Cell Cycle-Dependent Transcription in Yeast: Promoters, Transcription Factors, and Transcriptomes

Cell cycle-dependent transcription in yeast: promoters, transcription factors, and transcriptomes

Curt Wittenberg1 and Steven I Reed*,2

1Department of Molecular Biology, MB-3, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; 2Department of Molecular Biology, MB-7, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

In the budding yeast, Saccharomyces cerevisiae,a 1983) allowed for the testing of a large number of genes significant fraction of genes (>10%) are transcribed with for cell cycle regulation of transcription. Not surpris- cell cycle periodicity. These genes encode critical cell cycle ingly, many genes involved in specific cell cycle processes regulators as well as proteins with no direct connection to were found to be expressed maximally during specific cell cycle functions. Cell cycle-regulated genes can be cell cycle intervals where synthesis of encoded products organized into ‘clusters’ exhibiting similar patterns of could be rationalized in terms of function (Wittenberg regulation. In most cases periodic transcription is achieved and Reed, 1991). Based on single-gene analyses, it could via both repressive and activating mechanisms. Fine- be predicted that a significant but limited set of genes tuning appears to have evolved by the juxtaposition of encoding proteins directly involved in periodic cell cycle regulatory motifs characteristic of more than one cluster functions would be subject to cell cycle regulation. within the same promoter. Recent reports have provided However, more recently, large-scale array analysis of the significant newinsight into the role of the cyclin-dependent yeast transcriptome has revealed that a much larger set kinase Cdk1 (Cdc28) in coordination of transcription with of genes (minimally 800 or >10% of protein-encoding cell cycle events. In early G1, the transcription factor genes) exhibit cell cycle regulation and that many of the complex known as SBF is maintained in a repressed state encoded proteins are not ostensibly linked to cell cycle by association of the Whi5 protein. Phosphorylation of functions (Cho et al., 1998; Spellman et al., 1998). This Whi5 by Cdk1 in late G1 leads to dissociation from SBF presumably reflects a more intricate and subtle relation- and transcriptional derepression. G2/M-specific transcrip- ship between cellular physiology and cell cycle progres- tion is achieved by converting the repressor Fkh2 into an sion than has been appreciated. Furthermore, high- activator. Fkh2 serves as a repressor during most of the resolution scrutiny has revealed that even within so- cell cycle. However, phosphorylation of a cofactor, Ndd1, called clusters of coordinately-regulated genes, there are by Cdk1 late in the cell cycle promotes binding to Fkh2 significant differences observable between individual and conversion into a transcriptional activator. Such members, suggesting individualized optimization of insights derived from analysis of specific genes when transcriptional programming according to function combined with genome-wide analysis provide a more (Cho et al., 1998; Spellman et al., 1998). In this article, detailed and integrated viewof cell cycle-dependent we will review advances based on detailed analysis of transcription. transcriptional control of individual genes, but also Oncogene (2005) 24, 2746–2755. doi:10.1038/sj.onc.1208606 discuss the implications of findings based on genome- wide analysis. Keywords: transcription; cell cycle; cyclin-dependent protein kinase

The G1 gene cluster

Cell cycle initiation during G1 phase is the consequence Introduction of transcriptional activation of greater than 200 genes, many of which participate in events specifically asso- With the advent of modern molecular biology, some 30 ciated with cell cycle progression (Cho et al., 1998; years ago, it became possible to efficiently analyse Spellman et al., 1998). The primary cis-acting regulators transcription patterns of individual genes. In yeast, the of the G1-specific gene cluster are targeted by two convergence of relatively straightforward cell cycle alternative heterodimeric transcription factors com- synchronization protocols (Amon, 2002) with rapid prised of unique DNA-binding components, Swi4 and efficient mutational cloning techniques (Struhl, (SBF) or Mbp1 (MBF), and a common component, Swi6, that participates in transactivation (Breeden, 1996). The optimal binding site for MBF is called the *Correspondence: SI Reed; E-mail: [email protected] Mlu Cell cycle Box (MCB), due to the presence of an Cell cycle-dependent transcription in yeast C Wittenberg and SI Reed 2747 MluI restriction site in the consensus sequence, and that of at least a portion of SBF and MBF target genes, for SBF is the Swi4 Cell cycle Box (SCB) (Breeden, 1996; including CLN1 and CLN2 (Epstein and Cross, 1994; Di Lee et al., 2002; Kato et al., 2004). Como et al., 1995; Wijnen and Futcher, 1999). The target genes for SBF and MBF have been The identification of a central role for Cln3/Cdk1 traditionally defined by the presence of redundant SCB in transcriptional activation (Cross and Tinklenberg, and MCB sequences in their promoters. Functionally, 1991; Dirick and Nasmyth, 1991), and the finding that SBF and MBF targets, respectively, fall roughly into Cdk1 is likely to act directly rather than via its effect on two classes. MBF targets include those involved in the the accumulation of other proteins (Marini and Reed, control or execution of DNA replication and repair 1992), has led to a protracted search for CDK targets (POL2, CDC2, RNR1, CLB5/6), whereas SBF targets involved in activation. Swi6 and Swi4 are established include those involved in cell morphogenesis, spindle targets in vitro and Swi6 is known to be phosphorylated pole body duplication and other growth-related func- in a Cdk1-dependent manner in vivo (Sidorova et al., tions (CLN1/2, PCL1/2, GIN4, FKS1/2). This apparent 1995; Ubersax et al., 2003; Geymonat et al., 2004). differentiation into distinct functional classes may have Elimination of Cdk sites by mutation leads to a defect in adaptive significance due to regulatory differences nuclear import of Swi6 (Sidorova et al., 1995; Geymonat between the two transcription factors. However, each et al., 2004). However, those mutations appear not to group also includes many members that do not fall have an effect on the timing, extent of transcriptional neatly into these categories. Consequently, it is possible activation or Cdk dependence of transcriptional activa- that this separation is merely a reflection of the tion, suggesting that phosphorylation of Swi6 is not rate evolutionary history of these transcriptional regulatory limiting for access to the nucleus. modules (see below). The lack of a functional link between phosphoryla- Whereas the optimal binding sites for SBF and MBF tion of the transcription factors and transcriptional are distinct, there is considerable overlap in their activation has led to the suggestion that Cdk1 acts specificity both in terms of the DNA sequence require- indirectly via phosphorylation of another factor (Wijnen ments for binding and the occurrence of such sites et al., 2002), perhaps a previously unrecognized tran- within promoters. Consequently, there is considerable scriptional activator or repressor. Although a number of ambiguity concerning the role of each factor in proteins have been shown to interact with Swi6 (e.g., regulating specific genes (Partridge et al., 1997). This is Hrr25 (Ho et al., 1997), Skn7 (Morgan et al., 1995), and apparent from the global analysis of the binding of the Stb1 (Costanzo et al., 2003)), none appears to play a specific transcription factors to promoters (Iyer et al., significant role in the Cln/Cdk-dependent activation of 2001; Simon et al., 2001; Lee et al., 2002) and may transcription. Recently, the search for additional factors explain the modest effect of inactivation of SBF or MBF regulating G1 transcription led to the discovery of Whi5 on the expression of G1-specific genes (Koch et al., as an SBF-associated transcriptional repressor (Costan- 1993; de Bruin and Wittenberg, unpublished). zo et al., 2004; de Bruin et al., 2004). Inactivation of Whi5, previously shown to cause cell cycle initiation at a Regulation of G1-specific transcription small cell size (Jorgensen et al., 2002; Zhang et al., 2002), suppresses the requirement for Cln3in the absence of G1-specific transcription is promoted, in large part, by Bck2 (Costanzo et al., 2004; de Bruin et al., 2004). G1 cyclin-associated CDK activity. In fact, deregulation Consistent with that phenotype, inactivation of WHI5 of that process was the basis for the discovery of the G1 leads to premature activation of G1-specific transcrip- cyclin Cln3(Cross, 1988; Nash et al., 1988). Mutations tion during the G1 phase. The Whi5 protein associates leading to stabilization of Cln3result in premature with G1-specific promoters in an SBF-dependent expression of G1-specific genes and, thereby, small cell manner and is released from DNA coincident with size and resistance to G1-phase arrest by mating transcriptional activation. It is phosphorylated at Cdk pheromone. Any of the three G1 cyclins (Cln1, Cln2, consensus sites in vivo and in vitro and can be or Cln3) in conjunction with Cdk1 (Cdc28) were later phosphorylated in vitro by both G1- and B-type shown to be sufficient to mobilize Cdk1-dependent cyclin-associated Cdk1. Consistent with a role for activation of G1-specific transcription (Cross and Cdk1 in activation of SBF-dependent transcription, Tinkelenberg, 1991; Dirick and Nasmyth, 1991; Marini phosphorylation of SBF/Whi5 complexes in vitro and Reed, 1992). However, whereas Cln1- and Cln2- promotes dissociation of the complex. The regulation containing CDK complexes are sufficient for transcrip- of SBF by Whi5 bears striking resemblance to the tional activation, Cln3/Cdk1 is the primary activator regulation of the metazoan G1/S transcription factor, under physiological conditions (Tyers et al., 1993; E2F, by the retinoblastoma tumor suppressor protein, Dirick et al., 1995; Stuart and Wittenberg, 1995). Rb (Dyson, 1998; Nevins, 2001) (Figure 1). This is Consistent with that role, Cln3is largely nuclear despite the absence of detectable primary structure whereas the other G1 cyclins are distributed to the homology between the protein components of the two nucleus and cytoplasm (Miller and Cross, 2000; transcriptional regulatory systems, suggesting that they Edgington and Futcher, 2001). Although Cln3is not evolved independently. essential for viability, it becomes essential in cells Although inactivation of Whi5 leads to premature lacking Bck2, a protein of unknown molecular function expression of G1-specific genes and, thereby, premature but that is required for basal transcriptional activation cell cycle initiation, the strongly periodic expression of

Oncogene Cell cycle-dependent transcription in yeast C Wittenberg and SI Reed 2748 Repressed Activated This suggests that Cln/Cdk1 activates MBF via a Whi5- complex complex independent mechanism. MBF activation may involve Cln3/Cdk1 direct phosphorylation of Swi6 or phosphorylation of Y Whi5 E Swi6 Swi6 A Swi4 Swi4 Cln1 & 2, Clb5 & 6 another MBF-associated protein. Although Stb1 ap- S and T S phase genes pears to activate MBF and to be a target of Cln1/2/ P P Cdk1, phosphorylation does not appear to be required Whi5 for its activity (Costanzo et al., 2003). G1 S The primary determinant of the timing of G1-specific M E CycD/Cdk4/6 transcription is the activity of Cln3/Cdk1 toward the T Rb A E2F E2F Cyclin E & A inactive SBF and MBF transcription factors (Tyers Z and O S phase genes A et al., 1993; Dirick et al., 1995; Stuart and Wittenberg, N P P 1995). As such, it is also the primary determinant of cell Rb size. Several regulatory mechanisms appear to play a Figure 1 Regulation of G1-specific transcription by cyclin- role in determining Cln3/Cdk1 activity. First, the CLN3 dependent protein kinase in yeast and metazoans. In yeast, Cln3- gene is subject to cell cycle-regulated expression as a associated Cdk1 phosphorylates Whi5, promoting its release from SBF and leading to derepression of SBF-dependent transcription. member of the M/G1 phase gene cluster (McInerny Activation of SBF-dependent genes leads to accumulation of G1- et al., 1997; MacKay et al., 2001) (see below). In and S-phase cyclins which promote S-phase entry. Accumulation of addition, CLN3 gene expression and protein abundance Cln1- and Cln2-associated Cdk1 may provide additional regulation are induced by glucose (Hall et al., 1998; Parviz et al., of Whi5 and SBF. In metazoans cyclin D/Cdk4/6 phosphorylates 1998). Next, CLN3 mRNA must be efficiently trans- Rb, relieving its inhibition of E2F/DP1 and leading to derepression of G1/S-phase gene expression. Activation of E2F-responsive genes lated. That process appears to be subject to both leads to the accumulation of cyclins E and A, which promote S- transcript-specific and general translational controls phase entry. Accumulation of cyclin E/Cdk2 further phosphor- (Gallego et al., 1997; Polymenis and Schmidt, 1997). ylates Rb, contributing to the activation of E2F-responsive genes Recent evidence suggests a rather surprising regulatory mechanism whereby CLN3 mRNA and Cdk1 are tethered together in the cytoplasm by the Whi3protein, perhaps as a mechanism governing complex assembly both SBF- and MBF-dependent genes in whi5 mutants and transport (Gari et al., 2001; Wang et al., 2004). belies the existence of other mechanisms regulating this Furthermore, Cln3is known to be a highly unstable gene family (de Bruin et al., 2004). This is, in part, a protein targeted for degradation by an SCF ubiquitin consequence of the periodic accumulation of Clb2 (see ligase (Tyers et al., 1992). However, the details of that below), which antagonizes the binding of Swi4 to SBF- regulation are unclear. Finally, CLN3 appears to be the dependent promoters (Amon et al., 1993; Siegmund and target of a still mysterious free-running oscillator that Nasmyth, 1996) leading to transcriptional repression. underlies the timing of cell cycle events (Haase and Although it is not known precisely how localization of Reed, 1999). Swi6 is regulated, it is likely that both SBF and MBF levels are influenced by the lack of availability of nuclear Swi6 outside the G1 phase (Sidorova et al., 1995; Queralt and Igual, 2003). However, it is unclear whether The S-phase gene clusters limitation of nuclear Swi6 is a factor in the periodic expression of target genes in whi5 mutants. It has been Two clusters of genes characterized by the histone genes suggested that nucleocytoplasmic cycling of Swi6 is a and the MET genes, respectively, are expressed im- requirement for transcriptional activation (Queralt and mediately following the wave of the G1-specific tran- Igual, 2003). Finally, Cln3/Cdk can promote transcrip- scription as cells progress into S phase (Cho et al., 1998; tional activation even in the absence of Whi5 (de Bruin Spellman et al., 1998). The nine histone genes comprise and Wittenberg, unpublished). It is possible that this the entire histone cluster. Their expression is tightly activation involves the direct phosphorylation of Swi6 coordinated and slightly earlier than the MET gene by Cln/Cdk. Consistent with that notion, the loss of Cdk cluster (Cho et al., 1998; Spellman et al., 1998). Based on target sites on Whi5 and Swi6 appears to be lethal genome-wide ChIP analysis, the histone gene promoters (Costanzo et al., 2004). are bound by SBF and possibly MBF (Iyer et al., 2001; Despite the capacity of Whi5 to bind to MBF under Simon et al., 2001; Ng et al., 2002), consistent with the some conditions, activation of MBF-dependent genes presence of putative Swi4-binding motifs in their appears largely independent of Whi5. Strikingly, MBF- promoters (Osley et al., 1986; Lee et al., 2002; Kato dependent genes remain dependent upon Cln/Cdk for et al., 2004). In addition, each of the histone gene activation when Whi5 is inactive. Thus, whereas SBF- promoters binds to one or both of the transcriptional dependent genes are strongly derepressed in cells lacking repressors, Hir1 and Hir2 (Sherwood et al., 1993), which both G1 cyclin and Whi5 function (de Bruin et al., may act by recruitment of the RSC chromatin remodel- 2004), MBF-dependent genes fail to be expressed (de ing complex (Ng et al., 2002). Binding of the forkhead Bruin and Wittenberg, unpublished). In contrast, like transcription factors Fkh1/2 (see below) has also been SBF-dependent genes, MBF-dependent genes are fully documented. Although this constellation of regulators activated when Cln1 and Cln2 functions are retained. suggests that histone promoters are G1-specific genes

Oncogene Cell cycle-dependent transcription in yeast C Wittenberg and SI Reed 2749 that are constrained in their expression by the action of (Kumar et al., 2000; Pic et al., 2000; Hollenhorst et al., transcriptional repressors, inactivation of Hir1 and Hir2 2001). Deletion of either FKH1 or FKH2 does not renders the genes constitutive rather than simply shifting severely disrupt periodic expression of Clb2 cluster the timing of their expression (Sherwood et al., 1993). genes. However, simultaneous disruption of both genes Thus, it remains unclear whether expression outside the leads to constitutive basal transcription that is un- G1/S is mediated by SBF (positive control) or whether coupled from the cell cycle, suggesting that these Hir proteins are responsible for the difference in the transcription factors are functionally redundant (Zhu timing of transcription relative to other members of the et al., 2000; Hollenhorst et al., 2001). Yet, in vivo G1-specific gene cluster (negative control). The mechan- analysis of the CLB2 and other Clb2 cluster promoters ism by which Hir proteins are linked to the cell cycle is suggests that SFF sites are for the most part occupied by also unclear. One reasonable suggestion is that the Hir Fkh2 and not Fkh1 (Koranda et al., 2000; Hollenhorst proteins are direct CDK targets, like their mammalian et al., 2001). This is likely explained by the presence of homolog HirA (Hall et al., 2001), and that phosphor- binding sites for the MADS-box transcription factor ylation regulates their cell cycle-dependent function. Mcm1 in Clb2 cluster promoters and the cooperativity However, neither yeast repressor contains a full between Fkh2 and Mcm1 binding, a property not shared consensus Cdk phosphorylation site nor are they known with Fkh1 (Hollenhorst et al., 2001). The binding of to be direct targets for Cdk1. Fkh2 and Mcm1 to Clb2 cluster promoters does not The functional significance of S-phase-specific expres- explain the periodic expression of these genes since sion of genes of the MET cluster remains enigmatic (but occupancy by these factors is not cell cycle regulated. see below). The members of this family differ substan- The key to cell cycle regulation is Ndd1, a protein that tially both in degree of cell cycle-dependent fluctuation serves as a co-activator with Fkh2 (Loy et al., 1999; and their precise timing. Many members of the family Koranda et al., 2000) (Figure 2). appear to be targeted by the Met4, Met 28, Met32 and Ndd1 is expressed periodically during S phase and is Cbf1 transcription factors. All of these factors are turned over during mitosis (Loy et al., 1999). Without clearly associated with the transcriptional induction of an apparent DNA-binding domain, Ndd1 transactiva- MET genes in response to low adenosyl methionine tion activity depends on binding to the forkhead- (Thomas and Surdin-Kerjan, 1997). However, whether associated (FHA) domain of Fkh2 (Koranda et al., those factors, or some other regulator, mediate cell 2000). The timing of this binding, which correlates with cycle-dependent expression is unclear. transcription of Clb2 cluster genes, is mediated by Cdk1-dependent phosphorylation of Ndd1 (Reynolds et al., 2003). The primary cyclin responsible is Clb2 itself (Reynolds et al., 2003), leading to an apparent paradox: Regulation of the Clb2 cluster if activation of Clb2 transcription by Ndd1 depends on Clb2, how is the process initiated? Two simple Transcriptional array analysis has defined a group of 35 nonexclusive models provide a potential explanation. yeast genes that are transcribed roughly from the end of First, the basal level of Clb2 expression might be S phase until nuclear division (Cho et al., 1998; sufficient to prime the process, which would then Spellman et al., 1998). This set of genes has been termed rapidly accelerate due to a positive feedback loop. the ‘Clb2 cluster’ based on the CLB2 mitotic cyclin gene, Second, the process could be primed by another B-type which had previously been shown to exhibit these cyclin, not under Ndd1 control, for example, Clb3or mRNA kinetics (Ghiara et al., 1991; Surana et al., Clb4. Termination of Clb2 cluster transcription pre- 1991). Other members of this family include CLB1 sumably occurs when Ndd1 is degraded during mitosis (another mitotic cyclin gene), CDC5 (the yeast polo-like (Loy et al., 1999). Interestingly, whereas FKH1 and kinase homolog), CDC20 (a mitotic specificity factor for FKH2 are not essential (the double deletion is viable), the APC protein–ubiquitin ligase), and SWI5 and ACE2 Ndd1 is essential (Loy et al., 1999). However, deletion of (transcription factors required for late M-early G1- FKH2 renders Ndd1 nonessential (Koranda et al., 2000; specific gene expression). Interestingly, it was through Reynolds et al., 2003). This strongly suggests that, in analysis of the SWI5 promoter that insight into mediating the periodic expression of Clb2 cluster genes, regulation of this gene cluster was first obtained. A Fkh2 possesses both repressive and transactivating protein complex known as Swi5 factor (SFF) was shown activities, with Ndd1 binding serving as a functional to be capable of binding to specific elements in the SWI5 switch (Figure 2). Deletion analysis of Fkh2 has promoter (Lydall et al., 1991). More recent work on suggested a mechanism for Ndd1-mediated relief from SWI5 and other cluster genes, notably CLB2, has repression by Fkh2. Compared to Fkh1, Fkh2 contains revealed that SFF sites are binding sites for members a carboxy-terminal extension of approximately 280 of the forkhead family of transcription factors (Pic et al., amino acids. Deletion of this extension, similarly to 2000; Kumar et al., 2000). deletion of the entire FKH2 gene, renders Ndd1 Yeast contains several members of the ubiquitous nonessential (Reynolds et al., 2003). Therefore, this C- forkhead family of transcription factors, classified based terminal domain of Fkh2 presumably critical to repres- on their forkhead (winged-helix) DNA-binding do- sion can be neutralized by conformational changes mains. Two of these, Fkh1 and Fkh2, have been shown associated with Ndd1 binding. How the C-terminus of to be capable of binding to SFF sites in vitro and in vivo Fkh2 establishes repression of Clb2 cluster genes and

Oncogene Cell cycle-dependent transcription in yeast C Wittenberg and SI Reed 2750

? APC SBF/MBF Ndd1 Ndd1 Clb2/Cdk1 proteasome

Ndd1 P

FHA FHA FHA FHA Mcm1 Fkh2Mcm1 Fkh2Mcm1 Fkh2 Mcm1 Fkh2

Repressor complex Repressor complex Activator complex Repressor complex

G1/S S/G2 G2/M M/G1 Figure 2 Regulation of Clb2 cluster genes. Clb2 cluster genes are characterized by binding sites for Mcm1 and Fkh2 that are occupied continuously through the cell cycle. Genetic evidence suggests that this minimal complex is repressive. Activation depends on the co- activator Ndd1, which is synthesized during during late G1 and/or S phase, possibly through the action of SBF or MBF. However, binding of Ndd1 to Fkh2 depends on phosphorylation by the Clb2/Cdk1 kinase complex during G2. Genetic data suggest that binding of phosphorylated Ndd1 to the FHA domain of Fkh2 leads to a conformational change that converts Fkh2 from a repressor to a transcriptional activator. APC-mediated ubiquitylation and degradation of Ndd1 during mitosis restores the complex to its repressive state, terminating transcription of Clb2 cluster genes

how Ndd1 antogonizes this remains to be determined. In Spellman et al., 1998)). However, some proteins with no addition, the mechanism whereby, in the absence of ostensible G1-specific function (e.g., genes of the PHO Fkh2 and Ndd1, Fkh1 mediates cell cycle-regulated regulon) also exhibit this expression pattern (Cho et al., periodic expression of Clb2 cluster genes is not under- 1998; Spellman et al., 1998). stood (Reynolds et al., 2003). Presumably, a cell cycle- The largest group of genes exhibiting M-G1 periodi- regulated Fkh1 co-activator other than Ndd1 must be city are those driven by the MADS-box transcription involved, but this protein has not yet been identified. factor, Mcm1 (Nurrish and Treisman, 1995; Shore and The respective functions of Fkh1 and Fkh2 in regulation Sharrocks, 1995). Unlike the Clb2 cluster of genes that of Clb2 cluster transcription are further complicated by also require Mcm1 binding, the so-called MCM cluster the finding that these proteins have general nonredun- genes do not contain sites for Fkh1/2 binding. As dant roles in transcriptional elongation of a broad expected, therefore, deletion of FKH1 and FKH2 has no spectrum of yeast genes (Morillon et al., 2003). effect on these genes (Zhu et al., 2000). Instead, most of Although mutation of FKH1 and/or FKH2 leads to these genes contain a site for binding the homeodomain defects in phosphorylation of the large subunit of RNA repressors Yox1 and Yhp1 (Pramila et al., 2002) in close polymerase II, in polymerase movement from the 50–30 proximity to a palindromic site capable of binding an ends of ORFs, and in transcriptional termination, the Mcm1 dimer, known as an ECB for ‘early cell cycle box’ molecular basis for this is not known. Nevertheless, (McInerny et al., 1997) (although there is some post-initiation transcriptional functions of Fkh1 and disagreement on this issue; see Horak and Snyder, Fkh2 might contribute to Clb2 cluster periodic expres- 2002). In addition, Yox1 and Yhp1 can bind directly to sion. Interestingly, an Fkh1/Fkh2 homolog in mamma- Mcm1 (Pramila et al., 2002), presumably promoting lian cells, FoxM1, has been shown to have a role in cooperativity. It is the occupancy of these repressive transcription of the cyclin B1 gene (the Clb2 homolog) sites that blocks Mcm1-mediated transcription through as well as genes encoding other mitotic regulators most of the cell cycle, and it is the periodic transcription (Leung et al., 2001; Wang et al., 2002). Therefore, the of the genes encoding these presumably unstable role of transcription factors containing forkhead DNA- repressors that determines their functional interval. binding domains and their cognate DNA-binding sites Yox1 is expressed in mid-G1 through early S phase may constitute an ancient and highly conserved (Spellman et al., 1998; Horak and Snyder, 2002; Pramila regulatory motif for late cell cycle transcription. et al., 2002), whereas Yhp1 appears to be expressed later in the cell cycle (Cho et al., 1998; Spellman et al., 1998), leaving an interval from M phase to early G1 where these repressors are absent. During this window, Mcm1 Regulation of M-G1 genes can apparently drive transcription of these genes without DNA binding of additional activating proteins. A large number of yeast genes are expressed from M The second class of M-G1 expressed genes is referred phase to early G1 (Cho et al., 1998; Spellman et al., to as the Sic1 cluster. Interestingly, periodic expression 1998). Many of these are required for G1 functions (e.g., of these genes is affected by deletion of FKH1 and FKH2 MCM proteins involved in prereplication complex (Zhu et al., 2000). Yet, Fkh1 and Fkh2 do not bind the assembly, transcription factors required for G1 gene promoters of these genes. The basis for the indirect expression and proteins involved in the yeast mating requirement for FKH genes is the assignment of response, which occurs during G1 (Cho et al., 1998; transcription factors Swi5 and Ace2 to the Clb2 cluster

Oncogene Cell cycle-dependent transcription in yeast C Wittenberg and SI Reed 2751 (Nasmyth et al., 1987; Spellman et al., 1998). Genes of by activating the Cdk inhibitor Pho81 under low- the Sic1 cluster contain related consensus sites that bind phosphate conditions (Lenburg and O’Shea, 1996). Ace2 and Swi5 (Zhu et al., 2000). The relationship Inactivation of Pho85 allows nuclear retention of between Ace2 and Swi5, however, is complex. Although transcription factor Pho4, which, alone or in concert both transcription factors recognize the same set of sites with the homeodomain protein Pho2, drives transcrip- on target genes, their effect on such genes can vary from tion of more than 20 PHO regulon genes (Komeili and activation to repression (Dohrmann et al., 1996; O’Shea, 1999). M/G1 activation of PHO regulon genes McBride et al., 1999). The explanation for this requires the Pho85 inhibitor Pho81 and transcription differential behavior appears to reside in the ability of factors Pho2 and Pho4, indicating that the phosphate these factors to bind co-activators and co-repressors sensing pathway is involved (Neef and Kladde, 2003). that are also targeted to genes of the Sic1 cluster. For Further analysis has suggested that the timing is a result example, the gene encoding the endonuclease HO of periodic storage and depletion of polyphosphate involved in mating type switching appears to be specific pools, ultimately leading to low intracellular phosphate for Swi5, presumably because it binds cooperatively levels at the end of the cell cycle that trigger the with the homeodomain protein Pho2, which also binds phosphate starvation response (Neef and Kladde, 2003). to the HO promoter (McBride et al., 1999). Ace2 cannot interact with Pho2. Conversely, the CTS1 gene, which is specifically activated by Ace2, contains a putative repressor-binding site that prevents activation by Swi5 Hybrid promoters but not Ace2 (McBride et al., 1999; Doolin et al., 2001). A third group of genes that are activated during the A significant number of cell cycle-regulated genes do not M-G1 window are those that are normally induced by precisely fit the criteria used to define any of the major mating pheromone (the MAT cluster in Spellman et al., clusters. In most cases, this is because the promoters of 1998). It has been shown that the periodic expression of these genes contain binding sites that are characteristic these genes depends on a DNA sequence known as the of more than one cluster (Kato et al., 2004). pheromone response element (PRE) and a PRE-binding Although the majority of promoters activated speci- transcription factor, Ste12 (Oehlen et al., 1996). Under fically during G1 are bound by SBF, MBF, or both, it is conditions of receptor-mediated activation of the clear that many of those genes are also bound by other pheromone response pathway, the MAP kinase homo- transcription factors including the fork-head transcrip- log Fus3promotes activation of Ste12 by phosphorylat- tion factors Fkh1 and Fkh2, and Stb1 (Lee et al., 2002). ing and inactivating two co-repressors of Ste12 known As the data supporting these interactions is derived as Dig1 and Dig2 (Bardwell et al., 1998; van Drogen largely from genome-wide analysis of asynchronous et al., 2001; Breitkreutz et al., 2003; Kusari et al., 2004). populations, their impact on the pattern of expression of However, whether Dig1/Dig2 targeting by Fus3is those genes remains unclear. Establishing whether the involved in M-G1 activation of Ste12 is not known. binding of these factors has regulatory significance or One plausible idea is that genes encoding elements rate- whether they act simultaneously or sequentially awaits limiting in the signal transduction pathway, many of analysis in synchronized populations of wild-type and which have PRE elements themselves, also contain other mutant cells. cell cycle-regulated elements, which would prime the Hybrid promoters have also been observed in the system and set up a positive feedback loop. In effect, an Clb2 cluster. For example, CDC20 is grouped in the increase in basal signaling would then increase the Clb2 cluster based upon its pattern of transcription in transcription of rate-limiting signaling genes, further wild-type cells and its well-documented Fkh2-binding amplifying the basal signal. Intriguingly, STE2, the sites (Spellman et al., 1998; Hollenhorst et al., 2001). alpha-factor receptor gene at the apex of the pheromone However, CDC20 differs from most other Clb2 cluster response pathway, contains a hybrid promoter (see genes in several important respects. First, the interval of below), with PRE elements (driven by Ste12) and ECBs mRNA accumulation is more restricted than most Clb2 (driven by Mcm1) (Hwang-Shum et al., 1991). It is cluster genes, with peak expression coming relatively therefore possible that transcription of STE2 driven by late (Zhu et al., 2000). Second, CDC20 maintains Mcm1 initiates an increase in basal signaling down the periodic transcription even in an fkh1 fkh2 null back- pheromone response pathway leading to increased ground, although the kinetics of expression are shifted Ste12-mediated STE2 transcription. In effect, Mcm1 to an M/G1 pattern characteristic of the MCM cluster priming of STE2 transcription would set up a Ste12- (Zhu et al., 2000). Analysis of the sequences surrounding driven positive feedback loop affecting all PRE-contain- the CDC20 Mcm1-binding sites (ECBs) reveals at least ing promoters. one consensus and possibly several for binding of the A fourth group of M-G1-activated genes are surpris- Yox1 repressor (Pramila et al., 2002). Thus, transcrip- ingly those of the PHO regulon (Cho et al., 1998; tion of CDC20 is most likely positively regulated at the Spellman et al., 1998). These genes encoding proteins G2–M boundary by Fkh2 in conjunction with Ndd1 and involved in scavenging and transporting phosphate are Mcm1, but negatively regulated by Yox1 and Yhp1 until coordinately induced by phosphate starvation (Lenburg the M phase. and O’Shea, 1996). The phosphate-sensing pathway Another example of a hybrid promoter is that of the ultimately impinges on a cyclin-dependent kinase Pho85 FAR1 gene. Far1, a protein required for both cell cycle

Oncogene Cell cycle-dependent transcription in yeast C Wittenberg and SI Reed 2752 arrest and polarized growth in response to mating in PMA1 expression during G2 is most likely assoc- pheromone, accumulates from mitosis through G1 in iated with potentiation of a transient increase in non-mating cells (Oehlen et al., 1996). Examination of intracellular Ca2 þ required for mitosis in many organ- the FAR1 promoter reveals both an ECB, presumably isms (Whitaker, 1997). In conclusion, it appears that cell regulated by Mcm1, Yox1, and Yhp1, and PREs cycle regulation of transcription of genes not directly regulated by Ste12 (Oehlen et al., 1996). Elimination linked to cell cycle functions can usually be rationalized of Mcm1 results in loss of mitotic expression of FAR1, in the context of supporting or potentiating cell cycle whereas elimination of Ste12 results in loss of G1 functions indirectly. expression (Oehlen et al., 1996). Thus, utilizing two transactivating systems allows FAR1 expression to be extended over a broader cell cycle interval than would Maintaining cell cycle organization: interactions between be permitted by either system alone. The gene encoding transcriptional programs the alpha-factor pheromone receptor STE2 appears to be regulated in a similar fashion (Hwang-Shum et al., Sequential waves of transcription are critical for 1991; Spellman et al., 1998). maintaining the organization of cell cycle events. The genome-wide analysis of transcription factor binding, gene expression arrays, and bioinformatics analysis of Cell cycle regulation of genes not ostensibly involved in promoter motifs along with gene-specific studies of cell cycle functions transcriptional regulation have led to the visualization of the network of regulatory interactions that promote Arguably, virtually all metabolic and structural func- and maintain cell cycle-regulated transcription (Futcher, tions in microbial organisms either directly or indirectly 2002; Lee et al., 2002; Kato et al., 2004). Our current impinge on proliferation. Since the success of most understanding of those interactions is depicted in microorganisms is measured in terms of utilizing Figure 3. Whereas many of these regulatory interactions available resources to outcompete other organisms, the are established by experimental observation, some are link between nutrient acquisition, nutrient assimilation, simply inferred from genome-wide location analysis and energy metabolism, and cell cycle control should not be require experimental confirmation. surprising. In some cases, the periodic nature of gene Integration of the cell cycle-dependent transcriptional expression can be rationalized in this context. For regulatory network appears to be imposed via three example, genes of the PHO regulon are induced at the general mechanisms. First, the gene encoding a M–G1 boundary (Cho et al., 1998; Spellman et al., 1998). Most of the proteins encoded by PHO regulon genes are directly involved in scavenging phosphate from the environment and internalizing it. Since the availability of phosphate is one of the determinants that yeast cells use to make a decision on initiating a new cell cycle during G1, perhaps expression of PHO regulon genes at the M/G1 boundary, thereby mobilizing all available environmental phosphate, constitutes a means of optimizing the reliability of this decision-making process. The MET regulon genes serve as another example. These genes, encoding enzymes of the methionine biosynthetic pathway, are normally induced in response to methionine depletion in the environment (Thomas and Surdin-Kerjan, 1997). However, they are also induced periodically during the S phase (see above) (Cho et al., 1998; Spellman et al., 1998). The most likely rationale for this pattern is that methionine is the precursor for S-adenosyl methionine, the one-carbon donor essential for the biosynthesis of dTTP required for genome replication during the S phase. Other periodically expressed genes are more difficult to rationalize. The gene encoding the plasma membrane ATPase PMA1 is expressed periodically during G2, Figure 3 Integration of transcriptional regulatory networks similarly to Clb2 cluster genes (Cho et al., 1998; during the cell cycle. This diagram depicts the regulatory Spellman et al., 1998). Pma1 functions as an ATP- interactions occurring between cell cycle-specific gene clusters as driven proton efflux pump, presumably regulating discussed in the text. Transcription factors are denoted by ovals, intracellular pH, a parameter not obviously linked to cell cycle regulatory proteins are denoted by circles, and transcriptional repressors are denoted by squares. Dashed lines represent cell cycle processes. However, Pma1 has also been gene expression and solid lines represent direct regulatory 2 þ shown to be required for maintaining intracellular Ca interactions. Gray areas indicate intervals of transcription factor levels (Withee et al., 1998). Therefore, an increase activity

Oncogene Cell cycle-dependent transcription in yeast C Wittenberg and SI Reed 2753 transcription factor (activator or repressor) can be appears to be minimal and most of the cell cycle is directly regulated in the context of the cell cycle- devoid of cell cycle-regulated transcription (Rustici et al., dependent transcription program. For example, 2004). Why two yeasts inhabiting similar environmental NDD1, encoding an essential component of the active niches should exhibit such disparate transcriptional form of the Fkh2 transcription complex, is a member of programs is difficult to rationalize, but suggests the the G1-specific gene cluster (Spellman et al., 1998; Loy advantages gained by extensive cell cycle programming et al., 1999) and is required for activation of the Clb2 of transcription can be achieved by other regulatory gene cluster (Loy et al., 1999; Koranda et al., 2000). strategies. Interestingly, it was found that a core set of Similarly, SWI4, which encodes the DNA-binding approximately 40 genes exhibited cell cycle regulation of component of the G1-specific transcriptional activator transcription in both organisms, suggesting that this SBF, is expressed as a member of the M/G1 gene cluster constitutes a set of genes for which cell cycle regulation (McInerny et al., 1997; Spellman et al., 1998). is critical rather than simply advantageous (Rustici et al., The second potential mode of coupling is via cell 2004). On the other hand, genome-wide analysis of cycle-dependent expression of an enzymatic activity transcription through the cell cycle in human cells required for regulation of transcription of another gene indicates that minimally 800 genes are cell cycle-regulated, cluster. Regulated expression of cyclin genes provides similarly to S. cerevisiae (Cho et al., 2001; Whitfield et al., the best examples of this mechanism. Expression of the 2002). Although, as expected, most of the 40 or so core CLB2 gene, the canonical member of the G2 phase gene genes that exhibit cell cycle regulation in both S. cerevisiae family, leads to activation of Clb2/Cdk1 which binds to and S. pombe are also cell cycle regulated in proliferating Swi4 and, thereby, inactivates SBF (Amon et al., 1993; human cells, the human data suggest that, as in S. Siegmund and Nasmyth, 1996). The same Cdk complex cerevisiae, many functions only peripherally related to the phosphorylates Ndd1 to activate expression of the Clb2 cell cycle at best are part of the cell cycle transcriptional cluster (Reynolds et al., 2003). Mcm1-dependent ex- program, indeed constituting the majority of cell cycle- pression of CLN3 during M/G1 provides another regulated genes (Cho et al., 2001; Whitfield et al., 2002). example of this form of regulation (McInerny et al., These processes include signal transduction, mRNA 1997; Pramila et al., 2002). The activation of Cln3/Cdk1 metabolism, protein translation, energy metabolism, ionic promotes phosphorylation and inactivation of Whi5, homeostasis and many other categories. Therefore, it thereby activating G1-specific transcription (Costanzo would appear that transcriptionally toggling diverse et al., 2004; de Bruin et al., 2004). cellular processes and functions to the cell cycle program Finally, a third observed mechanism involves the is the rule rather than the exception. regulation of one cluster of genes by a transcriptional In addition to the pervasiveness of cell cycle-regulated repressor expressed as a member of another gene transcription, there are interesting mechanistic parallels cluster. Perhaps the best example of this form of regu- that imply that conservative pressures are at work. The lation is the restriction of Mcm1-mediated gene expres- recent discovery of Whi5 as a G1-specific transcriptional sion to M/G1 by the transcriptional repressors Yox1 repressor belies a mechanistic conservation of transcrip- and Yhp1, which are expressed during G1/S and G2/M tional regulatory mechanisms. Although Whi5 shares no phases, respectively. Yox1 is most likely a target of recognizable sequence homology to any of the, so- SBF (Spellman et al., 1998; Horak and Snyder, 2002; called, ‘pocket proteins’ (pRb, p107 and p130), as a Pramila et al., 2002). repressor of G1-specific transcription that is antago- nized by G1 cyclin/Cdk, it can be considered a functional analog arising through convergent evolution (Figure 1). Similarly, the utilization of forkhead domain Conclusions transcription factors for G2-specific transcription in both yeasts and metazoans underscores the conservative Yeast genomic analysis has provided a wealth of data, tendancy of mechanisms governing cell cycle-regulated including the observation that a large number of genes transcription. As yeast lacks some of the extensive are transcribed according to a cell cycle program (Cho redundancy that plagues the study of transcriptional et al., 1998; Spellman et al., 1998). While the fact that processes in animal cells, it will likely continue to prove expression of a significant number of genes is regulated a useful model for the dissection of mechanisms in this fashion is not surprising, perhaps the magnitude governing cell cycle-mediated transcriptional control. of this cohort (>10%) is. Whether this is a phenomenon related specifically to budding yeast and its lifestyle remains to be determined. Indeed recent genome-wide Acknowledgements analysis of cell cycle transcription in fission yeast (S. We thank all of the investigators whose research has pombe) indicates that a much lower percentage of genes contributed to our understanding of cell cycle-regulated are cell cycle regulated in that organism (136 compared transcription in yeast, and apologize to those who have been overlooked in the interest of brevity. We thank Rob de Bruin to 800) (Rustici et al., 2004). Furthermore, cell cycle for comments on the manuscript and members of the TSRI regulation of transcription appears less complex and Cell Cycle Group for their interest and support. Research relegated to a limited interval. Whereas in S. cerevisiae, concerning cell cycle-regulated transcription in our own waves of transcription around the cell cycle are linked, laboratories is supported by funding from the National forming a closed circuit (see above), in S. pombe, linkage Institutes of Health.

Oncogene Cell cycle-dependent transcription in yeast C Wittenberg and SI Reed 2754 References

Amon A. (2002). Methods Enzymol., 351, 457–467. Kato M, Hata N, Banerjee N, Futcher B and Zhang MQ. Amon A, Tyers M, Futcher B and Nasmyth K. (1993). Cell, (2004). Genome Biol., 5, R56. 74, 993–1007. Koch C, Moll T, Neuberg M, Ahorn H and Nasmyth K. Bardwell L, Cook JG, Zhu-Shimoni JX, Voora D and Thorner (1993). Science, 261, 1551–1557. J. (1998). Proc. Natl. Acad. Sci. USA, 95, 15400–15405. Komeili A and O’Shea EK. (1999). Science, 284, 977–980. Breeden L. (1996). Curr. Top. Microbiol. Immunol., 208, 95– Koranda M, Schleiffer A, Endler L and Ammerer G. (2000). 127. Nature, 406, 94–98. Breitkreutz A, Boucher L, Breitkreutz BJ, Sultan M, Jurisica I Kumar R, Reynolds DM, Shevchenko A, Goldstone SD and and Tyers M. (2003). Genetics, 165, 997–1015. Dalton S. (2000). Curr. Biol., 10, 896–906. Cho RJ, Campbell MJ, Winzeler EA, Steinmetz L, Conway A, Kusari AB, Molina DM, Sabbagh Jr W, Lau CS and Bardwell Wodicka L, Wolfsberg TG, Gabrielian AE, Landsman D, L. (2004). J. Cell Biol., 164, 267–277. Lockhart DJ and Davis RW. (1998). Mol. Cell, 2, 65–73. Lee TI, Rinaldi NJ, Robert F, Odom DT, Bar-Joseph Z, Cho RJ, Huang M, Campbell MJ, Dong H, Steinmetz L, Gerber GK, Hannett NM, Harbison CT, Thompson CM, Sapinoso L, Hampton G, Elledge SJ, Davis RW and Simon I, Zeitlinger J, Jennings EG, Murray HL, Gordon Lockhart DJ. (2001). Nat. Genet., 27, 48–54. DB, Ren B, Wyrick JJ, Tagne JB, Volkert TL, Fraenkel E, Costanzo M, Nishikawa JL, Tang X, Millman JS, Schub O, Gifford DK and Young RA. (2002). Science, 298, 799–804. Breitkreuz K, Dewar D, Rupes I, Andrews B and Tyers M. Lenburg ME and O’Shea EK. (1996). Trends Biochem. Sci., 21, (2004). Cell, 117, 899–913. 383–387. Costanzo M, Schub O and Andrews B. (2003). Mol. Cell. Biol., Leung TW, Lin SS, Tsang AC, Tong CS, Ching JC, Leung 23, 5064–5077. WY, Gimlich R, Wong GG and Yao KM. (2001). FEBS Cross FR. (1988). Mol. Cell. Biol., 8, 4675–4684. Lett., 507, 59–66. Cross FR and Tinklenberg AH. (1991). Cell, 65, 875–883. Loy CJ, Lydall D and Surana U. (1999). Mol. Cell. Biol., 19, de Bruin RA, McDonald WH, Kalashnikova TI, Yates III J 3312–3327. and Wittenberg C. (2004). Cell, 117, 887–898. Lydall D, Ammerer G and Nasmyth K. (1991). Genes Dev., 5, Di Como CJ, Chang H and Arndt KT. (1995). Mol. Cell. Biol., 2405–2419. 15, 1835–1846. MacKay VL, Mai B, Waters L and Breeden LL. (2001). Mol. Dirick L, Bohm T and Nasmyth K. (1995). EMBO J., 14, Cell. Biol., 21, 4140–4148. 4803–4813. Marini NJ and Reed SI. (1992). Genes Dev., 6, 557–567. Dirick L and Nasmyth K. (1991). Nature, 351, 754–757. McBride HJ, Yu Y and Stillman DJ. (1999). J. Biol. Chem., Dohrmann PR, Voth WP and Stillman DJ. (1996). Mol. Cell. 274, 21029–21036. Biol., 16, 1746–1758. McInerny CJ, Partridge JF, Mikesell GE, Creemer DP and Doolin MT, Johnson AL, Johnston LH and Butler G. (2001). Breeden LL. (1997). Genes Dev., 11, 1277–1288. Mol. Microbiol., 40, 422–432. Miller ME and Cross FR. (2000). Mol. Cell. Biol., 20, 542–555. Dyson N. (1998). Genes Dev., 12, 2245–2262. Morgan BA, Bouquin N, Merrill GF and Johnston LH. Edgington NP and Futcher B. (2001). J. Cell Sci., 114, 4599– (1995). EMBO J., 14, 5679–5689. 4611. Morillon A, O’Sullivan J, Azad A, Proudfoot N and Mellor J. Epstein CB and Cross FR. (1994). Mol. Cell. Biol., 14, 2041– (2003). Science, 300, 492–495. 2047. Nash R, Tokiwa G, Anand S, Erickson K and Futcher AB. Futcher B. (2002). Curr. Opin. Cell. Biol., 14, 676–683. (1988). EMBO J., 7, 4335–4346. Gallego C, Gari E, Colomina N, Herrero E and Aldea M. Nasmyth K, Seddon A and Ammerer G. (1987). Cell, 49, 549– (1997). EMBO J., 16, 7196–7206. 558. Gari E, Volpe T, Wang H, Gallego C, Futcher B and Aldea M. Neef DW and Kladde MP. (2003). Mol. Cell. Biol., 23, 3788– (2001). Genes Dev., 15, 2803–2808. 3797. Geymonat M, Spanos A, Wells GP, Smerdon SJ and Sedgwick Nevins JR. (2001). Hum. Mol. Genet., 10, 699–703. SG. (2004). Mol. Cell. Biol., 24, 2277–2285. Ng HH, Robert F, Young RA and Struhl K. (2002). Genes Ghiara JB, Richardson HE, Sugimoto K, Henze M, Lew DJ, Dev., 16, 806–819. Wittenberg C and Reed SI. (1991). Cell, 65, 163–174. Nurrish SJ and Treisman R. (1995). Mol. Cell. Biol., 15, 4076– Haase SB and Reed SI. (1999). Nature, 401, 394–397. 4085. Hall C, Nelson DM, Ye X, Baker K, DeCaprio JA, Seeholzer Oehlen LJ, McKinney JD and Cross FR. (1996). Mol. Cell. S, Lipinski M and Adams PD. (2001). Mol. Cell. Biol., 21, Biol., 16, 2830–2837. 1854–1865. Osley MA, Gould J, Kim S, Kane MY and Hereford L. (1986). Hall DD, Markwardt DD, Parviz F and Heideman W. (1998). Cell, 45, 537–544. EMBO J., 17, 4370–4378. Partridge JF, Mikesell GE and Breeden LL. (1997). J. Biol. Ho Y, Mason S, Kobayashi R, Hoekstra M and Andrews B. Chem., 272, 9071–9077. (1997). Proc. Natl. Acad. Sci. USA, 94, 581–586. Parviz F, Hall DD, Markwardt DD and Heideman W. (1998). Hollenhorst PC, Pietz G and Fox CA. (2001). Genes Dev., 15, J. Bacteriol., 180, 4508–4515. 2445–2456. Pic A, Lim FL, Ross SJ, Veal EA, Johnson AL, Sultan MR, Horak CE and Snyder M. (2002). Funct. Integr. Genomics, 2, West AG, Johnston LH, Sharrocks AD and Morgan BA. 171–180. (2000). EMBO J., 19, 3750–3761. Hwang-Shum JJ, Hagen DC, Jarvis EE, Westby CA and Polymenis M and Schmidt EV. (1997). Genes Dev., 11, 2522– Sprague Jr GF. (1991). Mol. Gen. Genet., 227, 197–204. 2531. Iyer VR, Horak CE, Scafe CS, Botstein D, Snyder M and Pramila T, Miles S, GuhaThakurta D, Jemiolo D and Breeden Brown PO. (2001). Nature, 409, 533–538. LL. (2002). Genes Dev., 16, 3034–3045. Jorgensen P, Nishikawa JL, Breitkreutz BJ and Tyers M. Queralt E and Igual JC. (2003). Mol. Cell. Biol., 23, 3126– (2002). Science, 297, 395–400. 3140.

Oncogene Cell cycle-dependent transcription in yeast C Wittenberg and SI Reed 2755 Reynolds D, Shi BJ, McLean C, Katsis F, Kemp B and Dalton Tyers M, Tokiwa G, Nash R and Futcher B. (1992). EMBO J., S. (2003). Genes Dev., 17, 1789–1802. 11, 1773–1784. Rustici G, Mata J, Kivinen K, Lio P, Penkett CJ, Burns G, Ubersax JA, Woodbury EL, Quang PN, Paraz M, Blethrow Hayles J, Brazma A, Nurse P and Bahler J. (2004). Nat. JD, Shah K, Shokat KM and Morgan DO. (2003). Nature, Genet., 36, 809–817. 425, 859864. Sherwood PW, Tsang SV and Osley MA. (1993). Mol. Cell. van Drogen F, Stucke VM, Jorritsma G and Peter M. (2001). Biol., 13, 28–38. Nat. Cell Biol., 3, 1051–1059. Shore P and Sharrocks AD. (1995). Eur. J. Biochem., 229, 1– Wang H, Gari E, Verges E, Gallego C and Aldea M. (2004). 13. EMBO J., 23, 180–190. Sidorova JM, Mikesell GE and Breeden LL. (1995). Mol. Biol. Wang X, Kiyokawa H, Dennewitz MB and Costa RH. (2002). Cell, 6, 1641–1658. Proc. Natl. Acad. Sci. USA, 99, 16881–16886. Siegmund RF and Nasmyth KA. (1996). Mol. Cell. Biol., 16, Whitaker M. (1997). Prog. Cell Cycle Res., 3, 261–269. 2647–2655. Whitﬁeld ML, Sherlock G, Saldanha AJ, Murray JI, Ball CA, Simon I, Barnett J, Hannett N, Harbison CT, Rinaldi NJ, Alexander KE, Matese JC, Perou CM, Hurt MM, Brown Volkert TL, Wyrick JJ, Zeitlinger J, Gifford DK, Jaakkola PO and Botstein D. (2002). Mol. Biol. Cell, 13, 1977–2000. TS and Young RA. (2001). Cell, 106, 697–708. Wijnen H and Futcher B. (1999). Genetics, 153, 1131–1143. Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Wijnen H, Landman A and Futcher B. (2002). Mol. Cell. Biol., Eisen MB, Brown PO, Botstein D and Futcher B. (1998). 22, 4402–4418. Mol. Biol. Cell, 9, 3273–3297. Withee JL, Sen R and Cyert MS. (1998). Genetics, 149, 865– Struhl K. (1983). Nature, 305, 391–397. 878. Stuart D and Wittenberg C. (1995). Genes Dev., 9, 2780–2794. Wittenberg C and Reed SI. (1991). Crit. Rev. Eukaryot. Gene Surana U, Robitsch H, Price C, Schuster T, Fitch I, Futcher Expr., 1, 189–205. AB and Nasmyth K. (1991). Cell, 65, 145–161. Zhang J, Schneider C, Ottmers L, Rodriguez R, Day A, Thomas D and Surdin-Kerjan Y. (1997). Microbiol. Mol. Biol. Markwardt J and Schneider BL. (2002). Curr. Biol., 12, Rev., 61, 503–532. 1992–2001. Tyers M, Tokiwa G and Futcher B. (1993). EMBO J., 12, Zhu G, Spellman PT, Volpe T, Brown PO, Botstein D, Davis 1955–1968. TN and Futcher B. (2000). Nature, 406, 90–94.

Oncogene