Transcription patterns shift dramatically as cells transit from one phase of the cell cycle to another. To better define this transcriptional circuitry, we collected new microarray data across the cell cycle of budding yeast. The combined analysis of these data with three other cell cycle data sets identifies hundreds of new highly periodic transcripts and provides a weighted average peak time for each transcript. Using these data and phylogenetic comparisons of promoter sequences, we have identified a late S-phase-specific promoter element. This element is the binding site for the forkhead protein Hcm1, which is required for its cell cycle-specific activity. Among the cell cycle-regulated genes that contain conserved Hcm1-binding sites, there is a significant enrichment of genes involved in chromosome segregation, spindle dynamics, and budding. This may explain why Hcm1 mutants show 10-fold elevated rates of chromosome loss and require the spindle checkpoint for viability. Hcm1 also induces the M-phase-specific transcription factors FKH1, FKH2, and NDD1, and two cell cycle-specific transcriptional repressors, WHI5 and YHP1. As such, Hcm1 fills a significant gap in our understanding of the transcriptional circuitry that underlies the cell cycle.[Keywords: Hcm1; cell cycle; transcription; genome stability; forkhead; yeast] Supplemental material is available at http://www.genesdev.org.

Cyclin-dependent kinases (Cdks) drive the cell cycle in all eukaryotic cells. In budding yeast, Cdk1 (Cdc28) expression is constant, but cyclin transcription, stability, and activity are regulated across the cell cycle (Miller and Cross 2001). These multiple levels of regulation result in the ordered appearance of different G1 (Cln)-and B-type (Clb) cyclins, which direct the phase-specific localization and/or substrate specificity of the kinase. There is a critical distinction between G1 phase and the rest of the cell cycle, in that G1 is expandable in response to the environment (Rupe 2002). The length of G1 is influenced by age, growth conditions, and the size of the cell (Hartwell and Unger 1977;Johnston et al. 1979). In contrast, once the cells exit G1, the length of the rest of the cycle is fairly constant (Jagadish and Carter 1977), even after severe nutrient limitation (Johnston et al. 1977). Accumulation of G1 cyclins (Clns) is rate-limiting for the G1 to S transition, and Clns are regulated at virtually every level (Wittenberg et al. 1990;Gallego et al. 1997;Polymenis and Schmidt 1997;MacKay et al. 2001;Newcomb et al. 2002). However, one of the great remaining mysteries is what triggers the rapid accumulation of Clns and causes the irreversible transition into S phase in the normal mitotic cycle.Entry into G1 requires that Clb kinase activity be eliminated (Zachariae and Nasmyth 1999). Clb kinase activity decays due to cessation of CLB transcription, targeted proteolysis of the Clbs by the anaphase-promoting complex (APC), and the M/G1-specific expression of an inhibitory subunit, Sic1, which inactivates Clb/Cdk complexes. Low Clb kinase activity allows the nuclear localization and assembly of Cdc6 and Mcm2-7 onto origin DNA to form the prereplication complexes (PRCs; Tye 1999). These PRC components are transcribed coordinately at the M/G1 boundary, and the assembly of this highly conserved complex sets the stage for DNA replication. Once the PRCs are formed, Clb kinases are required to initiate replication. This is brought about by the accumulation of Cln/Cdk complexes, which phosphorylate and promote the degradation of Sic1 (Schneider et al. 1996;Tyers 1996;Nash et al. 2001) and restore Clb kinase activity.Accumulation of the G1 cyclins requires the activation of Cln3/Cdk. This kinase is uniquely capable of activating two late G1-specific transcription complexes (SBF and MBF;Dirick et al. 1995;Stuart and Wittenberg 1996). Once activated, SBF and MBF cause a burst of transcription of the late G1 cyclins CLN1 and CLN2, and many other genes required for S phase. The burst of CLN1 and CLN2 transcription is delayed under conditions that prolong G1 (Sillje et al. 1997). This indicates that Cln3/Cdk and/or the transcription factors (SBF and MBF) are the likely targets of G1 regulation. Cold Spring Harbor Laboratory Press on May 9, 2018 -Published by genesdev.cshlp.org Downloaded from

The transition to quiescence in budding yeast involves highly asymmetric cell divisions and elaborate cell wall fortifications that can be followed by flow cytometry. Posttranscriptional regulators Ssd1V, Mpt5, and Lsm1 are important for this transition.

Pure populations of quiescent yeast can be obtained from stationary phase cultures that have ceased proliferation after exhausting glucose and other carbon sources from their environment. They are uniformly arrested in the G1 phase of the cell cycle, and display very high thermo-tolerance and longevity. We find that G1 arrest is initiated before all the glucose has been scavenged from the media. Maintaining G1 arrest requires transcriptional repression of the G1 cyclin, CLN3, by Xbp1. Xbp1 is induced as glucose is depleted and it is among the most abundant transcripts in quiescent cells. Xbp1 binds and represses CLN3 transcription and in the absence of Xbp1, or with extra copies of CLN3, cells undergo ectopic divisions and produce very small cells. The Rad53-mediated replication stress checkpoint reinforces the arrest and becomes essential when Cln3 is overproduced. The XBP1 transcript also undergoes metabolic oscillations under glucose limitation and we identified many additional transcripts that oscillate out of phase with XBP1 and have Xbp1 binding sites in their promoters. Further global analysis revealed that Xbp1 represses 15% of all yeast genes as they enter the quiescent state and over 500 of these transcripts contain Xbp1 binding sites in their promoters. Xbp1-repressed transcripts are highly enriched for genes involved in the regulation of cell growth, cell division and metabolism. Failure to repress some or all of these targets leads xbp1 cells to enter a permanent arrest or senescence with a shortened lifespan.

The transcription factor Mcm1 is regulated by adjacent binding of a variety of different factors regulating the expression of cell-type-specific, cell cycle-specific, and metabolic genes. In this work, we investigate a new class of Mcm1-regulated promoters that are cell cycle regulated and peak in late M-early G 1 phase of the cell cycle via a promoter element referred to as an early cell cycle box (ECB). Gel filtration experiments indicate that the ECB-specific DNA binding complex is over 200 kDa in size and includes Mcm1 and at least one additional protein. Using DNase I footprinting in vitro, we have observed protection of the ECB elements from the CLN3, SWI4, CDC6, and CDC47 promoters, which includes protection of the 16-bp palindrome to which Mcm1 dimers are known to bind as well as protection of extended flanking sequences. These flanking sequences influence the stability and the variety of complexes that form on the ECB elements, and base substitutions in the protected flank affect transcriptional activity of the element. Chromatin immunoprecipitations show that Mcm1 binds in vivo to ECB elements throughout the cell cycle and that binding is sensitive to carbon source changes.Mcm1, its mammalian counterpart (serum response factor), and other members of the MADS box family of transcription factors share a 56-amino-acid MADS box, carrying a conserved DNA binding and dimerization domain (42). They are combinatorial transcription factors in that they typically bind adjacent to and derive their regulatory specificity from other DNA binding and/or accessory factors (3,20,42). Figure 1 depicts four classes of transcription complexes that include Mcm1. Depending upon the context, Mcm1 can be a component of an activating or a repressing complex, and these complexes can be regulated by specific events in the cell cycle or in response to internal or external cues. For example, repression of a-specific genes in ␣ haploid and a/␣ diploid cells involves cooperative binding of ␣2 and Mcm1 to adjacent binding sites (22,39). Then ␣2 recruits the Ssn6/Tup1 repressor complex to the site and prevents transcription (16). In contrast, ␣-specific genes are induced by Mcm1 and another ␣-specific transcription factor, ␣1 (43). In a cells, ␣2 is not produced, so the repressing complex cannot be formed. In that case, Mcm1 cooperates with another haploid-specific transcription factor, Ste12, to activate transcription and confer pheromone responsiveness to these promoters (12,14).Mcm1 is also involved in the regulation of arginine metabolism (not shown), where it forms a complex with Arg82 and two other DNA binding proteins: Arg80 and -81 (13, 33). The presence of arginine is sensed by Arg82 and results in the activation of transcription of catabolic enzymes and repression of anabolic enzymes, depending upon the context. Arg82 has recently been identified as an inositol-1,4,5-triphosphate kinase, and this discovery suggests a direct connection between the lipid signaling cascade and gene regulation in yeast (35).Mcm1 also plays a rol...

Development, tissue renewal and long term survival of multi-cellular organisms is dependent upon the persistence of stem cells that are quiescent, but retain the capacity to re-enter the cell cycle to self-renew, or to produce progeny that can differentiate and re-populate the tissue. Deregulated release of these cells from the quiescent state, or preventing them from entering quiescence, results in uncontrolled proliferation and cancer. Conversely, loss of quiescent cells, or their failure to re-enter cell division, disrupts organ development and prevents tissue regeneration and repair. Understanding the quiescent state and how cells control the transitions in and out of this state is of fundamental importance. Investigations into the mechanics of G1 arrest during the transition to quiescence continue to identify striking parallels between the strategies used by yeast and mammals to regulate this transition. When cells commit to a stable but reversible arrest, the G1/S genes responsible for promoting S phase must be inhibited. This process, from yeast to humans, involves the formation of quiescence-specific complexes on their promoters. In higher cells these so-called DREAM complexes of E2F4/DP/RBL/MuvB recruit the highly conserved histone deacetylase HDAC1, which leads to local histone deacetylation and repression of S phase-promoting transcripts. Quiescent yeast cells also show pervasive histone deacetylation by the HDAC1 counterpart Rpd3. In addition, these cells contain quiescence-specific regulators of G1/S genes: Msa1 and Msa2, which can be considered components of the yeast equivalent of the DREAM complex. Despite a lack of physical similarities, the goals and the strategies used to achieve a reversible transition to quiescence are highly conserved. This motivates a detailed study of this process in the simple model organism: budding yeast.

Budding yeast begin the transition to quiescence by prolonging G1 and accumulating limited nutrients. They undergo asymmetric cell divisions, slow cellular expansion, acquire significant stress tolerance and construct elaborate cell walls. These morphologic changes give rise to quiescent (Q) cells, which can be distinguished from three other cell types in a stationary phase culture by flow cytometry. We have used flow cytometry to screen for genes that are required to obtain the quiescent cell fraction. We find that cell wall integrity is critical and these genes may help define quiescence-specific features of the cell wall. Genes required to evade the host innate immune response are common. These may be new targets for antifungal drugs. Acquired thermotolerance is also a common property, and we show that the stress-response transcription factors Msn2 and Msn4 promote quiescence. Many other pathways also contribute, including a subset of genes involved in autophagy, ubiquitin-mediated proteolysis, DNA replication, bud site selection, and cytokinesis.

Yeast that naturally exhaust their glucose source can enter a quiescent state that is characterized by reduced cell size, and high cell density, stress tolerance and longevity. The transition to quiescence involves highly asymmetric cell divisions, dramatic reprogramming of transcription and global changes in chromatin structure and chromosome topology. Cells enter quiescence from G1 and we find that there is a positive correlation between the length of G1 and the yield of quiescent cells. The Swi4 and Swi6 transcription factors, which form the SBF transcription complex and promote the G1 to S transition in cycling cells, are also critical for the transition to quiescence. Swi6 forms a second complex with Mbp1 (MBF), which is not required for quiescence. These are the functional analogues of the E2F complexes of higher eukaryotes. Loss of the RB analogue, Whi5, and the related protein Srl3/Whi7, delays G1 arrest, but it also delays recovery from quiescence. Two MBF- and SBF-Associated proteins have been identified that have little effect on SBF or MBF activity in cycling cells. We show that these two related proteins, Msa1 and Msa2, are specifically required for the transition to quiescence. Like the E2F complexes that are quiescence-specific, Msa1 and Msa2 are required to repress the transcription of many SBF target genes, including SWI4, the CLN2 cyclin and histones, specifically after glucose is exhausted from the media. They also activate transcription of many MBF target genes. msa1msa2 cells fail to G1 arrest and rapidly lose viability upon glucose exhaustion. msa1msa2 mutants that survive this transition are very large, but they attain the same thermo-tolerance and longevity of wild type quiescent cells. This indicates that Msa1 and Msa2 are required for successful transition to quiescence, but not for the maintenance of that state.