Overexpression limits of fission yeast cell‐cycle regulators in vivo and in silico
2011; Springer Nature; Volume: 7; Issue: 1 Linguagem: Inglês
10.1038/msb.2011.91
ISSN1744-4292
AutoresHisao Moriya, Ayako Chino, Orsolya Kapuy, Attila Csikász‐Nagy, Béla Novák,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle6 December 2011Open Access Overexpression limits of fission yeast cell-cycle regulators in vivo and in silico Hisao Moriya Corresponding Author Hisao Moriya Research Core for Interdisciplinary Sciences, Okayama University, Okayama, Japan Search for more papers by this author Ayako Chino Ayako Chino Research Core for Interdisciplinary Sciences, Okayama University, Okayama, Japan Search for more papers by this author Orsolya Kapuy Orsolya Kapuy Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK Search for more papers by this author Attila Csikász-Nagy Attila Csikász-Nagy The Microsoft Research—University of Trento Centre for Computational and Systems Biology, Trento, Italy Search for more papers by this author Béla Novák Béla Novák Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK Search for more papers by this author Hisao Moriya Corresponding Author Hisao Moriya Research Core for Interdisciplinary Sciences, Okayama University, Okayama, Japan Search for more papers by this author Ayako Chino Ayako Chino Research Core for Interdisciplinary Sciences, Okayama University, Okayama, Japan Search for more papers by this author Orsolya Kapuy Orsolya Kapuy Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK Search for more papers by this author Attila Csikász-Nagy Attila Csikász-Nagy The Microsoft Research—University of Trento Centre for Computational and Systems Biology, Trento, Italy Search for more papers by this author Béla Novák Béla Novák Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK Search for more papers by this author Author Information Hisao Moriya 1, Ayako Chino1, Orsolya Kapuy2, Attila Csikász-Nagy3 and Béla Novák2 1Research Core for Interdisciplinary Sciences, Okayama University, Okayama, Japan 2Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK 3The Microsoft Research—University of Trento Centre for Computational and Systems Biology, Trento, Italy *Corresponding author. Research Core for Interdisciplinary Sciences, Okayama University, Tsushimanaka 3-1-1, Kita-ku, Okayama 700-8530, Japan. Tel.: +81 86 251 8712; Fax: +81 86 251 8717; E-mail: [email protected] Molecular Systems Biology (2011)7:556https://doi.org/10.1038/msb.2011.91 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Cellular systems are generally robust against fluctuations of intracellular parameters such as gene expression level. However, little is known about expression limits of genes required to halt cellular systems. In this study, using the fission yeast Schizosaccharomyces pombe, we developed a genetic 'tug-of-war' (gTOW) method to assess the overexpression limit of certain genes. Using gTOW, we determined copy number limits for 31 cell-cycle regulators; the limits varied from 1 to >100. Comparison with orthologs of the budding yeast Saccharomyces cerevisiae suggested the presence of a conserved fragile core in the eukaryotic cell cycle. Robustness profiles of networks regulating cytokinesis in both yeasts (septation-initiation network (SIN) and mitotic exit network (MEN)) were quite different, probably reflecting differences in their physiologic functions. Fragility in the regulation of GTPase spg1 was due to dosage imbalance against GTPase-activating protein (GAP) byr4. Using the gTOW data, we modified a mathematical model and successfully reproduced the robustness of the S. pombe cell cycle with the model. Synopsis A genetic tug-of-war system is used to explore the sensitivity of the fission yeast cell cycle to changes in gene dosage, revealing a deeply conserved fragile core. A mathematical model can reproduce the robustness of the cell cycle, and points to currently unknown regulatory mechanisms. An experimental genetic tug-of-war (gTOW) system is developed for fission yeast that allows quantitation of the robustness of the cell to gene overexpression. The limits of gene overexpression were measured for 31 fission yeast cell-cycle regulators. The robustness of the cell cycle seems to be conserved between distantly related eukaryotes (the budding and fission yeasts). These data are used to build an integrative mathematical model of the fission yeast cell cycle, which can reproduce the robustness of this system. Introduction Intracellular parameters such as gene expression require optimization, such that cellular functions may be performed effectively (Alon et al, 1999; Zaslaver et al, 2004; Dekel and Alon, 2005; Wagner, 2005). Fluctuations in these parameters lead to various cellular defects. Overexpression of genes involved in proliferation of cancer cells due to gene amplification is a prime example (Albertson, 2006). On the other hand, in order to maintain cellular functions despite environmental change, mutation, and noise in intracellular biochemical reactions, these parameters may have certain permissible ranges, a characteristic termed robustness, which is commonly observed in various cellular systems (Barkai and Leibler, 1997; Little et al, 1999; von Dassow et al, 2000). We previously reported a method designated genetic 'tug-of-war (gTOW),' by which we can measure the limit of gene overexpression in the budding yeast Saccharomyces cerevisiae (Moriya et al, 2006). In gTOW, a target gene with its native regulatory regions is cloned into a particular plasmid, and the copy number of the plasmid is increased by genetic selection. Next, the copy number is measured just before the cellular system halts (i.e., the cell dies), such that the overexpression limit of the target gene is evaluated as the gene copy number limit. As the gene copy number increases, relative overexpression of the gene is expected. If we can measure the copy number limit of gene overexpression, we can evaluate the degree to which the cellular system resists overexpression of the target gene (namely, robustness versus gene overexpression). Using gTOW, we previously measured the copy number limits of 30 cell-cycle regulators in budding yeast (Moriya et al, 2006). The data were used to reveal the robustness profile of the cell-cycle regulatory system, and to evaluate and refine the integrative mathematical model of the budding yeast cell cycle (Moriya et al, 2006; Kaizu et al, 2010). The fission yeast Schizosaccharomyces pombe is distantly related to S. cerevisiae (Sipiczki, 2000), and like S. cerevisiae, is an established model eukaryote for the study of the molecular biology of the cell cycle (Egel, 2004). In this study, we developed a gTOW method using S. pombe and determined the copy number limits of 31 cell-cycle regulators. The data thus obtained were used to compare the robustness profiles of budding and fission yeasts in order to reveal the conserved/non-conserved properties of the eukaryotic cell cycle. The data were also used to constitute an integrative mathematical model of the fission yeast cell cycle. Results Development of plasmid vectors used for S. pombe gTOW The scheme of S. pombe gTOW is described in Figure 1A. The plasmid for gTOW must have the following properties: (1) the copy number in each cell is multiple; (2) the copy number is diverse in each cell; and (3) the plasmid contains a gene with a selection bias for increasing the plasmid copy number (leu2d in the case of S. cerevisiae gTOW). Because S. pombe possesses no plasmid with the above properties, we first constructed plasmid vectors for use in S. pombe gTOW. Figure 1.gTOW in Schizosaccharomyces pombe. (A) The four steps in the gTOW experiment. (1) Each target gene is cloned into a plasmid for gTOW. (2) The plasmid is introduced into the leu1Δura4Δ cell (first selection is uracil). The plasmid copy number becomes ∼10 per cell due to ars3002 × 2 ORI. If the copy number limit of the target gene is 100 (Figure 4), indicating that the cell-cycle regulatory system has a different robustness against overexpression of each gene, as observed in S. cerevisiae cell-cycle regulation (Moriya et al, 2006). The frameshift mutants generally showed much higher copy number limits than the wild types (orange bars in Figure 4), but the frameshift mutants of wee1 and rum1 had lower limits than the pTOWsp-H vector alone, probably because their frameshifts do not completely shut down protein expression. The morphology of cells with high copy frameshift mutant genes was similar to that of cells with high copies of their wild types (Supplementary Figure S6). Figure 4.Copy number limits of 30 cell-cycle regulators in S. pombe. The green bar indicates the plasmid copy number obtained in the gTOW experiment with each vector. The blue bar indicates the maximum plasmid copy number for each target gene obtained in the gTOW experiment with three different vectors. The orange bar indicates the maximum plasmid copy number for each target gene with frameshift mutation. The original data are shown in Supplementary Table S6. Because the cell has an endogenous copy of the target gene, the copy number limit for the target gene is the plasmid copy number plus 1. Download figure Download PowerPoint Comparison of robustness profiles of cell-cycle regulation in S. cerevisiae and S. pombe We next compared the copy number limits of the cdc genes determined in this study with that of the S. cerevisiae homologs determined previously (Moriya et al, 2006; Figure 5). S. pombe genes generally had lower copy number limits (average=52) than those of S. cerevisiae (average=87). We previously reported that cdc genes with lower limits constituted a fragile core that directly regulates the activity of B-type cyclin Cdk (Cyclin-dependent kinase) complexes (Moriya et al, 2006). The orthologs that constitute the core structure of S. pombe (Cdc13, Rum1, and Wee1) also demonstrated low limits, which may indicate the presence of a conserved fragile core in eukaryotic cell-cycle regulation. In contrast, the orthologous components of the mitotic exit network (MEN) of S. cerevisiae showed very different limits from those in the septation-initiation network (SIN) of S. pombe, although both networks have conserved architecture (Bardin and Amon, 2001; Krapp and Simanis, 2008). This might reflect the different physiologic functions in the two yeasts (namely, budding and fission), although these components have the same origin. These results suggest that comparison of robustness profiles is a useful way to reveal the conserved/non-conserved properties of cellular systems. Figure 5.Comparison of robustness profiles of cell-cycle regulation between S. cerevisiae and S. pombe. The dotted blue line connects the functional orthologs within the 'fragile core' conserved in both yeasts (left). The dotted orange line connects the functional orthologs involved in the cytokinesis regulatory pathway of both yeasts (MEN and SIN). Some components involved in both MEN and SIN are omitted from the diagrams because their copy number limits were not measured. Data for S. cerevisiae are obtained from a previous study (Moriya et al, 2006). The original data are shown in Supplementary Table S8. Download figure Download PowerPoint Dosage imbalance causes very low limit of spg1 We recently demonstrated that the M-phase phosphatase gene CDC14 has a very low limit is because of the dosage imbalance between CDC14 and its inhibitor gene, NET1 (Kaizu et al, 2010). Hence, we investigated whether the low limit of spg1, the lowest copy number gene in S. pombe cell-cycle regulators evaluated in this study, was also due to dosage imbalance. spg1 encodes a small GTPase involved in the initiation of cellular septation; this activity is regulated by the bipartite GTPase-activating protein (GAP), which is encoded by both cdc16 and byr4 (Furge et al, 1998; Figure 6A). The copy number limit of cdc16 is quite high (Figure 4), and cells with high copy cdc16 did not show any obvious phenotype (data not shown). In contrast, the limit of byr4 was very low (1.8±0.2, measured by pTOWsp-L in the leucine+ condition). And cellular lethality due to overexpression of spg1 is nullified by the simultaneous overexpression of byr4 (Furge et al, 1998). We thus assume that proper activation of Spg1 is in delicate balance with Byr4, and hence, the limits of both spg1 and byr4 are quite low. To investigate whether spg1 and byr4 were in dosage balance, we performed two-dimensional (2D) gTOW (Kaizu et al, 2010). As shown in Figure 6B, byr4 limits increased only when multicopy spg1 was supplied by another plasmid. Moreover, their copy numbers were balanced (Figure 6B, dotted line). Microscopic observation also confirmed our assumption that cells can divide normally only when spg1 and byr4 expressions are balanced (Figure 6C). These results strongly support the theory that spg1 has a very low limit because of the sensitive balance required for GTPase activity. Figure 6.The very low limit for spg1 is due to dosage imbalance with byr4. (A) To trigger cellular septation, GTPase Spg1 and its GAP Byr4 function in an antagonistic manner. The molecular interactions are given with Systems Biology Graphical Notation (SBGN) using CellDesigner 4.1 (http://www.celldesigner.org). (B) 2D-gTOW experiment between spg1 and byr4. The copy numbers of spg1 and byr4 can be increased only when both gene copy numbers are balanced (dotted line). Extra copies of spg1+ are supplied by the pA6R plasmid, and extra copies of byr4+ are supplied by the pTOWsp-M plasmid. (C) Microscopic image of cells by 2D-gTOW experiment involving spg1 and byr4. Sp286h+ cells with pTOWsp-M with byr4 and pA6R with spg1 were cultivated in EMM with leucine. GFP (reflecting the byr4 copy number), RFP (reflecting the spg1 copy number), the nucleus, and the septum were observed. Expected phenotypes of the cells within the dosage balance between spg1 and byr4, and the indices within the image are shown. Download figure Download PowerPoint Development of an integrative mathematical model that reproduces the robustness of cell-cycle regulation in S. pombe Robustness can be a measure of plausibility in mathematical models of biological networks (Morohashi et al, 2002). We previously used copy number limits obtained for S. cerevisiae gTOW to evaluate and refine an integrative mathematical model of the S. cerevisiae cell cycle (Chen et al, 2004; Moriya et al, 2006; Kaizu et al, 2010). We thus evaluated and refined the integrative mathematical model, such that the model reproduces robustness (copy number limits of genes) of the S. pombe cell cycle obtained from this study. We previously published a mathematical model for S. pombe cell-cycle regulation (Novak and Tyson, 1997; Sveiczer et al, 2000), and have independently modified the model from the gTOW experiment, such that the model reproduces more published experimental results. Here, we designated the model 'basic model,' the whole structure of which is shown in Figure 7A (green components; the simplified structure is shown in Supplementary Figure S8). We first investigated to what degree the core model reproduced the copy number limits obtained in this study (see Supplementary information for details). Although we did not use the gTOW data for development of the model, the model predicted the experimental data well (Figure 7B, green circles), and appeared to capture the robustness of fission yeast cell-cycle regulation. To describe the gTOW data more extensively, we modified the basic model by adding some important regulators (M-phase phosphatase Clp1, cyclins Cig1 and Puc1) and regulations, and the parameters were optimized by-hand parameter adjustments (we designated the model 'gTOW model,' shown in red in Figure 7A; see also Supplementary Figure S8). The model successfully reproduced the copy number limits obtained in this study (Figure 7B, orange squares). In addition, the model could also reproduce 42 already published mutant behaviors (Supplementary Table S10). The result of time-course simulation of the model is shown in Figure 7C. As a result, we can claim that the presented 'gTOW' model is the most detailed model of fission yeast cell-cycle regulation so far. Figure 7.Mathematical model reproducing gTOW data. (A) Whole structure of the mathematical model of the fission yeast cell cycle developed in this study (gTOW model) given with Systems Biology Graphical Notation (SBGN) using CellDesigner 4.1. Pink colored components are the ones added to the 'basic model' to make the 'gTOW model'. CellDesigner file and Systems Biology Markup Language (SBML) file are provided in Supplementary information. (B) Comparison of the copy number limits of cell-cycle regulators between the data obtained by gTOW and prediction of the mathematical model. Scale of the axis=log2. (C) Time-course simulation result of the gTOW model. Download figure Download PowerPoint Increase of each Cdc2/cyclin complex, but not the depletion of Cdc2/Cdc13 through the competition for Cdc2, determines the upper limits of cyclins (cig1, cig2, and puc1) S. pombe contains four cyclins (Cdc13, Cig1, Cig2, and Puc1), all of which bind to Cdc2 to form a Cdc2/cyclin complex that performs regulatory functions on Cdc2 (Figure 8A). Cdk's (like Cdc2) are thought to be in excess of cyclins, but in S. cerevisiae it is not tremendously so (Cross et al, 2002). It is thus possible that if cyclins compete with each other for Cdc2, overexpression of one of the cyclins would interfere in formation of other Cdc2/cyclin complexes. Especially since deletion of Cdc13 can lead to lethality, there is a chance that overexpression of the other cyclins is deleterious for the cells because Cdc2 is titrated away from Cdc13 (Figure 8A-2, case2). In other words, the overexpression limit of each cyclin may be determined by the increased Cdc2/cyclin activity itself (Figure 8A-2, case1) or by the reduction of Cdc2/Cdc13 complex. To answer this question, we first used the mathematical model to investigate which conditions involve cyclins in competitive situations, and the experimental means to test these conditions. In the gTOW model (and in the basic model), each cyclin is considered to form a Cdc2/
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