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• W2003089698 abstract "In budding yeast, commitment to cell division corresponds to activating the positive feedback loop of G1 cyclins controlled by the transcription factors SBF and MBF. This pair of transcription factors has over 200 targets, implying that cell-cycle commitment coincides with genome-wide changes in transcription. Here, we find that genes within this regulon have a well-defined distribution of transcriptional activation times. Combinatorial use of SBF and MBF results in a logical OR function for gene expression and partially explains activation timing. Activation of G1 cyclin expression precedes the activation of the bulk of the G1/S regulon, ensuring that commitment to cell division occurs before large-scale changes in transcription. Furthermore, we find similar positive feedback-first regulation in the yeasts S. bayanus and S. cerevisiae, as well as human cells. The widespread use of the feedback-first motif in eukaryotic cell-cycle control, implemented by nonorthologous proteins, suggests its frequent deployment at cellular transitions. In budding yeast, commitment to cell division corresponds to activating the positive feedback loop of G1 cyclins controlled by the transcription factors SBF and MBF. This pair of transcription factors has over 200 targets, implying that cell-cycle commitment coincides with genome-wide changes in transcription. Here, we find that genes within this regulon have a well-defined distribution of transcriptional activation times. Combinatorial use of SBF and MBF results in a logical OR function for gene expression and partially explains activation timing. Activation of G1 cyclin expression precedes the activation of the bulk of the G1/S regulon, ensuring that commitment to cell division occurs before large-scale changes in transcription. Furthermore, we find similar positive feedback-first regulation in the yeasts S. bayanus and S. cerevisiae, as well as human cells. The widespread use of the feedback-first motif in eukaryotic cell-cycle control, implemented by nonorthologous proteins, suggests its frequent deployment at cellular transitions. Targets of the same transcription factor can have different activation times Positive feedback genes are activated before other co-regulated genes Transcriptional activation and inactivation shows a logical OR-gate behavior Feedback-first regulation is conserved in yeasts and human cells Order may be produced in a sequence of biochemical events through feedback control mechanisms or substrate-specific chemical kinetics. In the cell cycle, regulatory checkpoints ensure the proper order of many essential events through feedback control. DNA replication must be finished and damage repaired before mitosis, while anaphase is initiated only after complete spindle assembly (Morgan, 2007Morgan D.O. The Cell Cycle: Principles of Control. New Science Press; Sinauer Associates, London; Sunderland, MA2007Google Scholar). Checkpoints use designated regulatory molecules to restrain cell-cycle progression until a set of criteria are satisfied (Hartwell and Weinert, 1989Hartwell L.H. Weinert T.A. Checkpoints: controls that ensure the order of cell cycle events.Science. 1989; 246: 629-634Crossref PubMed Scopus (2418) Google Scholar). However, order without checkpoint control is observed in Xenopus embryos as cell-cycle events are entrained by oscillations in cyclin dependent kinase (CDK) activity. Furthermore, addition of CDK substrates to Xenopus egg extracts in different stages of mitosis revealed that the order of substrate phosphorylation is independent of cell-cycle phase (Georgi et al., 2002Georgi A.B. Stukenberg P.T. Kirschner M.W. Timing of events in mitosis.Curr. Biol. 2002; 12: 105-114Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Thus, temporal order of phosphorylation in mitosis is likely the result of substrate-specific kinetics. Here, we investigate the integration of chemical kinetics and feedback control at the Start transition in budding yeast. Start marks the point of commitment to the mitotic cell cycle, which is located between cell division and DNA replication (Hartwell et al., 1974Hartwell L.H. Culotti J. Pringle J.R. Reid B.J. Genetic control of the cell division cycle in yeast.Science. 1974; 183: 46-51Crossref PubMed Scopus (731) Google Scholar). Prior to Start, cells integrate internal (e.g., cell size) and external (e.g., mating pheromone) signals to make an all-or-none decision to divide. Beyond Start, cells are committed to divide regardless of changes in extracellular signals. In another article in this issue, we show that passage through Start corresponds precisely to the activation of the G1 cyclin-positive feedback loop (Dončić et al., 2011Dončić A. Falleur-Fettig M. Skotheim J.M. Distinct interactions select and maintain a specific cell fate.Mol. Cell. 2011; 43 (this issue): 528-539Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Thus, Start is a member of a growing list of cellular and developmental transitions driven by positive feedback (Pomerening et al., 2003Pomerening J.R. Sontag E.D. Ferrell Jr., J.E. Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2.Nat. Cell Biol. 2003; 5: 346-351Crossref PubMed Scopus (587) Google Scholar, Xiong and Ferrell, 2003Xiong W. Ferrell Jr., J.E. A positive-feedback-based bistable ‘memory module’ that governs a cell fate decision.Nature. 2003; 426: 460-465Crossref PubMed Scopus (600) Google Scholar, Holt et al., 2008Holt L.J. Krutchinsky A.N. Morgan D.O. Positive feedback sharpens the anaphase switch.Nature. 2008; 454: 353-357Crossref PubMed Scopus (149) Google Scholar, Justman et al., 2009Justman Q.A. Serber Z. Ferrell Jr., J.E. El-Samad H. Shokat K.M. Tuning the activation threshold of a kinase network by nested feedback loops.Science. 2009; 324: 509-512Crossref PubMed Scopus (45) Google Scholar, López-Avilés et al., 2009López-Avilés S. Kapuy O. Novák B. Uhlmann F. Irreversibility of mitotic exit is the consequence of systems-level feedback.Nature. 2009; 459: 592-595Crossref PubMed Scopus (77) Google Scholar). Positive feedback at Start is initiated by the G1 cyclin, Cln3 in complex with the cyclin dependent kinase Cdc28 (Figure 1A ). The primary target of Cln3 is the transcriptional inhibitor Whi5, whose inactivation is rate limiting for the expression of the G1/S regulon (Costanzo et al., 2004Costanzo M. Nishikawa J.L. Tang X. Millman J.S. Schub O. Breitkreuz K. Dewar D. Rupes I. Andrews B. Tyers M. CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast.Cell. 2004; 117: 899-913Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, de Bruin et al., 2004de Bruin R.A. McDonald W.H. Kalashnikova T.I. Yates 3rd, J. Wittenberg C. Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5.Cell. 2004; 117: 887-898Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Cln3-Cdc28 phosphorylates and initiates Whi5 inactivation, which allows some transcription of two additional G1 cyclins, CLN1 and CLN2 (Tyers et al., 1993Tyers M. Tokiwa G. Futcher B. Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins.EMBO J. 1993; 12: 1955-1968Crossref PubMed Scopus (392) Google Scholar). The downstream G1 cyclins then complete the positive feedback loop through the inactivation and nuclear exclusion of Whi5 and the full activation of the transcription factors SBF (Swi4-Swi6) and MBF (Mbp1-Swi6) (Andrews and Herskowitz, 1989Andrews B.J. Herskowitz I. Identification of a DNA binding factor involved in cell-cycle control of the yeast HO gene.Cell. 1989; 57: 21-29Abstract Full Text PDF PubMed Scopus (123) Google Scholar, Nasmyth and Dirick, 1991Nasmyth K. Dirick L. The role of SWI4 and SWI6 in the activity of G1 cyclins in yeast.Cell. 1991; 66: 995-1013Abstract Full Text PDF PubMed Scopus (251) Google Scholar, Koch et al., 1993Koch C. Moll T. Neuberg M. Ahorn H. Nasmyth K. A role for the transcription factors Mbp1 and Swi4 in progression from G1 to S phase.Science. 1993; 261: 1551-1557Crossref PubMed Scopus (313) Google Scholar, de Bruin et al., 2004de Bruin R.A. McDonald W.H. Kalashnikova T.I. Yates 3rd, J. Wittenberg C. Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5.Cell. 2004; 117: 887-898Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, Skotheim et al., 2008Skotheim J.M. Di Talia S. Siggia E.D. Cross F.R. Positive feedback of G1 cyclins ensures coherent cell cycle entry.Nature. 2008; 454: 291-296Crossref PubMed Scopus (261) Google Scholar). Surprisingly, the transcription factors at the center of the positive feedback loop, SBF and MBF, are also responsible for the transcription of over 200 additional genes (Ferrezuelo et al., 2010Ferrezuelo F. Colomina N. Futcher B. Aldea M. The transcriptional network activated by Cln3 cyclin at the G1-to-S transition of the yeast cell cycle.Genome Biol. 2010; 11: R67Crossref PubMed Scopus (48) Google Scholar). Indeed, cell-cycle commitment appears to coincide with the coordinated transcriptional activation of approximately 5% of all genes (Spellman et al., 1998Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization.Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3898) Google Scholar). Although Whi5 phosphorylation is rate limiting for activation of positive feedback, it is also likely to be rate limiting for the transcription of all SBF regulated genes due to the direct Whi5-SBF interaction (de Bruin et al., 2004de Bruin R.A. McDonald W.H. Kalashnikova T.I. Yates 3rd, J. Wittenberg C. Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5.Cell. 2004; 117: 887-898Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). The concurrent activation of the related heterodimeric transcription factor MBF also requires CDK activity, possibly through phosphorylation of the shared component Swi6 (Wijnen et al., 2002Wijnen H. Landman A. Futcher B. The G(1) cyclin Cln3 promotes cell cycle entry via the transcription factor Swi6.Mol. Cell. Biol. 2002; 22: 4402-4418Crossref PubMed Scopus (71) Google Scholar). Thus, given the integrated nature of the regulatory circuit and the ability of the upstream cyclin Cln3 to activate SBF- and MBF-dependent transcription in cln1Δ cln2Δ cells (Dirick et al., 1995Dirick L. Böhm T. Nasmyth K. Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae.EMBO J. 1995; 14: 4803-4813Crossref PubMed Scopus (278) Google Scholar, Stuart and Wittenberg, 1995Stuart D. Wittenberg C. CLN3, not positive feedback, determines the timing of CLN2 transcription in cycling cells.Genes Dev. 1995; 9: 2780-2794Crossref PubMed Scopus (174) Google Scholar), it is unclear if genome-wide changes in transcription occur after commitment to division. Although G1/S transcription is largely regulated by SBF and MBF, single-cell studies have revealed significant differences in transcriptional activation of the three regulon members CLN2, RAD27, and RFA1 (Skotheim et al., 2008Skotheim J.M. Di Talia S. Siggia E.D. Cross F.R. Positive feedback of G1 cyclins ensures coherent cell cycle entry.Nature. 2008; 454: 291-296Crossref PubMed Scopus (261) Google Scholar). A rapid, feedback-driven increase in CDK activity drives the coherent and nearly simultaneous induction of these three genes in WT cells. However, significant differences in transcriptional activation timing are revealed in cln1Δ cln2Δ cells lacking positive feedback. CLN2 is induced earlier than two other regulon members, which suggests a model in which full regulon expression would only occur after feedback loop activation to avoid detrimental transcription in cases where the cell does not commit to the mitotic cell cycle. Therefore, we hypothesized that the G1 cyclins CLN1 and CLN2, involved in positive feedback, would be activated earlier than other genes in the G1/S regulon to ensure that commitment precedes the genome-wide change in transcription. In this study, we observed that the two SBF/MBF-regulated G1 cyclins, namely CLN1 and CLN2, are among the earliest activated genes of the G1/S regulon, which supports the hypothesis that genome-wide changes in transcription occur after a cell is committed to division. By comparing sets of genes regulated by SBF, MBF, or by both factors together, we found that both transcriptional activation and inactivation can be approximated as logical OR functions. Furthermore, CLN1 and CLN2 remain among the earliest activated cell cycle-regulated genes in the related yeast, S. bayanus, which has significantly diverged gene expression (Tirosh et al., 2006Tirosh I. Weinberger A. Carmi M. Barkai N. A genetic signature of interspecies variations in gene expression.Nat. Genet. 2006; 38: 830-834Crossref PubMed Scopus (229) Google Scholar, Guan et al., 2010Guan Y. Dunham M. Caudy A. Troyanskaya O. Systematic planning of genome-scale experiments in poorly studied species.PLoS Comput. Biol. 2010; 6: e1000698Crossref PubMed Scopus (20) Google Scholar). A similar analysis of human tissue culture cells revealed that functionally analogous feedback loop components E2F1, Skp2, and the cyclins E1 and E2 (Blagosklonny and Pardee, 2002Blagosklonny M.V. Pardee A.B. The restriction point of the cell cycle.Cell Cycle. 2002; 1: 103-110PubMed Google Scholar, Yung et al., 2007Yung Y. Walker J.L. Roberts J.M. Assoian R.K. A Skp2 autoinduction loop and restriction point control.J. Cell Biol. 2007; 178: 741-747Crossref PubMed Scopus (40) Google Scholar) are among the earliest activated cell cycle-regulated targets of the E2F family of transcription factors. Taken together, our results demonstrate that feedback-first regulation, which places genome-wide changes in transcription downstream of positive feedback-dependent cell-cycle commitment, is a common feature of G1/S control across eukaryotes. To test our model that induction of positive feedback and concomitant cell-cycle commitment precedes large-scale transcriptional change, we first need to accurately define the G1/S regulon. We are interested in the set of genes whose transcription is initiated due to increasing cyclin activity rather than upstream cyclin-independent processes (MacKay et al., 2001MacKay V.L. Mai B. Waters L. Breeden L.L. Early cell cycle box-mediated transcription of CLN3 and SWI4 contributes to the proper timing of the G(1)-to-S transition in budding yeast.Mol. Cell. Biol. 2001; 21: 4140-4148Crossref PubMed Scopus (45) Google Scholar, Di Talia et al., 2009Di Talia S. Wang H. Skotheim J.M. Rosebrock A.P. Futcher B. Cross F.R. Daughter-specific transcription factors regulate cell size control in budding yeast.PLoS Biol. 2009; 7: e1000221Crossref PubMed Scopus (86) Google Scholar). The set of cell cycle-regulated genes was defined as the 800 genes with the largest amplitude messenger RNA (mRNA) concentration oscillation through the cell cycle (Spellman et al., 1998Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization.Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3898) Google Scholar). To identify the set of G1 cyclin regulated genes, we relied on a second experiment by Spellman et al., 1998Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization.Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3898) Google Scholar, which identified a set of genes responding to exogenous Cln3 induction in G1 arrested cln1Δ cln2Δ cln3Δ cells. We took the top 413 as the set of G1 cyclin inducible genes. The intersection of these two sets defines the 362-gene regulon (Figure 1B and Table S1 available online). Next, we developed an algorithm to determine the time at which a specific gene is induced during the cell cycle. We analyzed seven previously published microarray time-course data sets with 5 min temporal resolution (Di Talia et al., 2009Di Talia S. Wang H. Skotheim J.M. Rosebrock A.P. Futcher B. Cross F.R. Daughter-specific transcription factors regulate cell size control in budding yeast.PLoS Biol. 2009; 7: e1000221Crossref PubMed Scopus (86) Google Scholar). All experiments were performed on cdc20Δ GALLpr-CDC20 cells that were synchronized by mitotic arrest. Cells were released by switching to media containing galactose resulting in CDC20 expression and a synchronous first cell cycle (Figure 1C). Although manually identifying activation points of cell cycle-regulated genes is not difficult, we developed an automated algorithm to both avoid potential bias and increase throughput. Our algorithm is robust to noisy data, which can produce incorrect estimates for the activation time. We normalized all the time series and assumed that the time scale for changing transcript concentration is greater than 10 min. We therefore remove data points associated with large concentration changes on shorter timescales. Data points further than 20% of the dynamic range of the time series (maximum – minimum) from adjacent points are removed. We discarded time series with two or more removed data points. The mRNA level is then estimated with smoothing splines. We selected the point where the first derivative first reaches 10% of its maximum. The smoothing parameter is optimized to minimize variation in biological replicates and the first derivative method is shown to be superior in estimating activation times relative to other methods (Figures S1A–S1C). Figure 1D shows the activation times for seven independent CLN2 expression profiles and their standard deviation and standard error of the mean. Because we have multiple time courses, our error in estimating the activation time is low, e.g., for CLN2 we find the activation time to be 13 min after galactose addition with a standard deviation of 1.9 min and a standard error of the mean of 0.7 min. For genes within the G1/S regulon, we find that the average standard deviation is 4.7 min and the average standard error of the mean is 2.1 min. Despite regulation by the same transcription factors, the activation times of G1/S regulon members has a defined distribution (mean = 17.2 min, standard deviation = 5.9 min; Figures 1E–1H, Figure S1D, and Table S2). To test our model that feedback activation precedes regulon induction, we averaged the activation times from all seven data sets for each gene (Figures 1G and 1H). These results were consistent with induction times measured in real-time PCR time courses (see Figure S1E). The positive feedback genes CLN1 and CLN2 are activated significantly earlier than the bulk of the G1/S regulon. Indeed, within error, CLN1 is the earliest activated gene, 5 min earlier than CLN2, suggesting a different temporal role even though these two genes are generally thought to be functionally redundant. However, it has been shown that CLN1, but not CLN2, transcription affects cell size (Flick et al., 1998Flick K. Chapman-Shimshoni D. Stuart D. Guaderrama M. Wittenberg C. Regulation of cell size by glucose is exerted via repression of the CLN1 promoter.Mol. Cell. Biol. 1998; 18: 2492-2501PubMed Google Scholar), which our data suggests is due to timing. We note that for the feedback-first model to work it is sufficient to express either G1 cyclin, not necessarily both, prior to the majority of the regulon. Thus, we see that induction of the G1 cyclin positive feedback loop, which coincides with cell-cycle commitment, precedes large-scale changes in the transcriptional program. Interestingly, NRM1, the negative feedback element responsible for inactivating MBF regulated genes (de Bruin et al., 2006de Bruin R.A. Kalashnikova T.I. Chahwan C. McDonald W.H. Wohlschlegel J. Yates 3rd, J. Russell P. Wittenberg C. Constraining G1-specific transcription to late G1 phase: the MBF-associated corepressor Nrm1 acts via negative feedback.Mol. Cell. 2006; 23: 483-496Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), is activated 15 min later than CLN1 (Figures 1G and 1H) even though both genes are MBF targets (Ferrezuelo et al., 2010Ferrezuelo F. Colomina N. Futcher B. Aldea M. The transcriptional network activated by Cln3 cyclin at the G1-to-S transition of the yeast cell cycle.Genome Biol. 2010; 11: R67Crossref PubMed Scopus (48) Google Scholar). Thus, distinct temporal regulation allows positive feedback sufficient time for regulon transcription prior to NRM1-dependent inactivation. To examine the functional consequences of feedback timing, we integrated a CLN2 allele regulated by the NRM1 promoter into a cln1Δ cln2Δ cell containing MET3pr-CLN2, CLN2pr-GFPpest, and RAD27-mCherry. Cells were grown overnight on media lacking methionine (MET3pr-CLN2 on) prior to switching to media containing methionine (MET3pr-CLN2 off) for single-cell analysis of one cell cycle (Skotheim et al., 2008Skotheim J.M. Di Talia S. Siggia E.D. Cross F.R. Positive feedback of G1 cyclins ensures coherent cell cycle entry.Nature. 2008; 454: 291-296Crossref PubMed Scopus (261) Google Scholar). Cells exhibited similarly incoherent gene expression (time between CLN2pr and RAD27pr induction) and cell size defect as cln1Δ cln2Δ cells (Figures 2A and 2B and Figure S2). However, the fitness defect was partially reduced (Figure 2C). This indicates the importance of running the positive feedback loop from an early-activated promoter. To further test our feedback-first model, we examined the effects of varying carbon source and synchronization method, which are both known to affect gene expression (Flick et al., 1998Flick K. Chapman-Shimshoni D. Stuart D. Guaderrama M. Wittenberg C. Regulation of cell size by glucose is exerted via repression of the CLN1 promoter.Mol. Cell. Biol. 1998; 18: 2492-2501PubMed Google Scholar, Levy et al., 2007Levy S. Ihmels J. Carmi M. Weinberger A. Friedlander G. Barkai N. Strategy of transcription regulation in the budding yeast.PLoS ONE. 2007; 2: e250Crossref PubMed Scopus (60) Google Scholar, Brauer et al., 2008Brauer M.J. Huttenhower C. Airoldi E.M. Rosenstein R. Matese J.C. Gresham D. Boer V.M. Troyanskaya O.G. Botstein D. Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast.Mol. Biol. Cell. 2008; 19: 352-367Crossref PubMed Scopus (399) Google Scholar). We performed a microarray time course after synchronizing cells with mating pheromone in media with either glucose or galactose. Carbon source does not have a large effect, as differences in activation times were similar to experimental replicates (Figure 3A ). To analyze the effect of synchronization method, we examined cells lacking endogenous G1 cyclins (cln1Δ cln2Δ cln3Δ) but containing an integrated MET3pr-CLN2 construct (see the Experimental Procedures). Cells were arrested in G1 before being transferred to media with a low level of methionine to activate exogenously controlled CLN2 transcription at physiological levels. We then compared activation times between the cyclin blocked and the pheromone blocked cells (Figure 3B). Our three G1 block-release experiments varying carbon source and synchronization method produced similar timing profiles. We examined the distribution of activation times pooled from the three separate G1 block experiments (Figure 3C). Although transcriptional order is affected by the arrest phase (Figure 3D and Figure S3), CLN1 is activated at the first possible time point (5 min after release) in agreement with the feedback-first model. Since transcriptional order changes with the arrest phase, we decided to investigate which block is more similar to the free-running cell cycle using time-lapse fluorescence microscopy (Skotheim et al., 2008Skotheim J.M. Di Talia S. Siggia E.D. Cross F.R. Positive feedback of G1 cyclins ensures coherent cell cycle entry.Nature. 2008; 454: 291-296Crossref PubMed Scopus (261) Google Scholar). We analyzed protein accumulation in ten strains expressing C-terminal GFP fusion proteins from the endogenous loci (Ghaemmaghami et al., 2003Ghaemmaghami S. Huh W.K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Global analysis of protein expression in yeast.Nature. 2003; 425: 737-741Crossref PubMed Scopus (2993) Google Scholar) and two strains containing an integrated CLN1 or CLN2 promoter driving the expression of a destabilized VenusPEST (Mateus and Avery, 2000Mateus C. Avery S.V. Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry.Yeast. 2000; 16: 1313-1323Crossref PubMed Scopus (128) Google Scholar). We selected this group of strains to span the distribution of activation times. Automated cell segmentation allows us to analyze the fluorescent intensity change in single cells through the cell cycle (Figure 4A ). We detected activation timing relative to bud emergence (Figures 4B and 4C and Table S3). We found that the mean single-cell activation times in the unperturbed cell cycle correlated more with the mitotic block experiments (R2 = 0.72; Figure 4D) than the G1 block experiments (R2 = 0.21; Figure 4E). This result also implies that the order of mRNA transcription is largely reflected in protein accumulation. Thus, the mitotic block experiments are more representative of freely cycling cells. Since transcription activation times change with the phase of the block used, we decided to analyze previously published cell cycle-synchronized microarray time courses (Spellman et al., 1998Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization.Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3898) Google Scholar, Pramila et al., 2006Pramila T. Wu W. Miles S. Noble W.S. Breeden L.L. The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle.Genes Dev. 2006; 20: 2266-2278Crossref PubMed Scopus (230) Google Scholar, Orlando et al., 2008Orlando D.A. Lin C.Y. Bernard A. Wang J.Y. Socolar J.E. Iversen E.S. Hartemink A.J. Haase S.B. Global control of cell-cycle transcription by coupled CDK and network oscillators.Nature. 2008; 453: 944-947Crossref PubMed Scopus (229) Google Scholar). Although quantitative comparisons of individual genes are difficult because of either poor temporal resolution or the lack of experimental replicates, we are able to detect correlations of genes within the G1/S regulon. We found that G1 blocks, including elutriation, correlate with our G1 block data (see Table S4). Interestingly, the cdc15ts data from Spellman et al., 1998Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization.Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3898) Google Scholar correlates with our G1 block experiments rather than the mitotic block experiments even though this is an anaphase block, indicating that an event occurring in cells blocked downstream of Cdc20 may be responsible for differences in gene activation timing. We note that release from G1 arrest and free cycling are both likely to be physiologically relevant. We hypothesized that the observed differences in gene activation time in different blocks might be due to differential regulation of specific transcription factors. The majority of genes in what we defined as the G1/S regulon are regulated by the transcription factors SBF and MBF (Ferrezuelo et al., 2010Ferrezuelo F. Colomina N. Futcher B. Aldea M. The transcriptional network activated by Cln3 cyclin at the G1-to-S transition of the yeast cell cycle.Genome Biol. 2010; 11: R67Crossref PubMed Scopus (48) Google Scholar). For our analysis, we divided the activation times of the G1/S genes into three categories: 136 SBF-only targets, 63 MBF-only targets, and 36 dual-regulated SBF and MBF targets. Since combinatorial use of transcription factors may yield differential activation timing, we analyzed the activation times of the SBF only, MBF only, and dual-regulated genes. For our G1 arrest data, we find that MBF-only targets are activated earlier than SBF-only targets (p < 0.01). Furthermore, the distribution of the dual regulated targets is more similar to the earlier-activated MBF-only targets (p = 0.90) than the more tardy SBF-only targets (p = 0.01; Figure 5A ). In the mitotic block-release, the SBF-only targets are activated earlier than the MBF-only targets (p = 0.08). This is the opposite order than in the G1-block experiments and consistent with the lack of correlation between activation times of individual G1/S regulon members (Figure 3D). Furthermore, we find that the common targets are much more likely to follow the SBF-only distribution (p = 0.79) than the MBF-only distribution (p = 0.06; Figure 5B). We note that the SBF distribution is broader so that the late-activated SBF genes are activated later than the late-activated MBF genes. However, the late-activated dual-regulated genes now appear to follow MBF. Taken together, our results from the G1 and mitotic block-release experiments suggest that the dual-regulated targets are activated by the earliest active transcription factor. In th" @default.
• W2003089698 created "2016-06-24" @default.
• W2003089698 creator A5019414569 @default.
• W2003089698 creator A5055818341 @default.
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• W2003089698 date "2011-08-01" @default.
• W2003089698 modified "2023-09-27" @default.
• W2003089698 title "Commitment to a Cellular Transition Precedes Genome-wide Transcriptional Change" @default.
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