Search tips
Search criteria

Results 1-25 (130)

Clipboard (0)

Select a Filter Below

Year of Publication
more »
Document Types
1.  Identification of cell cycle–regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors 
Molecular Biology of the Cell  2013;24(23):3634-3650.
Characterization of the cell cycle–regulated transcripts in U2OS cells yielded 1871 unique genes. FOXM1 targets were identified via ChIP-seq, and novel targets in G2/M and S phases were verified using a real-time luciferase assay. ChIP-seq data were used to map cell cycle transcriptional regulators of cell cycle–regulated gene expression in U2OS cells.
We identify the cell cycle–regulated mRNA transcripts genome-wide in the osteosarcoma-derived U2OS cell line. This results in 2140 transcripts mapping to 1871 unique cell cycle–regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identify genomic loci bound by the G2/M transcription factor FOXM1 by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associate these with cell cycle–regulated genes. FOXM1 is bound to cell cycle–regulated genes with peak expression in both S phase and G2/M phases. We show that ChIP-seq genomic loci are responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly activates promoters of G2/M phase genes and weakly activates those induced in S phase. Analysis of ChIP-seq data from a panel of cell cycle transcription factors (E2F1, E2F4, E2F6, and GABPA) from the Encyclopedia of DNA Elements and ChIP-seq data for the DREAM complex finds that a set of core cell cycle genes regulated in both U2OS and HeLa cells are bound by multiple cell cycle transcription factors. These data identify the cell cycle–regulated genes in a second cancer-derived cell line and provide a comprehensive picture of the transcriptional regulatory systems controlling periodic gene expression in the human cell division cycle.
PMCID: PMC3842991  PMID: 24109597
2.  Key events during the transition from rapid growth to quiescence in budding yeast require posttranscriptional regulators 
Molecular Biology of the Cell  2013;24(23):3697-3709.
The transition to quiescence in budding yeast involves highly asymmetric cell divisions and elaborate cell wall fortifications that can be followed by flow cytometry. Posttranscriptional regulators Ssd1V, Mpt5, and Lsm1 are important for this transition.
Yeast that naturally exhaust the glucose from their environment differentiate into three distinct cell types distinguishable by flow cytometry. Among these is a quiescent (Q) population, which is so named because of its uniform but readily reversed G1 arrest, its fortified cell walls, heat tolerance, and longevity. Daughter cells predominate in Q-cell populations and are the longest lived. The events that differentiate Q cells from nonquiescent (nonQ) cells are initiated within hours of the diauxic shift, when cells have scavenged all the glucose from the media. These include highly asymmetric cell divisions, which give rise to very small daughter cells. These daughters modify their cell walls by Sed1- and Ecm33-dependent and dithiothreitol-sensitive mechanisms that enhance Q-cell thermotolerance. Ssd1 speeds Q-cell wall assembly and enables mother cells to enter this state. Ssd1 and the related mRNA-binding protein Mpt5 play critical overlapping roles in Q-cell formation and longevity. These proteins deliver mRNAs to P-bodies, and at least one P-body component, Lsm1, also plays a unique role in Q-cell longevity. Cells lacking Lsm1 and Ssd1 or Mpt5 lose viability under these conditions and fail to enter the quiescent state. We conclude that posttranscriptional regulation of mRNAs plays a crucial role in the transition in and out of quiescence.
PMCID: PMC3842996  PMID: 24088570
3.  The nucleolus stress response is coupled to an ATR-Chk1–mediated G2 arrest 
Molecular Biology of the Cell  2013;24(9):1334-1342.
HeLa cells engineered with the fluorescent ubiquitinylation-based cell cycle indicator are used to study the connection between nucleolar stress and cell cycle progression. The results demonstrate a feedforward mechanism that leads to G2 arrest and identify ATR and Chk1 as molecular agents of the requisite checkpoint.
We report experiments on the connection between nucleolar stress and cell cycle progression, using HeLa cells engineered with the fluorescent ubiquitinylation-based cell cycle indicator. Nucleolar stress elicited by brief exposure of cells to a low concentration of actinomycin D that selectively inhibits rRNA synthesis had no effect on traverse of G1 or S, but stalled cells in very late interphase. Additional experiments revealed that a switch occurs during a specific temporal window during nucleolar stress and that the subsequent cell cycle arrest is not triggered simply by the stress-induced decline in the synthesis of rRNA or by a ribosome starvation phenomenon. Further experiments revealed that this nucleolus stress-induced cell cycle arrest involves the action of a G2 checkpoint mediated by the ataxia telangiectasia and Rad3-related protein (ATR)–checkpoint kinase 1 (Chk1) pathway. Based on analysis of the cell cycle stages at which this nucleolar stress effect is put into action, to become manifest later, our results demonstrate a feedforward mechanism that leads to G2 arrest and identify ATR and Chk1 as molecular agents of the requisite checkpoint.
PMCID: PMC3639045  PMID: 23447702
4.  The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks 
Molecular Biology of the Cell  2013;24(9):1343-1353.
The activation of Chk1 in response to stalled replication forks involves a pathway containing ATR, TopBP1, Rad17, and Claspin. We show that the Mre11-Rad50-Nbs1 (MRN) complex also has an important role in this pathway that is distinct from its role in response to double-stranded DNA breaks. These studies reveal a novel insight into the functions of the MRN complex.
The activation of Chk1 in response to stalled replication forks in Xenopus egg extracts involves a complex pathway containing ATM and Rad3-related (ATR), topoisomerase IIβ-binding protein 1 (TopBP1), Rad17, the Rad9-Hus1-Rad1 (9-1-1) complex, and Claspin. We have observed that egg extracts lacking the Mre11-Rad50-Nbs1 (MRN) complex show greatly, although not completely, reduced activation of Chk1 in response to replication blockages. Depletion of both Rad17 and MRN leads to a further, essentially complete, reduction in the activation of Chk1. Thus, Rad17 and MRN act in at least a partially additive manner in promoting activation of Chk1. There was not an obvious change in the binding of RPA, ATR, Rad17, or the 9-1-1 complex to chromatin in aphidicolin (APH)-treated, MRN-depleted extracts. However, there was a substantial reduction in the binding of TopBP1. In structure–function studies of the MRN complex, we found that the Mre11 subunit is necessary for the APH-induced activation of Chk1. Moreover, a nuclease-deficient mutant of Mre11 cannot substitute for wild-type Mre11 in this process. These results indicate that the MRN complex, in particular the nuclease activity of Mre11, plays an important role in the activation of Chk1 in response to stalled replication forks. These studies reveal a previously unknown property of the MRN complex in genomic stability.
PMCID: PMC3639046  PMID: 23468519
5.  Feedback control of Swe1p degradation in the yeast morphogenesis checkpoint 
Molecular Biology of the Cell  2013;24(7):914-922.
The morphogenesis checkpoint stabilizes the mitotic inhibitor Swe1p and prevents mitosis following stresses that affect bud formation. It is shown that, following some stresses, Swe1p stabilization is an indirect effect of cyclin-dependent kinase inhibition.
Saccharomyces cerevisiae cells exposed to a variety of physiological stresses transiently delay bud emergence or bud growth. To maintain coordination between bud formation and the cell cycle in such circumstances, the morphogenesis checkpoint delays nuclear division via the mitosis-inhibitory Wee1-family kinase, Swe1p. Swe1p is degraded during G2 in unstressed cells but is stabilized and accumulates following stress. Degradation of Swe1p is preceded by its recruitment to the septin scaffold at the mother-bud neck, mediated by the Swe1p-binding protein Hsl7p. Following osmotic shock or actin depolymerization, Swe1p is stabilized, and previous studies suggested that this was because Hsl7p was no longer recruited to the septin scaffold following stress. However, we now show that Hsl7p is in fact recruited to the septin scaffold in stressed cells. Using a cyclin-dependent kinase (CDK) mutant that is immune to checkpoint-mediated inhibition, we show that Swe1p stabilization following stress is an indirect effect of CDK inhibition. These findings demonstrate the physiological importance of a positive-feedback loop in which Swe1p activity inhibits the CDK, which then ceases to target Swe1p for degradation. They also highlight the difficulty in disentangling direct checkpoint pathways from the effects of positive-feedback loops active at the G2/M transition.
PMCID: PMC3608501  PMID: 23389636
6.  Mitogen-activated protein kinase phosphatase 2 regulates histone H3 phosphorylation via interaction with vaccinia-related kinase 1 
Molecular Biology of the Cell  2013;24(3):373-384.
Vaccinia-related kinase 1 (VRK1) is a histone kinase that phosphorylates histone H3 at Thr-3 and Ser-10. This study shows that mitogen-activated protein kinase phosphatase 2 regulates this phosphorylation negatively via interaction with VRK1, regardless of VRK1’s phosphatase activity.
Mitogen-activated protein kinase phosphatase 2 (MKP2) is a member of the dual-specificity MKPs that regulate MAP kinase signaling. However, MKP2 functions are still largely unknown. In this study, we showed that MKP2 could regulate histone H3 phosphorylation under oxidative stress conditions. We found that MKP2 inhibited histone H3 phosphorylation by suppressing vaccinia-related kinase 1 (VRK1) activity. Moreover, this regulation was dependent on the selective interaction with VRK1, regardless of its phosphatase activity. The interaction between MKP2 and VRK1 mainly occurred in the chromatin, where histones are abundant. We also observed that the protein level of MKP2 and its interaction with histone H3 increased from G1 to M phase during the cell cycle, which is similar to the VRK1 profile. Furthermore, MKP2 specifically regulated the VRK1-mediated histone H3 phosphorylation at M phase. Taken together, these data suggest a novel function of MKP2 as a negative regulator of VRK1-mediated histone H3 phosphorylation.
PMCID: PMC3564537  PMID: 23223570
7.  Phosphoinositide 3-kinase β regulates chromosome segregation in mitosis 
Molecular Biology of the Cell  2012;23(23):4526-4542.
The phosphoinositide 3-kinase (PI3K) pathway is mutated in approximately half of tumors; it is therefore important to define its functions. This study shows that PI3Kα activity regulates mitotic entry and spindle orientation; in contrast, PI3Kβ controls dynein/dynactin and Aurora B activation at kinetochores and, in turn, chromosome segregation.
Class IA phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP3). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110α and p110β. Whereas p110α is activated at G1-phase entry and promotes protein synthesis and gene expression, p110β activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110α and p110β function in mitosis. p110α was activated at mitosis entry and regulated early mitotic events, such as PIP3 generation, prometaphase progression, and spindle orientation. In contrast, p110β was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110β function in preserving genomic stability during mitosis.
PMCID: PMC3510015  PMID: 23051731
8.  Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle 
Molecular Biology of the Cell  2012;23(21):4203-4211.
This study identifies the degradation pathway for the F-box protein Dia2, which plays an important role in maintaining genomic integrity. The Hect domain E3 ligase Tom1 recognizes a stretch of positively charged residues in Dia2, leading to Dia2 degradation by the ubiquitin proteasome system. Failure to degrade Dia2 disrupts cell cycle dynamics.
The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.
PMCID: PMC3484099  PMID: 22933573
9.  Dual effect of heat shock on DNA replication and genome integrity 
Molecular Biology of the Cell  2012;23(17):3450-3460.
The data presented here suggest that in an asynchronous cell culture, heat shock might affect DNA integrity both directly and via arrest of replication fork progression and that the phosphorylation of histone H2AX has a protective effect on the arrested replication forks in addition to its known DNA damage signaling function.
Heat shock (HS) is one of the better-studied exogenous stress factors. However, little is known about its effects on DNA integrity and the DNA replication process. In this study, we show that in G1 and G2 cells, HS induces a countable number of double-stranded breaks (DSBs) in the DNA that are marked by γH2AX. In contrast, in S-phase cells, HS does not induce DSBs but instead causes an arrest or deceleration of the progression of the replication forks in a temperature-dependent manner. This response also provoked phosphorylation of H2AX, which appeared at the sites of replication. Moreover, the phosphorylation of H2AX at or close to the replication fork rescued the fork from total collapse. Collectively our data suggest that in an asynchronous cell culture, HS might affect DNA integrity both directly and via arrest of replication fork progression and that the phosphorylation of H2AX has a protective effect on the arrested replication forks in addition to its known DNA damage signaling function.
PMCID: PMC3431931  PMID: 22787276
10.  Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control 
Molecular Biology of the Cell  2012;23(16):3079-3093.
A periodic luciferase reporter system from cell cycle–regulated promoters in synchronous U2OS cells measures periodic, cell cycle–regulated gene expression in live cells. This assay is used to identify Forkhead transcription factors that control periodic gene expression, and it identifies FOXK1 as an activator of key cell cycle genes.
We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.
PMCID: PMC3418304  PMID: 22740631
11.  Identification of She3 as an SCFGrr1 Substrate in Budding Yeast 
PLoS ONE  2012;7(10):e48020.
The highly orchestrated progression of the cell cycle depends on the degradation of many regulatory proteins at different cell cycle stages. One of the key cell cycle ubiquitin ligases is the Skp1-cullin-F-box (SCF) complex. Acting in concert with the substrate-binding F-box protein Grr1, SCFGrr1 promotes the degradation of cell cycle regulators as well as various metabolic enzymes. Using a yeast two-hybrid assay with a Grr1 derivative as the bait, we identified She3, which is an adaptor protein in the asymmetric mRNA transport system, as a novel Grr1 substrate. We generated stabilized She3 mutants, which no longer bound to Grr1, and found that the degradation of She3 is not required for regulating asymmetric mRNA transport. However, She3 stabilization leads to slower growth compared to wild-type cells in a co-culture assay, demonstrating that the degradation of She3 by Grr1 is required for optimal cell growth.
PMCID: PMC3483296  PMID: 23144720
12.  Identification of Anaphase Promoting Complex Substrates in S. cerevisiae 
PLoS ONE  2012;7(9):e45895.
The Anaphase-Promoting Complex/Cyclosome (APC/C) is an essential ubiquitin ligase that targets numerous proteins for proteasome-mediated degradation in mitosis and G1. To gain further insight into cellular pathways controlled by APC/CCdh1, we developed two complementary approaches to identify additional APC/CCdh1 substrates in budding yeast. First, we analyzed the stabilities of proteins that were expressed at the same time in the cell cycle as known APC/C substrates. Second, we screened for proteins capable of interacting with the Cdh1 substrate-binding protein in a yeast two-hybrid system. Here we characterize five potential APC/C substrates identified using these approaches: the transcription factors Tos4 and Pdr3; the mRNA processing factor Fir1; the spindle checkpoint protein kinase Mps1; and a protein of unknown function, Ybr138C. Analysis of the degradation motifs within these proteins revealed that the carboxyl-terminal KEN box and D-boxes of Tos4 are important for its interaction with Cdh1, whereas the N-terminal domain of Ybr138C is required for its instability. Functionally, we found that a stabilized form of Mps1 delayed cell division upon mild spindle disruption, and that elevated levels of Ybr138C reduced cell fitness. Interestingly, both Tos4 and Pdr3 have been implicated in the DNA damage response, whereas Mps1 regulates the spindle assembly checkpoint. Thus, the APC/CCdh1-mediated degradation of these proteins may help to coordinate re-entry into the cell cycle following environmental stresses.
PMCID: PMC3458821  PMID: 23049888
13.  Gcn5p-dependent acetylation induces degradation of the meiotic transcriptional repressor Ume6p 
Molecular Biology of the Cell  2012;23(9):1609-1617.
Acetyltransferases induce transcription by enhancing the activity of transcriptional activators and opening chromatin domains. A third avenue is described by which gene activation is accomplished by acetylation through the targeted destruction of the Ume6p repressor.
Ume6p represses early meiotic gene transcription in Saccharomyces cerevisiae by recruiting the Rpd3p histone deacetylase and chromatin-remodeling proteins. Ume6p repression is relieved in a two-step destruction process mediated by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. The first step induces partial Ume6p degradation when vegetative cells shift from glucose- to acetate-based medium. Complete proteolysis happens only upon meiotic entry. Here we demonstrate that the first step in Ume6p destruction is controlled by its acetylation and deacetylation by the Gcn5p acetyltransferase and Rpd3p, respectively. Ume6p acetylation occurs in medium lacking dextrose and results in a partial destruction of the repressor. Preventing acetylation delays Ume6p meiotic destruction and retards both the transient transcription program and execution of the meiotic nuclear divisions. Conversely, mimicking acetylation induces partial destruction of Ume6p in dextrose medium and accelerates meiotic degradation by the APC/C. These studies reveal a new mechanism by which acetyltransferase activity induces gene expression through targeted destruction of a transcriptional repressor. These findings also demonstrate an important role for nonhistone acetylation in the transition between mitotic and meiotic cell division.
PMCID: PMC3338428  PMID: 22438583
14.  Punctuated cyclin synthesis drives early embryonic cell cycle oscillations 
Molecular Biology of the Cell  2012;23(2):284-296.
Cyclin B accumulation in the early Xenopus embryo drives CDK1 oscillations. CDK1 directs a negative-feedback loop upon cyclin B synthesis through the repression of global protein translation, and this attenuation of cyclin B synthesis could improve the efficiency and robustness of this cell cycle oscillator.
Cyclin B activates cyclin-dependent kinase 1 (CDK1) at mitosis, but conflicting views have emerged on the dynamics of its synthesis during embryonic cycles, ranging from continuous translation to rapid synthesis during mitosis. Here we show that a CDK1-mediated negative-feedback loop attenuates cyclin production before mitosis. Cyclin B plateaus before peak CDK1 activation, and proteasome inhibition caused minimal accumulation during mitosis. Inhibiting CDK1 permitted continual cyclin B synthesis, whereas adding nondegradable cyclin stalled it. Cycloheximide treatment before mitosis affected neither cyclin levels nor mitotic entry, corroborating this repression. Attenuated cyclin production collaborates with its destruction, since excess cyclin B1 mRNA accelerated cyclin synthesis and caused incomplete proteolysis and mitotic arrest. This repression involved neither adenylation nor the 3′ untranslated region, but it corresponded with a shift in cyclin B1 mRNA from polysome to nonpolysome fractions. A pulse-driven CDK1–anaphase-promoting complex (APC) model corroborated these results, revealing reduced cyclin levels during an oscillation and permitting more effective removal. This design also increased the robustness of the oscillator, with lessened sensitivity to changes in cyclin synthesis rate. Taken together, the results of this study underscore that attenuating cyclin synthesis late in interphase improves both the efficiency and robustness of the CDK1-APC oscillator.
PMCID: PMC3258173  PMID: 22130797
15.  Coupling of T161 and T14 phosphorylations protects cyclin B–CDK1 from premature activation 
Molecular Biology of the Cell  2011;22(21):3971-3985.
Whether CDK1 phosphorylations occur on the same molecules could be determined using two-dimensional gel electrophoresis. The activating T161 phosphorylation was found to be tightly coupled to the T14 inhibitory phosphorylation in cyclin B1–CDK1, suggesting that the mitotic timer is endowed with an intrinsic mechanism protecting it from premature activation by CAK.
Mitosis is triggered by the abrupt dephosphorylation of inhibitory Y15 and T14 residues of cyclin B1–bound cyclin-dependent kinase (CDK)1 that is also phosphorylated at T161 in its activation loop. The sequence of events leading to the accumulation of fully phosphorylated cyclin B1–CDK1 complexes remains unclear. Two-dimensional gel electrophoresis allowed us to determine whether T14, Y15, and T161 phosphorylations occur on same CDK1 molecules and to characterize the physiological occurrence of their seven phosphorylation combinations. Intriguingly, in cyclin B1–CDK1, the activating T161 phosphorylation never occurred without the T14 phosphorylation. This strict association could not be uncoupled by a substantial reduction of T14 phosphorylation in response to Myt1 knockdown, suggesting some causal relationship. However, T14 phosphorylation was not directly required for T161 phosphorylation, because Myt1 knockdown did uncouple these phosphorylations when leptomycin B prevented cyclin B1–CDK1 complexes from accumulating in cytoplasm. The coupling mechanism therefore depended on unperturbed cyclin B1–CDK1 traffic. The unexpected observation that the activating phosphorylation of cyclin B1–CDK1 was tightly coupled to its T14 phosphorylation, but not Y15 phosphorylation, suggests a mechanism that prevents premature activation by constitutively active CDK-activating kinase. This explained the opposite effects of reduced expression of Myt1 and Wee1, with only the latter inducing catastrophic mitoses.
PMCID: PMC3204060  PMID: 21900495
16.  Anaphase promoting complex–dependent degradation of transcriptional repressors Nrm1 and Yhp1 in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2011;22(13):2175-2184.
The anaphase-promoting complex is a ubiquitin ligase that promotes the degradation of numerous cell cycle regulators during mitosis and in G1. This report identifies two transcriptional repressors—Nrm1 and Yhp1—as novel APC substrates in budding yeast. In the absence of their degradation, target genes are misexpressed and cell fitness is reduced.
The anaphase-promoting complex/cyclosome (APC/C) is an essential ubiquitin ligase that targets cell cycle proteins for proteasome-mediated degradation in mitosis and G1. The APC regulates a number of cell cycle processes, including spindle assembly, mitotic exit, and cytokinesis, but the full range of its functions is still unknown. To better understand cellular pathways controlled by the APC, we performed a proteomic screen to identify additional APC substrates. We analyzed cell cycle–regulated proteins whose expression peaked during the period when other APC substrates were expressed. Subsequent analysis identified several proteins, including the transcriptional repressors Nrm1 and Yhp1, as authentic APC substrates. We found that APCCdh1 targeted Nrm1 and Yhp1 for degradation in early G1 through Destruction-box motifs and that the degradation of these repressors coincided with transcriptional activation of MBF and Mcm1 target genes, respectively. In addition, Nrm1 was stabilized by phosphorylation, most likely by the budding yeast cyclin–dependent protein kinase, Cdc28. We found that expression of stabilized forms of Nrm1 and Yhp1 resulted in reduced cell fitness, due at least in part to incomplete activation of G1-specific genes. Therefore, in addition to its known functions, APC-mediated targeting of Nrm1 and Yhp1 coordinates transcription of multiple genes in G1 with other cell cycle events.
PMCID: PMC3128521  PMID: 21562221
17.  Mes1 controls the meiosis I to meiosis II transition by distinctly regulating the anaphase-promoting complex/cyclosome coactivators Fzr1/Mfr1 and Slp1 in fission yeast 
Molecular Biology of the Cell  2011;22(9):1486-1494.
Although meiosis is extremely important not only for sexual reproduction but also for creating diversity, very little is known about meiotic regulation. Five APC/C coactivators have been found in the fission yeast genome. This study investigates how all the coactivators are involved in the meiosis I/meiosis II transition.
Meiosis is a specialized form of cell division generating haploid gametes and is dependent upon protein ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C). Accurate control of the APC/C during meiosis is important in all eukaryotic cells and is in part regulated by the association of coactivators and inhibitors. We previously showed that the fission yeast meiosis-specific protein Mes1 binds to a coactivator and inhibits APC/C; however, regulation of the Mes1-mediated APC/C inhibition remains elusive. Here we show how Mes1 distinctively regulates different forms of the APC/C. We study all the coactivators present in the yeast genome and find that only Slp1/Cdc20 is essential for meiosis I progression. However, Fzr1/Mfr1 is a critical target for Mes1 inhibition because fzr1Δ completely rescues the defect on the meiosis II entry in mes1Δ cells. Furthermore, cell-free studies suggest that Mes1 behaves as a pseudosubstrate for Fzr1/Mfr1 but works as a competitive substrate for Slp1. Intriguingly, mutations in the D-box or KEN-box of Mes1 increase its recognition as a substrate by Fzr1, but not by Slp1. Thus Mes1 interacts with two coactivators in a different way to control the activity of the APC/C required for the meiosis I/meiosis II transition.
PMCID: PMC3084671  PMID: 21389117
18.  Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited 
Molecular Biology of the Cell  2011;22(8):1191-1206.
Activation of Cdk1 is rapid and switch-like due to positive feedback mechanisms. When Cdk1 is fully on, cells are capable of M-to-G1 transition. Inhibition of positive feedback prevents rapid Cdk1 activation and induces a mitotic “collapse” phenotype characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis.
Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic “collapse,” a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.
PMCID: PMC3078080  PMID: 21325631
19.  RB deletion disrupts coordination between DNA replication licensing and mitotic entry in vivo 
Molecular Biology of the Cell  2011;22(7):931-939.
The retinoblastoma tumor suppressor (RB) is functionally inactivated at high frequency in nearly all tumor types. Herein the acute deletion of RB in the liver reveals an immediate and profound dysregulation of spatiotemporal coordination in cell-cycle phases, resulting in robust DNA damage and aneuploidy that is not found in other tissues.
The integrity of the retinoblastoma tumor suppressor (RB) pathway is critical for restraining inappropriate proliferation and suppressing tumor development in a plethora of tissues. Here adenovirus-mediated RB deletion in the liver of adult mice led to DNA replication in the absence of productive mitotic condensation. The replication induced by RB loss was E2F-mediated and associated with the induction of DNA damage and a nontranscriptional G2/M checkpoint that targeted the accumulation of Cyclin B1. In the context of RB deletion or E2F activation, there was an increase in hepatocyte ploidy that was accompanied by hyperphysiological assembly of prereplication complexes. In keeping with this dysregulation, initiation of DNA replication was readily observed in hepatocytes that were phenotypically in G2/M. Under such conditions, uncoupling of replication initiation from mitotic progression led to altered genome ploidy in the liver. Interestingly, these findings in hepatocytes were not recapitulated in the basally proliferative tissues of the gastrointestinal tract, where RB deletion, while increasing DNA replication, did not lead to a profound uncoupling from mitosis. Combined, these findings demonstrate the critical role of RB in controlling cell-cycle transitions and underscore the importance of intrinsic tissue environments in resultant phenotypes.
PMCID: PMC3069018  PMID: 21289097
20.  Ama1p-activated anaphase-promoting complex regulates the destruction of Cdc20p during meiosis II 
Molecular Biology of the Cell  2011;22(3):315-326.
During meiosis, the APC/C is activated by either Cdc20 or the meiosis-specific activator Ama1. Upon exit from meiosis II, APC/CAma1 mediates Cdc20 destruction using Db1 and GxEN degrons. The amino terminus of Ama1, which contains the Cdc20-binding domain, is sufficient for Cdc20 degradation but not spore formation.
The execution of meiotic divisions in Saccharomyces cerevisiae is regulated by anaphase-promoting complex/cyclosome (APC/C)–mediated protein degradation. During meiosis, the APC/C is activated by association with Cdc20p or the meiosis-specific activator Ama1p. We present evidence that, as cells exit from meiosis II, APC/CAma1 mediates Cdc20p destruction. APC/CAma1 recognizes two degrons on Cdc20p, the destruction box and destruction degron, with either domain being sufficient to mediate Cdc20p destruction. Cdc20p does not need to associate with the APC/C to bind Ama1p or be destroyed. Coimmunoprecipitation analyses showed that the diverged amino-terminal region of Ama1p recognizes both Cdc20p and Clb1p, a previously identified substrate of APC/CAma1. Domain swap experiments revealed that the C-terminal WD region of Cdh1p, when fused to the N-terminal region of Ama1p, could direct most of Ama1p functions, although at a reduced level. In addition, this fusion protein cannot complement the spore wall defect in ama1Δ strains, indicating that substrate specificity is also derived from the WD repeat domain. These findings provide a mechanism to temporally down-regulate APC/CCdc20 activity as the cells complete meiosis II and form spores.
PMCID: PMC3031463  PMID: 21118994
21.  MiR-322/424 and -503 Are Induced during Muscle Differentiation and Promote Cell Cycle Quiescence and Differentiation by Down-Regulation of Cdc25A 
Molecular Biology of the Cell  2010;21(13):2138-2149.
This article describes a novel role of Cdc25A down-regulation during differentiation of proliferating myoblasts.
Induction of a G1 phase cell cycle arrest, caused primarily by the inhibition of cyclin-dependent-kinase 2 (cdk2), is a critical step in the differentiation of myoblasts into myotubes. Here, we report that two microRNAs, miR-322/424 and miR-503, are induced and promote cdk2 inhibition during myogenesis. These microRNAs down-regulate Cdc25A, the phosphatase responsible for removing inhibitory phosphorylation of cdk2, both in myoblasts differentiating into myotubes and in nonmuscle cells. Cdc25A is down-regulated during muscle differentiation by multiple pathways: action of these two microRNAs, proteasomal degradation of Cdc25A protein and transcriptional repression. Overexpression of Cdc25A or of cdk2 with mutations on T14 and Y15 (cdk2-AF), so that it cannot be inhibited by phosphorylation, decreases differentiation and differentiation-induced cell cycle quiescence. Introduction of miR-322/424 and miR-503 in heterologous cancer cells induces G1 arrest, which is also attenuated by overexpression of the cdk2-AF mutant. Until now Cdc25A and the inhibitory phosphorylation on T14 and Y15 of cdk2 have only been implicated in the intra-S phase checkpoint pathway after DNA damage. Our results reveal an unexpected role of Cdc25A down-regulation and the inhibitory phosphorylation of cdk2 T14 and Y15 in cell cycle quiescence during muscle differentiation and implicate two muscle differentiation-induced microRNAs in the process.
PMCID: PMC2893979  PMID: 20462953
22.  Dissecting the M Phase–specific Phosphorylation of Serine–Proline or Threonine–Proline Motifs 
Molecular Biology of the Cell  2010;21(9):1470-1481.
Mitotic induction tightly associates with increased reactivity to mitotic phosphoprotein antibody MPM-2. Here we report that phosphorylation of TP motifs surrounded by hydrophobic residues at the −1 and +1 positions plays a dominant role in M phase–associated MPM-2 reactivity. The majority of these motifs are not phosphorylated by mitotic Cdk or MAPK.
M phase induction in eukaryotic cell cycles is associated with a burst of protein phosphorylation, primarily at serine or threonine followed by proline (S/TP motif). The mitotic phosphoprotein antibody MPM-2 recognizes a significant subset of mitotically phosphorylated S/TP motifs; however, the required surrounding sequences of and the key kinases that phosphorylate these S/TP motifs remain to be determined. By mapping the mitotic MPM-2 epitopes in Xenopus Cdc25C and characterizing the mitotic MPM-2 epitope kinases in Xenopus oocytes and egg extracts, we have determined that phosphorylation of TP motifs that are surrounded by hydrophobic residues at both −1 and +1 positions plays a dominant role in M phase–associated burst of MPM-2 reactivity. Although mitotic Cdk and MAPK may phosphorylate subsets of these motifs that have a basic residue at the +2 position and a proline residue at the −2 position, respectively, the majority of these motifs that are preferentially phosphorylated in mitosis do not have these features. The M phase–associated burst of MPM-2 reactivity can be induced in Xenopus oocytes and egg extracts in the absence of MAPK or Cdc2 activity. These findings indicate that the M phase–associated burst of MPM-2 reactivity represents a novel type of protein phosphorylation in mitotic regulation.
PMCID: PMC2861607  PMID: 20219976
23.  Emi2 Inhibition of the Anaphase-promoting Complex/Cyclosome Absolutely Requires Emi2 Binding via the C-Terminal RL Tail 
Molecular Biology of the Cell  2010;21(6):905-913.
Both the D-box and the zinc-binding region (ZBR) of Emi2 are implicated in APC/C inhibition. This article shows that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. The RL tail apparently promotes the inhibitory interactions of the D-box and the ZBR with the APC/C. The RL tail thus serves as a docking site for the APC/C.
Emi2 (also called Erp1) inhibits the anaphase-promoting complex/cyclosome (APC/C) and thereby causes metaphase II arrest in unfertilized vertebrate eggs. Both the D-box and the zinc-binding region (ZBR) of Emi2 have been implicated in APC/C inhibition. However, it is not well known how Emi2 interacts with and hence inhibits the APC/C. Here we show that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. When expressed in Xenopus oocytes and egg extracts, Emi2 lacking the RL tail fails to interact with and inhibit the APC/C. The RL tail itself can directly bind to the APC/C, and, when added to egg extracts, either an excess of RL tail peptides or anti-RL tail peptide antibody can dissociate endogenous Emi2 from the APC/C, thus allowing APC/C activation. Furthermore, and importantly, the RL tail–mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C. Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail. We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR.
PMCID: PMC2836971  PMID: 20089832
24.  NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans 
Molecular Biology of the Cell  2010;21(5):712-724.
Cellular and genetic analysis supports the notion that NPP-16/NUP50 and CDK-1 function to reversibly arrest prophase blastomeres in Caenorhabditis elegans embryos exposed to anoxia. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.
Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type–arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.
PMCID: PMC2828959  PMID: 20053678
25.  Genomic Study of Replication Initiation in Human Chromosomes Reveals the Influence of Transcription Regulation and Chromatin Structure on Origin Selection 
Molecular Biology of the Cell  2010;21(3):393-404.
DNA replication in metazoans initiates from multiple chromosomal loci called origins. This study identifies 150 new origins of replication that were confirmed by two methods of nascent strand purification. We discern the role of transcription initiation and regulation, as well as chromatin signatures in determining origin selection in human genome.
DNA replication in metazoans initiates from multiple chromosomal loci called origins. Currently, there are two methods to purify origin-centered nascent strands: lambda exonuclease digestion and anti-bromodeoxyuridine immunoprecipitation. Because both methods have unique strengths and limitations, we purified nascent strands by both methods, hybridized them independently to tiling arrays (1% genome) and compared the data to have an accurate view of genome-wide origin distribution. By this criterion, we identified 150 new origins that were reproducible across the methods. Examination of a subset of these origins by chromatin immunoprecipitation against origin recognition complex (ORC) subunits 2 and 3 showed 93% of initiation peaks to localize at/within 1 kb of ORC binding sites. Correlation of origins with functional elements of the genome revealed origin activity to be significantly enriched around transcription start sites (TSSs). Consistent with proximity to TSSs, we found a third of initiation events to occur at or near the RNA polymerase II binding sites. Interestingly, ∼50% of the early origin activity was localized within 5 kb of transcription regulatory factor binding region clusters. The chromatin signatures around the origins were enriched in H3K4-(di- and tri)-methylation and H3 acetylation modifications on histones. Affinity of origins for open chromatin was also reiterated by their proximity to DNAse I-hypersensitive sites. Replication initiation peaks were AT rich, and >50% of the origins mapped to evolutionarily conserved regions of the genome. In summary, these findings indicate that replication initiation is influenced by transcription initiation and regulation as well as chromatin structure.
PMCID: PMC2814785  PMID: 19955211

Results 1-25 (130)