after YS110 treatment; in addition, cell cycle regulator p21 cip/waf1 was increased and cyclin B1 was decreased after YS110 treatment. Inhibitory phosphorylation of both cdc2 (Tyr15) and cdc25C (Ser216) were elevated. Furthermore, activating phosphorylation of p38 MAPK (Thr180/Tyr182) and ERK1/2 (Thr202/Tyr204) were augmented at 24 h after YS110 treatment. PMX rapidly induced CD26 expression on cell surface and the treatment with both YS110 and PMX inhibited in vivo tumor growth accompanied by a synergistic reduction in the MIB-1 index. CONCLUSION: This is a first report of a novel anti-proliferative mechanism of the humanized anti-CD26 monoclonal antibody YS110, which resulted in G2/M cell cycle delay through regulation of quantity and activity of various cell cycle regulating molecules.

p21 is Responsible for Ionizing Radiation-induced Bypass of Mitosis.

OBJECTIVE: To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest. METHODS: Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence- associated-ß-galactosidase (SA-ß-gal) staining combined with Ki67 staining, a cell proliferation marker. RESULTS: Accompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells. CONCLUSION: Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis.

Fission yeast nucleolar protein Dnt1 regulates G2/M transition and cytokinesis by downregulating Wee1 kinase.

Cytokinesis involves temporally and spatially coordinated action of the cell cycle, cytoskeletal and membrane systems to achieve separation of daughter cells. The septation initiation network (SIN) and mitotic exit network (MEN) signaling pathways regulate cytokinesis and mitotic exit in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Previously, we have shown that in fission yeast, the nucleolar protein Dnt1 negatively regulates the SIN pathway in a manner that is independent of the Cdc14-family phosphatase Clp1/Flp1, but how Dnt1 modulates this pathway has remained elusive. By contrast, it is clear that its budding yeast relative, Net1/Cfi1, regulates the homologous MEN signaling pathway by sequestering Cdc14 phosphatase in the nucleolus before mitotic exit. In this study, we show that dnt1(+) positively regulates G2/M transition during the cell cycle. By conducting epistasis analyses to measure cell length at septation in double mutant (for dnt1 and genes involved in G2/M control) cells, we found a link between dnt1(+) and wee1(+). Furthermore, we showed that elevated protein levels of the mitotic inhibitor Wee1 kinase and the corresponding attenuation in Cdk1 activity is responsible for the rescuing effect of dnt1Δ on SIN mutants. Finally, our data also suggest that Dnt1 modulates Wee1 activity in parallel with SCF-mediated Wee1 degradation. Therefore, this study reveals an unexpected missing link between the nucleolar protein Dnt1 and the SIN signaling pathway, which is mediated by the Cdk1 regulator Wee1 kinase. Our findings also define a novel mode of regulation of Wee1 and Cdk1, which is important for integration of the signals controlling the SIN pathway in fission yeast.

DNA topoisomerase II-dependent control of the cell cycle progression in root meristems of Allium cepa.

The catalytic ability of DNA topoisomerases (Topo) to generate short-term DNA breaks allow these enzymes to play crucial functions in managing DNA topology during S-phase replication, transcription, and chromatin-remodelling processes required to achieve commitment for the onset and transition through mitosis. Our experiments on root meristem cells of onion (Allium cepa) were designed to gain insight into the contribution of Topo II to plant-specific progression throughout interphase and mitosis. Irrespective of the position of the cell in interphase, the immunofluorescence of Topo II revealed similar nuclear labelling pattern with well defined signals dispersed in the nucleoplasm and the cortical zone of the nucleolus. Only weak labelling was detected in metaphase and anaphase chromosomes. Experiments with two potent anti-Topo II agents, doxorubicin (DOX, an anthracycline) and a bisdioxopiperazine derivative, ICRF-193, suggest that the inhibition-mediated increase in Topo II immunofluorescence may represent a compensatory mechanism, by which an up-regulated expression of the enzyme tends to counteract the drug-induced loss of indispensable catalytic and relaxation functions. γ-H2AX immunolabelling seems to indicate that both DOX- and ICRF-193-induced alterations in cell cycle progression reflect primarily the activity of the G2/M DNA damage checkpoint. Our findings provide evidence for the plant-specific cell cycle control mechanism induced by Topo II inhibitors under DNA stress conditions.