scholarly journals Identification of DHX9 as a cell cycle regulated nucleolar recruitment factor for CIZ1

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Urvi Thacker ◽  
Tekle Pauzaite ◽  
James Tollitt ◽  
Maria Twardowska ◽  
Charlotte Harrison ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Zinc Finger Protein ◽  
S Phase ◽  
Cycle Progression ◽  
Inactive X Chromosome ◽  
Functional Characterisation ◽  
Interaction Partners

Abstract CIP1-interacting zinc finger protein 1 (CIZ1) is a nuclear matrix associated protein that facilitates a number of nuclear functions including initiation of DNA replication, epigenetic maintenance and associates with the inactive X-chromosome. Here, to gain more insight into the protein networks that underpin this diverse functionality, molecular panning and mass spectrometry are used to identify protein interaction partners of CIZ1, and CIZ1 replication domain (CIZ1-RD). STRING analysis of CIZ1 interaction partners identified 2 functional clusters: ribosomal subunits and nucleolar proteins including the DEAD box helicases, DHX9, DDX5 and DDX17. DHX9 shares common functions with CIZ1, including interaction with XIST long-non-coding RNA, epigenetic maintenance and regulation of DNA replication. Functional characterisation of the CIZ1-DHX9 complex showed that CIZ1-DHX9 interact in vitro and dynamically colocalise within the nucleolus from early to mid S-phase. CIZ1-DHX9 nucleolar colocalisation is dependent upon RNA polymerase I activity and is abolished by depletion of DHX9. In addition, depletion of DHX9 reduced cell cycle progression from G1 to S-phase in mouse fibroblasts. The data suggest that DHX9-CIZ1 are required for efficient cell cycle progression at the G1/S transition and that nucleolar recruitment is integral to their mechanism of action.

scholarly journals Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression.

1994 ◽  
Vol 125 (4) ◽  
pp. 705-719 ◽  
Author(s):  
S Kornbluth ◽  
M Dasso ◽  
J Newport
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Nuclear Structure ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
A Cell ◽  
Xenopus Egg Extracts

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP-bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.

scholarly journals A Mammalian Bromodomain Protein, Brd4, Interacts with Replication Factor C and Inhibits Progression to S Phase

2002 ◽  
Vol 22 (18) ◽  
pp. 6509-6520 ◽  
Author(s):  
Tetsuo Maruyama ◽  
Andrea Farina ◽  
Anup Dey ◽  
JaeHun Cheong ◽  
Vladimir P. Bermudez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Replication Factor C ◽  
Cycle Progression ◽  
Vitro Analysis ◽  
Factor C ◽  
In Vitro Analysis

ABSTRACT Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G1 upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G1 to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.

scholarly journals Day/Night Separation of Oxygenic Energy Metabolism and Nuclear DNA Replication in the Unicellular Red AlgaCyanidioschyzon merolae

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Shin-ya Miyagishima ◽  
Atsuko Era ◽  
Tomohisa Hasunuma ◽  
Mami Matsuda ◽  
Shunsuke Hirooka ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Dna Replication ◽  
Energy Metabolism ◽  
Cell Cycle Progression ◽  
Nuclear Dna ◽  
S Phase ◽  
Cycle Progression ◽  
Content Type

ABSTRACTThe transition from G1to S phase and subsequent nuclear DNA replication in the cells of many species of eukaryotic algae occur predominantly during the evening and night in the absence of photosynthesis; however, little is known about how day/night changes in energy metabolism and cell cycle progression are coordinated and about the advantage conferred by the restriction of S phase to the night. Using a synchronous culture of the unicellular red algaCyanidioschyzon merolae, we found that the levels of photosynthetic and respiratory activities peak during the morning and then decrease toward the evening and night, whereas the pathways for anaerobic consumption of pyruvate, produced by glycolysis, are upregulated during the evening and night as reported recently in the green algaChlamydomonas reinhardtii. Inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) largely reduced respiratory activity and the amplitude of the day/night rhythm of respiration, suggesting that the respiratory rhythm depends largely on photosynthetic activity. Even when the timing of G1/S-phase transition was uncoupled from the day/night rhythm by depletion of retinoblastoma-related (RBR) protein, the same patterns of photosynthesis and respiration were observed, suggesting that cell cycle progression and energy metabolism are regulated independently. Progression of the S phase under conditions of photosynthesis elevated the frequency of nuclear DNA double-strand breaks (DSB). These results suggest that the temporal separation of oxygenic energy metabolism, which causes oxidative stress, from nuclear DNA replication reduces the risk of DSB during cell proliferation inC. merolae.IMPORTANCEEukaryotes acquired chloroplasts through an endosymbiotic event in which a cyanobacterium or a unicellular eukaryotic alga was integrated into a previously nonphotosynthetic eukaryotic cell. Photosynthesis by chloroplasts enabled algae to expand their habitats and led to further evolution of land plants. However, photosynthesis causes greater oxidative stress than mitochondrion-based respiration. In seed plants, cell division is restricted to nonphotosynthetic meristematic tissues and populations of photosynthetic cells expand without cell division. Thus, seemingly, photosynthesis is spatially sequestrated from cell proliferation. In contrast, eukaryotic algae possess photosynthetic chloroplasts throughout their life cycle. Here we show that oxygenic energy conversion (daytime) and nuclear DNA replication (night time) are temporally sequestrated inC. merolae. This sequestration enables “safe” proliferation of cells and allows coexistence of chloroplasts and the eukaryotic host cell, as shown in yeast, where mitochondrial respiration and nuclear DNA replication are temporally sequestrated to reduce the mutation rate.

scholarly journals Cdc25A Serine 123 Phosphorylation Couples Centrosome Duplication with DNA Replication and Regulates Tumorigenesis

2008 ◽  
Vol 28 (24) ◽  
pp. 7442-7450 ◽  
Author(s):  
Sathyavageeswaran Shreeram ◽  
Weng Kee Hee ◽  
Dmitry V. Bulavin
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Phosphorylation Site ◽  
Centrosome Duplication ◽  
Cycle Progression ◽  
Cdc25a Phosphatase

ABSTRACT The cell division cycle 25A (Cdc25A) phosphatase is a critical regulator of cell cycle progression under normal conditions and after stress. Stress-induced degradation of Cdc25A has been proposed as a major way of delaying cell cycle progression. In vitro studies pointed toward serine 123 as a key site in regulation of Cdc25A stability after exposure to ionizing radiation (IR). To address the role of this phosphorylation site in vivo, we generated a knock-in mouse in which alanine was substituted for serine 123. The Cdc25 S123A knock-in mice appeared normal, and, unexpectedly, cells derived from them exhibited unperturbed cell cycle and DNA damage responses. In turn, we found that Cdc25A was present in centrosomes and that Cdc25A levels were not reduced after IR in knock-in cells. This resulted in centrosome amplification due to lack of induction of Cdk2 inhibitory phosphorylation after IR specifically in centrosomes. Further, Cdc25A knock-in animals appeared sensitive to IR-induced carcinogenesis. Our findings indicate that Cdc25A S123 phosphorylation is crucial for coupling centrosome duplication to DNA replication cycles after DNA damage and therefore is likely to play a role in the regulation of tumorigenesis.

scholarly journals Lyn is activated during late G1 of stem-cell-factor-induced cell cycle progression in haemopoietic cells

1999 ◽  
Vol 342 (1) ◽  
pp. 163-170 ◽  
Author(s):  
Sherry MOU ◽  
Diana LINNEKIN
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Family Member ◽  
Stem Cell Factor ◽  
Cell Cycle Progression ◽  
Kinetic Studies ◽  
S Phase ◽  
Cycle Progression ◽  
Haemopoietic Cells

Stem cell factor (SCF) binds the receptor tyrosine kinase c-Kit and is critical in haemopoiesis. Recently we found that the Src family member Lyn is highly expressed in SCF-responsive cells, associates with c-Kit and is activated within minutes of the addition of SCF. Here we show that SCF activates Lyn a second time, hours later, during SCF-induced cell cycle progression. In cells arrested at specific phases of the cell cycle with the drugs mimosine, aphidicolin and nocodazole, maximal Lyn kinase activity occurred in late G1 and through the G1/S transition. Similarly, kinetic studies of SCF-induced cell cycle progression found that activation of Lyn preceded the G1/S transition and was maintained into early S-phase. Activation of Lyn was paralleled by two events critical for the G1/S transition, increases in cyclin-dependent kinase 2 (Cdk2) activity and phosphorylation of the retinoblastoma gene product (Rb). Lyn was associated with Cdk2; Cdk2-associated Lyn was heavily phosphorylated on serine and threonine residues both in vitro and in situ during S-phase. Inhibition of Lyn activity with PP1 disrupted association with Cdk2 and decreased the numbers of cells entering S-phase. The degree of phosphorylation of Rb in PP1-treated cells suggested an increased number of cells arrested in the middle of G1. These findings demonstrate that SCF activates the Src family member Lyn before the G1/S transition of the cell cycle and suggest that Lyn is involved in SCF-induced cell cycle progression.

scholarly journals A Cds1-Mediated Checkpoint Protects the MBF Activator Rep2 from Ubiquitination by Anaphase-Promoting Complex/Cyclosome-Ste9 at S-Phase Arrest in Fission Yeast

2009 ◽  
Vol 29 (18) ◽  
pp. 4959-4970 ◽  
Author(s):  
Zhaoqing Chu ◽  
Majid Eshaghi ◽  
Suk Yean Poon ◽  
Jianhua Liu
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Transcriptional Activity ◽  
S Phase ◽  
Anaphase Promoting Complex ◽  
Cell Recovery ◽  
Cycle Progression ◽  
Phase Arrest ◽  
S Phase Arrest

ABSTRACT Transcription of the MluI cell cycle box (MCB) motif-containing genes at G1 phase is regulated by the MCB-binding factors (MBF) (also called DSC1) in Schizosaccharomyces pombe. Upon S-phase arrest, the MBF transcriptional activity is induced through the accumulation of the MBF activator Rep2. In this study, we show that the turnover of Rep2 is attributable to ubiquitin-mediated proteolysis. Levels of Rep2 oscillate during the cell cycle, with a peak at G1 phase, coincident with the MBF activity. Furthermore, we show that Rep2 ubiquitination requires the function of the E3 ligase anaphase-promoting complex/cyclosome (APC/C). Ste9 can be phosphorylated by the checkpoint kinase Cds1 in vitro, and its inhibition/phosphorylation at S-phase arrest is dependent on the function of Cds1. Our data indicate that the Cds1-dependent stabilization of Rep2 is achieved through the inhibition/phosphorylation of APC/C-Ste9 at the onset of S-phase arrest. Stabilization of Rep2 is important for stimulating transcription of the MBF-dependent genes to ensure a sufficient supply of proteins essential for cell recovery from S-phase arrest. We propose that oscillation of Rep2 plays a role in regulation of periodic transcription of the MBF-dependent genes during cell cycle progression.

scholarly journals The F-Box Protein Dia2 Regulates DNA Replication

2006 ◽  
Vol 17 (4) ◽  
pp. 1540-1548 ◽  
Author(s):  
Deanna M. Koepp ◽  
Andrew C. Kile ◽  
Swarna Swaminathan ◽  
Veronica Rodriguez-Rivera
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Protein Complexes ◽  
S Phase ◽  
Cycle Progression ◽  
Origin Binding Protein ◽  
Cold Sensitive ◽  
F Box Protein

Ubiquitin-mediated proteolysis plays a key role in many pathways inside the cell and is particularly important in regulating cell cycle transitions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases whose specificity is determined by a substrate-binding F-box protein. Dia2 is a Saccharomyces cerevisiae F-box protein previously described to play a role in invasive growth and pheromone response pathways. We find that deletion of DIA2 renders cells cold-sensitive and subject to defects in cell cycle progression, including premature S-phase entry. Consistent with a role in regulating DNA replication, the Dia2 protein binds replication origins. Furthermore, the dia2 mutant accumulates DNA damage in both S and G2/M phases of the cell cycle. These defects are likely a result of the absence of SCFDia2 activity, as a Dia2 ΔF-box mutant shows similar phenotypes. Interestingly, prolonging G1-phase in dia2 cells prevents the accumulation of DNA damage in S-phase. We propose that Dia2 is an origin-binding protein that plays a role in regulating DNA replication.

scholarly journals DNA replication times the cell cycle and contributes to the mid-blastula transition in Drosophila embryos

2009 ◽  
Vol 187 (1) ◽  
pp. 7-14 ◽  
Author(s):  
Mark L. McCleland ◽  
Antony W. Shermoen ◽  
Patrick H. O'Farrell
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Cycle Progression ◽  
Replication Checkpoint ◽  
Drosophila Embryogenesis ◽  
Zygotic Transcription ◽  
Phase Cycle ◽  
Drosophila Embryos

We examined the contribution of S phase in timing cell cycle progression during Drosophila embryogenesis using an approach that deletes S phase rather than arresting its progress. Injection of Drosophila Geminin, an inhibitor of replication licensing, prevented subsequent replication so that the following mitosis occurred with uninemic chromosomes, which failed to align. The effect of S phase deletion on interphase length changed with development. During the maternally regulated syncytial blastoderm cycles, deleting S phase shortened interphase, and deletion of the last of blastoderm S phase (cycle 14) induced an extra synchronous division and temporarily deferred mid-blastula transition (MBT) events. In contrast, deleting S phase after the MBT in cycle 15 did not dramatically affect mitotic timing, which appears to retain its dependence on developmentally programmed zygotic transcription. We conclude that normal S phase and replication checkpoint activities are important timers of the undisturbed cell cycle before, but not after, the MBT.

scholarly journals Endothelial cells isolated from caveolin-2 knockout mice display higher proliferation rate and cell cycle progression relative to their wild-type counterparts

2010 ◽  
Vol 298 (3) ◽  
pp. C693-C701 ◽  
Author(s):  
Leike Xie ◽  
Philippe G. Frank ◽  
Michael P. Lisanti ◽  
Grzegorz Sowa
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Inhibitors ◽  
Fold Increase ◽  
S Phase ◽  
Lower Percentage ◽  
Wild Type ◽  
Cycle Progression ◽  
Molecular Events

The goal of this study was to determine whether caveolin-2 (Cav-2) is capable of controlling endothelial cell (EC) proliferation in vitro. To realize this goal, we have directly compared proliferation rates and cell cycle-associated signaling proteins between lung ECs isolated from wild-type (WT) and Cav-2 knockout (KO) mice. Using three independent proliferation assays, we have determined that Cav-2 KO ECs proliferate by ca. 2-fold faster than their WT counterparts. Cell cycle analysis by flow cytometry of propidium iodide-stained cells showed a relatively higher percentage of Cav-2 KO ECs in S and G2/M and lower percentage in Go/G1 phases of cell cycle relative to their WT counterparts. Furthermore, an over 2-fold increase in the percentage of S phase-associated Cav-2 KO relative to WT ECs was independently determined with bromodeoxyuridine incorporation assay. Mechanistically, the increase in proliferation/cell cycle progression of Cav-2 KO ECs correlated well with elevated expression levels of predominantly S phase- and G2/M phase-associated cyclin A and B1, respectively. Further mechanistic analysis of molecular events controlling cell cycle progression revealed increased level of hyperphosphorylated (inactive) form of G1 to S phase transition inhibitor, the retinoblastoma protein in hyperproliferating Cav-2 KO ECs. Conversely, the expression level of the two cyclin-dependent kinase inhibitors p16INK4 and p27Kip1 was reduced in Cav-2 KO ECs. Finally, increased phosphorylation (activation) of proproliferative extracellular signal-regulated kinase 1/2 was observed in hyperproliferating Cav-2 KO ECs. Overall, our data suggest that Cav-2 negatively regulates lung EC proliferation and cell cycle progression.

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