topology and control of the cell-cycle-regulated ...cell-cycle-regulated genes has grown, slowly at...

YEASTBOOK

Topology and Control of the Cell-Cycle-RegulatedTranscriptional CircuitrySteven B. Haase* and Curt Wittenberg†,1

*Department of Biology and Duke Center for Systems Biology, Duke University, Durham, North Carolina 27708, and yDepartment of Celland Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT Nearly 20% of the budding yeast genome is transcribed periodically during the cell division cycle. The precise temporalexecution of this large transcriptional program is controlled by a large interacting network of transcriptional regulators, kinases, andubiquitin ligases. Historically, this network has been viewed as a collection of four coregulated gene clusters that are associated witheach phase of the cell cycle. Although the broad outlines of these gene clusters were described nearly 20 years ago, new technologieshave enabled major advances in our understanding of the genes comprising those clusters, their regulation, and the complexregulatory interplay between clusters. More recently, advances are being made in understanding the roles of chromatin in the controlof the transcriptional program. We are also beginning to discover important regulatory interactions between the cell-cycletranscriptional program and other cell-cycle regulatory mechanisms such as checkpoints and metabolic networks. Here we reviewrecent advances and contemporary models of the transcriptional network and consider these models in the context of eukaryotic cell-cycle controls.

TABLE OF CONTENTS

Abstract 65

Introduction 66

Evolution of Experimental Approaches and Models 67The single-gene approach 67

Genomic approaches 67

Caveats associated with population-based approaches 68

Algorithmic approaches to population studies 69

Single-cell approaches 69

Cell-Cycle-Regulated Gene Clusters: The Logicand Strategy for Expression 69The G1/S gene cluster 70

Activation of G1/S transcription factors 70

Repression of G1/S transcription factors 71

Temporal regulation within the G1/S gene cluster 72

Coupling of G1/S transcription to the rest of the transcriptional program 72

Continued

Copyright © 2014 by the Genetics Society of Americadoi: 10.1534/genetics.113.152595Manuscript received May 9, 2013; accepted for publication September 16, 20131Corresponding author: Department of Molecular Biology, MB-3, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail: [email protected]

Genetics, Vol. 196, 65–90 January 2014 65

mailto:[email protected]

CONTENTS, continued

The S phase gene cluster 72

The G2/M gene cluster 73

The M/G1 gene cluster 74

The Mcm1 gene cluster 74

The Sic1 gene cluster 75

The MAT gene cluster 76

Emerging Views of Cell-Cycle-Regulated Transcription 77CDK’s role in cell-cycle-regulated transcriptional program 77

Checkpoint control of cell-cycle-regulated transcription 78

Yeast metabolic cycle control of cell cycle and transcription 80The YMC-regulated transcriptional program: 80YMC control of cell cycle: 80

What is the role of chromatin incell-cycle-regulated transcription? 81

Rationale for Cell-Cycle-Regulated Transcription: Need to Have or Nice to Have? 82

The Topology of Cell-Cycle-Regulated Transcriptional Circuitry is Conserved 83

THE cell cycle is the sum of the processes by which cellsreplicate. That process, involving the replication and re-

distribution of all cellular components, requires the synthesisof many proteins, including constituents of cellular struc-tures and organelles, enzymes that catalyze the many ana-bolic and catabolic processes required for their replicationand distribution, as well as the regulatory proteins that gov-ern those events. Although the genes encoding many ofthose proteins are expressed constitutively, a large numberare expressed at or near the interval when they are needed.Consequently, progression through the cell cycle is accom-panied by dramatic reorganization of gene expression thatwe refer to collectively as “cell-cycle-regulated transcrip-tion.” The nature and mechanism of that program of geneexpression is the subject of this review.

The first cell-cycle-regulated genes were observed inyeast more than three decades ago (Hereford et al. 1981)when the mRNA encoding histone genes was observed toaccumulate periodically during the course of the cell cycle ina synchronized population of cells. Since then, the list ofcell-cycle-regulated genes has grown, slowly at first, onegene at a time, and then very rapidly, largely as a conse-quence of genome-wide approaches, to encompass betweenas much as 20% of the genome (Cho et al. 1998; Spellmanet al. 1998; Pramila 2002; Orlando et al. 2008; Guo et al.2013). Despite the relatively large number of individualgenes that are periodically expressed, it has become clearthat they fall into a relatively small number of gene familiesthat are coregulated. Consequently, the entire programappears to be controlled by a relatively small set of specifictranscriptional regulatory factors.

This general topic has been extensively reviewed (Bähler2005; Wittenberg and Reed 2005; McInerny 2011) and in-

depth reviews covering specific transcription factor familiesand cell-cycle-regulated gene clusters have been presented(Murakami et al. 2010; Cross et al. 2011; Eriksson et al.2012). We will introduce the constituents and regulatorylogic of the cell-cycle transcriptional circuitry with discus-sion weighted toward more recent contributions.

A general understanding of both the pattern of geneexpression and the regulation of the cell-cycle transcrip-tional program is, in many cases, emerging. When viewedin its entirety, the program appears as a continuum oftranscriptional activation and deactivation. However, wenow appreciate that waves of gene expression are coupled toobservable cell-cycle events, which, in most cases, dependon the activity of the cyclin-dependent protein kinase Cdk1(Cdc28, see below). The transcriptional program guides theactivity of Cdk1 by initiating the properly timed expressionof specific cyclin genes. In turn, cyclin/Cdk1 complexesphosphorylate transcription factors and regulate their activ-ity. Thus, there is a complex dynamic interplay between thetranscriptional program, CDK activity, and cell-cycle progres-sion (Figure 1). Waves of gene expression are associatedwith (i) cell-cycle initiation late in G1 phase prior to theinitiation of S phase (G1/S transcription), (ii) S phase(S phase transcription), (iii) the transition from G2 phaseinto M phase (G2/M transcription), and (iv) the transition ofcells from M phase back into G1 phase (M/G1 transcrip-tion). Genes within a coregulated cluster are not all acti-vated at the same time but appear to be turned on ina precise order during an interval that can span 20% ofthe cell cycle (Eser et al. 2011; Guo et al. 2013). The con-sequence of this highly regulated pattern of transcription isthe sequential periodic expression of upwards of 1000genes.

66 S. B. Haase and C. Wittenberg

http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000000364




The existence of this intricately orchestrated sequence oftranscriptional activity raises a number of important ques-tions that form the basis for contemporary studies. Whichgenes constitute the full cell-cycle-regulated set? How is thetranscriptional activation within a coregulated cluster or-dered, and how is the temporal order of clusters maintained?How is the cell-cycle-regulated transcriptional program influ-enced by regulatory pathways, including checkpoints and theyeast metabolic cycle (YMC)? Recent findings that suggestthe transcriptional program can be uncoupled from cell-cycleprogression leave in question the mechanisms that maintainsynchrony between the transcriptional program and cell-cycleevents. Finally, why is the temporal order of this transcrip-tional program important for execution of the yeast cell cycleand to what degree is this program conserved amongeukaryotes? In the sections that follow, we will review theliterature regarding these issues with a focus on the emergingmodels.

Evolution of Experimental Approaches and Models

Models of biological phenomena are necessarily constrainedby the approaches we use to observe them. Views of cell-cycle-regulated transcription have certainly evolved as newtechnologies and approaches have been applied.

The single-gene approach

Most early studies identified cell-cycle-regulated genes oneat a time. Typically, populations of cells were synchronized

by arresting with mating pheromone, released from thearrest, and sampled over time. RNAs were isolated from thetime series and subjected to Northern blot analysis usingradioactively labeled probes from the gene of interest. Takentogether, these early studies revealed that many of the genesidentified by genetic methods as important cell-cycle regu-lators were themselves transcriptionally controlled duringthe cell cycle. Cell-cycle regulators, like the cyclins, weregenerally found to be transcribed in the phase in which theyfunctioned (Wittenberg et al. 1990; Surana et al. 1991;Richardson et al. 1992; Kuhne and Linder 1993; Schwoband Nasmyth 1993). These findings pointed to a model inwhich the cell cycle is driven, at least in part, by successivewaves of expression of regulatory proteins (Amon et al.1993).

Genomic approaches

In 1998, the first global view of the cell-cycle-regulatedtranscription program was revealed when several groupsexamined synchronized populations of cells in time-seriesexperiments using microarrays (Cho et al. 1998; Spellmanet al. 1998). To date, five studies (using different types ofmicroarrays and different means of synchronizing popula-tions) have reported global transcriptional expression pro-files in wild-type yeast cells as they progress through the cellcycle (Cho et al. 1998; Spellman et al. 1998; Pramila et al.2006; Orlando et al. 2008; Granovskaia et al. 2010). Thesestudies, and comparable studies in other systems (Rusticiet al. 2004; Oliva et al. 2005; Peng et al. 2005), delineated

Figure 1 The cell-cycle transcriptional circuitry. Thistranscriptional circuit depicts the major interactions be-tween transcriptional activators and repressors andtheir regulation by cyclin/CDK and APC discussed inthe course of this article. This circuit is not meant tobe exhaustive but rather to provide a reference for theinteraction between the cluster regulators that aredepicted in the subsequent figures. Many other inter-actions are discussed in the body of the article. Sub-units in green are those with activating activity,subunits in red are those with repressing or inhibitoryactivity, and subunits in blue represent those that re-quire a regulatory subunit. Arrows in green representactivating activities, those in red represent repressingactivities, and those in black represent transitions in theprocess.

Cell-Cycle-Regulated Transcription 67

the scope of the problem, identifying several hundred geneswhose transcript levels oscillate during the cell cycle. Theprecise number of periodic genes is still a somewhat conten-tious issue, ranging from 600 to 1500 or between 10 and20% of all yeast genes (discussed further below). This frac-tion is higher than expected, given the number of knowncell-cycle regulators identified by genetics, and suggests thatprecise regulation of a large fraction of the genome mightcontribute to proper progression through the cell cycle.

The emerging picture from these studies also seemed tosuggest genes are not transcribed in a small number ofdiscrete intervals, but are instead turned on and off ina continuum as cells passed through the cycle. Nonetheless,the program could be divided into large blocks of genes(clusters) found to be transcriptionally active in specific cell-cycle phases. The lists of periodic genes enabled searches toidentify promoter motifs governing cell-cycle-regulatedtranscription. This effort benefited greatly from the genomesequences of related Saccharomyces species, which allowedfor comparisons that highlighted conserved promoter ele-ments (Cherry et al. 2004; Teixeira et al. 2006). Bioinfor-matic analyses determined that genes within clusters sharedcommon promoter elements and were likely coregulated byspecific subsets of transcription factors (Spellman et al.1998; Pramila et al. 2006; Orlando et al. 2008).

At the turn of the century, high-throughput chromatinimmunoprecipitation-microarray chip (ChIP-chip) technolo-gies enabled researchers to collectively identify the bindingsites of all known transcription factors across the buddingyeast genome (Ren et al. 2000; Simon et al. 2001; Lee et al.2002; Harbison et al. 2004). These ChIP-chip experiments,like other early generation high-throughput technologies,likely produced false positives and false negatives at rela-tively high rates. Thus, functional experimental evidence isneeded to confidently assign regulatory roles to specifictranscription factor/gene combinations. Nonetheless, thesestudies provided physical evidence that clusters of genesidentified by microarray experiments were indeed coregu-lated by specific transcriptional regulators expressed in dis-crete cell-cycle phases. It also became clear that the genesencoding transcriptional regulators were themselves lo-cated in these clusters (Simon et al. 2001). When theresults of genome-wide transcription factor localizationwere combined with transcript dynamics from microarrayexperiments, it became apparent that the cell-cycle tran-scriptional program could be regulated by a relatively smallnetwork of serially activated transcription factors (Simonet al. 2001; Lee et al. 2002; Pramila et al. 2006). Further-more, this limited network of factors can function indepen-dently or combinatorially to enhance the complexity of thepattern of transcriptional regulation (Kato et al. 2004).The idea that a transcription factor network could controlthe cell-cycle transcriptional program was a paradigm shiftin the field (discussed below), and this shift was madepossible only through the development and application ofgenomic technologies.

A qualitatively similar view of the cell-cycle transcrip-tional program emerged in Schizosaccharomyces pombewhen microarray approaches were applied to synchronouspopulations of fission yeast (Rustici et al. 2004; Oliva et al.2005; Peng et al. 2005). Cell-cycle-regulated transcripts inS. pombe are expressed in waves that can be subdivided intoclusters of temporally coregulated genes. Many recognizableorthologs in Saccharomyces cerevisiae and in S. pombe exhibitsimilar transcript periodicity, suggesting that transcriptionalregulatory mechanisms are conserved (Oliva et al. 2005;Orlando et al. 2007). Despite the fact that many of the tran-scription factors that mediate cell-cycle-regulated transcrip-tion and some specific genes regulated by those factors areconserved, there is considerable divergence among cell-cycle-regulated genes and programs, even among closelyrelated yeasts (Eser et al. 2011; Guan et al. 2013). Fur-thermore, computational models indicate some alteredbehaviors that likely reflect unique aspects of cell-cycleprogression in these highly diverged yeasts (Orlando et al.2007). Comparison with other yeasts and more highlydiverged metazoan systems has led to the concept of con-servation of the general topology, but not the specifics, ofthe cell-cycle-regulated transcriptional circuitry (discussedbelow).

Caveats associated with population-based approaches

Taken together, the single-gene and genomic approacheshave been phenomenally successful in providing a globalview of how transcript levels are regulated during cell-cycleprogression. However, it seems likely that some of the moresubtle aspects of transcriptional regulation remain hidden,due to the nature of the experiments. The approachesdescribed above rely on synchronous populations of cellsto produce enough RNA to be interrogated by Northern blot,microarray, qPCR, or RNA-Seq. Because populations areused, measurements of mRNA levels necessarily representthe average level across the population. In an ideal case, allof the cells in the population would be in the exact samecell-cycle phase, and thus the measured values represent anaverage over cell-to-cell variance (biological noise). Al-though budding yeast is well known for the ease ofsynchronization using a variety of methods (mating phero-mone, conditional mutants in cell-cycle genes, elutriation),synchronized populations are never fully synchronous, andthey lose synchrony over time. Loss of synchrony isespecially severe in budding yeast where the division isasymmetric (Hartwell and Unger 1977; Lord and Wheals1980, 1981; Woldringh et al. 1993; Bean et al. 2006;Orlando et al. 2007). Thus, the measured values fromtime-series experiments using synchronized populations rep-resent a convolution of values from cells distributed acrosscell-cycle phases. The result is that transcript oscillationsappear to dampen over time. Convolution due to loss ofsynchrony can obscure the true amplitude of transcript oscil-lations as well as the precise timing of the appearance anddisappearance of a specific transcript.


Algorithmic approaches to population studies

To better identify more subtle aspects of the cell-cycle-regulated transcriptional program, several groups set out touse algorithmic approaches to deconvolve the measuredvalues of transcript levels from microarray experiments(Bar-Joseph et al. 2004; Qiu et al. 2006; Rowicka et al.2007; Guo et al. 2013). The goal of these approaches wasto better approximate the dynamic behavior of transcripts inthe average cell in the population. The groups made variousassumptions about the underlying models that drive syn-chrony loss in time-series experiments, and each reportedan increased performance in accurately inferring both thetiming and amplitude of transcript levels during the cellcycle. Additionally, Guo et al. (2013) explicitly modeledasymmetric division using a branching process model thatallowed them to infer differences in expression dynamicsbetween mother and daughter cells. Thus, they were ableto identify 82 genes that were expressed exclusively indaughter cells (see below). Taken together, these algorith-mic approaches permit a more accurate view of the timing oftranscript dynamics and suggest that the amplitude of tran-scriptional responses is, in many cases, substantially higherthan previously recognized.

Although these algorithmic approaches likely providea sharper view of transcript dynamics during the cell cycle,the values they return are still estimates rather than truemeasurements. Of course, the estimates are only as good asthe underlying models for population asynchrony. Addition-ally, the algorithms report values for the average cell in thepopulation and do not tell us much about the distribution ofbehaviors across cells in the population.

Single-cell approaches

While there are computational methods to address theissues associated with approaches using synchronized pop-ulations, arguably the most direct way to address the issuesis to measure transcript abundance in single cells. Ideally,one would be able to monitor the abundance of a variety oftranscripts in living cells as they progress through the cellcycle. However, current protocols are limited to measuringonly a few transcripts per experiment in fixed cells or tousing the expression of destabilized proteins as a proxy formRNA abundance in living cells.

Single-cell fluorescence in situ hybdrization (FISH) stud-ies (Femino et al. 1998) can be used in yeast to measure thelevels of a specific transcript. Because cells are fixed duringthe hybridization procedure, measurements of transcriptfluctuations over time can only be made on a populationof synchronized cells, and thus this approach would sufferfrom some of the same issues related to population syn-chrony. However, single-cell FISH was used effectively todemonstrate that individual yeast cells in an unsynchronizedculture of slow growing yeast cells exhibit metabolic cyclingof gene expression (Silverman et al. 2010). This approach,examining the coincidence of expression of several gene

pairs, enabled investigators to establish positive correlationbetween genes expressed in the same phase of the metabolicoscillation and negative correlation between those ex-pressed in opposite phases. Earlier studies relied on sam-pling synchronized populations of cells from a chemostatfor microarray analysis (Klevecz et al. 2004; Tu et al.2005). This approach also worked well when applied tocell-cycle-regulated genes by Silverman et al. (2010).Single-molecule fluorescence techniques, which have beenapplied successfully to monitor the initiation and elongationrates, as well as the stability of cell-cycle-regulated mRNAsin living cells, may be applicable in studies of the dynamicsof cell-cycle-regulated transcription (Larson et al. 2011;Trcek et al. 2011).

The dynamics of periodic transcripts can also be inferredby time-lapse microscopy in live cells using strains with cell-cycle-regulated promoters fused to unstable variants ofgreen or red fluorescent proteins (GFP/RFP) (Skotheimet al. 2008; Eser et al. 2011). Time-lapse microscopy onsingle cells does not suffer from population effects, and incases where other markers are available, coherence betweenthe expression of a fluorescent protein and other events canbe determined. However, this approach does not directlymeasure transcript levels, and the kinetics of the appearanceand disappearance of transcripts can only be estimated fromthe accumulation of the GFP protein. Precise estimation isblurred by variations in the maturation time of the GFP/RFP, and in the protein half-life, especially for short-livedtranscripts. Furthermore, detection of GFP/RFP fluorescenceis insufficiently sensitive to be useful for all but the mostpowerful promoters. Despite the low-throughput nature ofthe approach, transcriptional reporters with better signal tonoise (e.g., luciferase) and advancements in high-resolutiontime-lapse microscopy should expand its utility (Howellet al. 2012).

Cell-Cycle-Regulated Gene Clusters: The Logicand Strategy for Expression

Despite the observation that transcripts appear and disap-pear in a continuum throughout the cycle, the cell-cycletranscription program has historically been viewed as fourmajor waves of gene expression that define the dominantcell-cycle-regulated gene clusters including the G1/S cluster,the S phase cluster, the G2/M cluster, and the M/G1 cluster,based upon the coincidence of the timing of expressionrelative to the easily discernible cell-cycle events (reviewedby Bähler 2005; Wittenberg and Reed 2005; McInerny2011). Despite their apparently coherent expression, thegenes within a specific cluster are often coordinately regu-lated by distinct transcription factors or mechanisms. In ad-dition to the prominent clusters, there are smaller geneclusters or single genes with distinct patterns of cell-cycle-regulated expression sometimes mediated by multiple tran-scription factors acting in concert. Our goal in this section isto discuss the major clusters and their modes of regulation.


The G1/S gene cluster

The G1/S gene cluster (also called simply the G1 genecluster) comprises the largest and, arguably, the best-characterized family of cell-cycle-regulated genes in yeast.This family of upwards of 200 genes is expressed largely inG1 phase cells coincident with START and prior to theinitiation of DNA replication (reviewed by Bähler 2005;Wittenberg and Reed 2005; McInerny 2011). These genesare controlled by two heterodimeric transcription factors,SCB-binding factor (SBF) and MCB-binding factor (MBF),which bind to distinct but related DNA sequence motifs inthe promoters of their target genes (Figure 2). SBF binds toSCB elements (SBF cell-cycle box) and MBF to MCB ele-ments (MBF cell-cycle box), respectively (Lee et al. 2002;Teixeira et al. 2006). The distinct DNA-binding properties ofthose factors is attributable to their DNA-binding proteins,Swi4 for SBF and Mbp1 for MBF. Both factors bind DNA viaa classical winged helix-turn-helix DNA-binding fold andassociate with the common subunit Swi6. Although bothDNA-binding proteins and Swi6 share a common domainstructure, Swi4 and Mbp1 bind distinct DNA sequences,whereas Swi6 does not bind directly to DNA (McIntoshet al. 2000; Xu et al. 1997; Nair et al. 2003; Taylor et al.1997, 2000, 2010).

SBF and MBF bind and regulate partially overlappingsubsets of genes in the G1/S cluster because of the presenceof both consensus sequence motifs in some of the promoters(Spellman et al. 1998; Iyer et al. 2001; Simon et al. 2001;Teixeira et al. 2006; Ferrezuelo et al. 2010). The extent towhich the overlap is reflected in coordinate or differentialregulation is only beginning to be understood (see below)(Bean et al. 2005; Eser et al. 2011; de Oliveira et al. 2012;Smolka et al. 2012; Travesa and Wittenberg 2012; Travesaet al. 2012). Finally, many potential and actual binding siteshave been identified in promoters that do not appear topromote cell-cycle periodicity but may, in some cases, havefunctional consequences (Iyer et al. 2001; Horak et al. 2002;Bean et al. 2005; also see Haber 2012; Eriksson et al. 2012).

The G1/S gene cluster is rich in genes involved inprocesses associated with progression from G1 into S phase.Passage through START is particularly important for initiat-ing the many events required for the new cell division cycle.Those include dramatic changes in morphogenesis leadingto the formation of the bud, the nascent daughter cell withits associated septin ring marking the future site of cytoki-nesis (reviewed in Bi and Park 2012; Howell and Lew2012). Additionally, the duplication of the spindle pole body,the first structures associated with assembly of the mitoticspindle (reviewed by Winey and Bloom 2012), and the ini-tiation of DNA replication are triggered following START(reviewed by Remus and Diffley 2009). Although the sepa-ration of function is by no means absolute, in general, manygenes involved in morphogenesis, including enzymes re-quired for cell wall biosynthesis and septin ring components,are regulated by SBF, whereas genes for DNA replication

and repair, including nucleotide biosynthetic enzymes andproteins acting at the replication fork, are regulated by MBF.The rationale for segregating SBF and MBF targets basedupon function has, until recently, lacked explanation becausethe two transcription factors are coordinately regulated duringthe cell cycle. However, it has become apparent that seg-regation of targets into two groups can have dramatic con-sequences under specific physiological conditions (see below)(Doncic et al. 2011; Eser et al. 2011; Smolka et al. 2012;Travesa et al. 2012).

The hallmark of these two transcriptional regulators istheir capacity to abruptly activate their target genes duringG1 phase in response to the activation of G1 cyclin-associated Cdk1 (Figure 2) (Costanzo et al. 2004; Di Taliaet al. 2007; Taberner et al. 2009). That regulation is disrup-ted by cdc28-ts mutants (Marini and Reed 1992). We nowunderstand that the activation of G1/S genes is a conse-quence of the regulated accumulation of the G1 cyclinCln3 (Tyers et al. 1993; Dirick et al. 1995; Stuart andWittenberg 1995; Garí et al. 2001). The accumulation, acti-vation, and localization of Cln3/CDK are critical determi-nants of the timing of START and are responsive to cellgrowth and cell size (Nash et al. 1988; Tyers et al. 1993;Baroni et al. 1994; Polymenis and Schmidt 1997; Miller andCross 2000; Edgington and Futcher 2001; Jorgensen et al.2002; Di Talia et al. 2007; Taberner et al. 2009; Ferrezueloet al. 2012; Thorburn et al. 2013). Cln3, once accumulatedto a critical level in the nucleus, activates Cdk1, leading tothe phosphorylation of the SBF-bound transcriptional re-pressor, Whi5 (Costanzo et al. 2004; de Bruin et al. 2004;Schaefer and Breeden 2004). Upon phosphorylation, Whi5,which binds to the carboxy terminus of Swi6 through itsGTB-containing domain, dissociates from promoter-boundSBF and is relocalized to the cytoplasm (Figure 2) (Costanzoet al. 2004; de Bruin et al. 2004; Di Talia et al. 2007;Skotheim et al. 2008; Travesa et al. 2013). Phosphorylationof Swi6 by Cln/CDK may also contribute to the regulation ofWhi5 binding (Sidorova et al. 1995; Costanzo et al. 2004;Wagner et al. 2009). Dissociation and nuclear export ofWhi5, in turn, leads to the activation of SBF and the expres-sion of its target genes. Although Cln3/CDK-dependentWhi5 phosphorylation appears to promote both dissociationfrom promoters and export from the nucleus, those twophenomena have not been separated in terms of their effecton the activation of G1/S transcription (Costanzo et al.2004; de Bruin et al. 2004; Di Talia et al. 2007; Skotheimet al. 2008; Travesa et al. 2013). A recent study has estab-lished that release of the Whi5 repressor is the critical de-terminant of the event historically referred to as START byHartwell and colleagues (Doncic et al. 2011; Eser et al.2011).

Activation of G1/S transcription factors

Our current understanding of SBF derives from its earlyidentification (by the Nasmyth and Herskowitz laboratories)among the genes required for expression of the HO gene,

























which encodes the endonuclease that mediates switching ofmating types via DNA rearrangements at the MAT locus(Breeden and Nasmyth 1987; Andrews and Herskowitz1989; see Haber 2012). The known targets of SBF, whichnow number �100, include those encoding the G1 cyclins,Cln1 and Cln2, which, like Cln3, promote Cdk1-dependentactivation of G1/S transcription (Cross 1988; Wittenberget al. 1990; Nasmyth and Dirick 1991; Tyers et al. 1993;Wijnen et al. 2002). Although earlier studies supportedthe autonomy of Cln3/Cdk1 in the timely activation ofG1/S genes (Dirick et al. 1995; Stuart and Wittenberg1995), the application of single-cell techniques to the anal-ysis of START led to the realization that the rapid accumu-lation of Cln1 and Cln2, and their potent activation of Cdk1,leads to a dramatic acceleration of nuclear export of Whi5.This positive feedback greatly enhances the coherency ofactivation of G1/S genes (Skotheim et al. 2008), confirminga model advanced many years prior (Cross and Tinkelen-berg 1991; Dirick and Nasmyth 1991). Thus, the combinedactivity of the G1 cyclin-associated forms of Cdk1 is respon-sible for the switch-like behavior and irreversibility of theSTART event.

The expression of MBF targets, like those regulated bySBF, occurs during late G1 phase and depends upon Cln3/Cdk1 activity (Tyers et al. 1993; Dirick et al. 1995; Stuart

and Wittenberg 1995; Wittenberg and Reed 2005). MBFtargets include genes encoding DNA replication and repairproteins and the cyclin Clb5, which is critical for timelyactivation of DNA replication. Interestingly, the SWI4 genewas recently shown to be a target of MBF, which is sufficientfor much of its expression during G1 phase (Harris et al.2013), suggesting there is crosstalk between the two majorG1/S transcription factors. The mechanism of activation ofMBF-regulated genes remains to be established. We doknow that, unlike SBF, MBF acts, in part, as a repressor ofits target genes outside of G1 phase, suggesting that Cdk1acts to relieve its repressive activity (de Bruin et al. 2006).However, as of yet, no protein playing an equivalent role toWhi5 has been identified. This has led to the prevailinghypothesis that the direct phosphorylation of the transcrip-tion factor by Cdk1 is the critical event in transcriptionalactivation. Although both Swi6 and Mbp1 are known to besubstrates of Cdk1 in vitro and are phosphoproteins in vivo,there is currently no evidence indicating that those phos-phorylation events are required for activation of MBF targets(Sidorova et al. 1995; Siegmund and Nasmyth 1996; Ubersaxet al. 2003; Geymonat et al. 2004; Holt et al. 2008).

Repression of G1/S transcription factors

Like SBF targets, MBF-regulated genes play important rolesin the circuitry regulating G1/S transcription (Figure 2).One of its targets is the gene encoding the MBF-specifictranscriptional repressor, Nrm1 (de Bruin et al. 2006). Un-like Whi5, which represses transcription from M phasethrough late G1 phase, Nrm1 acts only after G1/S transcrip-tion is strongly activated, leading to Nrm1 accumulation.Nrm1 binds to the carboxy terminus of Swi6 at MBF targetpromoters via interaction between its GTB-containing do-main, a motif conserved with Whi5 and its fungal orthologs(Ofir et al. 2012; Travesa et al. 2013). This leads to thereestablishment of MBF as a transcriptional repressor suchthat expression of MBF target genes, including that of NRM1itself, is terminated by negative feedback. Subsequently, ascells exit mitosis, the Nrm1 protein is targeted for degrada-tion by the APCCdh1 ubiquitin ligase, preparing cells to reac-tivate MBF during the subsequent G1 phase (Ostapenko andSolomon 2011). In addition to Nrm1, MBF also promotesthe expression of two B-type cyclins, Clb5 and Clb6.Whereas Clb5 ultimately activates Cdk1 to drive entry intoS phase, Clb6/Cdk1 has been reported to phosphorylateSwi6, promoting its accumulation in the cytoplasm (Geymonatet al. 2004). It has been suggested that Swi6 is constitu-tively cycling between the cytoplasm and the nucleus suchthat the phosphorylation-dependent inhibition of nuclearuptake results in accumulation in the cytoplasm that is thenreversed upon dephosphorylation subsequent to mitosis bythe Cdc14 phosphatase (Queralt and Igual 2003; Geymonatet al. 2004). Interestingly, cycling of Swi6 between the cy-toplasm and nucleus appears to be important for SBF-dependent transcriptional activity, although the molecularbasis for that requirement has not been established (Queralt

Figure 2 Transcriptional regulation of the G1/S gene cluster. The regu-latory circuitry for the G1/S gene cluster is depicted. Two transcriptionalregulators, SBF and MBF, bind to SCB and MCB elements, respectively, inthe repressed promoters of G1/S genes as cells enter G1 phase. SBFrepresses its targets due to the binding of Whi5. MBF appears to act, inpart, as a repressor, during early G1 phase. Once accumulated to appro-priate levels, Cln3/Cdk1 phosphorylates Whi5, leading to Whi5 dissocia-tion from SBF and activation of the G1/S gene promoters. Cln3/Cdk1 alsoactivates MBF-regulated promoters via an unknown mechanism. Activa-tion of the CLN1 and CLN2 promoters by SBF leads to activation of theCln1/Cdk1 and Cln2/Cdk1, both of which feed back to further activateSBF-regulated promoters, as well as activating MBF-regulated promoters.The activation of MBF-regulated promoters leads to the accumulation ofNrm1, a specific repressor of MBF that, when accumulated sufficiently,feeds back to bind MBF, thereby repressing MBF targets. MBF also acti-vates Swi4, thereby increasing the abundance and activity of SBF. Acti-vation of SBF targets promotes cell-cycle progression, leading ultimatelyto the accumulation of Clb2/Cdk1, which binds and phosphorylates Swi4,leading to its dissociation from SBF-regulated promoters. Clb2/Cdk1 canalso promote repression of MBF-regulated promoters via an unknownmechanism. The color of subunits and arrows is as in Figure 1.












































and Igual 2003). Like Swi6, nuclear localization of Whi5 ispromoted by Cdc14-dependent dephosphorylation upon Mphase exit. The capacity of Whi5 to relocalize to the nucleusif CDK activity is reduced during other cell cycle phasessuggests that phosphatases other than Cdc14 must also becompetent to dephosphorylate it (Charvin et al. 2010). Nu-clear exclusion of Whi5 is also promoted by CDK-dependentphosphorylation, albeit earlier in the cell cycle than Swi6(Costanzo et al. 2004; Di Talia et al. 2007; Taberner et al.2009).

The repression of SBF does not seem to act throughNrm1-like negative feedback. The primary mechanism ter-minating expression of SBF targets is the accumulation ofthe B-type cyclin, Clb2 (Figure 2). Clb2/Cdk1 binds stably toSwi4 through its conserved ankyrin repeat and phosphory-lates Swi4, leading to its dissociation from DNA and exportto the cytoplasm, terminating SBF-dependent transcription(Amon et al. 1993; Koch et al. 1993; Siegmund and Nasmyth1996). SBF-dependent expression of the genes encoding theYox1 and Yhp1 transcriptional repressors may also contrib-ute, albeit modestly, to the repression of SBF by repressingSWI4 expression (Pramila 2002). Although a similar mech-anism has not been demonstrated for MBF, it is clear that inthe absence of Nrm1, MBF can also be inactivated by a mech-anism that depends upon B cyclin-Cdk1 (de Bruin et al. 2006).

Temporal regulation within the G1/S gene cluster

The application of algorithmic approaches to microarrayanalysis of the cell-cycle transcriptome has revealed anordered timing in the activation of G1/S genes that hadpreviously appeared to be expressed simultaneously (Eseret al. 2011; Guo et al. 2013). Although some hints of tem-poral resolution of gene expression within that cluster hadbeen observed in prior analyses, the lack of reproducibilityand the problems associated with differing amplitudes ofexpression had obscured those differences. Using high-throughput analysis of multiple array experiments coupledwith single-cell fluorescence analyses, Eser and colleaguesfound that CLN1 and CLN2 were among the earliestexpressed genes of the G1/S cluster, leading to the conceptof “feedback first,” wherein the first targets to be expressedare those that promote further transcriptional activation ofG1/S targets. That mechanism ensures the irreversibility ofthe process and is generally conserved in both closely re-lated yeast species and in systems as divergent as humans.Their findings also revealed that NRM1, encoding the re-pressor of MBF-regulated transcription, is among the lastgenes of the cluster to be expressed, thereby ensuring thatMBF targets are not repressed before they are adequatelyexpressed. Different synchronization regimens can cause se-quential waves of MBF-then-SBF or SBF-then-MBF activitysuch that genes containing both SBF- and MBF-binding sitesare activated by whichever of the MBF or SBF factors isactivated first and inactivated by whichever transcriptionfactor is repressed first (Eser et al. 2011). Similar resultswere obtained using a mathematical deconvolution ap-

proach that increases the temporal resolution of microarrayexperiments (Guo et al. 2013). These findings suggests thatcells can adapt their sequential program of gene activationin response to environmental or physiological conditions,perhaps resulting in increased fitness.

Coupling of G1/S transcription to the rest of thetranscriptional program

The targets of the G1/S transcription factors are a large anddiverse set of genes involved in a broad spectrum of events,many of which are initiated during the interval of G1/S geneexpression. Of particular interest, in the context of un-derstanding cell-cycle-regulated gene expression, are genesthat act to limit G1/S transcription and those that inducesubsequent waves of transcription. Nrm1 is perhaps the bestexample of a G1/S target that acts to limit G1/S transcrip-tion. Clb2 similarly limits SBF-regulated transcription, butit is not itself a G1/S target (see below). Hcm1 is the tran-scriptional activator of a large group of S phase genes (dis-cussed below), and is expressed during G1/S from apromoter that is bound by both SBF and MBF (Iyer et al.2001; Pramila 2002). Thus, Hcm1 targets are activated asa consequence of the burst of G1/S transcription, thereby,participating as a link in the cell-cycle-transcriptional circuit(Figure 1).

The S phase gene cluster

The G1/S transition, characterized by the initiation of DNAreplication, is accompanied by the activation of two clustersof S phase genes. One cluster is composed of the genesencoding the histones, which make up the nucleosomes thatpackage newly replicated DNA. Histone genes are regulatedby an, as yet, poorly understood transcriptional regulatorymechanism involving SBF, Spt10, histone chaperones, andother factors. That S phase cluster has been recentlyreviewed by Eriksson et al. (2012) and will not be discussedfurther here. The second, much larger, cluster of genesexpressed during S phase is that regulated by Hcm1 (Figure3). Although less thoroughly studied than transcription fac-tors controlling the other major bursts of cell-cycle-regulatedgene expression, Hcm1 plays a central role in that regulatorycircuitry. It is one of four members of the forkhead family oftranscription factors found in budding yeast (Hcm1, Fkh1,Fkh2, and Fhl1 (Forkhead-like) (reviewed in Murakamiet al. 2010). Like the other members of that family, whichinclude mammalian Fox transcription factors, Hcm1 bindsDNA directly via a winged helix DNA-binding motif. Thepresence of putative CDK phosphorylation sites in Hcm1and its capacity to be phosphorylated by Clb/Cdk1 in vitromake it a likely target of the Cdk1 protein kinase (Ubersaxet al. 2003).

The Hcm1 transcription factor appears to regulate �180genes based upon the timing of their expression and thepresence of the Hcm1-binding site in their promoters(Pramila et al. 2006). However, only a small fraction of thosegenes were found to bind Hcm1 by genome-wide chromatin









































immunoprecipitation (Horak et al. 2002), likely a conse-quence of the low reliability of the early studies using thattechnique. Among the targets are �10 genes involved inbudding and morphogenesis and nearly 100 involved insome aspect of chromosome segregation. Unsurprisingly,these include protein components of chromosomes and ele-ments of the chromosome segregation machinery, much ofwhich is assembled during S and G2 phases. However, a rolein the regulation of genes involved in budding was unex-pected because bud morphogenesis is a well-establishedfunction of many genes in the G1/S gene cluster. Neverthe-less, under most circumstances, budding occurs coincidentwith initiation of DNA replication so expression during Sphase under the control of Hcm1 is timely. That said,Hcm1 is dispensable for budding but is required for theproper timing and execution of mitosis (Pramila et al.2006). The synthetic lethality of hcm1 mutations with muta-tions in a number of genes involved in chromosome segre-gation is consistent with its importance in the orchestrationof mitotic functions.

The relatively recent discovery of Hcm1 makes it some-thing of a “missing link” in the transcriptional circuitry of thecell cycle. Its targets include regulators of all of the othermajor bursts of cell-cycle-regulated gene expression: thetranscriptional repressors Whi5 (G1/S transcription) andYhp1 (M/G1 transcription) as well as Fkh1, Fkh2, andNdd1, the central regulators of the G2/M gene cluster (seebelow). Although these factors do not play a direct role inlimiting G1/S transcription via feedback, Hcm1 indirectlyactivates Clb2, the feedback repressor of SBF genes, viaits role in the expression of Fkh2 and Ndd1 activators ofthe G2/M gene cluster (Figure 1). Despite the central roleof Hcm1 in the regulation of genes involved in cell-cycle-regulated transcription, hcm1 mutants are viable and retaindetectable periodicity of S phase gene expression (Pramilaet al. 2006; Simmons Kovacs et al. 2012). This suggests theinvolvement of additional, as yet unknown, transcriptionfactors in the regulation of this gene family.

The G2/M gene cluster

Progression from S phase into G2 phase is accompanied byactivation of a family of �35 genes falling into the G2/Mcluster, also called the CLB2 cluster (Cho et al. 1998; Spellmanet al. 1998). As the latter name implies, this clusterincludes the genes encoding the B-type cyclin Clb2, alongwith its paralog Clb1 and other important cell cycle reg-ulatory factors including Cdc5, the yeast polo-like kinase,Cdc20, a specificity subunit of the APC ubiquitin ligase,and the Ace2 and Swi5 transcription factors. The primaryregulator of this gene cluster is the MADS box transcrip-tion factor Mcm1, in conjunction with the forkhead familymember Fkh2 and the coactivator Ndd1, both members ofthe S phase gene cluster regulated by Hcm1 (Figure 3)(Loy et al. 1999; Kumar et al. 2000).

Mcm1 plays diverse roles that derive from its capacity tomultimerize with a diverse set of coregulators. This allows itto regulate distinct gene families including the M/G1 clustergenes (see below), the MAT locus genes (Tuch et al. 2008),and even to play a role in mating-type switching, which isapparently transcription independent (Coïc et al. 2006).The collaboration of Mcm1 with Fkh2 in the context of tran-scriptional regulation of the G2/M cluster is, in part, aconsequence of the occurrence of adjacent Mcm1- andFkh2-binding sites in G2/M cluster promoters (Lydall et al.1991; Boros et al. 2003). The association of that hetero-dimer with the adjacent binding sites on those genes createsa platform for association of the coactivator Ndd1 to activategene expression. Whereas other forkhead family memberspossess the capacity to bind to the DNA via their conservedwinged helix motif, only Fkh2 can also associate directlywith Mcm1 and thereby exert its effect on gene expressionin that context. Interestingly, this binding interface has beenconserved sufficiently for Fkh2 to bind to the human serumresponse factor (SRF), the homolog of Mcm1 (Boros et al.2003). Fkh1, which lacks the Mcm1 interaction domain ofFkh2, binds to the same DNA motif as Fkh2 at CLB2 cluster

Figure 3 Transcriptional regulation of the S phase andG2/M gene clusters. The regulatory circuitry for the Sphase gene cluster (top) and the G2/M gene cluster(bottom) is depicted. The Hcm1 transcription factor isthe major activator of S phase genes. It is uncertainwhether additional components are required to pro-mote gene expression and little is known about theactivation or repression mechanisms. The G2/M genecluster is regulated by Mcm1 in complex with Fkh2,which act as a repressor during S phase. Ndd1 bindsto Fkh2, and activates G2/M genes including thoseencoding Clb2 and Cdc5. Clb2 and Cdc5 kinases actvia positive feedback to phosphorylate Ndd1 to pro-mote binding to Fkh2 and transcriptional activation.Clb2/Cdk1 activates the APCCdh1 ubiquitin ligase,which targets Ndd1 for degradation, terminating theexpression of the G2/M gene cluster as cells completeM phase. The color of subunits and arrows is as inFigure 1.











































promoters, but only in the absence of Mcm1 (Koranda et al.2000; Hollenhorst et al. 2001).

The general view of regulation of the CLB2 cluster positsthat Mcm1 and Fkh2 remain bound to DNA, and to eachother, throughout the cell cycle. They repress transcriptionduring G1 and S phases, but subsequent recruitment ofNdd1 activates those same genes as cells progress throughG2 and into M phase. FKH2 and NDD1 are coexpressed ascomponents of the S phase gene cluster. Ndd1 is destroyedduring late M phase, presumably as a consequence of ubiq-uitylation by the APC (Loy et al. 1999). In contrast, Fkh2 isstable, persisting throughout the cell cycle (Koranda et al.2000). Although association of Ndd1 with Fkh2 is the lim-iting event for activation of G2/M cluster expression, itsaccumulation alone is reported to be insufficient for associ-ation and transcriptional activation (Reynolds et al. 2003).Phosphorylation of Ndd1 by both Clb2/Cdk1 and Cdc5 polo-like kinase appears necessary for efficient recruitment to theFkh2 FHA domain and robust activation of Clb2 clustergenes (Reynolds et al. 2003; Pic-Taylor et al. 2004; Darievaet al. 2006, 2012).

The requirement for Ndd1 phosphorylation constitutesa positive feedback loop, wherein both Clb2 and Cdc5, whenexpressed as G2/M cluster genes, feedback and activatetheir own gene expression. This posed a problem because,if these two protein kinases are required for activation oftheir own expression, it is unclear how they would be acti-vated to initiate the feedback loop. This is particularly vex-ing in the case of Cdc5, which, unlike Clb2, has no paralogsin yeast and which appears to be the only kinase that phos-phorylates S85 of Ndd1 (Darieva et al. 2006). However, thisproblem was recently addressed by the report that at leasta subset of the Clb2 cluster genes (including CLB2) can bepartially activated, even in the complete absence of B-typecyclins (Orlando et al. 2008). This finding suggests a mech-anism by which the feedback loop can be activated in theabsence of phosphorylation by Clb2.

In addition, phosphorylation of Ndd1 by Pkc1 (proteinkinase C) has recently been shown to delay its associationwith Mcm1/Fkh2 and, thereby, prevent activation of Clb2cluster genes by Ndd1 until conditions are appropriate(Darieva et al. 2012). Precisely what conditions are moni-tored via Pkc1 is unclear but it seems likely that it is relatedto cell wall synthesis or stress (reviewed in Levin 2011).Finally, it is believed that upon destruction of Ndd1 late inM phase, Mcm1/Fkh2-bound CLB2 cluster promoters arereturned to their repressed state, prepared to initiatea new cell cycle (Loy et al. 1999). It remains unclearwhether Ndd1 destruction alone is sufficient for repressionof CLB2 cluster genes or whether that repression alsoinvolves a specific transcriptional repressor that acts eithervia negative feedback or some other mechanism.

This view of regulation of G2/M cluster genes, althoughsufficient to explain expression of CLB2 and other members,does not explain the regulation of the entire gene cluster.In fact, some genes within the cluster exhibit different

behaviors in the mitotic cyclin mutants, suggesting that morethan one regulatory mechanism exists for triggering transcrip-tion (Orlando et al. 2008). It has recently been recognizedthat other members of the family, including SPO12, despitebeing Mcm1/Fkh2 targets, are subject to an additional levelof regulation by the homeodomain-containing repressor pro-tein, Yox1 (Pramila 2002; Darieva et al. 2010). Yox1 waspreviously recognized as an important repressor in the regu-lation of the M/G1 gene cluster (see below). However, inaddition to having a consensus DNA-binding site for Fkh2downstream of that for Mcm1, some G2/M genes havea Yox1-binding site upstream, suggesting that Yox1 also playsa role in their transcriptional regulation (Darieva et al. 2010).In fact, occupancy of that Yox1-binding site precludes thebinding of Fkh2. Consequently, Yox1 represses those geneshaving a Yox1-binding site in a manner similar to its effect onmembers of the M/G1 gene cluster. The basis for the mutualexclusivity of binding sites for Yox1 and Fkh2 remains unclearbut presumably involves competition between Yox1 and Fkh2for binding sites on Mcm1 and not the geometry of the DNA-binding sites themselves (Boros et al. 2003; Darieva et al.2010). Regardless, this regulatory circuit is clearly effectivein repressing gene expression at those loci and shifting theexpression of those genes later in the cell cycle relative to theother members of the G2/M gene cluster (Pramila 2002).Importantly, this regulation does not affect Clb2 or Cdc5,which act early during the transcriptional burst to promoteactivation via positive feedback. This is similar to the“feedback first” phenomenon observed earlier in the cycle,wherein Cln1 and Cln2, encoded by the earliest expressedgenes of the G1/S cluster, feedback to promote SBF- andMBF-regulated transcription.

The M/G1 gene cluster

As cells exit M phase and transition into G1 phase of a newcell cycle, cells activate four families of genes, distinguish-able based upon either the function of their products or thetranscription factors that regulate them. Together theycomprise the M/G1 gene cluster. That cluster includes genesencoding the cyclin Cln3 and the Swi4 transcription factor,two proteins responsible for promoting G1/S transcription(SWI4 is also activated by MBF; see above). Components ofthe prereplication complex that prepare cells to initiate DNAreplication during S phase, elements of the mating responsepathway, and genes of the PHO regulon that are required formobilization of phosphate are also members of that cluster(Haber 2012; Ljungdahl and Daignan-Fornier 2012).

The Mcm1 gene cluster

The largest family of genes in the M/G1 gene cluster, like theG2/M cluster, is regulated by Mcm1 but, in this case, with-out the involvement of Ndd1 (Zhu et al. 2000). Mcm1 isbound to the promoters of target genes, including Cln3,Swi4, and seven members of the prereplicative complex(Cdc6 and Mcm2–7) (Spellman et al. 1998), via an elementreferred to as an early cell-cycle box (ECB) (McInerny et al.










































































1997; MacKay et al. 2001). Mcm1 is regulated by one of twotranscriptional repressors, the homeodomain proteins Yox1or Yhp1, that hold expression of this cluster of genes incheck outside of the M/G1 interval by binding directly toMcm1 and a DNA element that flanks the ECB (Mai et al.2002; Pramila 2002) (Figure 4). Their interaction with bothMcm1 and DNA is required to stabilize the repressive com-plex (Darieva et al. 2010). Consistent with the need to re-press M/G1 cluster expression from G1 through M phase,YOX1 is expressed as a G1/S cluster gene during G1 andYHP1 somewhat later, perhaps as a consequence of the pres-ence of Fkh2 sites in its promoter (Spellman et al. 1998;Pramila 2002). Expression of Mcm1 target genes does notoccur until the Yox1 and Yhp1 repressor proteins are elim-inated, presumably by APC-dependent ubiquitylation, dur-ing late M phase (Pramila 2002). Whereas Yox1 appears torepress gene expression at some G2/M cluster promoters bydisplacing Fkh2-Ndd1, no similar mechanism is known forM/G1 cluster genes (Darieva et al. 2010). Nevertheless,renewed accumulation of Yox1 and Yhp1 at the beginningof the next cell cycle certainly contributes to M/G1 generepression.

The Sic1 gene cluster

The second family of M/G1 cluster genes, often referred toas the Sic1 cluster, is regulated by the paralogous transcrip-tion factors Ace2 and Swi5 (Dohrmann et al. 1992, 1996;McBride et al. 1999; Laabs et al. 2003) (Figure 4). Thesefactors bind identical DNA sequences via zinc finger domains

in vitro but regulate groups of genes in vivo that are onlypartially overlapping. CTS1 is a classical Ace2-regulatedgene encoding chitinase, which is expressed only in daugh-ter cells and mediates cell separation following mitosis(reviewed in Bi and Park 2012). In contrast, the best-studiedSwi5 target, HO, is expressed only in mother cells (reviewedin Haber 2012). Both factors participate in the activation ofSIC1, encoding a Clb-specific CDK inhibitor for which thecluster is named. Sic1 is expressed very late in M phase,where it contributes to the inhibition of Clb/CDK activityduring exit from mitosis. Recent analyses suggest that theSIC1 transcription occurs primarily in daughter cells duringG1 phase (Guo et al. 2013), where it prevents prematureinitiation of DNA replication (reviewed in Enserink andKolodner 2010). Consequently, regulating the activity ofSwi5 and Ace2 and their partitioning between mother anddaughter cells is critical for proper cell-cycle regulation.

The differential regulation of Sic1 cluster genes by paral-ogous transcription factors that not only bind to the sameDNA-binding site in vitro but, in many cases, bind to thesame genes in vivo poses a conundrum (Dohrmann et al.1992, 1996). By evaluating the requirement for Ace2 andSwi5 for expression of M/G1 genes and correlating that withthe binding of Ace2 and Swi5 to the promoters of thosegenes, it became clear that the promoters of genes regulatedsolely by Swi5 are bound only by Swi5, whereas those reg-ulated by both factors or by Ace2 alone bind both factors(Voth et al. 2007). Why, then, are some genes that bind bothfactors only dependent upon Ace2 for expression, whereas

Figure 4 Transcriptional regulation of the M/G1 geneclusters. The regulatory circuitry of the M/G1 genecluster is depicted, including the Mcm1 cluster (top)and the Sic1 cluster (bottom). Mcm1 binds theMcm1 cluster promoters along with the repressorsYox1 and Yhp1, repressing those genes prior to Mphase. As cells exit M phase, Yox1 and Yhp1 are tar-geted for degradation by the APCCdh1 ubiquitin ligaseand Mcm1 targets are activated. Expression of thosetwo repressors as products of G1/S genes restores re-pression of Mcm1 cluster genes as cells exit G1 phase.Sic1 cluster genes are regulated by the Swi5 and Ace2transcription factors, which share the same bindingsequence in target gene promoters. Swi5 and Ace2are both excluded from the nucleus when Clb/Cdk1is active. As cells progress through mitosis, Clb proteinsare degraded and the Cdc14 phosphatase is activated,promoting accumulation of the transcription factors innuclei. Whereas Swi5 accumulates in both mother anddaughter nuclei, Ace2 accumulates only in daughternuclei. One class of genes characterized by SIC1 isexpressed in both mothers and daughters. At thosegenes, either Swi5 or Ace2 can bind and activate. An-other class of genes, characterized by CTS1, expressedonly in daughters, can bind both Swi5 and Ace2, butSwi5 binds at those promoters along with Fkh1 or

Fkh2, which act as “antiactivators.” Consequently, in mother cells, the binding of Swi5 fails to activate transcription. In daughter nuclei, the samepromoters are activated by Ace2, despite the presence of Swi5. Whether Swi5/Fkh protein complexes also bind to those promoters but fail to activate isnot known. Swi5-specific and daughter-specific genes are not depicted. Finally, all of these genes are inactivated as cells pass START as a consequenceof the nuclear degradation of Swi5 and the nuclear export of Ace2. The color of subunits and arrows is as in Figure 1.







































others can be activated by either one? This conundrum is, inpart, resolved by the finding that promoters regulated onlyby Ace2 bind Swi5 along with Fkh1 and Fkh2, which act as“antiactivators” for Swi5 at those promoters (Figure 4). Incontrast, genes that are activated by both factors or only bySwi5, including SIC1, CDC6, and HO, lack the Fkh1/2 bind-ing site. Consequently, it appears that Swi5 binds to thepromoters of all Sic1 cluster genes but only activates thoseat which Fkh1 or Fkh2 is not bound. Whether the Swi5/Fkh1/2 binds but fails to activate the same promoters indaughters, as well as in mothers, is unclear.

Another aspect of the differential regulation of genes bythese transcription factors involves the asymmetric distribu-tion of Ace2 to daughter nuclei following mitosis. Thatasymmetry is a consequence of the failure to retain Ace2in mother cell nuclei following mitosis (Colman-Lerneret al. 2001; Weiss et al. 2002; Mazanka and Weiss 2010).Daughter-specific retention of Ace2 is regulated, at least inpart, by the NDR/LATS family protein kinase Cbk1, a com-ponent of the so-called RAM complex (regulation of Ace2and morphogenesis). A deconvolution algorithm developedby Guo and colleagues revealed the existence of a largegroup of genes subject to the daughter-specific transcriptionprogram (Guo et al. 2013). In addition to those genes thathad been reported previously (Colman-Lerner et al. 2001; DiTalia et al. 2009), the high temporal resolution afforded bythe deconvolution approach led to the identification of atleast four temporally distinct waves of daughter-specifictranscripts and identified several new daughter-specificgenes. Transcription factor enrichment analyses suggestthe earliest waves are regulated by Swi5 and Ace2, whereasthe later waves appear to be regulated by Mcm1. Theseanalyses also indicate a role for several other transcriptionfactors in that regulation, including Sok2, Phd1, Ste12,Cin5, Yap6, and Tec1 (Guo et al. 2013).

One consequence of asymmetry is that chitinase, encodedby the Ace2 target CTS1, is daughter specific. This pattern ofexpression is responsible for the characteristic retention ofthe bud scar on mother cells, but not daughter cells, sub-sequent to cytokinesis. Although Ace2 and Swi5 can bothbind to the CTS1 promoter, when Swi5 is bound, it fails toactivate due to the binding of Fkh1/2 (Voth et al. 2007)(Figure 4). Only when Ace2 is bound, can it activateCTS1. Another consequence of the daughter-specific locali-zation of Ace2 is the characteristic delay in budding indaughter cells relative to mother cells. This is, in part, a con-sequence of the Ace2-dependent delay in CLN3 expressionand, thereby, delayed activation of G1/S transcription indaughter cells (Laabs et al. 2003; Di Talia et al. 2009). Al-though this impacts the range of cell size at which daughtercells bud under various nutrient conditions, it does not ex-plain the phenomenon of cell size control (reviewed byTurner et al. 2012).

Finally, the Swi5 transcription factor exhibits specificityfor binding to some cell-cycle-regulated promoters (e.g.,CDC6 and HO), a property that is apparently not shared

by Ace2 (Voth et al. 2007). Swi5 binding to the HO pro-moter, which occurs late in M phase, licenses that promoterfor activation only in mother cells during the subsequent G1phase. Similar activation does not occur in daughter cellsdue to the daughter-specific localization of the transcrip-tional repressor Ash1, which prevents Swi5 binding (Bobolaet al. 1996; Sil and Herskowitz 1996; Cosma 2004; Shenet al. 2009). Thus, the selective binding of Swi5 at pro-moters in the absence of the Fkh1 and Fkh2 transcriptionfactors defines genes that are uniquely regulated by Swi5.

Clb/CDK dependent phosphorylation is another criticalcontributor to Ace2 and Swi5 regulation during the cellcycle because it impacts their nuclear localization (Mollet al. 1992) (Figure 4). Phosphorylation within the nuclearlocalization sequences (NLSs) of Swi5 and Ace2 from Sphase through M phase, catalyzed by increasingly activeCDK, prevents transcriptional activation by preventing theirentry into the nucleus. Only when B-type cyclins aredestroyed during M phase and the Cdc14 phosphatase isactivated upon exit from mitosis, are their NLS motifsdephosphorylated, allowing entry into the nucleus and pro-moter binding (Visintin et al. 1998). Thus, activation ofSwi5 and Ace2 targets in early G1 is dependent on exit frommitosis. The asymmetric loss of Ace2 from mother cell nucleidepends upon its nuclear export sequence (NES), a motifthat is not shared with Swi5 (Sbia et al. 2008). Inactivationof Ace2 by nuclear exclusion is necessary because it is stablethroughout the cell cycle, whereas the bulk of Swi5 is ap-parently rapidly degraded upon localization to the nucleus(Tebb et al. 1993). Together, these mechanisms account forthe selective inactivation of the M/G1 transcription factorsin mother and daughter cells.

The MAT gene cluster

Another cluster of M/G1 genes that is expressed differen-tially based upon cell type is the so-called MAT cluster ofhaploid-specific genes regulated by the Ste12 transcriptionfactor (Oehlen et al. 1996; Spellman et al. 1998). AlthoughSte12 participates in multiple transcription factor com-plexes, some as a heterodimer with Tec1 or Mcm1, regula-tion of the MAT gene cluster by mating pheromone occursvia binding to pheromone response elements (PREs) in thetarget promoters as a homodimer (Hwang-Shum et al.1991). Genes in the MAT cluster encode many proteins in-volved in the response to mating pheromone, includingthe Fus3 MAP kinase and Far1, both of which are criticalfor induction of G1 phase cell-cycle arrest (Roberts et al.2000). Whereas Ste12-regulated transcription is stronglyinduced in response to mating pheromone signaling, its tar-get genes are expressed at a basal level in haploid cells ofboth mating types (Oehlen et al. 1996; Spellman et al.1998).

Both basal and pheromone-induced expression of Ste12-regulated genes are restricted to the pre-START intervalof G1 phase (reviewed by Dohlman and Thorner 2001;Bardwell 2005). Confining mating to G1 phase prevents











































































inappropriate ploidy of the mating cells and proper organi-zation of the apparatus for nuclear fusion. Transcriptionaland post-transcriptional controls contribute to the G1 phaserestriction. In particular, Far1, an inhibitor of G1 cyclin-associated CDK and regulator of cell polarity, is expressedas a target of both Mcm1 and Ste12 and, thereby, accumu-lates from M phase through early G1 phase (Oehlen et al.1996). Fus3 activates both Ste12 and Far1 by phosphorylation,thereby, promoting gene expression and CDK inhibition.Conversely, pheromone signaling is inhibited and Far1 istargeted for destruction by ubiquitin-mediated proteolysisby G1 cyclin/CDK, which accumulates as cells pass START(McKinney et al. 1993; McKinney and Cross 1995; Henchozet al. 1997). Cln/CDK also attenuates mating pheromonesignal transduction by phosphorylating the MAPK cascadescaffold protein Ste5 (Oehlen and Cross 1994; Strickfadenet al. 2007; Bhaduri and Pryciak 2011). Because the sub-stantial activation of Far1 by Fus3-dependent phosphory-lation occurs only when the mating pheromone-signalingpathway is induced by mating factor, stable arrest resultingfrom Far1 inhibition of Cln/CDK does not occur in the absenceof pheromone. Instead, under those conditions, Cln/CDKattenuates Ste12-dependent gene expression and promotescell proliferation. Consequently, cycling cells are only sensitiveto mating pheromone-induced cell-cycle arrest during earlyG1 phase.

The mechanism of periodic expression of Ste12-dependentgenes in cycling cells is associated with basal signalingthrough the pheromone response pathway leading to basalactivation of Ste12. Like pheromone-inducible expression,basal expression of the MAT cluster genes occurs only duringG1 phase because the pathway is repressed by CDK, whichremains active from START until the Clb/CDK inhibitor Sic1is expressed as cells exit M phase. Although basal pheromonesignaling is insufficient to cause cell cycle arrest, it is likelyresponsible for the enhanced rate of cell proliferation ob-served in pheromone signaling mutants even in the absenceof pheromone (Lang et al. 2009).

The stability of both the START transition and phero-mone arrest creates a tension between the mating pathwayand cell proliferation. Both the stability and reversibility ofthose pathways is a consequence of the architecture of thecell-cycle- and pheromone-regulated transcriptional net-works (Doncic et al. 2011; Doncic and Skotheim 2013).Pheromone arrest is stable due to pheromone-induced ex-pression of Far1, Fus3, and other factors in the pathway. Adrop in pheromone leads rapidly to mitotic growth due bothto the rapid decay of signaling through the pathway and theassociated activation of Cln/CDK activity resulting from thedecreased expression of Far1. Cln/CDK activity promotesproliferation by attenuating the basal activity of the phero-mone pathway and thereby Ste12-dependent transcriptionby promoting Far1 proteolysis and by activating G1/S tran-scription via positive feedback. Together, these responses flipthe bistable switch governing START (reviewed by Ferrell2011).

Emerging Views of Cell-Cycle-RegulatedTranscription

The broad outlines of cell-cycle-regulated gene expressionhave been worked out for almost a decade. Yet, as can beseen from our discussion of the regulation of the cell-cyclegene clusters, substantial fleshing out and numerous revi-sions of that view have led to a much greater understanding.In the following vignettes, we introduce some of the recentdevelopments in the general area of cell-cycle-regulatedgene expression and a number of specific issues that are justbeginning to influence our understanding of this importantproblem. Important topics impacting our understanding ofcell-cycle-regulated gene expression, including the influenceof nutrients, pheromones, stress responses, cell size, andother factors, are discussed in other Reviews and Yeastbookarticles previously published and to come.

CDK’s role in cell-cycle-regulated transcriptional program

CDKs are widely believed to be the core mechanismcontrolling cell-cycle-regulated transcription. Support forthe idea that periodic transcription occurs as a consequenceof periodic cyclin/Cdk activity came from experiments inwhich specific groups of transcripts were found to bemisregulated in cells mutated for specific cyclins (Dirickand Nasmyth 1991; Amon et al. 1993; Stuart and Wittenberg1994; Koch et al. 1996). An attractive model was proposed byNasmyth and colleagues in which successive waves of cyclinexpression drive progression through the cell cycle as well ascontrol periodic transcription (Amon et al. 1993; Koch et al.1996). When cells commit to a new cell cycle, Cln3 activatesa wave of transcription peaking in late G1. Clb2 then down-regulates a subset of those transcripts and simultaneouslypromotes the accumulation of a later wave of transcriptspeaking in G2/M. A subsequent wave of transcripts peakingin late M/G1 is triggered by the destruction of Clb2 as cellsexit mitosis.

Textbook models suggest that the temporal program oftranscription is regulated by an interconnected regulatorynetwork containing both CDKs and transcription factors. Inthese models, CDKs act as the key regulatory componentsthat control the activity of cell-cycle transcription factors(Morgan 2007). There is good motivation for proposing thisregulatory relationship, as many of the 10–15 transcriptionfactors that are commonly placed in the cell-cycle networkare phosphorylated by CDKs. CDK-mediated phosphoryla-tion regulates both nuclear localization and the formationof transcription factor complexes at promoters (Ho et al.1999; Costanzo et al. 2004; de Bruin et al. 2004). Conse-quently, they may serve as critical molecular switches forthe interconnected network. In particular, G1-cyclin/Cdksactivate SBF and MBF through phosphorylation of Whi5(Costanzo et al. 2004; de Bruin et al. 2004) and, perhaps,Swi6 (Sidorova et al. 1995), triggering accumulation ofmany late G1 transcripts; SBF is then inhibited by the mi-totic B-cyclin/Cdk complexes (Amon et al. 1993; Koch et al.



























1996); B-cyclin/Cdks also phosphorylate and activate Ndd1and Fkh2 to promote complex formation and trigger accu-mulation of many G2/M transcripts (Pic-Taylor et al. 2004);B-cyclins inhibit nuclear localization of Swi5 (Moll et al.1991) and Ace2 (O’Conallain et al. 1999) during mitosisand, it is likely that B-cyclin destruction at the end of mitosistriggers late M/early G1 transcript accumulation (Breedenand Nasmyth 1987; Knapp et al. 1996; Kovacech et al. 1996;Toyn et al. 1997). Thus, successive waves of transcriptioncould arise due to changing cyclin/CDK activities that actpost-translationally to turn key factors on and off.

By contrast, in network models arising from genomicapproaches, the temporal program of transcription can ariselargely independent of CDK regulation (Simon et al. 2001;Lee et al. 2002; Pramila et al. 2006). In these models, suc-cessive waves of transcription emerge from oscillations intranscription factor abundance, rather than regulation oftranscription factor activity. In a simplified model, transcrip-tion factors activate the expression of gene clusters that in-clude genes encoding other transcription factors. Thus, theprogram could be controlled intrinsically by a network ofserially activated transcription factors.

At the time when the first network models were pro-posed, the CDK and network models seemed incompatible.Both existing models had widely acknowledged gaps. Thenetwork models did not incorporate much of the post-translational regulation of transcription factors by CDKcomplexes that had been established by directed studies ofspecific transcription factors. However, although the CDKmodel was supported by rigorous experiments interrogatingsmall subsets of target genes, this model had not been testedon a genome-wide scale.

The extent to which the cell-cycle-regulated transcrip-tional program was controlled by oscillations of specificcyclin/CDK complexes or by a network of serially activatedtranscription factors was examined directly by Orlando et al.(2008). Using budding yeast cells in which all six B-typecyclin genes had been inactivated (clb1,2,3,4,5,6Δ), theyshowed that nearly 70% of the cell-cycle-regulated genescontinued to be expressed at the proper time. In addition,the program of transcription repeated in a second cycle, de-spite the fact that the cells were arrested at the G1/S borderby all conventional cell cycle measures. In a later study,similar results were observed in cdc28-4 cells at restrictivetemperature, although the number and amplitude of theperiodic genes was substantially reduced (Simmons Kovacset al. 2012).

Taken together, these results suggested that intercon-nected networks of transcription factors could drive most ofthe periodic transcriptional program in the absence of Clb/CDK activity and function as an autonomous oscillator(Orlando et al. 2008; Simmons Kovacs et al. 2008, 2012).Nonetheless, while many transcripts continue to oscillate inthe cells depleted for CDK activities, there were clear alter-ations in the behaviors of others. Thus, post-transcriptionalregulation by CDKs may act more like a rheostat in the

control of periodic transcription rather than simply function-ing as an “on–off” switch. In general, as CDK activities de-creased, the number of identifiable periodic genes alsodecreased (Simmons Kovacs et al. 2012). Furthermore, de-creasing CDK activity was associated with diminished tran-script amplitudes as well as increasing oscillatory periods.The regulatory mechanisms responsible for the loss of am-plitude and increased period may be positive feedback loopsin which cyclin expression is promoted by a transcriptionfactor that is then phosphorylated by the cyclin/CDK complex,which increases the transcription factor’s activity (SimmonsKovacs et al. 2012). This regulatory network motif affects bothG1 (SBF and Cln2; see section titled “Activation of G1/S tran-scription factors”) (Cross and Tinkelenberg 1991; Dirick andNasmyth 1991; Skotheim et al. 2008) and G2/M gene clusters(Pic-Taylor et al. 2004).

Although CDKs may not be required as a core mechanismfor generating the temporally ordered program of transcrip-tion during the cell cycle, they clearly play a role inmaintaining the robust character of the program. Throughpositive feedback loops, CDKs provide the basis for impor-tant switch-like behavior observed at G1/S and G2/Mtransitions. Furthermore, the inhibition of Swi5/Ace2-regulatedgenes by mitotic cyclin/CDK ensures that the initiation of a newprogram of G1 transcription is dependent on exit from mitosisand the subsequent loss of Clb2/CDK activity. Thus, transcrip-tion factor networks and CDKs collaborate to coordinate theexecution of the transcriptional program with the cell cycle(Simmons Kovacs et al. 2012).

Checkpoint control of cell-cycle-regulated transcription

Cell-cycle checkpoints maintain the appropriate order ofcell-cycle progression by placing prerequisites on progres-sion into a particular cell-cycle phase or on the execution ofphase-specific events (Hartwell et al. 1994). For example,a checkpoint mechanism restricts progress into M phasewhen replication is incomplete, either due to ongoing repli-cation or stalling of DNA replication forks. Similarly, anothercheckpoint mechanism restricts mitotic spindle elongationwhen chromosomes are not appropriately attached to themitotic spindle. To elicit such responses, checkpoint path-ways must impinge upon cell-cycle-regulatory mechanisms(Elledge 1996). Furthermore, they must allow the cell timeto correct the problem yet retain the capacity to proceedonce that problem is corrected.

The observation that the cell-cycle-regulated transcrip-tional program can proceed (albeit less robustly) when CDKactivity is lost suggests that checkpoint arrest, which blocksCDK cycling, might allow the transcription program to bedecoupled from cell-cycle events. This problem would beresolved if checkpoint pathways directly regulate the tran-scription network to maintain synchrony between transcrip-tion and cell-cycle progression. Some evidence suggests thatcheckpoints do, in fact, regulate transcription to enforcerobust cell-cycle arrest (Gardner et al. 1999; Chu et al.2007). Several global transcript-profiling studies indicate












that the DNA replication and DNA damage checkpoint path-ways trigger a checkpoint-dependent transcription programcontrolled by Dun1 kinase (Figure 5) (reviewed in Fu et al.2008). The Dun1 kinase induces a well-described transcrip-tional response involving genes (such as RNR2 and RNR3)that mediate recovery from DNA damage or blocks to DNAreplication (Elledge et al. 1993; Zhou and Elledge 1993;Allen et al. 1994; Gasch et al. 2001). Dun1 activity is stim-ulated by Rad53. Crt1, a repressor of DNA damage responsegenes, is phosphorylated by Dun1 upon checkpoint activa-tion and released from the DNA, thereby, allowing transcrip-tional activators access to promoter regions of Crt1-targetgenes (Huang et al. 1998). In addition to its role in recoveryfrom checkpoint stresses, genetic evidence suggests thatDun1 also plays a role in enforcing a robust checkpointarrest (Gardner et al. 1999). Although some of these studieshint at regulation of cell-cycle genes (Gasch et al. 2001),a specific role for Dun1-regulated transcription in cell-cyclearrest has yet to be described.

In addition to activating the Dun1 pathway, Rad53also directly phosphorylates Nrm1, the repressor of MBF-regulated transcription, preventing its association with MBFat target promoters (Figure 5) (de Oliveira et al. 2012; Travesaet al. 2012). Consequently, MBF targets, which would nor-mally be repressed when DNA replication is underway, re-main active, leading to the accumulation of their products.Because many of those targets are required for DNA replica-tion and repair, this positions cells to correct the problemsleading to induction of the checkpoint and to progressthrough S phase once that problem has been eliminated.A similar mechanism is observed in fission yeast where theRad53 ortholog, Cds1, phosphorylates both Nrm1 and Cdc10,the S. pombe ortholog of Swi6. The result is persistent acti-vation of transcription by MBF, the sole G1/S transcriptionfactor in that organism (de Bruin et al. 2008b; Dutta et al.2008; Aligianni et al. 2009; Gómez-Escoda et al. 2011). Finally,Rad53 also phosphorylates Swi6 in S. cerevisiae (Sidorova andBreeden 1997). Although the importance of that phosphoryla-tion has not been established, it has been suggested that itdelays expression of CLN1 and CLN2 in G1 cells responding toDNA damage.

The phosphorylation of Nrm1 by Rad53 specificallyaffects MBF, allowing SBF to be repressed, like it is in un-treated cells (Travesa et al. 2012). Consequently, repressionof SBF targets by Clb2/CDK occurs normally because Clb2 isstabilized in response to down-regulation of APCCdc20 activ-ity by the checkpoint. Thus, inappropriate accumulationtranscripts associated with morphogenesis and other pro-cesses promoted by targets of SBF is prevented. The differ-ential regulation of the SBF and MBF transcription factors,and thereby their specific targets by the DNA replicationcheckpoint, provides one clear rationale for the existenceof independent transcription factors regulating the twogroups of G1/S cluster genes (de Oliveira et al. 2012;Smolka et al. 2012; Travesa et al. 2012). This dual regula-tion is dispensable in fission yeast where MBF, the sole G1/S

transcription factor, is also subject to checkpoint regulationvia phosphorylation of the transcriptional repressor Nrm1(de Bruin et al. 2006, 2008b; Dutta et al. 2008). In thatsystem, proliferation does not occur via budding and, con-sequently, morphogenesis is not regulated during G1 phase.

In addition to providing a mechanism by which thecheckpoint can activate only a subset of G1/S genes, thedifferential regulation of SBF and MBF by the DNA re-plication checkpoint also poses a conundrum. SBF- andMBF-regulated genes can be repressed by the accumulationof Clb/CDK activity during the unperturbed cell cycle (Amonet al. 1993; de Bruin et al. 2006). Why, then, is SBF re-pressed normally during S phase in cells responding to thecheckpoint signal, whereas MBF remains unaffected? Thisobservation suggests that a mechanism invoked by thecheckpoint signal allows MBF to evade the repressive activ-ity of Clb/CDK. Although the mechanism of that evasion isunknown, it is possible that the checkpoint-induced phos-phorylation of Swi6 by Rad53 makes MBF refractile to inhi-bition by Clb/CDK (Sidorova and Breeden 1997). Whateverthe mechanism, this difference in their response to Clb/CDKprovides another example of differential regulation of SBFand MBF during the cell cycle.

Although, the differential regulation of SBF and MBFby the DNA replication checkpoint provides a seemingly

Figure 5 Control of MBF-regulated transcription by the DNA replicationcheckpoint. The influence of the DNA replication checkpoint on the MBFtranscriptional regulatory circuit is depicted. MBF-regulated promoters arenormally repressed as cells progress into S phase as a consequence of theexpression of the NRM1 gene and the accumulation of its product, theMBF-specific repressor, Nrm1 (dashed line, graph). When the DNA repli-cation checkpoint is activated as a consequence of stalling of DNA rep-lication forks, the DNA replication checkpoint pathway leads ultimately tothe activation of the Rad53 protein kinase, which can phosphorylate thedownstream protein kinase Dun1 and the Nrm1 protein. Phosphorylationactivates Dun1, which can then phosphorylate the transcriptional repres-sor Crt1, leading to the activation of DNA damage response (DDR) genes.Phosphorylation of Nrm1 inactivates it as a transcriptional repressor, lead-ing to persistent activation of MBF target genes (solid line, graph) tosupport repair of DNA and restoration of DNA replication. The color ofsubunits and arrows is as in Figure 1.



























straightforward rationale for the involvement of two tran-scription factors in G1/S transcription, several observationssuggest that the situation is really much more complex. First,many G1/S genes have multiple binding sites for bothtranscription factors that appear to be functional in vivo (Iyeret al. 2001; Horak et al. 2002; Bean et al. 2005; de Bruinet al. 2006). Second, the expression of many G1/S genes isimpacted by inactivation of either of the two transcriptionfactors, suggesting that both factors can contribute to theirregulation (Bean et al. 2005; de Bruin et al. 2006). Finally,some genes undergo a switch from binding SBF to bindingMBF during the course of the G1/S transcriptional burst asa consequence of the presence overlapping binding sites forthe two transcription factors (de Oliveira et al. 2012). Thosegenes seem to be activated by SBF, dependent upon inacti-vation of Whi5, and repressed by MBF, via the activity ofNrm1. One rationale that has been proposed for this regula-tory switch is to enable inducibility of those genes by theDNA replication checkpoint and to ensure that the genesare not expressed inappropriately if MBF is inactivated bymutation or other insult (reviewed by Smolka et al. 2012).Regardless of the reason, it is clear that the regulation of G1/Sgenes by the combined activities of SBF and MBF providesopportunities for more diverse modes of regulation than doesthe involvement of either transcription factor on its own.

We are just beginning to understand the complex mech-anisms by which checkpoint effector pathways control cell-cycle-regulated transcription of the G1/S cluster. It alsoseems likely that checkpoints may regulate other clusters ofperiodic genes. Thus, it is likely that checkpoint control of cell-cycle-regulated transcription will be a rich area of futureinvestigation.

Yeast metabolic cycle control of cell cycle and transcription

It has been recognized for some time that metabolicprocesses in yeast are periodically regulated (Futcher 2006and references within) and may be coordinated with the cellcycle (e.g., Creanor 1978; Novak and Mitchison 1986,1990). When budding yeast cells are grown in continuousculture conditions at appropriate densities and growth rates,the cells synchronize in robust metabolic cycles that can bemonitored by periodic changes in dissolved oxygen. Thereported periods of the YMC can vary substantially (Kleveczet al. 2004; Tu et al. 2005; Slavov and Botstein 2011), andseem to be linked to growth rate (Brauer et al. 2008; Slavovand Botstein 2011). The YMC regulates a large transcrip-tional program and appears to be coordinated with the cellcycle under slow growth conditions.

The YMC-regulated transcriptional program: By samplingcontinuous cultures of metabolically synchronous popula-tions of budding yeast over time, investigators have identi-fied oscillations in gene expression that are coincident withthe periodicity of the YMC. In one of the initial reports, cellswere sampled over the course of three metabolic cycles andby microarray analyses found that the bulk of the genome

exhibited oscillations in transcript abundance (Klevecz et al.2004). The amplitudes of these oscillations were modest,with peak-to-trough ratios of �2. In a subsequent study,the McKnight group (Tu et al. 2005) also sampled metabol-ically synchronous cells growing in a chemostat over threemetabolic cycles. Their findings suggested approximatelyhalf of the genes in the genome exhibited transcript oscilla-tions coincident with the YMC. The amplitudes of thesetranscript oscillations were in the range of 10-fold (Tuet al. 2005), substantially higher than observed by Kleveczand colleagues.

Using clustering approaches, both groups were able toidentify sets of transcripts that appeared to be coregulated,and many of the coregulated genes shared related functions(Klevecz et al. 2004; Tu et al. 2005). Analysis of the datafrom Tu and colleagues revealed three “super clusters” ofgenes that were expressed in relatively discrete temporalintervals that correspond to three phases of the YMC: oxida-tive (Ox), reductive building (R/B), and reductive charging(R/C) (Tu et al. 2005). Within these clusters of oscillatingtranscripts were several cell-cycle-regulated genes, suggestingthat the YMC transcriptional program and the cell-cycle tran-scriptional program are interconnected (Klevecz et al. 2004;Tu et al. 2005).

Botstein and colleagues approached YMC control oftranscription from a different angle. They used continuousculture conditions to investigate the transcriptional responseof yeast to a wide variety of growth limiting conditions inwhich cells were starved for a variety of different nutrients(Brauer et al. 2008). Under this broad range of growth con-ditions, they found that the expression of approximatelyone-fourth of the genes in the genome is linearly correlated(either positively or negatively) with growth rate. Surpris-ingly, this set of growth rate responsive (GRR) genesexhibited substantial overlap with a set of genes previouslyidentified as environmental stress response (ESR) genes(Gasch et al. 2000), suggesting a functional relationshipbetween the gene sets. Genes that exhibited positive corre-lation with growth rate tended to be genes inhibited bystress, while genes that were down-regulated in fast growthconditions were often activated by stress (Brauer et al.2008). As well, there was considerable overlap betweenthe GRR gene set and the YMC responsive gene sets identi-fied by Tu et al. (2005) (Brauer et al. 2008). In a recentstudy using continuous culture conditions and a variety ofnutritional limitations, Slavov and Botstein (2011) demon-strated that all of the GRR transcripts were, in fact, peri-odic during the YMC. Strikingly, negatively and positivelycorrelated GRR transcripts oscillate in opposing YMCphases. Taken together, these findings suggest that themechanisms controlling oscillation in transcript abundancemay be integrating signals from stress, growth rate, YMC,and cell cycle.

YMC control of cell cycle: The identification of cell-cycle-regulated genes as part of the YMC transcriptional program




suggested that these genes were regulated by multipleconditions or that the YMC and the cell cycle may becoordinated (Klevecz et al. 2004; Tu et al. 2005). DNA con-tent and budding analyses both suggest that the YMC andthe cell cycle are indeed synchronized in continuous cultureconditions (Klevecz et al. 2004; Tu et al. 2005). Both groupssuggest that only a fraction of cells in the population [Tuet al. (2005) estimates 50%] enter the cell cycle during eachmetabolic cycle. In addition, it appeared that the YMCphases might be coherent with cell-cycle phases, with Sphase being restricted from occurring during the Ox phaseof the YMC (Klevecz et al. 2004; Tu et al. 2005).

Both groups proposed that the YMC gates the cell cycle sothat the execution of S phase is restricted to the reductivephase and that this restriction is important for preventingoxidative damage of DNA. Supporting evidence for thishypothesis was reported in a subsequent study from theMcKnight group (Chen et al. 2007). In mutant cells where Sphase was allowed to proceed during the Ox phase of theYMC, they observed an increase in the spontaneous muta-tion rates. However, the mutant genes found to disrupt thephase coherence of the YMC and the cell cycle were them-selves genes controlling the cell division cycle (Chen et al.2007), suggesting the increase in mutation rate observed inthese mutants could be related to improper execution ofcell-cycle functions rather than replicating DNA during theoxidative phase of YMC.

In a recent study, the connection between the cell cycleand the YMC was directly tested (Slavov and Botstein2011). By varying nutrient availability, and thereby alteringthe growth rate of continuous cultures, it was demonstratedthat the restriction of S phase from the Ox phase of the YMCwas dependent on the growth rate. Under specific growthrate conditions, synchrony between the cell cycle and YMCcould be maintained, but execution of S phase occurredduring the Ox phase of the YMC (Slavov and Botstein2011). Interestingly, the cell cycle mutants that disruptedthe coordination between S phase with the reductive phasesof the YMC were also the mutants that exhibited the mostsubstantial alterations in the growth rate and YMC period(Chen et al. 2007).

One interpretation of these data is that the duration ofthe cell division cycle influences the YMC period. Consistentwith this possibility, it has been proposed that the coherenceof cell-cycle phases with YMC phases may reflect the storageof carbohydrates under slow-growth conditions and a sud-den “burn” of these carbohydrates during G1 phase (Futcher2006). This rapid utilization of carbohydrates provides en-ergy and drives rapid protein synthetic rates, providinga “finishing kick” for cells to pass through START and enterS phase (Futcher 2006). This coupling of cell-cycle and YMCprograms seems to be restricted to conditions of slowgrowth, and oscillation of YMC or GRR transcripts has notbeen observed in microarray studies performed using rap-idly growing cell populations synchronized in the cell cycle(Cho et al. 1998; Spellman et al. 1998; Pramila et al. 2006;

Brauer et al. 2008; Orlando et al. 2008). This may be be-cause the rapidly growing cells have abundant glucose anddo not store substantial amounts of carbohydrates.

Many open questions remain regarding how and whybudding yeast cells coordinate the YMC with the cell-cycleprogram. The interaction between the YMC and the cellcycle is likely to be more complex than a simple gating of thecell cycle by a metabolic oscillator. Although the mecha-nisms driving the YMC remain undiscovered, like the cellcycle, it is clearly capable of producing oscillations in anabundance of transcripts on a genome-level scale. Theproposal that the cell-cycle period may be controlled bya transcriptional network oscillator suggests that the YMCand the cell cycle may be coupled via coordinated controlof key transcriptional regulators (Orlando et al. 2008;Simmons Kovacs et al. 2008, 2012).

What is the role of chromatin incell-cycle-regulated transcription?

Transcription takes place in the context of chromatin and, asa consequence, is influenced by it (Lenstra et al. 2011;Rando and Winston 2012). Cell-cycle-regulated genes haveproven useful for understanding both general, and gene-specific contributions of chromatin to the regulation of ex-pression. The specific roles of chromatin in the control ofcell-cycle-regulated gene expression and the relationship be-tween chromatin and the transcription factors that controlthe cell-cycle gene clusters are only beginning to be under-stood. In many cases, the specific vs. general roles of chro-matin in the control of cell-cycle-regulated gene expressionhave yet to be established.

The earliest studies of the contribution of chromatinmodification and remodeling to gene expression and, morespecifically, cell-cycle-regulated gene expression were based,in large part, upon the pioneering studies of the G1/S gene,HO, by the Nasmyth and Herskowitz laboratories (reviewedby Stillman 2013; see Haber 2012). Most of the studies ofthe role of chromatin in the regulation of HO expressionfocused upon licensing for differential expression in mothercells, mediated by an upstream regulatory region known asURS1. However, the balance of the regulatory functions,mediated by URS2, are largely indistinguishable from ex-pression of other SBF target genes (Peterson and Herskowitz1992; Cosma et al. 1999, 2001). Consequently, most of theobservations regarding URS2 can be generalized to CLN2and other G1/S promoters.

Recent study of the CLN2 and HO promoters has focused,in part, upon the role for histone deacetylation by the Rpd3(L) HDAC complex in repression of SBF and MBF genes (deBruin et al. 2008a; Takahata et al. 2009a,b, 2011; Wanget al. 2009). Rpd3(L) recruitment to SBF promoters dependsupon the binding of the Sin3-associated protein Stb1 to pro-moters in a manner that depends upon the SBF-specifictranscriptional repressor Whi5 (Wang et al. 2009; Takahataet al. 2011). At MBF genes, Stb1 binding occurs, but is, bynecessity, Whi5 independent (Costanzo et al. 2003; de Bruin














et al. 2008a; Takahata et al. 2009a). Stb1 binding is accom-panied by the binding of Cdk1, which, via some combinationof phosphorylation of Whi5, Stb1, and the transcriptionfactors, promotes activation of the promoters. Cln/Cdk1-dependent phosphorylation of Whi5 and Stb1 leads to thedissociation and export of Whi5 from the nucleus and thedissociation of Rpd3(L) from promoters. Promoter activa-tion occurs upon the loss of Rpd3(L) binding, presumablyaccompanied by the restoration of histone acetylation. Sim-ilar changes in histone acetylation during G1 and S phasesare observed at both SBF and MBF target promoters. Al-though Rpd3(L) is an important factor in the regulation ofhistone acetylation state and involved in the repression ofmany genes, Stb1 may affect only a small subset of genes,including those in the G1/S gene cluster.

Activation of both CLN2 and HO expression, and likelythe entire G1/S gene cluster, also involves the FACT com-plex, which promotes nucleosome eviction at those pro-moters (Takahata et al. 2009a,b; Wang et al. 2009). FACTacts just prior to, or at the time of, transcriptional activationand is required for G1/S expression. The binding of FACT toboth CLN2 and HO promoters depends upon the Swi6 sub-unit of SBF along with Whi5 and Stb1. The G1/S genesprovide the only example of a promoter-associated role forFACT, which is most often associated with transcriptionalelongation (Rando and Winston 2012). FACT has also beenimplicated in CLN3 transcription, although whether thatoccurs via a direct or indirect mechanism remains obscure(Morillo-Huesca et al. 2010).

At least some of these factors play well-established rolesat genes that are expressed independent of cell-cycleposition, but the extent to which these transcription-factor-dependent mechanisms for chromatin modification andremodeling play specific roles at cell-cycle genes is not yetclear. However, those factors are clearly distinct from themore general role of nucleosome-depleted regions (NDRs)that encompass the SBF-binding sites at the CLN2 promoterand are required for the reliability of cell-cycle-dependenttranscriptional activation (Bai et al. 2010, 2011). Consistentwith that, the formation of NDRs at the CLN2 promoter doesnot require any of the known G1/S-specific transcriptionalregulators. Additionally, histone H3 K79 methylation hasalso been associated with the function of SBF, Whi5, andNrm1, although the relevance of that association to the reg-ulation of G1/S promoters is not clear (Schulze et al. 2009).

There are many studies suggesting a role for chromatin inthe regulation of other cell-cycle-regulated gene clusters,although most are less well studied. For example, Fkh2/Mcm1 promotes recruitment of Rpd3(L) leading to repres-sion of G2/M cluster genes (Veis et al. 2007). Interestingly,in a two-step process, Rpd3(L) dissociates from G2/M genepromoters at the onset of S phase in a manner that dependsupon Cln/CDK and, only then, does the combined activationof Clb2/CDK and Cdc5 promote eviction of a nucleosomefrom those promoters and binding of Ndd1 to Fkh2. Recentstudies describing a role for histone chaperones and chro-

matin boundary proteins in chromatin remodeling at thehistone gene cluster are reviewed elsewhere (Eriksson et al.2012). Together, these studies suggest both direct and indirectroles for cluster-specific transcription factors in the recruit-ment and regulation of chromatin modifying and remodelingproteins. Nevertheless, the degree of uncertainty regardingthe specificity of these processes makes this a fertile groundfor future research.

Rationale for Cell-Cycle-Regulated Transcription:Need to Have or Nice to Have?

To understand the rationale for regulating the expression ofgenes during the cell cycle, it is helpful to examine whichgenes are controlled. Deciding whether or not a gene is cell-cycle-regulated is not as trivial as it might seem. Manydefinitions of “cell-cycle regulated” can and have been ap-plied, yielding a variety of estimates of the number andidentities of cell-cycle-regulated genes in S. cerevisiae (Choet al. 1998; Spellman et al. 1998; de Lichtenberg et al. 2005;Pramila et al. 2006; Orlando et al. 2008; Granovskaia et al.2010; Guo et al. 2013). The number of transcripts reportedas cell-cycle regulated has varied from 416 to 1270. A con-sensus set of 440 genes (Orlando et al. 2008) was identifiedby each of three distinct studies (Spellman et al. 1998;Pramila et al. 2006; Orlando et al. 2008). The remaininggenes, which are unique to each of the studies, could resultfrom differences in strain backgrounds or experimental con-ditions including synchrony procedures, growth medium,microarray platform, or the approach used to identify peri-odic genes.

So which genes are cell-cycle regulated? The best answeris: There is no single, identifiable set of cell-cycle-regulatedgenes. Because there is no precise and universally accepteddefinition of cell-cycle regulated, there is no single algorith-mic method available for identifying genes with periodicbehaviors over the cell cycle. In addition, each of the algo-rithms returns a rank-ordered list of genes with no obviousgap in the distribution that distinguishes periodic from non-periodic genes. Thus, all cut-offs chosen by investigators aresomewhat arbitrary, and it is likely more accurate to say thatthere is a continuum of cell-cycle-regulated genes.

Despite the difficulty in precisely defining a single set ofcell-cycle-regulated genes, it is likely that �20% of genes inthe yeast genome are expressed periodically during the cellcycle at a substantial energy cost to the cell. This observationsuggests that coordination of gene expression with cell-cycleevents is important for the well being of cells. Several dis-tinct though potentially overlapping rationales have beenadvanced as reasons for phase-specific expression of variousgenes.

First, some genes are expressed at the time they arerequired during the cell cycle. This is clearly true for a largenumber of genes whose function is required for, or is relatedto, morphogenesis, DNA replication, and chromosomesegregation. The logic for this pattern of expression is


































referred to as “just in time.” Whether genes are actuallyexpressed just in time is not always so clear. For example,because cytokinesis occurs subsequent to M phase, onemight expect the expression of genes encoding the structuralelements of the bud neck, including septins, to occur in Mphase rather than G1. However, those genes are, in fact,expressed just in time because the structures at the budneck, which play roles from G1 through M phases, are as-sembled during G1 phase prior to budding, precisely whenG1/S gene expression is maximal (reviewed in Bi and Park2012; Howell and Lew 2012). Similar scenarios may explainthe periodic expression of some of the genes for which norationale is currently apparent.

Many genes that fall into the just-in-time category,despite being expressed at the time of their essentialfunction, need not be expressed precisely at that time and,in some cases, need not be expressed periodically at all. Infact, several genome-wide studies have revealed the vastmajority of yeast genes cause no significant growth disad-vantage, at least when constitutively overexpressed incycling cells from the inducible GAL1 promoter (Liu et al.1992; Sopko et al. 2006; Douglas et al. 2012).

In addition to those genes that are expressed just in time,there are a large number of genes expressed at a specificcell-cycle interval during which they have no apparentfunction. In some cases, the apparent lack of phase-specificfunction may be a consequence of our lack of knowledge,but in other cases the genes may just “tag along” with othergenes expressed during the same phase. That is, the patternof expression may be adequate but not necessarily advanta-geous. However, some of those genes might be better de-scribed as just in case, because their expression may preparethe cell to deal with a sporadic event that does not occur inall cell cycles. For example, whereas many DNA repair pro-teins are expressed prior to every S phase, damage mayoccur only in a subset of cells during any round of DNAreplication.

Finally, some genes that are expressed just in time aredeleterious when expressed outside of the interval duringwhich they function. Those genes often encode proteinswith regulatory functions that are temporally coupled totheir accumulation. For example, the genes encoding theB-type cyclins Clb2 and Clb5 and others encoding the ana-phase regulators Cdc20 and Pds1 perturb cell-cycle progres-sion when they are expressed continuously throughout thecycle, with the caveat that the studies in question involveoverexpression (Nasmyth 1993; Sopko et al. 2006). How-ever, even in the context of overexpression, such genes aresurprisingly rare.

So, why express a gene just in time when that patternof expression is unnecessary? One possibility is that just-in-time expression is simply a matter of economy. Often,proteins expressed outside of the interval during which theyfunction are disposed of by proteolysis or other mechanisms(e.g., those encoding the G1 cyclins Cln1–3 and the CDKinhibitor Sic1) (Lanker et al. 1996; Willems et al. 1996;

Verma et al. 1997). Consequently, there is a large energycost to the cells of expressing those genes outside of theirfunctional interval. Although expressing those proteinsthroughout the cycle may not produce a noticeable or de-bilitating phenotype, it may result in a slight competitivedisadvantage under optimal or suboptimal conditions. Con-sequently, such changes would not be noticed in typical lab-oratory growth conditions. A definitive answer to thesequestions is lacking because, except for studies involvingoverexpression, the impact of continuous expression ofcell-cycle-regulated genes on overall fitness has not beensystematically investigated.

The Topology of Cell-Cycle-Regulated TranscriptionalCircuitry is Conserved

One of the overarching questions arising in the study ofmodel systems is the degree to which their regulatorycircuitry is conserved with humans. Comparative analysisof cell-cycle transcriptional programs across the Eukaryotahas provided one window through which to examine thatquestion. The general organization and structure of the cell-cycle apparatus is conserved among the organisms so farexamined. Finally, all of those organisms express a portion oftheir genes in a cell-cycle-dependent manner. Consequently,it has been anticipated that the conservation of the cell-cycle-dependent transcriptional machinery will be substan-tial. This supposition has been met with both confirmationand contradiction.

The cell-cycle transcription program has now beenexamined on a genomic scale in plants, a variety of yeastsincluding budding and fission yeast, and in humans (Spellmanet al. 1998; Whitfield et al. 2002; Menges et al. 2003; Bar-Joseph et al. 2004; Rustici et al. 2004; Pramila et al. 2006;Gauthier et al. 2008). Although it is often difficult to assessin light of the evolution of both sequence and function,comparative analysis of cell-cycle-regulated gene expressionhas led to the conclusion that the ontology of genes undercell-cycle control is often conserved (CDK regulators, chro-matin proteins, transcription factors, DNA replication andrepair proteins, mitotic proteins, etc.), but the expressionpattern of orthologs is not always conserved (Lu et al.2007; Orlando et al. 2007). The degree of divergence seemsto increase with the evolutionary distance between the spe-cies and is even obvious in closely related Saccharomycesspecies, cerevisiae and bayanus (Guan et al. 2010, 2013; Eseret al. 2011). This seems surprising, considering the similar-ities in cell-cycle organization between those organisms.However, the observation that the timing of expression ofcell-cycle-regulated genes in yeast is flexible, and, in somecases, completely dispensable (see above) suggests that suchchanges in expression might be easily accommodated. Ofcourse, the differences in periodicity at the transcriptionlevel do not account for the loss or acquisition of the broadarray of post-transcriptional mechanisms that might restoreperiodic regulation of many of those gene products. In fact,









differences in the periodicity of transcription that havecoevolved with changes in post-transcriptional regulationhave been documented (de Lichtenberg et al. 2005; Jensenet al. 2006). Another indication of the evolutionary flexibilityof cell-cycle-regulatory phenomenon is the finding that withinprotein complexes where one or more subunits is periodicallyexpressed, the identity of the periodically expressed subunitsmay differ between species (Jensen et al. 2006). In fact, itappears that just-in-time assembly may be important andconserved, whereas just-in-time expression may be dispens-able (de Lichtenberg et al. 2005). This notion is consistentwith the finding that within closely related yeast species,there is extensive repurposing of conserved transcription fac-tor subunits to form novel complexes with other factors andproduce entirely new transcriptional outcomes (Tuch et al.2008).

Like cell-cycle-regulated transcripts, comparison of theregulators of periodic gene expression reveals strong con-servation of the general topology of the systems anddramatic divergence of the details. This is well illustratedby analysis of the transcriptional machinery regulating theG1/S gene clusters of fungi and mammals (reviewed byWittenberg and Reed 2005; Cross et al. 2011; Bertoli et al.2013b). The activating members of the E2F family of tran-scriptional regulators, E2F1 being the best studied, exhibitmany parallels with SBF (Figure 6) (Dimova and Dyson2005). E2F1/DP1 binds to promoters in quiescent cells incomplex with a transcription repressor, in this case Rb, theretinoblastoma tumor suppressor protein, a functional ho-molog of Whi5. Upon entry into the cell cycle, Rb is phos-phorylated by a G1 cyclin/CDK, in this case CycD/Cdk4/6,functional analogs of Cln3/Cdk1, leading to activation of theE2F1/DP1 targets. Finally, the G1/S genes are repressed bythe binding of the E2F6, a repressive form of E2F, as cellsprogress into S phase, leading to the dissociation of E2F1/DP1 from promoters (Bertoli et al. 2013a). This preciselyparallels the regulation of SBF in budding yeast. However,examination of E2F1, DP1, and Rb, reveals no recognizable

protein sequence conservation, or even structural conserva-tion, with Swi4, Swi6, or Whi5 (Cross et al. 2011).

Whether this is an example of rapid evolution betweenfungi and humans or convergent evolution directed ata redundant function remains to be established. However,it is clear that the E2F/Rb family and not the SBF/Whi5family of regulators exists in the plant kingdom, which isevolutionarily distant from both fungi and mammals (Crosset al. 2011). Furthermore, despite the lack of relationshipbetween the transcriptional regulators, all of these organ-isms use a conserved, albeit diversified, family of CDKkinases to coordinate gene expression with the cell cycle(Enserink and Kolodner 2010).

In contrast to the lack of conservation of SBF, E2F, andtheir regulators, other cell-cycle-associated transcriptionalregulators from yeast exhibit strong sequence and structuralconservation with other eukaryotes. For example, both theMADS box transcription factor, Mcm1, and Forkhead tran-scription factors, Fkh1 and Fkh2, are relatively well con-served among eukaryotes. However, at least in the caseof the MADS box transcription factors, their role in cell-cycle regulation appears not to be conserved. SRF is awell-established regulator of growth-associated genes, butnot those expressed periodically during the cell cycle (Shoreand Sharrocks 1995). In plants, MADS box transcriptionfactors are prominently associated with developmentallyregulated gene expression (reviewed by Masiero et al.2011). Interestingly, although yeast Mcm1 plays a promi-nent role in the regulation of G2/M and M/G1 regulatedgenes, it is also involved in a number of functions that arenot related to the cell cycle.

In contrast, the human Forkhead protein, FoxM1, sharesmany regulatory properties with Fkh2. That regulationappears not to involve either a MADS box transcription fac-tor or an Ndd1 ortholog, as it does in yeast. However, like inyeast, the activity of FoxM1 is required for the proper ex-pression of a number of mitotic genes during G2/M and, likethe Mcm1/Fkh2/Ndd1 complex, it is subject to regulation by

Figure 6 Conservation of the G1/S regulatory circuitryin yeast and human. Comparison of the topology of theG1/S transcriptional circuitry between yeast and humanreveals a high degree of conservation. Both SBF and E2Fare heterodimeric transcription factors that bind to pro-moters in quiescent cells and repress transcription byvirtue of their interaction with repressors, Whi5 andRb, respectively. Both Whi5 and Rb, the retinoblastomatumor suppressor, are phosphorylated by a G1 cyclin-associated CDK, leading to their dissociation from thetranscription factor and activation of G1/S cluster pro-moters. Those genes are then repressed in S phase viamechanisms that promote the loss of the activatingtranscription factor from their target promoters. Despitethe high degree of conservation of the topology ofthese regulatory circuits, the transcription factors exhibitlittle or no relatedness in terms of either amino acidsequence or structure. The color of subunits and arrowsis as in Figure 1.


















both Cdk1 and the human Polo kinase, Plk1 (reviewed byMurakami et al. 2010).

Together these observations suggest a complex evolu-tionary relationship between the factors mediating cell-cycle-regulated gene expression and their targets. Per-haps unsurprisingly, it is the final product, the overallexecution of cell-cycle events, that is best conserved ratherthan the details of the regulatory pathway that controlsthat outcome.

Acknowledgments

The authors thank Anna Travesa, Danny Lew, Nick Buchler,Sara Bristow, Adam Leman, Christina Kelliher, Mark Chee,and anonymous reviewers for helpful discussion and com-ments on the manuscript. S.B.H. is supported by grants fromthe Defense Advanced Research Projects Agency and theNational Institutes of Health (NIH) P50-GM081883. C.W. issupported in part by grants R01 GM059441 and R01GM100354 from the National Institutes of Health.

Literature Cited

Aligianni, S., D. H. Lackner, S. Klier, G. Rustici, B. T. Wilhelm et al.,2009 The fission yeast homeodomain protein Yox1p binds toMBF and confines MBF-dependent cell-cycle transcription to G1-S via negative feedback. PLoS Genet. 5: e1000626.

Allen, J. B., Z. Zhou, W. Siede, E. C. Friedberg, and S. J. Elledge,1994 The SAD1/RAD53 protein kinase controls multiplecheckpoints and DNA damage-induced transcription in yeast.Genes Dev. 8: 2401–2415.

Amon, A., M. Tyers, B. Futcher, and K. Nasmyth,1993 Mechanisms that help the yeast cell cycle clock tick:G2 cyclins transcriptionally activate G2 cyclins and repressG1 cyclins. Cell 74: 993–1007.

Andrews, B. J., and I. Herskowitz, 1989 Identification of a DNAbinding factor involved in cell-cycle control of the yeast HOgene. Cell 57: 21–29.

Bähler, J., 2005 Cell-cycle control of gene expression in buddingand fission yeast. Annu. Rev. Genet. 39: 69–94.

Bai, L., G. Charvin, E. D. Siggia, and F. R. Cross,2010 Nucleosome-depleted regions in cell-cycle-regulatedpromoters ensure reliable gene expression in every cell cycle.Dev. Cell 18: 544–555.

Bai, L., A. Ondracka, and F. R. Cross, 2011 Multiple sequence-specific factors generate the nucleosome-depleted region onCLN2 promoter. Mol. Cell 42: 465–476.

Bardwell, L., 2005 A walk-through of the yeast mating phero-mone response pathway. Peptides 26: 339–350.

Bar-Joseph, Z., S. Farkash, D. K. Gifford, I. Simon, and R. Rosenfeld,2004 Deconvolving cell cycle expression data with complemen-tary information. Bioinformatics 20(Suppl 1): I23–I30.

Baroni, M. D., P. Monti, and L. Alberghina, 1994 Repression ofgrowth-regulated G1 cyclin expression by cyclic AMP in buddingyeast. Nature 371: 339–342.

Bean, J. M., E. D. Siggia, and F. R. Cross, 2005 High functionaloverlap between MluI cell-cycle box binding factor and Swi4/6cell-cycle box binding factor in the G1/S transcriptional pro-gram in Saccharomyces cerevisiae. Genetics 171: 49–61.

Bean, J. M., E. D. Siggia, and F. R. Cross, 2006 Coherence andtiming of cell cycle start examined at single-cell resolution. Mol.Cell 21: 3–14.

Bertoli, C., S. Klier, C. McGowan, C. Wittenberg, and R. A. M. deBruin, 2013a Chk1 inhibits E2F6 repressor function in re-sponse to replication stress to maintain cell-cycle transcription.Curr. Biol. 23: 1629–1637.

Bertoli, C., J. M. Skotheim, and R. A. M. de Bruin, 2013b Controlof cell cycle transcription during G1 and S phases. Nat. Rev. Mol.Cell Biol. 14: 518–528.

Bhaduri, S., and P. M. Pryciak, 2011 Cyclin-specific docking mo-tifs promote phosphorylation of yeast signaling proteins by G1/SCdk complexes. Curr. Biol. 21: 1615–1623.

Bi, E., and H.-O. Park, 2012 Cell polarization and cytokinesis inbudding yeast. Genetics 191: 347–387.

Bobola, N., R. P. Jansen, T. H. Shin, and K. Nasmyth,1996 Asymmetric accumulation of Ash1p in postanaphase nu-clei depends on a myosin and restricts yeast mating-type switch-ing to mother cells. Cell 84: 699–709.

Boros, J., F.-L. Lim, Z. Darieva, A. Pic-Taylor, R. Harman et al.,2003 Molecular determinants of the cell-cycle regulatedMcm1p-Fkh2p transcription factor complex. Nucleic Acids Res.31: 2279–2288.

Brauer, M. J., C. Huttenhower, E. M. Airoldi, R. Rosenstein, J. C.Matese et al., 2008 Coordination of growth rate, cell cycle,stress response, and metabolic activity in yeast. Mol. Biol. Cell19: 352–367.

Breeden, L., and K. Nasmyth, 1987 Cell cycle control of the yeastHO gene: cis- and trans-acting regulators. Cell 48: 389–397.

Charvin, G., C. Oikonomou, E. D. Siggia, and F. R. Cross,2010 Origin of irreversibility of cell cycle start in buddingyeast. PLoS Biol. 8: e1000284.

Chen, Z., E. A. Odstrcil, B. P. Tu, and S. L. McKnight,2007 Restriction of DNA replication to the reductive phase ofthe metabolic cycle protects genome integrity. Science 316:1916–1919.

Cherry, J. M., E. L. Hong, C. Amundsen, R. Balakrishnan, G. Binkleyet al., 2012 Saccharomyces Genome Database: the genomicsresource of budding yeast. Nucleic Acids Res. DOI: 10.1093/nar/gkr1029

Cho, R. J., M. J. Campbell, E. A. Winzeler, L. Steinmetz, A. Conwayet al., 1998 A genome-wide transcriptional analysis of the mi-totic cell cycle. Mol. Cell 2: 65–73.

Chu, Z., J. Li, M. Eshaghi, X. Peng, R. K. Karuturi et al.,2007 Modulation of cell cycle-specific gene expressions atthe onset of S phase arrest contributes to the robust DNA rep-lication checkpoint response in fission yeast. Mol. Biol. Cell 18:1756–1767.

Coïc, E., K. Sun, C. Wu, and J. E. Haber, 2006 Cell cycle-dependentregulation of Saccharomyces cerevisiae donor preference duringmating-type switching by SBF (Swi4/Swi6) and Fkh1. Mol. Cell.Biol. 26: 5470–5480.

Colman-Lerner, A., T. E. Chin, and R. Brent, 2001 Yeast Cbk1 andMob2 activate daughter-specific genetic programs to induceasymmetric cell fates. Cell 107: 739–750.

Cosma, M. P., 2004 Daughter-specific repression of Saccharomy-ces cerevisiae HO: Ash1 is the commander. EMBO Rep. 5: 953–957.

Cosma, M. P., T. Tanaka, and K. Nasmyth, 1999 Ordered recruit-ment of transcription and chromatin remodeling factors to a cellcycle- and developmentally regulated promoter. Cell 97: 299–311.

Cosma, M. P., S. Panizza, and K. Nasmyth, 2001 Cdk1 triggersassociation of RNA polymerase to cell cycle promoters onlyafter recruitment of the mediator by SBF. Mol. Cell 7: 1213–1220.

Costanzo, M., O. Schub, and B. Andrews, 2003 G1 transcriptionfactors are differentially regulated in Saccharomyces cerevisiaeby the Swi6-binding protein Stb1. Mol. Cell. Biol. 23: 5064–5077.



Costanzo, M., J. L. Nishikawa, X. Tang, J. S. Millman, O. Schubet al., 2004 CDK activity antagonizes Whi5, an inhibitor of G1/Stranscription in yeast. Cell 117: 899–913.

Creanor, J., 1978 Carbon dioxide evolution during the cell cycleof the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 33:385–397.

Cross, F. R., 1988 DAF1, a mutant gene affecting size control,pheromone arrest, and cell cycle kinetics of Saccharomyces cer-evisiae. Mol. Cell. Biol. 8: 4675–4684.

Cross, F. R., and A. H. Tinkelenberg, 1991 A potential positivefeedback loop controlling CLN1 and CLN2 gene expression atthe start of the yeast cell cycle. Cell 65: 875–883.

Cross, F. R., N. E. Buchler, and J. M. Skotheim, 2011 Evolution ofnetworks and sequences in eukaryotic cell cycle control. Philos.Trans. R. Soc. Lond. B Biol. Sci. 366: 3532–3544.

Darieva, Z., R. Bulmer, A. Pic-Taylor, K. S. Doris, M. Geymonatet al., 2006 Polo kinase controls cell-cycle-dependent tran-scription by targeting a coactivator protein. Nature 444: 494–498.

Darieva, Z., A. Clancy, R. Bulmer, E. Williams, A. Pic-Taylor et al.,2010 A competitive transcription factor binding mechanismdetermines the timing of late cell cycle-dependent gene expres-sion. Mol. Cell 38: 29–40.

Darieva, Z., N. Han, S. Warwood, K. S. Doris, B. A. Morgan et al.,2012 Protein kinase C regulates late cell cycle-dependent geneexpression. Mol. Cell. Biol. 32: 4651–4661.

de Bruin, R. A., W. H. McDonald, T. I. Kalashnikova, J. Yates, and C.Wittenberg, 2004 Cln3 activates G1-specific transcription viaphosphorylation of the SBF bound repressor Whi5. Cell 117:887–898.

de Bruin, R. A., T. I. Kalashnikova, C. Chahwan, W. H. McDonald, J.Wohlschlegel et al., 2006 Constraining G1-specific transcrip-tion to late G1 phase: the MBF-associated corepressor Nrm1 actsvia negative feedback. Mol. Cell 23: 483–496.

de Bruin, R. A., T. I. Kalashnikova, and C. Wittenberg, 2008a Stb1collaborates with other regulators to modulate the G1-specifictranscriptional circuit. Mol. Cell. Biol. 28: 6919–6928.

de Bruin, R. A., T. I. Kalashnikova, A. Aslanian, J. Wohlschlegel, C.Chahwan et al., 2008b DNA replication checkpoint promotesG1-S transcription by inactivating the MBF repressor Nrm1.Proc. Natl. Acad. Sci. USA 105: 11230–11235.

de Lichtenberg, U., L. J. Jensen, A. Fausboll, T. S. Jensen, P. Borket al., 2005 Comparison of computational methods for theidentification of cell cycle-regulated genes. Bioinformatics 21:1164–1171.

de Oliveira, F. M. B., M. R. Harris, P. Brazauskas, R. A. M. de Bruin,and M. B. Smolka, 2012 Linking DNA replication checkpointto MBF cell-cycle transcription reveals a distinct class of G1/Sgenes. EMBO J. 31: 1798–1810.

Di Talia, S., J. M. Skotheim, J. M. Bean, E. D. Siggia, and F. R.Cross, 2007 The effects of molecular noise and size control onvariability in the budding yeast cell cycle. Nature 448: 947–951.

Di Talia, S., H. Wang, J. M. Skotheim, A. P. Rosebrock, B. Futcheret al., 2009 Daughter-specific transcription factors regulatecell size control in budding yeast. PLoS Biol. 7: e1000221.

Dimova, D. K., and N. J. Dyson, 2005 The E2F transcriptionalnetwork: old acquaintances with new faces. Oncogene 24:2810–2826.

Dirick, L., and K. Nasmyth, 1991 Positive feedback in the activa-tion of G1 cyclins in yeast. Nature 351: 754–757.

Dirick, L., T. Böhm, and K. Nasmyth, 1995 Roles and regulation ofCln-Cdc28 kinases at the start of the cell cycle of Saccharomycescerevisiae. EMBO J. 14: 4803–4813.

Dohlman, H. G., and J. W. Thorner, 2001 Regulation of G protein-initiated signal transduction in yeast: paradigms and principles.Annu. Rev. Biochem. 70: 703–754.

Dohrmann, P. R., G. Butler, K. Tamai, S. Dorland, J. R. Greene et al.,1992 Parallel pathways of gene regulation: homologous regu-lators SWI5 and ACE2 differentially control transcription of HOand chitinase. Genes Dev 6: 93–104.

Dohrmann, P. R., W. P. Voth, and D. J. Stillman, 1996 Role ofnegative regulation in promoter specificity of the homologoustranscriptional activators Ace2p and Swi5p. Mol. Cell. Biol. 16:1746–1758.

Doncic, A., M. Falleur-Fettig, and J. M. Skotheim, 2011 Distinctinteractions select and maintain a specific cell fate. Mol. Cell 43:528–539.

Doncic, A., and J. M. Skotheim, 2013 Feedforward regulationensures stability and rapid reversibility of a cellular state. Mol.Cell 50: 856–868.

Douglas, A. C., A. M. Smith, S. Sharifpoor, Z. Yan, T. Durbic et al.,2012 Functional analysis with a barcoder yeast gene overex-pression system. G3 (Bethesda) 2: 1279–1289.

Dutta, C., P. K. Patel, A. Rosebrock, A. Oliva, J. Leatherwood et al.,2008 The DNA replication checkpoint directly regulates MBF-dependent G1/S transcription. Mol. Cell. Biol. 28: 5977–5985.

Edgington, N. P., and B. Futcher, 2001 Relationship between thefunction and the location of G1 cyclins in S. cerevisiae. J. CellSci. 114: 4599–4611.

Elledge, S. J., 1996 Cell cycle checkpoints: preventing an identitycrisis. Science 274: 1664–1672.

Elledge, S. J., Z. Zhou, J. B. Allen, and T. A. Navas, 1993 DNAdamage and cell cycle regulation of ribonucleotide reductase.Bioessays 15: 333–339.

Enserink, J. M., and R. D. Kolodner, 2010 An overview of Cdk1-controlled targets and processes. Cell Div. 5: 11.

Eriksson, P. R., D. Ganguli, V. Nagarajavel, and D. J. Clark,2012 Regulation of histone gene expression in budding yeast.Genetics 191: 7–20.

Eser, U., M. Falleur-Fettig, A. Johnson, and J. M. Skotheim,2011 Commitment to a cellular transition precedes genome-wide transcriptional change. Mol. Cell 43: 515–527.

Femino, A. M., F. S. Fay, K. Fogarty, and R. H. Singer,1998 Visualization of single RNA transcripts in situ. Science280: 585–590.

Ferrell, J. E., 2011 Simple rules for complex processes: new les-sons from the budding yeast cell cycle. Mol. Cell 43: 497–500.

Ferrezuelo, F., N. Colomina, B. Futcher, and M. Aldea, 2010 Thetranscriptional network activated by Cln3 cyclin at the G1-to-Stransition of the yeast cell cycle. Genome Biol. 11: R67.

Ferrezuelo, F., N. Colomina, A. Palmisano, E. Gari, C. Gallego et al.,2012 The critical size is set at a single-cell level by growth rateto attain homeostasis and adaptation. Nat. Commun. 3: 1012.

Fu, Y., L. Pastushok, and W. Xiao, 2008 DNA damage-inducedgene expression in Saccharomyces cerevisiae. FEMS Microbiol.Rev. 32: 908–926.

Futcher, B., 2006 Metabolic cycle, cell cycle, and the finishing kickto Start. Genome Biol. 7: 107.

Gardner, R., C. W. Putnam, and T. Weinert, 1999 RAD53, DUN1and PDS1 define two parallel G2/M checkpoint pathways inbudding yeast. EMBO J. 18: 3173–3185.

Garí, E., T. Volpe, H. Wang, C. Gallego, B. Futcher et al.,2001 Whi3 binds the mRNA of the G1 cyclin CLN3 to modu-late cell fate in budding yeast. Genes Dev. 15: 2803–2808.

Gasch, A. P., P. T. Spellman, C. M. Kao, O. Carmel-Harel, M. B.Eisen et al., 2000 Genomic expression programs in the re-sponse of yeast cells to environmental changes. Mol. Biol. Cell11: 4241–4257.

Gasch, A. P., M. Huang, S. Metzner, D. Botstein, S. J. Elledge et al.,2001 Genomic expression responses to DNA-damaging agentsand the regulatory role of the yeast ATR homolog Mec1p. Mol.Biol. Cell 12: 2987–3003.


Gauthier, N. P., M. E. Larsen, R. Wernersson, U. de Lichtenberg,L. J. Jensen et al., 2008 Cyclebase.org: a comprehensive multi-organism online database of cell-cycle experiments. NucleicAcids Res. 36: D854–D859.

Geymonat, M., A. Spanos, G. P. Wells, S. J. Smerdon, and S. G.Sedgwick, 2004 Clb6/Cdc28 and Cdc14 regulate phosphory-lation status and cellular localization of Swi6. Mol. Cell. Biol.24: 2277–2285.

Gómez-Escoda, B., T. Ivanova, I. A. Calvo, I. Alves-Rodrigues, E.Hidalgo et al., 2011 Yox1 links MBF-dependent transcriptionto completion of DNA synthesis. EMBO Rep. 12: 84–89.

Granovskaia, M. V., L. J. Jensen, M. E. Ritchie, J. Toedling, Y. Ninget al., 2010 High-resolution transcription atlas of the mitoticcell cycle in budding yeast. Genome Biol. 11: R24.

Guan, Y., M. Dunham, A. Caudy, and O. Troyanskaya,2010 Systematic planning of genome-scale experiments inpoorly studied species. PLOS Comput. Biol. 6: e1000698.

Guan, Y., M. J. Dunham, O. G. Troyanskaya, and A. A. Caudy,2013 Comparative gene expression between two yeast species.BMC Genomics 14: 33.

Guo, X., A. Bernard, D. A. Orlando, S. B. Haase, and A. J. Hartemink,2013 Branching process deconvolution algorithm reveals a de-tailed cell-cycle transcription program. Proc. Natl. Acad. Sci. USA110: E968–E977.

Haber, J. E., 2012 Mating-type genes and MAT switching in Sac-charomyces cerevisiae. Genetics 191: 33–64.

Harbison, C. T., D. B. Gordon, T. I. Lee, N. J. Rinaldi, K. D. Macisaacet al., 2004 Transcriptional regulatory code of a eukaryoticgenome. Nature 431: 99–104.

Harris, M. R., D. Lee, S. Farmer, N. F. Lowndes, and R. A. M. deBruin, 2013 Binding specificity of the G1/S transcriptional reg-ulators in budding yeast. PLoS ONE 8: e61059.

Hartwell, L., and M. Unger, 1977 Unequal division in Saccharo-myces cerevisiae and its implications for the control of cell di-vision. J. Cell Biol. 75: 422–435.

Hartwell, L., T. Weinert, L. Kadyk, and B. Garvik, 1994 Cell cyclecheckpoints, genomic integrity, and cancer. Cold Spring Harb.Symp. Quant. Biol. 59: 259–263.

Henchoz, S., Y. Chi, B. Catarin, I. Herskowitz, R. J. Deshaies et al.,1997 Phosphorylation- and ubiquitin-dependent degradationof the cyclin-dependent kinase inhibitor Far1p in budding yeast.Genes Dev. 11: 3046–3060.

Hereford, L. M., M. A. Osley, T. R. Ludwig, and C. S. McLaughlin,1981 Cell-cycle regulation of yeast histone mRNA. Cell 24:367–375.

Ho, Y., M. Costanzo, L. Moore, R. Kobayashi, and B. J. Andrews,1999 Regulation of transcription at the Saccharomyces cerevi-siae start transition by Stb1, a Swi6-binding protein. Mol. Cell.Biol. 19: 5267–5278.

Hollenhorst, P. C., G. Pietz, and C. A. Fox, 2001 Mechanismscontrolling differential promoter-occupancy by the yeast fork-head proteins Fkh1p and Fkh2p: implications for regulatingthe cell cycle and differentiation. Genes Dev. 15: 2445–2456.

Holt, L. J., A. N. Krutchinsky, and D. O. Morgan, 2008 Positivefeedback sharpens the anaphase switch. Nature 454: 353–357.

Horak, C. E., N. M. Luscombe, J. Qian, P. Bertone, S. Piccirrilloet al., 2002 Complex transcriptional circuitry at the G1/S tran-sition in Saccharomyces cerevisiae. Genes Dev. 16: 3017–3033.

Howell, A. S., M. Jin, C. F. Wu, T. R. Zyla, T. C. Elston et al.,2012 Negative feedback enhances robustness in the yeast po-larity establishment circuit. Cell 149: 322–333.

Howell, A. S., and D. J. Lew, 2012 Morphogenesis and the cellcycle. Genetics 190: 51–77.

Huang, M., Z. Zhou, and S. J. Elledge, 1998 The DNA replicationand damage checkpoint pathways induce transcription by inhi-bition of the Crt1 repressor. Cell 94: 595–605.

Hwang-Shum, J. J., D. C. Hagen, E. E. Jarvis, C. A. Westby, and G.F. Sprague, 1991 Relative contributions of MCM1 and STE12to transcriptional activation of a- and alpha-specific genes fromSaccharomyces cerevisiae. Mol. Gen. Genet. 227: 197–204.

Iyer, V. R., C. E. Horak, C. S. Scafe, D. Botstein, M. Snyder et al.,2001 Genomic binding sites of the yeast cell-cycle transcrip-tion factors SBF and MBF. Nature 409: 533–538.

Jensen, L. J., T. S. Jensen, U. de Lichtenberg, S. Brunak, and P.Bork, 2006 Co-evolution of transcriptional and post-transla-tional cell-cycle regulation. Nature 443: 594–597.

Jorgensen, P., J. L. Nishikawa, B. J. Breitkreutz, and M. Tyers,2002 Systematic identification of pathways that couple cellgrowth and division in yeast. Science 297: 395–400.

Kato, M., N. Hata, N. Banerjee, B. Futcher, and M. Q. Zhang,2004 Identifying combinatorial regulation of transcription fac-tors and binding motifs. Genome Biol. 5: R56.

Klevecz, R. R., J. Bolen, G. Forrest, and D. B. Murray, 2004 Agenomewide oscillation in transcription gates DNA replicationand cell cycle. Proc. Natl. Acad. Sci. USA 101: 1200–1205.

Knapp, D., L. Bhoite, D. J. Stillman, and K. Nasmyth, 1996 Thetranscription factor Swi5 regulates expression of the cyclin ki-nase inhibitor p40SIC1. Mol. Cell. Biol. 16: 5701–5707.

Koch, C., T. Moll, M. Neuberg, H. Ahorn, and K. Nasmyth, 1993 Arole for the transcription factors Mbp1 and Swi4 in progressionfrom G1 to S phase. Science 261: 1551–1557.

Koch, C., A. Schleiffer, G. Ammerer, and K. Nasmyth,1996 Switching transcription on and off during the yeast cellcycle: Cln/Cdc28 kinases activate bound transcription factorSBF (Swi4/Swi6) at start, whereas Clb/Cdc28 kinases displaceit from the promoter in G2. Genes Dev. 10: 129–141.

Koranda, M., A. Schleiffer, L. Endler, and G. Ammerer,2000 Forkhead-like transcription factors recruit Ndd1 to thechromatin of G2/M-specific promoters. Nature 406: 94–98.

Kovacech, B., K. Nasmyth, and T. Schuster, 1996 EGT2 gene tran-scription is induced predominantly by Swi5 in early G1. Mol.Cell. Biol. 16: 3264–3274.

Kuhne, C., and P. Linder, 1993 A new pair of B-type cyclins fromSaccharomyces cerevisiae that function early in the cell cycle.EMBO J. 12: 3437–3447.

Kumar, R., D. M. Reynolds, A. Shevchenko, A. Shevchenko, S. D.Goldstone et al., 2000 Forkhead transcription factors, Fkh1pand Fkh2p, collaborate with Mcm1p to control transcription re-quired for M-phase. Curr. Biol. 10: 896–906.

Laabs, T. L., D. D. Markwardt, M. G. Slattery, L. L. Newcomb, D. J.Stillman et al., 2003 ACE2 is required for daughter cell-specificG1 delay in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci.USA 100: 10275–10280.

Lang, G. I., A. W. Murray, and D. Botstein, 2009 The cost of geneexpression underlies a fitness trade-off in yeast. Proc. Natl.Acad. Sci. USA 106: 5755–5760.

Lanker, S., M. H. Valdivieso, and C. Wittenberg, 1996 Rapid deg-radation of the G1 cyclin Cln2 induced by CDK-dependent phos-phorylation. Science 271: 1597–1601.

Larson, D. R., D. Zenklusen, B. Wu, J. A. Chao, and R. H. Singer,2011 Real-time observation of transcription initiation andelongation on an endogenous yeast gene. Science 332: 475–478.

Lee, T. I., N. J. Rinaldi, F. Robert, D. T. Odom, Z. Bar-Joseph et al.,2002 Transcriptional regulatory networks in Saccharomycescerevisiae. Science 298: 799–804.

Lenstra, T. L., J. J. Benschop, T. Kim, J. M. Schulze, N. A. Braberset al., 2011 The specificity and topology of chromatin interac-tion pathways in yeast. Mol. Cell 42: 536–549.

Levin, D. E., 2011 Regulation of cell wall biogenesis in Saccharo-myces cerevisiae: the cell wall integrity signaling pathway. Ge-netics 189: 1145–1175.


Liu, H., J. Krizek, and A. Bretscher, 1992 Construction of a GAL1-regulated yeast cDNA expression library and its application tothe identification of genes whose overexpression causes lethalityin yeast. Genetics 132: 665–673.

Ljungdahl, P. O., and B. Daignan-Fornier, 2012 Regulation ofamino acid, nucleotide, and phosphate metabolism in Saccharo-myces cerevisiae. Genetics 190: 885–929.

Lord, P. G., and A. E. Wheals, 1980 Asymmetrical division ofSaccharomyces cerevisiae. J. Bacteriol. 142: 808–818.

Lord, P. G., and A. E. Wheals, 1981 Variability in individual cellcycles of Saccharomyces cerevisiae. J. Cell Sci. 50: 361–376.

Loy, C. J., D. Lydall, and U. Surana, 1999 NDD1, a high-dosagesuppressor of cdc28–1N, is essential for expression of a subset oflate-S-phase-specific genes in Saccharomyces cerevisiae. Mol.Cell. Biol. 19: 3312–3327.

Lu, Y., S. Mahony, P. V. Benos, R. Rosenfeld, I. Simon et al.,2007 Combined analysis reveals a core set of cycling genes.Genome Biol. 8: R146.

Lydall, D., G. Ammerer, and K. Nasmyth, 1991 A new role forMCM1 in yeast: cell cycle regulation of SW15 transcription.Genes Dev. 5: 2405–2419.

MacKay, V. L., B. Mai, L. Waters, and L. L. Breeden, 2001 Earlycell cycle box-mediated transcription of CLN3 and SWI4 con-tributes to the proper timing of the G(1)-to-S transition in bud-ding yeast. Mol. Cell. Biol. 21: 4140–4148.

Mai, B., S. Miles, and L. L. Breeden, 2002 Characterization of theECB binding complex responsible for the M/G(1)-specific tran-scription of CLN3 and SWI4. Mol. Cell. Biol. 22: 430–441.

Marini, N. J., and S. I. Reed, 1992 Direct induction of G1-specifictranscripts following reactivation of the Cdc28 kinase in theabsence of de novo protein synthesis. Genes Dev 6: 557–567.

Masiero, S., L. Colombo, P. E. Grini, A. Schnittger, and M. M. Kater,2011 The emerging importance of type I MADS box transcrip-tion factors for plant reproduction. Plant Cell 23: 865–872.

Mazanka, E., and E. L. Weiss, 2010 Sequential counteractingkinases restrict an asymmetric gene expression program to earlyG1. Mol. Biol. Cell 21: 2809–2820.

McBride, H. J., Y. Yu, and D. J. Stillman, 1999 Distinct regions ofthe Swi5 and Ace2 transcription factors are required for specificgene activation. J. Biol. Chem. 274: 21029–21036.

McInerny, C. J., 2011 Cell cycle regulated gene expression inyeasts. Adv. Genet. 73: 51–85.

McInerny, C. J., J. F. Partridge, G. E. Mikesell, D. P. Creemer, andL. L. Breeden, 1997 A novel Mcm1-dependent element in theSWI4, CLN3, CDC6, and CDC47 promoters activates M/G1-specifictranscription. Genes Dev. 11: 1277–1288.

McIntosh, P. B., I. A. Taylor, T. A. Frenkiel, S. J. Smerdon, and A. N.Lane, 2000 The influence of DNA binding on the backbonedynamics of the yeast cell-cycle protein Mbp1. J. Biomol. NMR16: 183–196.

McKinney, J. D., F. Chang, N. Heintz, and F. R. Cross,1993 Negative regulation of FAR1 at the Start of the yeast cellcycle. Genes Dev. 7: 833–843.

McKinney, J. D., and F. R. Cross, 1995 FAR1 and the G1 phasespecificity of cell cycle arrest by mating factor in Saccharomycescerevisiae. Mol. Cell. Biol. 15: 2509–2516.

Menges, M., L. Hennig, W. Gruissem, and J. A. Murray,2003 Genome-wide gene expression in an Arabidopsis cell sus-pension. Plant Mol. Biol. 53: 423–442.

Miller, M. E., and F. R. Cross, 2000 Distinct subcellular localiza-tion patterns contribute to functional specificity of the Cln2 andCln3 cyclins of Saccharomyces cerevisiae. Mol. Cell. Biol. 20:542–555.

Moll, T., G. Tebb, U. Surana, H. Robitsch, and K. Nasmyth,1991 The role of phosphorylation and the CDC28 protein ki-nase in cell cycle-regulated nuclear import of the S. cerevisiaetranscription factor SWI5. Cell 66: 743–758.

Moll, T., L. Dirick, H. Auer, J. Bonkovsky, and K. Nasmyth,1992 SWI6 is a regulatory subunit of two different cell cycleSTART-dependent transcription factors in Saccharomyces cere-visiae. J. Cell Sci. Suppl. 16: 87–96.

Morgan, D. O., 2007 The Cell Cycle: Principles of Control, NewScience Press, London.

Morillo-Huesca, M., D. Maya, M. C. Muñoz-Centeno, R. K. Singh, V.Oreal et al., 2010 FACT prevents the accumulation of freehistones evicted from transcribed chromatin and a subsequentcell cycle delay in G1. PLoS Genet. 6: e1000964.

Murakami, H., H. Aiba, M. Nakanishi, and Y. Murakami-Tonami,2010 Regulation of yeast forkhead transcription factors andFoxM1 by cyclin-dependent and polo-like kinases. Cell Cycle9: 3233–3242.

Nair, M., P. B. McIntosh, T. A. Frenkiel, G. Kelly, I. A. Taylor et al.,2003 NMR structure of the DNA-binding domain of the cellcycle protein Mbp1 from Saccharomyces cerevisiae. Biochemis-try 42: 1266–1273.

Nash, R., G. Tokiwa, S. Anand, K. Erickson, and A. B. Futcher,1988 The WHI1+ gene of Saccharomyces cerevisiae tetherscell division to cell size and is a cyclin homolog. EMBO J. 7:4335–4346.

Nasmyth, K., 1993 Regulating the HO endonuclease in yeast.Curr. Opin. Genet. Dev. 3: 286–294.

Nasmyth, K., and L. Dirick, 1991 The role of SWI4 and SWI6 inthe activity of G1 cyclins in yeast. Cell 66: 995–1013.

Novak, B., and J. M. Mitchison, 1986 Change in the rate of CO2production in synchronous cultures of the fission yeast Schizo-saccharomyces pombe: a periodic cell cycle event that persistsafter the DNA-division cycle has been blocked. J. Cell Sci. 86:191–206.

Novak, B., and J. M. Mitchison, 1990 Changes in the rate of ox-ygen consumption in synchronous cultures of the fission yeastSchizosaccharomyces pombe. J. Cell Sci. 96(Pt 3): 429–433.

O’Conallain, C., M. T. Doolin, C. Taggart, F. Thornton, and G.Butler, 1999 Regulated nuclear localisation of the yeasttranscription factor Ace2p controls expression of chitinase(CTS1) in Saccharomyces cerevisiae. Mol. Gen. Genet. 262:275–282.

Oehlen, L. J., and F. R. Cross, 1994 G1 cyclins CLN1 and CLN2repress the mating factor response pathway at Start in the yeastcell cycle. Genes Dev. 8: 1058–1070.

Oehlen, L. J., J. D. McKinney, and F. R. Cross, 1996 Ste12 andMcm1 regulate cell cycle-dependent transcription of FAR1. Mol.Cell. Biol. 16: 2830–2837.

Ofir, A., K. Hofmann, E. Weindling, T. Gildor, K. S. Barker et al.,2012 Role of a Candida albicans Nrm1/Whi5 homologue incell cycle gene expression and DNA replication stress response.Mol. Microbiol. 84: 778–794.

Oliva, A., A. Rosebrock, F. Ferrezuelo, S. Pyne, H. Chen et al.,2005 The cell cycle-regulated genes of Schizosaccharomycespombe. PLoS Biol. 3: e225.

Orlando, D. A., C. Y. Lin, A. Bernard, E. S. Iversen, A. J. Harteminket al., 2007 A probabilistic model for cell cycle distributions insynchrony experiments. Cell Cycle 6: 478–488.

Orlando, D. A., C. Y. Lin, A. Bernard, J. Y. Wang, J. E. S. Socolaret al., 2008 Global control of cell-cycle transcription by cou-pled CDK and network oscillators. Nature 453: 944–947.

Ostapenko, D., and M. J. Solomon, 2011 Anaphase promotingcomplex-dependent degradation of transcriptional repressorsNrm1 and Yhp1 in S. cerevisiae. Mol. Biol. Cell 22: 2175–2184.

Peng, X., R. K. Karuturi, L. D. Miller, K. Lin, Y. Jia et al.,2005 Identification of cell cycle-regulated genes in fissionyeast. Mol. Biol. Cell 16: 1026–1042.

Peterson, C. L., and I. Herskowitz, 1992 Characterization of theyeast SWI1, SWI2, and SWI3 genes, which encode a global ac-tivator of transcription. Cell 68: 573–583.


Pic-Taylor, A., Z. Darieva, B. A. Morgan, and A. D. Sharrocks,2004 Regulation of cell cycle-specific gene expression throughcyclin-dependent kinase-mediated phosphorylation of the fork-head transcription factor Fkh2p. Mol. Cell. Biol. 24: 10036–10046.

Polymenis, M., and E. V. Schmidt, 1997 Coupling of cell divisionto cell growth by translational control of the G1 cyclin CLN3 inyeast. Genes Dev. 11: 2522–2531.

Pramila, T., 2002 Conserved homeodomain proteins interact withMADS box protein Mcm1 to restrict ECB-dependent transcrip-tion to the M/G1 phase of the cell cycle. Genes Dev. 16: 3034–3045.

Pramila, T., W. Wu, S. Miles, W. S. Noble, and L. L. Breeden,2006 The Forkhead transcription factor Hcm1 regulates chro-mosome segregation genes and fills the S-phase gap in the tran-scriptional circuitry of the cell cycle. Genes Dev. 20: 2266–2278.

Qiu, P., Z. J. Wang, and K. J. R. Liu, 2006 Polynomial modelapproach for resynchronization analysis of cell-cycle gene ex-pression data. Bioinformatics 22: 959–966.

Queralt, E., and J. C. Igual, 2003 Cell cycle activation of theSwi6p transcription factor is linked to nucleocytoplasmic shut-tling. Mol. Cell. Biol. 23: 3126–3140.

Rando, O. J., and F. Winston, 2012 Chromatin and transcriptionin yeast. Genetics 190: 351–387.

Remus, D., and J. F. Diffley, 2009 Eukaryotic DNA replication con-trol: lock and load, then fire. Curr. Opin. Cell Biol. 21: 771–777.

Ren, B., F. Robert, J. J. Wyrick, O. Aparicio, E. G. Jennings et al.,2000 Genome-wide location and function of DNA binding pro-teins. Science 290: 2306–2309.

Reynolds, D., B. J. Shi, C. McLean, F. Katsis, B. Kemp et al.,2003 Recruitment of Thr 319-phosphorylated Ndd1p to theFHA domain of Fkh2p requires Clb kinase activity: a mechanismfor CLB cluster gene activation. Genes Dev 17: 1789–1802.

Richardson, H., D. J. Lew, M. Henze, K. Sugimoto, and S. I. Reed,1992 Cyclin-B homologs in Saccharomyces cerevisiae functionin S phase and in G2. Genes Dev. 6: 2021–2034.

Roberts, C. J., B. Nelson, M. J. Marton, R. Stoughton, M. R. Meyeret al., 2000 Signaling and circuitry of multiple MAPK path-ways revealed by a matrix of global gene expression profiles.Science 287: 873–880.

Rowicka, M., A. Kudlicki, B. P. Tu, and Z. Otwinowski, 2007 High-resolution timing of cell cycle-regulated gene expression. Proc.Natl. Acad. Sci. USA 104: 16892–16897.

Rustici, G., J. Mata, K. Kivinen, P. Lio, C. J. Penkett et al.,2004 Periodic gene expression program of the fission yeast cellcycle. Nat. Genet. 36: 809–817.

Sbia, M., E. J. Parnell, Y. Yu, A. E. Olsen, K. L. Kretschmann et al.,2008 Regulation of the yeast Ace2 transcription factor duringthe cell cycle. J. Biol. Chem. 283: 11135–11145.

Schaefer, J. B., and L. L. Breeden, 2004 RB from a bud’s eye view.Cell 117: 849–850.

Schulze, J. M., J. Jackson, S. Nakanishi, J. M. Gardner, T. Hentrichet al., 2009 Linking cell cycle to histone modifications: SBFand H2B monoubiquitination machinery and cell-cycle regula-tion of H3K79 dimethylation. Mol. Cell 35: 626–641.

Schwob, E., and K. Nasmyth, 1993 CLB5 and CLB6, a new pair ofB cyclins involved in DNA replication in Saccharomyces cerevi-siae. Genes Dev. 7: 1160–1175.

Shen, Z., N. Paquin, A. Forget, and P. Chartrand, 2009 Nuclearshuttling of She2p couples ASH1 mRNA localization to its trans-lational repression by recruiting Loc1p and Puf6p. Mol. Biol.Cell 20: 2265–2275.

Shore, P., and A. D. Sharrocks, 1995 The MADS-box family oftranscription factors. Eur. J. Biochem. 229: 1–13.

Sidorova, J. M., and L. L. Breeden, 1997 Rad53-dependent phos-phorylation of Swi6 and down-regulation of CLN1 and CLN2

transcription occur in response to DNA damage in Saccharomy-ces cerevisiae. Genes Dev. 11: 3032–3045.

Sidorova, J. M., G. E. Mikesell, and L. L. Breeden, 1995 Cell cycle-regulated phosphorylation of Swi6 controls its nuclear localiza-tion. Mol. Biol. Cell 6: 1641–1658.

Siegmund, R. F., and K. A. Nasmyth, 1996 The Saccharomycescerevisiae Start-specific transcription factor Swi4 interactsthrough the ankyrin repeats with the mitotic Clb2/Cdc28 kinaseand through its conserved carboxy terminus with Swi6. Mol.Cell. Biol. 16: 2647–2655.

Sil, A., and I. Herskowitz, 1996 Identification of asymmetricallylocalized determinant, Ash1p, required for lineage-specific tran-scription of the yeast HO gene. Cell 84: 711–722.

Silverman, S. J., A. A. Petti, N. Slavov, L. Parsons, R. Briehof et al.,2010 Metabolic cycling in single yeast cells from unsynchro-nized steady-state populations limited on glucose or phosphate.Proc. Natl. Acad. Sci. USA 107: 6946–6951.

Simmons Kovacs, L. A., D. A. Orlando, and S. B. Haase,2008 Transcription networks and cyclin/CDKs: the yin andyang of cell cycle oscillators. Cell Cycle 7: 2626–2629.

Simmons Kovacs, L. A., M. B. Mayhew, D. A. Orlando, Y. Jin, Q. Liet al., 2012 Cyclin-dependent kinases are regulators and effec-tors of oscillations driven by a transcription factor network. Mol.Cell 45: 669–679.

Simon, I., J. Barnett, N. Hannett, C. T. Harbison, N. J. Rinaldi et al.,2001 Serial regulation of transcriptional regulators in theyeast cell cycle. Cell 106: 697–708.

Skotheim, J. M., S. Di Talia, E. D. Siggia, and F. R. Cross,2008 Positive feedback of G1 cyclins ensures coherent cellcycle entry. Nature 454: 291–296.

Slavov, N., and D. Botstein, 2011 Coupling among growth rateresponse, metabolic cycle, and cell division cycle in yeast. Mol.Biol. Cell 22: 1997–2009.

Smolka, M. B., F. M. Bastos de Oliveira, M. R. Harris, and R. A. M.de Bruin, 2012 The checkpoint transcriptional response: makesure to turn it off once you are satisfied. Cell Cycle 11: 3166–3174.

Sopko, R., D. Huang, N. Preston, G. Chua, B. Papp et al.,2006 Mapping pathways and phenotypes by systematic geneoverexpression. Mol. Cell 21: 319–330.

Spellman, P. T., G. Sherlock, M. Q. Zhang, V. R. Iyer, K. Anderset al., 1998 Comprehensive identification of cell cycle-regulatedgenes of the yeast Saccharomyces cerevisiae by microarray hy-bridization. Mol. Biol. Cell 9: 3273–3297.

Stillman, D. J., 2013 Dancing the cell cycle two-step: regulationof yeast G1-cell-cycle genes by chromatin structure. Trends Bio-chem. Sci. 38: 467–475.

Strickfaden, S. C., M. J. Winters, G. Ben-Ari, R. E. Lamson, M. Tyerset al., 2007 A mechanism for cell-cycle regulation of MAP ki-nase signaling in a yeast differentiation pathway. Cell 128: 519–531.

Stuart, D., and C. Wittenberg, 1994 Cell cycle-dependent tran-scription of CLN2 is conferred by multiple distinct cis-actingregulatory elements. Mol. Cell. Biol. 14: 4788–4801.

Stuart, D., and C. Wittenberg, 1995 CLN3, not positive feedback,determines the timing of CLN2 transcription in cycling cells.Genes Dev. 9: 2780–2794.

Surana, U., H. Robitsch, C. Price, T. Schuster, I. Fitch et al.,1991 The role of CDC28 and cyclins during mitosis in thebudding yeast S. cerevisiae. Cell 65: 145–161.

Taberner, F. J., I. Quilis, and J. C. Igual, 2009 Spatial regulation ofthe start repressor Whi5. Cell Cycle 8: 3010–3018.

Takahata, S., Y. Yu, and D. J. Stillman, 2009a The E2F functionalanalogue SBF recruits the Rpd3(L) HDAC, via Whi5 and Stb1,and the FACT chromatin reorganizer, to yeast G1 cyclin pro-moters. EMBO J. 28: 3378–3389.


Takahata, S., Y. Yu, and D. J. Stillman, 2009b FACT and Asf1regulate nucleosome dynamics and coactivator binding at theHO promoter. Mol. Cell 34: 405–415.

Takahata, S., Y. Yu, and D. J. Stillman, 2011 Repressive chroma-tin affects factor binding at yeast HO (Homothallic Switching)promoter. J. Biol. Chem. 286: 34809–34819.

Taylor, I. A., M. K. Treiber, L. Olivi, and S. J. Smerdon, 1997 TheX-ray structure of the DNA-binding domain from the Saccharo-myces cerevisiae cell-cycle transcription factor Mbp1 at 2.1 Aresolution. J. Mol. Biol. 272: 1–8.

Taylor, I. A., P. B. McIntosh, P. Pala, M. K. Treiber, S. Howell et al.,2000 Characterization of the DNA-binding domains from theyeast cell-cycle transcription factors Mbp1 and Swi4. Biochem-istry 39: 3943–3954.

Taylor, I. A., D. C. Goldstone, P. Pala, L. F. Haire, and S. J. Smerdon,2010 Structure of the amino-terminal domain from the cell-cycle regulator Swi6. Proteins. 78: 2861–2865.

Tebb, G., T. Moll, C. Dowzer, and K. Nasmyth, 1993 SWI5 insta-bility may be necessary but is not sufficient for asymmetric HOexpression in yeast. Genes Dev. 7: 517–528.

Teixeira, M. C., P. Monteiro, P. Jain, S. Tenreiro, A. R. Fernandeset al., 2006 The YEASTRACT database: a tool for the analysisof transcription regulatory associations in Saccharomyces cere-visiae. Nucleic Acids Res. 34: D446–D451.

Thorburn, R. R., C. Gonzalez, G. A. Brar, S. Christen, T. M. Carlileet al., 2013 Aneuploid yeast strains exhibit defects in cellgrowth and passage through START. Mol. Biol. Cell 24: 1274–1289.

Toyn, J. H., A. L. Johnson, J. D. Donovan, W. M. Toone, and L. H.Johnston, 1997 The Swi5 transcription factor of Saccharomy-ces cerevisiae has a role in exit from mitosis through inductionof the cdk-inhibitor Sic1 in telophase. Genetics 145: 85–96.

Travesa, A., and C. Wittenberg, 2012 Turned on by genotoxicstress. Cell Cycle 11: 3145–3146.

Travesa, A., D. Kuo, R. A. M. de Bruin, T. I. Kalashnikova, M.Guaderrama et al., 2012 DNA replication stress differentiallyregulates G1/S genes via Rad53-dependent inactivation ofNrm1. EMBO J. 31: 1811–1822.

Travesa, A., T. I. Kalashnikova, R. A. M. de Bruin, S. R. Cass, C.Chahwan et al., 2013 Repression of G1/S transcription is me-diated via interaction of the GTB motifs of Nrm1 and Whi5 withSwi6. Mol. Cell. Biol. 33: 1476–1486.

Trcek, T., D. R. Larson, A. Moldon, C. C. Query, and R. H. Singer,2011 Single-molecule mRNA decay measurements reveal pro-moter- regulated mRNA stability in yeast. Cell 147: 1484–1497.

Tu, B. P., A. Kudlicki, M. Rowicka, and S. L. McKnight, 2005 Logicof the yeast metabolic cycle: temporal compartmentalization ofcellular processes. Science 310: 1152–1158.

Tuch, B. B., D. J. Galgoczy, A. D. Hernday, H. Li, and A. D. Johnson,2008 The evolution of combinatorial gene regulation in fungi.PLoS Biol. 6: e38.

Turner, J. J., J. C. Ewald, and J. M. Skotheim, 2012 Cell sizecontrol in yeast. Curr. Biol. 22: R350–R359.

Tyers, M., G. Tokiwa, and B. Futcher, 1993 Comparison of theSaccharomyces cerevisiae G1 cyclins: Cln3 may be an upstreamactivator of Cln1, Cln2 and other cyclins. EMBO J. 12: 1955–1968.

Ubersax, J. A., E. L. Woodbury, P. N. Quang, M. Paraz, J. D. Blethrowet al., 2003 Targets of the cyclin-dependent kinase Cdk1. Nature425: 859–864.

Veis, J., H. Klug, M. Koranda, and G. Ammerer, 2007 Activation ofthe G2/M-specific gene CLB2 requires multiple cell cycle signals.Mol. Cell. Biol. 27: 8364–8373.

Verma, R., R. S. Annan, M. J. Huddleston, S. A. Carr, G. Reynardet al., 1997 Phosphorylation of Sic1p by G1 Cdk required forits degradation and entry into S phase. Science 278: 455–460.

Visintin, R., K. Craig, E. S. Hwang, S. Prinz, M. Tyers et al.,1998 The phosphatase Cdc14 triggers mitotic exit by reversalof Cdk-dependent phosphorylation. Mol. Cell 2: 709–718.

Voth, W. P., Y. Yu, S. Takahata, K. L. Kretschmann, J. D. Liebet al., 2007 Forkhead proteins control the outcome of tran-scription factor binding by antiactivation. EMBO J. 26: 4324–4334.

Wagner, M. V., M. B. Smolka, R. A. M. de Bruin, H. Zhou, C.Wittenberg et al., 2009 Whi5 regulation by site specific CDK-phosphorylation in Saccharomyces cerevisiae. PLoS ONE 4: e4300.

Wang, H., L. B. Carey, Y. Cai, H. Wijnen, and B. Futcher,2009 Recruitment of Cln3 cyclin to promoters controls cellcycle entry via histone deacetylase and other targets. PLoS Biol.7: e1000189.

Weiss, E. L., C. Kurischko, C. Zhang, K. Shokat, D. G. Drubin et al.,2002 The Saccharomyces cerevisiae Mob2p-Cbk1p kinasecomplex promotes polarized growth and acts with the mitoticexit network to facilitate daughter cell-specific localization ofAce2p transcription factor. J. Cell Biol. 158: 885–900.

Whitfield, M. L., G. Sherlock, A. J. Saldanha, J. I. Murray, C. A. Ballet al., 2002 Identification of genes periodically expressed inthe human cell cycle and their expression in tumors. Mol. Biol.Cell 13: 1977–2000.

Wijnen, H., A. Landman, and B. Futcher, 2002 The G(1) cyclinCln3 promotes cell cycle entry via the transcription factor Swi6.Mol. Cell. Biol. 22: 4402–4418.

Willems, A., S. Lanker, E. Patton, K. Craig, T. Nason et al.,1996 Cdc53 targets phosphorylated G1 cyclins for degradationby the ubiquitin proteolytic pathway. Cell 86: 453–463.

Winey, M., and K. Bloom, 2012 Mitotic spindle form and function.Genetics 190: 1197–1224.

Wittenberg, C., and S. I. Reed, 2005 Cell cycle-dependent tran-scription in yeast: promoters, transcription factors, and tran-scriptomes. Oncogene 24: 2746–2755.

Wittenberg, C., K. Sugimoto, and S. I. Reed, 1990 G1-specificcyclins of S. cerevisiae: cell cycle periodicity, regulation by mat-ing pheromone, and association with the p34CDC28 proteinkinase. Cell 62: 225–237.

Woldringh, C. L., P. G. Huls, and N. O. Vischer, 1993 Volumegrowth of daughter and parent cells during the cell cycle ofSaccharomyces cerevisiae a/alpha as determined by image cy-tometry. J. Bacteriol. 175: 3174–3181.

Xu, R. M., C. Koch, Y. Liu, J. R. Horton, D. Knapp et al.,1997 Crystal structure of the DNA-binding domain of Mbp1,a transcription factor important in cell-cycle control of DNAsynthesis. Structure 5: 349–358.

Zhou, Z., and S. J. Elledge, 1993 DUN1 encodes a protein kinasethat controls the DNA damage response in yeast. Cell 75: 1119–1127.

Zhu, G., P. T. Spellman, T. Volpe, P. O. Brown, D. Botstein et al.,2000 Two yeast forkhead genes regulate the cell cycle andpseudohyphal growth. Nature 406: 90–94.

Communicating editor: M. Tyers


topology and control of the cell-cycle-regulated ...cell-cycle-regulated genes has grown, slowly at...

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