The budding yeast Saccharomyces cerevisiae is a eukaryotic organism with extensive genetic redundancy. Large-scale gene deletion analysis has shown that over 80% of the ~6200 predicted genes are nonessential and that the functions of 30% of all ORFs remain unclassified, implying that yeast cells can tolerate deletion of a substantial number of individual genes. For example, a class of zinc finger proteins containing C2H2 zinc fingers in tandem arrays of two or three is predicted to be transcription factors; however, seven of the thirty-one predicted genes of this class are nonessential, and their functions are poorly understood. In this study we completed a transcriptomic profiling of three mutants lacking C2H2 zinc finger proteins, ypr013cΔ,ypr015cΔ and ypr013cΔypr015cΔ.
Gene expression patterns were remarkably different between wild type and the mutants. The results indicate altered expression of 79 genes in ypr013cΔ, 185 genes in ypr015cΔ and 426 genes in the double mutant when compared with that of the wild type strain. More than 80% of the alterations in the double mutants were not observed in either one of the single deletion mutants. Functional categorization based on Munich Information Center for Protein Sequences (MIPS) revealed up-regulation of genes related to transcription and down-regulation of genes involving cell rescue and defense, suggesting a decreased response to stress conditions. Genes related to cell cycle and DNA processing whose expression was affected by single or double deletions were also identified.
Our results suggest that microarray analysis can define the biological roles of zinc finger proteins with unknown functions and identify target genes that are regulated by these putative transcriptional factors. These findings also suggest that both YPR013C and YPR015C have biological processes in common, in addition to their own regulatory pathways.
We have characterized the role of YPR128cp, the orthologue of human PMP34, in fatty acid metabolism and peroxisomal proliferation in Saccharomyces cerevisiae. YPR128cp belongs to the mitochondrial carrier family (MCF) of solute transporters and is localized in the peroxisomal membrane. Disruption of the YPR128c gene results in impaired growth of the yeast with the medium-chain fatty acid (MCFA) laurate as a single carbon source, whereas normal growth was observed with the long-chain fatty acid (LCFA) oleate. MCFA but not LCFA β-oxidation activity was markedly reduced in intact ypr128cΔ mutant cells compared to intact wild-type cells, but comparable activities were found in the corresponding lysates. These results imply that a transport step specific for MCFA β-oxidation is impaired in ypr128cΔ cells. Since MCFA β-oxidation in peroxisomes requires both ATP and CoASH for activation of the MCFAs into their corresponding coenzyme A esters, we studied whether YPR128cp is an ATP carrier. For this purpose we have used firefly luciferase targeted to peroxisomes to measure ATP consumption inside peroxisomes. We show that peroxisomal luciferase activity was strongly reduced in intact ypr128cΔ mutant cells compared to wild-type cells but comparable in lysates of both cell strains. We conclude that YPR128cp most likely mediates the transport of ATP across the peroxisomal membrane.
Yeasts used in the production of lagers contain complex allopolyploid genomes, resulting from the fusion of two different yeast species closely related to Saccharomyces cerevisiae and Saccharomyces bayanus. Recombination between the homoeologous chromosomes has generated a number of hybrid chromosomes. These recombination events provide potential for adaptive evolution through the loss or gain of gene function. We have examined the genotypic and phenotypic effects of one of the conserved recombination events that occurred on chromosome XVI in the region of YPR159W and YPR160W. Our analysis shows that the recombination event occurred within the YPR160W gene, which encodes the enzyme glycogen phosphorylase and generates a hybrid gene that does not produce mature mRNA and is nonfunctional due to frameshifts in the coding region. The loss of function of the hybrid gene leads to glycogen levels similar to those found in haploid yeast strains. The implications for the control of glycogen levels in fermentative yeasts are discussed.
The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.
Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly acting BUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed a G2 DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal.
The checkpoint mechanisms that delay cell cycle progression in response to DNA damage or inhibition of DNA replication are necessary for maintenance of genetic stability in eukaryotic cells. Potential targets of checkpoint-mediated regulation include proteins directly involved in DNA metabolism, such as the cellular single-stranded DNA (ssDNA) binding protein, replication protein A (RPA). Studies in Saccharomyces cerevisiae have revealed that the RPA large subunit (Rfa1p) is involved in the G1 and S phase DNA damage checkpoints. We now demonstrate that Rfa1p is phosphorylated in response to various forms of genotoxic stress, including radiation and hydroxyurea exposure, and further show that phosphorylation of Rfa1p is dependent on the central checkpoint regulator Mec1p. Analysis of the requirement for other checkpoint genes indicates that different mechanisms mediate radiation- and hydroxyurea-induced Rfa1p phosphorylation despite the common requirement for functional Mec1p. In addition, experiments with mutants defective in the Cdc13p telomere-binding protein indicate that ssDNA formation is an important signal for Rfa1p phosphorylation. Because Rfa1p contains the major ssDNA binding activity of the RPA heterotrimer and is required for DNA replication, repair and recombination, it is possible that phosphorylation of this subunit is directly involved in modulating RPA activity during the checkpoint response.
The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.
Deregulated CDK activity drives cell proliferation of the majority of human tumors, making CDKs highly relevant research subjects. Cdc28 controls cell cycle progression in the budding yeast Saccharomyces cerevisiae, but the identity of many genes that function in conjunction with CDC28 to regulate the cell cycle and cell viability remains obscure. In a recent study, we used a chemical-genetic screen to identify the genetic network of CDC28. Through this analysis, we discovered that the Rad6-Bre1 pathway functions in this network and links ubiquitin levels to cell cycle progression by increasing transcription of cyclin genes. Thus, Rad6 boosts the activity of the cell cycle machinery.
cell cycle; Rad6; Cdc28; Cdk1; Doa1; Cdc48; transcription; Whi5; cyclin; ubiquitin
In Saccharomyces cerevisiae, the ATF/CREB transcription factor Sko1 (Acr1) regulates the expression of genes induced by osmotic stress under the control of the high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway. By combining chromatin immunoprecipitation and microarrays containing essentially all intergenic regions, we estimate that yeast cells contain approximately 40 Sko1 target promoters in vivo; 20 Sko1 target promoters were validated by direct analysis of individual loci. The ATF/CREB consensus sequence is not statistically overrepresented in confirmed Sko1 target promoters, although some sites are evolutionarily conserved among related yeast species, suggesting that they are functionally important in vivo. These observations suggest that Sko1 association in vivo is affected by factors beyond the protein-DNA interaction defined in vitro. Sko1 binds a number of promoters for genes directly involved in defense functions that relieve osmotic stress. In addition, Sko1 binds to the promoters of genes encoding transcription factors, including Msn2, Mot3, Rox1, Mga1, and Gat2. Stress-induced expression of MSN2, MOT3, and MGA1 is diminished in sko1 mutant cells, while transcriptional regulation of ROX1 seems to be unaffected. Lastly, Sko1 targets PTP3, which encodes a phosphatase that negatively regulates Hog1 kinase activity, and Sko1 is required for osmotic induction of PTP3 expression. Taken together our results suggest that Sko1 operates a transcriptional network upon osmotic stress, which involves other specific transcription factors and a phosphatase that regulates the key component of the signal transduction pathway.
The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerevisiae plays an essential role in the progression of cells from the G1 to S phase of the cell division cycle. Using a high-copy suppression strategy, we have identified a yeast gene (UBS1) whose elevated expression suppresses the conditional cell cycle defects associated with cdc34 mutations. The UBS1 gene encodes a 32.2-kDa protein of previously unknown function and is identical in sequence to a genomic open reading frame on chromosome II (GenBank accession number Z36034). Several lines of evidence described here indicate that Ubs1 functions as a general positive regulator of Cdc34 activity. First, overexpression of UBS1 suppresses not only the cell proliferation and morphological defects associated with cdc34 mutants but also the inability of cdc34 mutant cells to degrade the general amino acid biosynthesis transcriptional regulator, Gcn4. Second, deletion of the UBS1 gene profoundly accentuates the cell cycle defect when placed in combination with a cdc34 temperature-sensitive allele. Finally, a comparison of the Ubs1 and Cdc34 polypeptide sequences reveals two noncontiguous regions of similarity, which, when projected onto the three-dimensional structure of a ubiquitin-conjugating enzyme, define a single region situated on its surface. While cdc34 mutations corresponding to substitutions outside this region are suppressed by UBS1 overexpression, Ubs1 fails to suppress amino acid substitutions made within this region. Taken together with other findings, the allele specificity exhibited by UBS1 expression suggests that Ubs1 regulates Cdc34 by interaction or modification.
In a cross-species overexpression approach, we used the pseudohyphal transition of Saccharomyces cerevisiae as a model screening system to identify human genes that regulate cell morphology and the cell cycle. Human enhancer of invasion-cluster (HEI-C), encoding a novel evolutionarily conserved coiled-coil protein, was isolated in a screen for human genes that induce agar invasion in S. cerevisiae. In human cells, HEI-C is primarily localized to the spindle during mitosis. Depletion of HEI-C in vivo with short interfering RNAs results in severe mitotic defects. Analysis by immunofluorescence, flow cytometry analysis, and videomicroscopy indicates that HEI-C-depleted cells form metaphase plates with normal timing after G2/M transition, although in many cases cells have disorganized mitotic spindles. Subsequently, severe defects occur at the metaphase-anaphase transition, characterized by a significant delay at this stage or, more commonly, cellular disintegration accompanied by the display of classic biochemical markers of apoptosis. These mitotic defects occur in spite of the fact that HEI-C-depleted cells retain functional cell cycle checkpoints, as these cells arrest normally following nocodazole or hydroxyurea treatment. These results place HEI-C as a novel regulator of spindle function and integrity during the metaphase-anaphase transition.
The Saccharomyces cerevisiae ubiquitin ligase SCFMet30 is essential for cell cycle progression. To identify and characterize SCFMet30-dependent cell cycle steps, we used temperature-sensitive met30 mutants in cell cycle synchrony experiments. These experiments revealed a requirement for Met30 during both G1/S transition and M phase, while progression through S phase was unaffected by loss of Met30 function. Expression of the G1-specific transcripts CLN1, CLN2, and CLB5 was very low in met30 mutants, whereas expression of CLN3 was unaffected. However, overexpression of Cln2 could not overcome the G1 arrest. Interestingly, overexpression of Clb5 could induce DNA replication in met30 mutants, albeit very inefficiently. Increased levels of Clb5 could not, however, suppress the cell proliferation defect of met30 mutants. Consistent with the DNA replication defects, chromatin immunoprecipitation experiments revealed significantly lower levels of the replication factors Mcm4, Mcm7, and Cdc45 at replication origins in met30 mutants than in wild-type cells. These data suggest that Met30 regulates several aspects of the cell cycle, including G1-specific transcription, initiation of DNA replication, and progression through M phase.
We have cloned a yeast gene, SKO1, which in high copy number suppresses lethal overexpression of cAMP-dependent protein kinase. SKO1 encodes a bZIP protein that binds to the CRE motif, TGACGTCA. We found that SKO1 also binds to a CRE-like site in SUC2, a yeast gene encoding invertase which is under positive control by cAMP. A disruption of the SKO1 gene causes a partial derepression of SUC2, indicating that SKO1 is a negative regulator of the SUC2 gene. SKO1 interacts positively with MIG1, a zinc finger protein that mediates glucose repression of SUC2. A kinetic analysis revealed a complex regulation of the SUC2 mRNA in response to glucose. First, MIG1 mediates a rapid and strong repression of SUC2, which is complete within 10 minutes. Second, a MIG1-independent process causes a further slow reduction in the mRNA. Third, in the absence of MIG1, there is also a rapid but transient glucose induction of the SUC2 mRNA. This induction is correlated with a transient loss of SKO1-dependent repression.
Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.
Double strand DNA breaks (DSBs) are dangerous chromosomal lesions that can lead to genome rearrangements, genetic instability, and cancer if not accurately repaired. Eukaryotes activate a surveillance mechanism, called DNA damage checkpoint, to arrest cell cycle progression and facilitate DNA repair. Several factors are physically recruited to DSBs, and specific kinases phosphorylate multiple targets leading to checkpoint activation. Budding yeast is a good model system to study checkpoint, and most of the factors involved in the DSBs response were originally characterized in this organism. Using the yeast Saccharomyces cerevisiae, we explored the functional role of polo kinase Cdc5 in regulating the DSB–induced checkpoint. Polo kinases have been previously involved in checkpoint inactivation in all the eukaryotes, and they are frequently overexpressed in cancer cells. We found that elevated levels of Cdc5 affect the cellular response to a DSB at different steps, altering DNA processing and overriding the signal triggered by checkpoint kinases. Our findings suggest that Cdc5 likely regulates multiple factors in response to a DSB and provide a rationale for a proteome-wide screening to identify targets of polo kinases in yeast and human cells. Such information may have a practical application to design specific molecular tools for cancer therapy. Two related papers published in PLoS Biology—by Vidanes et al., doi:10.1371/journal.pbio.1000286, and van Vugt et al., doi:10.1371/journal.pbio.1000287—similarly investigate the phenomenon of checkpoint adaptation/overriding.
Regulated gene expression is essential for a proper progression through the cell cycle. The transcription factor NF-Y has a fundamental function in transcriptional regulation of cell cycle genes, particularly of G2/M genes. In order to investigate common and distinct functions of NF-Y subunits in cell cycle regulation, NF-YA, NF-YB and NF-YC have been silenced by shRNAs in HCT116 cells. NF-YA loss led to a delay in S-phase progression, DNA damage and apoptosis: we showed the activation of the replication checkpoint, through the recruitment of Δp53 and of the replication proteins PCNA and Mcm7 to chromatin. Differently, NF-YB depletion impaired cells from exiting G2/M, but did not interfere with S-phase progression. Gene expression analysis of NF-YA and NF-YB inactivated cells highlighted a common set of hit genes, as well as a plethora of uncommon genes, unveiling a different effect of NF-Y subunits loss on NF-Y binding to its target genes. Chromatin extracts and ChIP analysis showed that NF-YA depletion was more effective than NF-YB in hitting NF-Y recruitment to CCAAT-promoters. Our data suggest a critical role of NF-Y expression, highlighting that the lack of the single subunits are differently perceived by the cells, which activate diverse cell cycle blocks and signaling pathways.
Calcium has long been recognized as an important regulator of cell cycle transitions although the mechanisms are largely unknown. A functional genomic screen has identified genes involved in the regulation of early cell cycle progression by calcium. These genes when overexpressed confer the ability to bypass the G1/S arrest induced by Ca2+- channel antagonists in mouse fibroblasts. Overexpression of the cystine-glutamate exchanger, xCT, had the greatest ability to evade calcium antagonist-induced cell cycle arrest. xCT carries out the rate limiting step of glutathione synthesis in many cell types and is responsible for the uptake of cystine in most human cancer cell lines. Functional analysis indicates that the cystine uptake activity of xCT overcomes the G1/S arrest induced by Ca2+- channel antagonists by bypassing the requirement for calcium signaling. Since cells overexpressing xCT were found to have increased levels and activity of the AP-1 transcription factor in G1, redox stimulation of AP-1 activity accounts for the observed growth of these cells in the presence of calcium channel antagonists. These results suggest that reduced calcium signaling impairs AP-1 activation and that xCT expression may directly affect cell proliferation.
calcium; glutathione; AP-1; redox; cell cycle; growth factor
Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, in most cases, it is not known whether the compounds tested alter the timing of particular cell cycle transitions. Here, we evaluated an FDA-approved drug library to identify pharmaceuticals that alter cell cycle progression in yeast, using DNA content measurements by flow cytometry. This approach revealed strong cell cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. Based on their effects on cell cycle progression, we also examined cell proliferation in the presence of both compounds. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. Combinations of interest among diverse pharmaceuticals are difficult to identify, due to the daunting number of possible combinations that must be evaluated. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation.
The yeast Fps1 protein is an aquaglyceroporin that functions as the major facilitator of glycerol transport in response to changes in extracellular osmolarity. Although the High Osmolarity Glycerol pathway is thought to have a function in at least basal control of Fps1 activity, its mode of regulation is not understood. We describe the identification of a pair of positive regulators of the Fps1 glycerol channel, Rgc1 (Ypr115w) and Rgc2 (Ask10). An rgc1/2Δ mutant experiences cell wall stress that results from osmotic pressure associated with hyper-accumulation of glycerol. Accumulation of glycerol in the rgc1/2Δ mutant results from a defect in Fps1 activity as evidenced by suppression of the defect through Fps1 overexpression, failure to release glycerol upon hypo-osmotic shock, and resistance to arsenite, a toxic metalloid that enters the cell through Fps1. Regulation of Fps1 by Rgc1/2 appears to be indirect; however, evidence is presented supporting the view that Rgc1/2 regulate Fps1 channel activity, rather than its expression, folding, or localization. Rgc2 was phosphorylated in response to stresses that lead to regulation of Fps1. This stress-induced phosphorylation was partially dependent on the Hog1 MAPK. Hog1 was also required for basal phosphorylation of Rgc2, suggesting a mechanism by which Hog1 may regulate Fps1 indirectly.
When challenged by changes in extracellular osmolarity, many fungal species regulate their intracellular glycerol concentration to modulate their internal osmotic pressure. Maintenance of osmotic homeostasis prevents either cellular collapse under hyper-osmotic stress or cell rupture under hypo-osmotic stress. In baker's yeast, the Fps1 glycerol channel functions as the main vent for glycerol. Proper regulation of Fps1 is critical to the maintenance of osmotic homeostasis. In this study, we identify a pair of proteins (Rgc1 and Rgc2) that function as positive regulators of Fps1 activity. Their absence results in hyper-accumulation of glycerol and consequent cell lysis due to impaired Fps1 channel activity. Additionally, we found that these glycerol channel regulators function between the Hog1 (High Osmolarity Glycerol response) signaling kinase and Fps1, defining a signaling pathway for control of glycerol efflux. Because members of the Rgc1/2 family are found among pathogenic fungal species, but not in humans, they represent potentially attractive targets for antifungal drug development.
Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints. We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure. We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2. The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component. Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function. The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure.
Budding yeast Saccharoymyces cerevisiae is a powerful model system for analyzing eukaryotic cell cycle regulation. Yeast cell cycle analysis is typically performed by visual analysis or flow cytometry, and both have limitations in the scope and accuracy of data obtained. This study demonstrates how Multispectral Imaging Flow Cytometry (MIFC) provides precise quantitation of cell cycle distribution and morphological phenotypes of yeast cells in flow.
Cell cycle analysis of wild-type yeast, nap1Δ, and yeast overexpressing NAP1, was performed visually, by flow cytometry and by MIFC. Quantitative morphological analysis employed measurements of cellular length, thickness and aspect ratio in an algorithm to calculate a novel feature, bud length.
MIFC demonstrated reliable quantification of the yeast cell cycle compared to morphological and flow cytometric analyses. By employing this technique we observed both the G2/M delay and elongated buds previously described in the nap1Δ strain.
Using MIFC, we demonstrate that overexpression of NAP1 causes elongated buds yet only a minor disruption in the cell cycle. The different effects of NAP1 expression level on cell cycle and morphology suggests that these phenotypes are independent. Unlike conventional yeast flow cytometry, MIFC generates complete cell cycle profiles and concurrently offers multiple parameters for morphological analysis.
Budding yeast; Cell cycle; MIFC; Multipectral Imaging Flow Cytometry; Nap1
Phosphorylation of the cellular single-stranded DNA-binding protein, replication protein A (RPA), occurs during normal mitotic cell cycle progression and also in response to genotoxic stress. In budding yeast, these reactions require the ATM homolog Mec1, a central regulator of the DNA replication and DNA damage checkpoint responses. We now demonstrate that the middle subunit of yeast RPA (Rfa2) becomes phosphorylated in two discrete steps during meiosis. Primary Rfa2 phosphorylation occurs early in meiotic progression and is independent of DNA replication, recombination and Mec1. In contrast, secondary Rfa2 phosphorylation is activated upon initiation of recombination and requires Mec1. While the primary Rfa2 phosphoisomer is detectable throughout most of meiosis, the secondary Rfa2 phosphoisomer is only transiently generated and begins to disappear soon after recombination is complete. Extensive secondary Rfa2 phosphorylation is observed in a recombination mutant defective for the pachytene checkpoint, indicating that Mec1-dependent Rfa2 phosphorylation does not function to maintain meiotic delay in response to DNA double-strand breaks. Our results suggest that Mec1-dependent RPA phosphorylation could be involved in regulating recombination rather than cell cycle or meiotic progression.
How cells determine when to initiate DNA replication is poorly understood. Here we report that in Saccharomyces cerevisiae overexpression of the dosage-dependent cell cycle regulator genes DCR2 (YLR361C) and GID8 (DCR1/YMR135C) accelerates initiation of DNA replication. Cells lacking both GID8 and DCR2 delay initiation of DNA replication. Genetic analysis suggests that Gid8p functions upstream of Dcr2p to promote cell cycle progression. DCR2 is predicted to encode a gene product with phosphoesterase activity. Consistent with these predictions, a DCR2 allele carrying a His338 point mutation, which in known protein phosphatases prevents catalysis but allows substrate binding, antagonized the function of the wild-type DCR2 allele. Finally, we report genetic interactions involving GID8, DCR2, and CLN3 (which encodes a G1 cyclin) or SWI4 (which encodes a transcription factor of the G1/S transcription program). Our findings identify two gene products with a probable regulatory role in the timing of initiation of cell division.
Tripeptidyl-peptidase II (TPPII) is a serine peptidase highly expressed in malignant Burkitt’s lymphoma cells (BL). We have previously shown that overexpression of TPPII correlates with chromosomal instability, centrosomal and mitotic spindle abnormalities and resistance to apoptosis induced by spindle poisons. Furthermore, TPPII knockdown by RNAi was associated with endoreplication and the accumulation of polynucleated cells that failed to complete cell division, indicating a role of TPPII in the cell cycle. Here we have applied a global approach of gene expression analysis to gain insights on the mechanism by which TPPII regulates this phenotype. mRNA profiling of control and TPPII knockdown BL cells identified one hundred and eighty five differentially expressed genes. Functional categorization of these genes highlighted major physiological functions such as apoptosis, cell cycle progression, cytoskeleton remodeling, proteolysis, and signal transduction. Pathways and protein interactome analysis revealed a significant enrichment in components of MAP kinases signaling. These findings suggest that TPPII influences a wide network of signaling pathways that are regulated by MAPKs and exerts thereby a pleiotropic effect on biological processes associated with cell survival, proliferation and genomic instability.
TPPII; MAPK signaling; centrosome; cell cycle; cytoskeleton
Histone mRNAs accumulate in the S phase and are rapidly degraded as cells progress into the G2 phase of the cell cycle. In Saccharomyces cerevisiae, fusion of the 3′ untranslated region and downstream sequences of the yeast histone gene HTB1 to a neomycin phosphotransferase open reading frame is sufficient to confer cell cycle regulation on the resulting chimera gene (neo-HTB1). We have identified a sequence element, designated the distal downstream element (DDE), that influences both the 3′-end cleavage site selection and the cell cycle regulation of the neo-HTB1 mRNA. Mutations in the DDE, which is located approximately 110 nucleotides downstream of the HTB1 gene, lead to a delay in the accumulation of the neo-HTB1 mRNA in the S phase and a lack of mRNA turnover in the G2 phase. The DDE is transcribed as part of the primary transcript and binds a protein factor(s). Maximum binding is observed in the S phase of the cell cycle, and mutations that affect the turnover of the HTB1 mRNA alter the binding activity. While located in the same general region, mutations that affect 3′-end cleavage site selection act independently from those that alter the cell cycle regulation.
Eukaryotic cell cycle is a complex process and is precisely regulated at many levels. Many genes specific to the cell cycle are regulated transcriptionally and are expressed just before they are needed. To understand the cell cycle process, it is important to identify the cell cycle transcription factors (TFs) that regulate the expression of cell cycle-regulated genes.
We developed a method to identify cell cycle TFs in yeast by integrating current ChIP-chip, mutant, transcription factor binding site (TFBS), and cell cycle gene expression data. We identified 17 cell cycle TFs, 12 of which are known cell cycle TFs, while the remaining five (Ash1, Rlm1, Ste12, Stp1, Tec1) are putative novel cell cycle TFs. For each cell cycle TF, we assigned specific cell cycle phases in which the TF functions and identified the time lag for the TF to exert regulatory effects on its target genes. We also identified 178 novel cell cycle-regulated genes, among which 59 have unknown functions, but they may now be annotated as cell cycle-regulated genes. Most of our predictions are supported by previous experimental or computational studies. Furthermore, a high confidence TF-gene regulatory matrix is derived as a byproduct of our method. Each TF-gene regulatory relationship in this matrix is supported by at least three data sources: gene expression, TFBS, and ChIP-chip or/and mutant data. We show that our method performs better than four existing methods for identifying yeast cell cycle TFs. Finally, an application of our method to different cell cycle gene expression datasets suggests that our method is robust.
Our method is effective for identifying yeast cell cycle TFs and cell cycle-regulated genes. Many of our predictions are validated by the literature. Our study shows that integrating multiple data sources is a powerful approach to studying complex biological systems.