Keywords: cell cycle, chromatin, cyclin genes, transcriptional regulation, yeast

SBF binds to CLN2 transiently during the cell cycle

We used ChIP assays to measure Swi4-Myc binding to theCLN2 promoter during the cell cycle (Figure 1A). Cells were synchronized byGALp∷CDC20 arrest and release, and samples were taken at various times to determine SBF binding. SBF binding begins at ∼20 min after release and peaks at 50 min. Late G1 in these synchronized cells is about 30 min, whenCLN2 mRNA levels start to rise;CLN2 RNA levels peak at about 50 min. Using synchronized cells, we see SBF binding to theCLN1 andCLN2 promoters during G1, but binding is not seen in G2-arrested cells (Figure 1B). SBF does not bind toCDC21, an MBF-activated gene. SBF association with the cyclin promoters is therefore transient and correlates with transcription of the genes.

Rpd3(L) is recruited to SBF- and MBF-dependent promoters by Swi6

SBF is functionally analogous to mammalian E2F, and E2F recruits HDACs to promoters (Iaquinta and Lees, 2007;van den Heuvel and Dyson, 2008). On the basis of this analogy, we asked whether the Rpd3(L) HDAC was recruited to theCLN2 promoter. We performed ChIP assays with synchronized cells to detect Sds3-Myc at promoters. Sds3 is a subunit of Rpd3(L), but absent from the Rpd3(S) complex (Carrozzaet al, 2005). Rpd3(L) and Rpd3(S) appear to have different functions, with Rpd3(L) localized primarily to promoter regions and Rpd3(S) at transcribed regions (Joshi and Struhl, 2005). Rpd3(L) binds toCLN2 with kinetics very similar to that observed for SBF (Figure 1A). However, Rpd3(L) binding decays more rapidly than does SBF. The similar kinetics of binding for SBF and Rpd3(L) suggest that SBF recruits Rpd3(L). To test this idea we examined Rpd3(L) binding to promoters inswi6 mutants (Figure 1C). Binding of the Sds3-Myc subunit of Rpd3(L) toCLN1 andCLN2 is lost in theswi6 mutant. Rpd3(L) also binds to the promoter of the MBF-dependentCDC21 gene, although later in the cell cycle. Swi6 is a subunit of both SBF and MBF, and Rpd3(L) binding toCDC21 is also lost in theswi6 mutant. Rpd3(L) also binds to theCTS1 promoter, recruited by the Fkh1 and Fkh2 factors (Vothet al, 2007). No defect in Rpd3(L) binding toCTS1 is seen in theswi6 mutant, and thus theswi6 mutation does not simply affect the integrity of the HDAC complex.

Whi5 and Stb1 are required for Rpd3(L) binding to SBF-dependent genes

Stb1 and Whi5 interact with SBF and are described as inhibitors of SBF (Costanzoet al, 2004;de Bruinet al, 2004,2008). The analogy between these inhibitors and mammalian Rb protein suggested that they could be the intermediaries that allow SBF to recruit the Rpd3(L) HDAC. We examined Rpd3(L) binding to theCLN1 andCLN2 promoters in strains with either astb1 or awhi5 mutation (Supplementary Figure S1). Astb1 mutation modestly reduces Rpd3(L) binding toCLN1 orCLN2, and awhi5 mutation has little effect on Rpd3(L) binding. We next examined Rpd3(L) binding to SBF-dependent promoters in a strain lacking both Stb1 and Whi5 (Figure 2A). Rpd3(L) binding to the SBF-dependentCLN1 andCLN2 promoters is essentially eliminated in thestb1 whi5 mutant. Importantly, Rpd3(L) binding toCTS1 is not affected in thestb1 whi5 double mutant (Figure 2C). Additionally, SBF binding toCLN1 andCLN2 is not affected by either astb1 or awhi5 mutation (Supplementary Figure S2). We conclude that Whi5 and Stb1 both link Rpd3(L) to SBF.

Whi5 has been described as a negative regulator of SBF-dependent genes (Costanzoet al, 2004;de Bruinet al, 2004), consistent with its ability to recruit Rpd3(L). This predicts that awhi5 mutation should result in increased expression of target genes. However, RNA measurements show nearly normal expression ofCLN1 andCLN2 inwhi5 mutants (Figure 2D). Stb1 has been described as promoting transcriptional activation (de Bruinet al, 2008), and consistent with this, we find decreased expression of SBF-dependent genes in astb1 mutant (Figure 2D). Interestingly, thestb1 defect in transcriptional activation is suppressed by awhi5 mutation. As a control, expression of theCYC1 gene is unaffected bywhi5 orstb1 mutations (Supplementary Figure S3). These results support the idea that Stb1 is an activator, whereas Whi5 is a transcriptional inhibitor, but additional mechanisms appear to limit the level ofCLN1 andCLN2 transcription to near the WT level in cells lacking only Whi5.

We expected thatCLN1 andCLN2 RNA levels would be significantly higher in thestb1 whi5 double mutant, as Rpd3(L) binding to these promoters is lost in this mutant. However, in the asynchronousstb1 whi5 cellsCLN2 RNA was at wild-type levels andCLN1 RNA was only slightly increased (Figure 2D). We therefore synchronized wild-type andstb1 whi5 cells and measuredCLN1 andCLN2 RNA during the cell cycle (Figure 2E). There is no significant effect on the kinetics or RNA accumulation, and only a modest increase in the levels ofCLN1 andCLN2 RNA.

Only Stb1 is required for Rpd3(L) binding to MBF-dependent genes

Rpd3(L) binds to the promoters of MBF-dependent genes, and this binding requires the Swi6 protein common to both SBF and MBF (Figure 1C). Similarly, Rpd3(L) binding toCDC6 andCDC21, which are MBF dependent, is lost in thestb1 whi5 double mutant (Figure 2B), like the SBF-dependentCLN1 andCLN2 promoters. Rpd3(L) binding to MBF target genes is not affected by awhi5 mutation (Supplementary Figure S1C), similar to SBF genes (Supplementary Figure S1A). However, a major difference in factor dependence was seen for Rpd3(L) binding to MBF-dependent and SBF-dependent promoters. Astb1 mutation essentially eliminates Rpd3(L) binding toCDC6 andCDC21 (Supplementary Figure S1D), whereas thestb1 mutation results in only a modest reduction in Rpd3(L) binding to the SBF-dependentCLN1 andCLN2 promoters (Supplementary Figure S1B). Expression of MBF-dependent genes is reduced in astb1 mutant (Supplementary Figure S3), as was seen for SBF-dependent genes (Figure 2D). However, although awhi5 mutation strongly suppresses thestb1 defect in the activation of MBF-dependent genes, there is less suppression at MBF-dependent genes bywhi5.

Although Stb1 interacts with the Swi6 subunit present in both SBF and MBF (de Bruinet al, 2008), there is discrepancy in the literature as to whether Whi5 interacts with both SBF and MBF or with SBF alone SBF (Costanzoet al, 2004;de Bruinet al, 2004). To address this question in our strain background we performed co-immunoprecipitation experiments with tagged alleles. The experiment performed with extracts prepared from a Whi5-Flag and Swi4-Myc strain show that Whi5 interacts with the SBF-specific Swi4 protein, irrespective of which antibody is used for the immunoprecipitation (Supplementary Figure S4A). In contrast, we saw no interaction between Whi5-Flag and the Mbp1-Myc subunit of MBF (Supplementary Figure S4B). We conclude that Whi5 does not interact with MBF, and this is consistent with our result that only Stb1 is required for recruiting Rpd3(L) to MBF-dependent genes.

Cdc28 is recruited to SBF- and MBF-dependent promoters

The Cdc28 CDK has long been implicated in transcriptional activation by SBF and MBF (Wittenberg and Reed, 2005). Cdc28 has been shown to be directly recruited to theCDC20 promoter when it is expressed in late G2 (Morriset al, 2003), so we performed ChIP experiments and found that Cdc28 is also recruited directly to both SBF- and MBF-dependent promoters (Supplementary Figure S5). Disruption of eitherSWI6 orWHI5 eliminates Cdc28 binding, whereas astb1 mutation only modestly reduces Cdc28 recruitment. Whi5 does not bind to MBF (Supplementary Figure S4;de Bruinet al, 2004), and thus it is surprising that Cdc28 binding is reduced in thewhi5 mutant. Awhi5 mutation affects cell cycle progression (Jorgensenet al, 2002), and thus it could indirectly affect Cdc28 recruitment in these asynchronous cells.CDC20 expression is independent of SBF or MBF (Simonet al, 2001), and Cdc28 recruitment toCDC20 is not affected byswi6,whi5 orstb1 mutations. We also examined Cdc28 binding toCLN2 in synchronized cells (Figure 1A). The kinetics of Cdc28 binding is similar to that seen for SBF, although Cdc28 binding decreases more rapidly after peak binding. This rapid decline in Cdc28 binding is consistent with this kinase phosphorylating Whi5 resulting in nuclear export of Whi5 (Costanzoet al, 2004;de Bruinet al, 2004), and the loss of Whi5 would end Cdc28 binding. We conclude that Cdc28 recruitment to SBF- and MBF-dependent promoters requires Swi6 and Whi5.

FACT and Swi6 are required for nucleosome eviction at CLN2

Chromatin disassembly at promoters often accompanies gene activation (Workman, 2006;Williams and Tyler, 2007). We determined earlier that nucleosome eviction at the SBF-dependentHO promoter requires both FACT and Asf1 histone chaperones (Takahataet al, 2009). FACT co-immunoprecipitates with Swi6 (Takahataet al, 2009), and we wanted to determine whether this interaction was direct. We expressed a GST-Swi6 fusion protein inEscherichia coli, and loaded the purified GST-Swi6 protein onto glutathione-agarose beads. Purified FACT was loaded onto the beads, washed and eluted. The Spt16 and Pob3 subunits of FACT bind to GST-Swi6, but not to the GST-only or the GST-Swi5 controls (Figure 3). We conclude that FACT and Swi6 interact directly.

We next examined nucleosome eviction from cyclin gene promoters during their activation, and any function for FACT and Asf1 in this process. We used synchronized cells for a ChIP experiment to measure histone H3 occupancy atCLN2 during the cell cycle in wild-type cells and in cells with mutations inASF1,SWI6 andPOB3 (Figure 4A). The Spt16 and Pob3 subunits of FACT are encoded by essential genes (Formosa, 2008), and in these experiments we used apob3(L78R) mutation that strongly decreases the level of Pob3 protein even at the permissive temperature (VanDemarket al, 2008). In wild-type cells, nucleosomes are transiently evicted from theCLN2 promoter at 30 min after release fromGALp∷CDC20 arrest. Nucleosome eviction atCLN2 is sharply reduced inpob3 orswi6 mutants, but theasf1 mutation has no effect. Thus,CLN2 differs fromPHO5 andHO in which Asf1 is required for chromatin disassembly (Adkinset al, 2004;Takahataet al, 2009).PIR1 is a cell cycle-regulated gene expressed in M/early G1, and nucleosomes are evicted from thePIR1 promoter transiently during the cell cycle (Figure 4B). Thepob3 andswi6 mutations have no effect on nucleosome loss atPIR1. Our data suggest that chromatin disassembly occurs at many cell cycle genes coincident with transcriptional activation, but the mechanism of eviction varies widely. Importantly, FACT and Swi6 are required for the changes in chromatin structure at theCLN2 promoter.

Nucleosome eviction atCLN2 is reduced, or possibly delayed, in strains with mutations affecting FACT or SBF. On the basis of this result, we examinedCLN2 mRNA levels during the cell cycle in synchronized wild-type,pob3 orswi6 strains. There is a marked delay in the appearance ofCLN2 mRNA in thepob3 andswi6 mutants (Supplementary Figure S6A), but transcription eventually occurs. Earlier work has suggested a function for Rme1 in activating CLN2 (Tooneet al, 1995). Overexpression of Rme1 increasesCLN2 expression and also permits growth of normally inviable cells lacking both SBF subunits. The Rme1 DNA-binding protein binds to theCLN2 promoterin vitro and activates through promoter sequences distinct from the SBF-binding sites. To address the function ofRME1 inCLN2 activation, we constructedrme1 mutant strains and also made double mutants withswi6 andpob3. RNA measurements with asynchronous cells show reducedCLN2 expression inrme1 mutants, and strong additive defects inswi6 rme1 andpob3 rme1 double-mutant strains (Supplementary Figure S7). Although theswi6 andpob3 mutations reduceHO expression, little additive effect was seen in theswi6 rme1 andpob3 rme1 double-mutant strains. Finally,PIR1 expression was unaffected byrme1,swi6 orpob3 mutations. These results are consistent with the idea that Rme1 promotes the residualCLN2 activation in strains with SBF or FACT mutations.

In contrast to the situation atCLN2 in which there is residual expression in theswi6 andpob3 mutants (Supplementary Figure S6A), expression of the SBF- and FACT-dependentHO gene is largely eliminated in bothpob3 andswi6 cells (Supplementary Figure S6B).HO gene transcription requires recruitment of coactivator complexes to the URS2 region of the promoter, and mutations affecting FACT or SBF eliminate this coactivator recruitment (Takahataet al, 2009). We therefore examined binding of the Gcn5 subunit of the SAGA coactivator toCLN2 using ChIP assays to see whether this mechanism is also used at this promoter (Supplementary Figure S6C). In wild-type cells Gcn5 binds transiently toCLN2 during the cell cycle, at about the same time as SBF that binds. Thepob3 andswi6 mutations do not eliminate Gcn5 binding toCLN2, but rather cause a delay similar to the delay in mRNA levels. We conclude that FACT and Swi6 are required to express theCLN2 promoter at the appropriate time because they allow eviction of nucleosomes and coactivator recruitment to occur rapidly. This is unlike the situation at theHO promoter in which the gene is completely transcriptionally inactive in FACT or SBF mutants. In contrast, nucleosome eviction, coactivator recruitment and gene expression eventually occur atCLN2 in these mutants, and thus FACT and Swi6 are not absolutely required here.

Swi6 is required for FACT binding and nucleosome eviction at SBF- and MBF-regulated genes

On the basis of the delay inCLN2 activation inswi6 mutants during the cell cycle, we decided to repeat the histone H3 ChIP experiment but with a longer time course, so that we could discriminate between events that are required forCLN2 activation and those that affect its rate. FACT binds toHO URS2 in aSWI6-dependent manner (Takahataet al, 2009), and so we measured FACT binding in wild-type cells (Figure 4C–H), and inswi6 mutants (Supplementary Figure S8). Both wild-type andswi6 cells were synchronized byGALp∷CDC20 arrest and release, and samples were taken at time points for H3 ChIP, FACT ChIP and RNA measurements; each measurement is therefore from the same culture of synchronized cells.

FACT binds toHO URS2 at 20 min, just before nucleosomes are evicted, with strong gene expression seen starting at 40 min (Figure 4C), consistent with our earlier experiments (Takahataet al, 2009). ThePIR1 gene, whose cell cycle expression is independent of Swi6, serves as a control (Figure 4D).PIR1 expression is not affected by a FACT mutation (Takahataet al, 2009), and FACT does not bind to this promoter. Nucleosomes are transiently evicted during the cell cycle as the gene is expressed, and these events are not affected in aswi6 mutant (Supplementary Figure S8). Thus, theswi6 mutation does not disrupt events at all cell cycle-regulated genes.

Expression of the SBF-dependent genesCLN1 andCLN2 is reduced in FACT mutants (Rowleyet al, 1991;Lycanet al, 1994;Takahataet al, 2009), and this is also seen in our experiments analysing RNA levels in synchronized cells (Figure 4;Supplementary Figure S8). There are two peaks of FACT binding at these promoters, one at 25 min after release and one at 50 min. (See also the independent ChIP experiments inFigure 5 showing this result is reproducible.) Nucleosome occupancy decreases first at 20 min, and then further at 40 min; return of nucleosomes to the promoters begins at 50 min. There is no FACT binding to these promoters in theswi6 mutant (Supplementary Figure S8C and D). However, theswi6 mutation causes only a modest change in nucleosome disassembly, despite the markedly reduced and delayed gene expression. Although no FACT binds to the promoters in theswi6 mutant, nucleosome eviction and gene transcription still occur, albeit more slowly. The delayed and reduced residual chromatin disassembly and transcription may be induced by Rme1 (Tooneet al, 1995). We conclude that although FACT recruitment is correlated with timely gene induction, it is not absolutely required for nucleosome disassembly at these promoters.

We also examined promoter events at two MBF-dependent promoters,CDC21 andPOL1. AtCDC21 there is a sharp peak of FACT binding at 20 min, followed by nucleosome loss and gene transcription (Figure 4G). All of these events are eliminated in aswi6 mutant (Supplementary Figure S8E). The kinetics atCDC21 resemble those seen atHO (Figure 4C), except that FACT binding and chromatin disassembly occur slightly sooner atHO. Events atPOL1 occur later in the cell cycle (Figure 4H). Major FACT binding is seen at 40 min after release, although there may be weak transient binding at 20–25 min. Nucleosome eviction and gene transcription also occur later, after the peak in FACT binding. FACT binding, chromatin disassembly, and gene activation are all lost in aswi6 mutant (Supplementary Figure S8F).

These studies allow us to make a number of conclusions regarding the common features of promoters that are expressed at different times during the cell cycle. FACT binds transiently during the cell cycle to promoters of SBF- and MBF-activated genes. This FACT binding is followed first by changes in chromatin structure at the promoter and subsequently by gene transcription. Swi6 is required for recruitment of FACT to these promoters and for chromatin disassembly, although there is significant nucleosome loss atCLN1 andCLN2 in aswi6 mutant so FACT is not required for all nucleosome loss, and nucleosome loss is not sufficient to cause increased transcription.

Whi5 and Stb1 inhibit FACT binding to SBF-dependent promoters

SBF recruits both Stb1 and Whi5 to promoters, and SBF also recruits FACT. We wondered whether Stb1 or Whi5 were required for recruiting FACT. We measured FACT binding in synchronized wild-type cells and astb1 whi5 double mutant. To our surprise, instb1 whi5 mutants we observed markedly increased FACT binding to three SBF-dependent genes,HO,CLN1 andCLN2 (Figure 5A). This suggests that Stb1 or Whi5, or both, inhibit FACT binding to SBF-dependent promoters. In contrast, FACT binding to three MBF-dependent promoters was not affected in thestb1 whi5 double mutants (Figure 5B). Whi5 binds to SBF but not to MBF (Supplementary Figure S4;de Bruinet al, 2004). We suggest that Whi5 antagonizes FACT binding to SBF-dependent promoters, consistent with Whi5's function as a transcriptional inhibitor.

stb1 and whi5 suppress FACT and cdc28 mutations

Consistent with an antagonistic relationship between FACT and Whi5, we identified genetic interactions between FACT,WHI5 andSTB1. Anspt16-11 mutation in FACT shows growth defects at higher temperatures, but this defect is suppressed bywhi5 (Figure 6A). In contrast, thespt16-11 stb1 double mutant shows an additive defect compared with either single mutant at 35°C or on media containing hydroxyurea (HU). This defect is suppressed in thespt16-11 stb1 whi5 triple mutant, and thuswhi5 is epistatic tostb1. Awhi5 mutation also suppresses both the temperature- and HU-sensitive phenotypes caused by twopob3 alleles (Figure 6B and C); a synthetic defect is not seen in thepob3 stb1 double mutants.

At the nonpermissive temperature,cdc28-13 mutants arrests in G1, before START (Mendenhallet al, 1988). We next examined whetherstb1 orwhi5 mutations could suppress thecdc28-13 defects. Althoughcdc28-13 cells do not form colonies at temperatures of 35°C or above, growth is restored by either astb1 orwhi5 mutation (Figure 6D). Suppression bywhi5 is more robust, and only thecdc28-13 whi5 strain is able to grow at 36°C, whereascdc28-13 stb1 cells cannot. Finally, suppression bystb1 andwhi5 is additive, as only thecdc28-13 stb1 whi5 triple mutant can grow at 37°C. Thisstb1 whi5 double mutant has a growth defect at elevated temperatures (Supplementary Figure S9), making this suppression ofcdc28-13 all the more impressive. These genetic results confirm that Whi5 antagonizes both FACT and CDK functions, but Stb1's function is more complex as it appears to promote FACT function but to antagonize CDK.

CLN2 overexpression suppresses pob3 swi6 synthetic lethality

CLN2 transcription is reduced in bothpob3 andswi6 mutants, and we wanted to see whetherCLN2 expression is further reduced in a double-mutant strain. A doubly heterozygous +/pob3 +/swi6 diploid strain was transformed with a YCp(URA3) plasmid with a wild-typePOB3 gene. After sporulation, a haploidpob3 swi6 double-mutant strain with the YCp(URA3):POB3 was isolated. We tested whether this strain could grow on medium containing 5-FOA, as only strains lacking theURA3 plasmid can grow on these plates. Thepob3 swi6 YCp(URA3):POB3 cells are unable to grow on 5-FOA (Figure 6E), showing thatpob3 andswi6 are synthetically lethal. We next transformed thesepob3 swi6 YCp(URA3):POB3 cells with multicopy YEp(LEU2) plasmids, and tested these transformants for the ability to grow on 5-FOA medium. As shown inFigure 6F, a multicopy plasmid withCLN2 allows cells to lose the YCp(URA3):POB3 plasmid and grow on 5-FOA; multicopyCLN1 also suppresses thepob3 swi6 synthetic lethality but to a lesser extent. This genetic experiment suggests that thepob3 andswi6 mutations are synthetically lethal because of insufficient G1 cyclin gene expression.

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