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. 2002 Nov 15;16(22):2935–2945. doi: 10.1101/gad.764102

Cell-cycle control of the establishment of mating-type silencing in S. cerevisiae

Anna Lau 1,1, Hannah Blitzblau 1, Stephen P Bell 1,2
PMCID: PMC187485  PMID: 12435634

Abstract

Transcriptional silencing in Saccharomyces cerevisiae involves the assembly of a heterochromatic domain that is heritable from generation to generation. To maintain the silenced state, the propagation of silent heterochromatin must be coordinated with the events of chromosome duplication and segregation. Here we present an in vivo analysis of the cell-cycle events required for the establishment of the silenced state at the HMR silent mating-type locus. We show that Sir protein recruitment to and spreading from the HMRE silencer is poor during S phase, but is robust during G2 and subsequent phases. Despite abundant Sir protein association in cells arrested in G2/M phase, silencing is not fully established by this stage of the cell cycle. Rather, robust silencing is not observed until telophase. Interestingly, the elimination of the cohesin subunit Scc1/Mcd1p allows the full establishment of silencing to occur in G2/M phase. Furthermore, expression of a noncleavable allele of Scc1/Mcd1p inhibits the establishment of silencing. Our findings reveal both S- and M-phase requirements for the establishment of silencing and implicate the loss of sister-chromatid cohesion as a critical event in this process.

Keywords: SIR, SCC1, MCD1, HMRE, transcriptional silencing, sister-chromatid cohesion

Transcriptional silencing is a specialized form of nonspecific gene repression characterized by its dependence on heterochromatin and its stable inheritance during cell division. Silencing occurs within heterochromatic regions in the genome that are thought to repress transcription both by limiting access to DNA modifying enzymes (Gottschling 1992; Loo and Rine 1994) and by blocking transcription elongation (Sekinger and Gross 2001). Since the silent state is heritable, silent heterochromatin must be assembled onto newly replicated DNA during each cell division. The cell-cycle events that regulate this complex assembly process are not well understood.

The budding yeast, Saccharomyces cerevisiae, possesses three classes of silent domains: the mating-type loci HML and HMR, the telomeres, and the ribosomal DNA (rDNA) repeats (for review, see Guarente 1999). The assembly of silent heterochromatin at these sites requires at least three steps: (1) the recognition of a target site, (2) the recruitment of the silencing proteins to the target, and (3) the spreading of the silencing complex along the DNA to form a heterochromatic domain (Laurenson and Rine 1992). HML and HMR, which contain donor copies of α- or a-mating-type information, respectively, are maintained in a transcriptionally silent state through the action of four short sequence elements. These E and I silencers flank each silenced domain (Abraham et al. 1984; Feldman et al. 1984) and are composed of binding sites for ORC, Rap1p, and Abf1p that nucleate silent heterochromatin assembly (Brand et al. 1987; Loo and Rine 1995). The Sir proteins, Sir1p–Sir4p, are components of the silent heterochromatin (Ivy et al. 1986). Through an interaction with ORC, Sir1p facilitates the recruitment of Sir2p, Sir3p, and Sir4p to each of the silencers (Triolo and Sternglanz 1996; Gardner et al. 1999). Sir3p and Sir4p also interact with Rap1p (Moretti et al. 1994). Once Sir protein association is nucleated at the silencer, the histone deacetylase activity of Sir2p is thought to facilitate the spreading of the Sir2/Sir3/Sir4 proteins along nucleosomal DNA via interactions with the N-terminal tails of histones H3 and H4 (Braunstein et al. 1993; Hecht et al. 1995; Imai et al. 2000; Tanny and Moazed 2001; Hoppe et al. 2002; Rusche et al. 2002).

The process of silencing has three phases: (1) establishment, when a domain becomes silent, (2) maintenance, when a silent domain is kept repressed through the cell cycle, and (3) inheritance, when the silent state is passed to each daughter chromosome. It has been demonstrated that Sir1p functions primarily in the establishment of silencing (Pillus and Rine 1989). The other Sir proteins function in both the establishment and maintenance of silencing, as mutations in Sir2p, Sir3p, or Sir4p result in the uniform and complete loss of silencing (Ivy et al. 1986).

Several lines of evidence point to cell-cycle regulation of the establishment of silencing. In an early study, derepressed cells were observed to require passage through S phase before the reestablishment of silencing (Miller and Nasmyth 1984). Based on this finding, it was proposed that DNA replication was responsible for this S-phase requirement. Recent studies have raised doubts concerning this hypothesis. Extrachromosomal circles containing HMR have been shown to silence even though replication of the circle is not detectable (Kirchmaier and Rine 2001; Li et al. 2001), indicating that the action of a replication fork is not linked to the requirement for S-phase passage.

Other studies have alluded to an involvement of mitotic events in determining silencing. When URA3 is subject to telomeric silencing, the function of its activator, PPR1, is able to overcome silencing when cells are arrested in G2/M phase but not when cells are arrested in G1 or early S phase (Aparicio and Gottschling 1994). This finding suggests that telomeric silencing is weak during this period of the cell cycle. In addition, Sir3p and Sir4p are observed to release from telomeres during G2/M phase (Laroche et al. 2000). A cis-acting boundary element that limits the spreading of the HMR silenced domain requires the activity of cohesin subunits Smc1p and Smc3p (Donze et al. 1999), suggesting that the cohesin complex limits propagation of the silenced domain. Another cohesin subunit, Scc1/Mcd1p, has been shown by chromatin immunoprecipitation (ChIP) to bind preferentially to the HMR boundary elements (Laloraya et al. 2000). Together, these observations suggest that factors involved in mitosis are linked to the silencing process and may participate in the establishment, maintenance, or inheritance of silencing.

In this study, we investigated the cell-cycle requirements for the establishment of silencing by examining Sir protein association with the HMR silenced domain and the impact of cell-cycle mutants on expression from the HMRa1 gene. We found that Sir2p, Sir3p, and Sir4p associate poorly with HMR during S phase. By G2/M phase, however, Sir proteins are abundantly associated with the silenced domain, yet silencing is not established. This failure to silence in G2/M-phase cells is dependent on the presence of the Scc1/Mcd1p cohesin subunit. When Scc1/Mcd1p is repressed or cleaved during the cell cycle, the establishment of silencing is permitted. Moreover, when Scc1/Mcd1p cleavage does not occur, the establishment of silencing is severely hindered. Our findings identify multiple cell-cycle requirements in the establishment of silencing and implicate sister-chromatid cohesion as a negative regulator of this process in M phase.

Results

Sir protein association with HMRE is weak during S phase, but robust during G2/M phase

Although earlier studies demonstrated that passage through S phase was necessary to establish silencing (Miller and Nasmyth 1984; Fox et al. 1995, 1997; Kirchmaier and Rine 2001; Li et al. 2001), it remains unclear which step of establishment is S-phase-dependent. One candidate event is the association of Sir proteins with the HMRE silencer. To address this possibility, we examined the abilities of Sir2p, Sir3p, and Sir4p to associate with the HMRE silencer at different cell-cycle stages. We constructed a strain carrying a temperature-sensitive allele of SIR3 (sir3-8) that allowed the control of silencing by temperature shift. In addition, a 3′ portion of the MATa1 gene was deleted so that a1 transcripts expressed from HMR could be distinguished from a1 transcripts constitutively expressed from the MAT locus.

To assess the ability of the Sir proteins to associate with the silencer during the cell cycle, cells were synchronized in G1 phase at 23°C with α-factor (αF), raised to 37°C for 2 h to disrupt silencing. These cells were then returned to the permissive temperature (23°C), released from the G1 arrest, and permitted to progress through a portion of the cell cycle. Cells were rearrested in one of three cell-cycle stages (at 23°C): (1) S phase by treatment with hydroxyurea (HU), (2) G2/M phase by treatment with nocodazole (noc), or (3) the next G1 phase by retreatment with αF after the appearance of buds (Fig. 1A). Throughout the experiment, cell-cycle progression was monitored by flow cytometric measurement of DNA content (Fig. 1B).

Figure 1.

Figure 1

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The establishment of silencing requires passage through G2/M phase, despite abundant Sir protein association with HMRE. (A) Experimental outline. sir3-8 cells (yAL170, see Table 1) were synchronized in G1 phase with αF, raised to 37°C, then released into fresh media at 23°C containing HU, nocodazole, or no block. After 1 h, when buds had appeared, αF was added to the “no block” sample. (B) DNA content. Samples were withdrawn at the indicated timepoints throughout the experiment, and DNA content was measured by propidium iodide staining of DNA and flow cytometry. (C) α-Sir ChIP assays. Samples were withdrawn at the indicated timepoints throughout the experiment. Chromatin-containing extracts were prepared from formaldehyde-treated sir3-8 cells and were immunoprecipitated with α-Sir3p (top, left), α-Sir2p (middle, left), or α-Sir4p (bottom, left) serum. DNA was amplified using primers specific to HMRE, the HMRa1 promoter, or URA3 (control). These primers were also used to amplify DNA isolated from extracts before immunoprecipitation (input). (D) HMRa1 expression. RNA was isolated at the times indicated throughout the experiment and subjected to both RT–PCR and real-time PCR. cDNA was amplified with primers to both HMRa1 and ACT1 (control). The primer set for HMRa1 does not detect MATa1 in this strain, because a portion of MATa1 where the reverse primer anneals is deleted. Shown below particular samples is the HMRa1/ACT1 ratio determined by real-time PCR. This ratio for the sample at 37°C is set to 1.0, because these cells are fully derepressed. In each case, the standard deviation is less than 10% of the average of two or three experiments.

We analyzed the association of Sir proteins with the HMRE silencer at each stage of the experiment described above using ChIP. Sir3p associated with the HMRE silencer at 23°C but not at 37°C, indicating that the sir3-8 allele resulted in Sir3p release from the silencer at the nonpermissive temperature (Fig. 1C). Interestingly, when cells were returned to the permissive temperature, Sir3p reassociated with the HMRE silencer regardless of the cell-cycle stage at which the cells were blocked, although its association was consistently weaker in S-phase-arrested cells (Fig. 1C). The limited association of Sir proteins with HMRE in S-phase-arrested cells was not due to incomplete recovery from incubation at 37°C, as the same levels of association were observed even if cells were allowed to recover for 2 h at 23°C before being released from the initial αF arrest (see Supplementary Fig. 1). Similar to Sir3p, Sir2p and Sir4p were shown to reassociate abundantly with HMRE in cells arrested in G2/M or the subsequent G1 phase but not in S phase (Fig. 1C).

The establishment of silencing requires passage through both S and M phases

To monitor the establishment of silencing, expression of the HMRa1 gene was determined by RT–PCR at all stages of the experiment described above. Cells arrested in G1 phase at 23°C showed no expression, whereas cells at 37°C showed strong expression, demonstrating that HMRa1 was expressed when Sir3p function was disrupted (Fig. 1D). Upon return to 23°C, HMRa1 expression remained high in cells blocked in HU and nocodazole, indicating that cells prevented from completing S and G2/M phases were unable to fully establish silencing (Fig. 1D). In contrast, cells arrested in the subsequent G1 phase showed the most substantial decrease in HMRa1 transcription, signifying a high degree of silencing by the time cells reached this stage of the cell cycle. Because our Sir protein ChIP analyses showed robust association of these proteins with HMRE, yet little silencing was observed during G2/M phase, we concluded that Sir protein association with a silencer (at least as detected by ChIP assays) was not sufficient to restore silencing at this cell-cycle stage.

To quantitatively verify our expression results, we used real-time sequence detection to determine the amount of transcription from HMRa1 (Heid et al. 1996). In all cases, the amount of HMRa1 transcript was normalized to that of ACT1 transcript. The HMRa1/ACT1 ratio for the sample incubated at 37°C was set to unity, because cells at the nonpermissive temperature are fully derepressed (determined by comparison with sir3Δ cells; data not shown). Compared to derepressed cells arrested in G1, cells blocked in S phase showed a slightly increased ratio of HMRa1 to ACT1 expression (HMRa1/ACT1 = 1.3). Cells blocked in G2/M phase showed lower HMRa1 expression, by slightly more than a half (HMRa1/ACT1 = 0.45). Cells that were allowed to reach the subsequent G1 phase, however, showed the most significant decrease in HMRa1 expression (HMRa1/ACT1 = 0.07). These results confirmed the RT–PCR results indicating that robust silencing was not established by G2/M, but was observed in the following G1 phase.

Spreading of Sir proteins to the HMRa1 promoter is not sufficient to establish silencing

We found that the association of Sir proteins with HMRE is not sufficient to bring about silencing of HMRa1. Because silencing requires both the nucleation and the spreading of Sir proteins from the silencer to the HMRa1 promoter, the presence of these proteins at HMRE, but not at the HMRa1 promoter, could explain the lack of silencing observed in G2/M-arrested cells. To test this hypothesis, we examined whether HMRa1 promoter DNA was enriched in Sir protein chromatin immunoprecipitates. As expected from the findings at HMRE, Sir proteins were only weakly associated with the HMRa1 promoter in the derepressed cells arrested in S phase (Fig. 1C, HMRa1). Equal and abundant association with promoter DNA was observed in both the derepressed G2/M-arrested and silenced G1-arrested cells. Thus, these findings showed that despite equivalent spreading of Sir proteins to the HMRa1 promoter in the G2/M- and G1-arrested cells, robust silencing was established only in cells arrested in the subsequent G1 phase.

Silencing is established in telophase-arrested cells

Our results indicated that silencing was established at some time between the nocodazole arrest point in G2/early M phase and the αF arrest point in the subsequent G1 phase. To further narrow the window of time during which silencing was established, we examined whether exit from mitosis was required for this event. The GTPase Tem1p is an essential component of the pathway that controls exit from mitosis, and in its absence, cells arrest in telophase. To enable Tem1p depletion in the sir3-8 strain, the TEM1 gene was placed under galactose control and fused to a ubiquitin-proline-lacI (UPL) epitope, which destabilizes attached proteins (Shou et al. 1999). Under depletion conditions (growth in glucose), TEM1 transcription is turned off, existing UPL-Tem1p is rapidly degraded, and cells arrest in telophase. We determined whether silencing could be established in telophase-arrested cells by monitoring silencing in the absence of Tem1p. G1-synchronized cells were raised to 37°C, and Tem1p depletion was initiated by the addition of glucose. Then, cells were washed and released into 23°C, glucose media containing HU, nocodazole, or no drug to arrest cells in S, G2/early M, or telophase, respectively (Fig. 2A). Cells arrested in S phase expressed HMRa1 at high levels (Fig. 2D; HMRa1/ACT1 = 1.1). G2/M-blocked cells showed a moderate decrease in HMRa1 expression (HMRa1/ACT1 = 0.25). Telophase-arrested cells, however, showed a more significant decrease in HMRa1 expression (HMRa1/ACT1 = 0.06), comparable to the decrease observed in cells that exit mitosis (cf. Figs. 2D and 1D). Telophase arrest was confirmed in the unblocked, Tem1p-depleted cells both by flow cytometric measurement of DNA content showing the accumulation of cells with 2C DNA content (Fig. 2B), and by indirect immunofluorescence of tubulin, showing that these cells accumulated with elongated spindles (Fig. 2C). These results indicated that silencing had been established in telophase-arrested cells, and therefore, exit from mitosis was not necessary for full repression.

Figure 2.

Figure 2

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Telophase-arrested cells can establish silencing. (A) Experimental outline. The scheme for this experiment is similar to that described in Figure 1, except that sir3-8 GAL-UPL-TEM1 cells (yAL218) were initially grown in galactose media to express Tem1p, then switched to glucose media to repress Tem1p. (B) DNA content. Samples were treated for flow cytometry exactly as described in Figure 1. (C) Long spindle count. Cells grown either in the presence (□) or absence (▪) of Tem1p were taken at the indicated times throughout the experiment, fixed, and stained with rat α-tubulin antibody and α-rat IgG conjugated to Cy3. At least 200 cells were counted at each timepoint, and the percent of these with elongated, telophase spindles was calculated. (D) HMRa1 expression. RNA was isolated from samples at the indicated times throughout the experiment, and analyzed as in Figure 1.

Loss of Scc1/Mcd1p enables the establishment of silencing in G2/early M phase

We sought to identify the event between early and late M phase that accounted for the different levels of silencing we observed. One candidate event is the dissolution of sister-chromatid cohesion during anaphase, a process that depends on cleavage of the cohesin subunit Scc1/Mcd1p by the Esp1p protease (Uhlmann et al. 1999). Because Scc1/Mcd1p is fully cleaved by telophase and has been shown to bind to the HMR locus, we examined whether loss of this cohesin subunit influenced the establishment of silencing. In the sir3-8 strain, we placed the SCC1/MCD1 gene under the control of the MET3 promoter so that its expression could be repressed by the addition of methionine. To monitor cohesion between sister-chromatids, we marked the Chromosome V centromere with a tetracyclin (tet) operator array and expressed a tetracyclin repressor-GFP fusion protein (tetR-GFP; Michaelis et al. 1997). By monitoring the number of spots of GFP-dependent fluorescence, the dissolution of sister-chromatid cohesion can be easily determined.

Cells were arrested in G1 phase, and then raised to 37°C with the addition of methionine to repress SCC1/MCD1 transcription. Because Scc1/Mcd1p is unstable during G1 phase, the existing protein is rapidly degraded (Uhlmann and Nasmyth 1998; data not shown). The cells were then washed and released into methionine-supplemented media containing HU, nocodazole, or αF added after the appearance of buds (Fig. 3A). In the absence of Scc1/Mcd1p, the majority of nocodazole-blocked cells accumulated with two GFP dots (60%; Fig. 3C), indicating a loss of cohesion in these cells. In contrast to the silencing profile observed for the sir3-8 SCC1/MCD1 strain, the strain lacking Scc1/Mcd1p showed comparable decreases in HMRa1 expression in cells arrested in G2/M or the subsequent G1 phase (Fig. 3D; HMRa1/ACT1 = 0.12 and 0.11 in G2/early M and G1, respectively). These results indicated that the absence of Scc1/Mcd1p alleviated the M-phase requirement to establish silencing, suggesting a negative role for this cohesin subunit or for sister-chromatid cohesion in the regulation of mating-type silencing. Interestingly, cells blocked in S phase lacking Scc1/Mcd1p did not establish silencing (Fig. 3D), indicating that the S- and M-phase requirements for the establishment of silencing were separable and that the S-phase requirement did not involve Scc1/Mcd1p function.

Figure 3.

Figure 3

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Silencing can be established in G2/M phase in the absence of Scc1/Mcd1p. (A) Experimental outline. The scheme for this experiment is similar to that described in Figure 1, except that sir3-8 MET-SCC1/MCD1 cells (yAL227) were initially grown in media without methionine to express Scc1/Mcd1p, then switched to media with methionine to repress Scc1/Mcd1p. (B) DNA content. Samples were treated for flow cytometry exactly as described in Figure 1. (C) GFP dot count. Binding of the tetR-GFP to an array of tet operators near the centromere of Chromosome V fluorescently tags the chromosome. In G2/M cells, a single GFP dot visible within a cell indicates cohesion between the two Chromosomes V (and presumably all the other chromosome pairs). Two GFP dots visible within a cell indicate the loss of cohesion. At least 200 cells grown either in the presence (▪) or absence (□) of Scc1/Mcd1p were counted for each timepoint, and the percent of cells with two GFP dots was calculated. (D) HMRa1 expression. RNA was isolated from samples at the indicated times throughout the experiment, and analyzed as in Figure 1.

Noncleavable Scc1/Mcd1p inhibits the establishment of silencing

We reasoned that if the loss of Scc1/Mcd1p promotes the establishment of silencing, then a mutant Scc1/Mcd1p that cannot be cleaved should inhibit the establishment of silencing. To test this hypothesis, we took advantage of an allele of SCC1/MCD1 (SCC1-RRDD; Uhlmann et al. 1999) that lacks both of its cleavage sites and is resistant to Esp1p protease. In the presence of the noncleavable form of Scc1/Mcd1p, cells exit from mitosis without releasing cohesion between sister chromosomes. To determine whether Scc1/Mcd1p cleavage is required to establish silencing, a galactose-inducible allele of SCC1-RRDD was introduced into the sir3-8 MET-SCC1/MCD1 strain. Cells were arrested in G1 phase, and raised to 37°C while repressing expression of the wild-type Scc1/Mcd1p with methionine and inducing expression of noncleavable Scc1-RRDDp with galactose. This resulted in cells that contained only the noncleavable form of Scc1/Mcd1p. Cells were washed and released into methionine/galactose media containing HU, nocodazole, or αF added after the appearance of buds (Fig. 4A). Consistent with the inability of Scc1-RRDDp to lose sister-chromatid cohesion, cells overexpressing the mutant protein and retreated with αF arrested with mostly 2C DNA content by flow cytometry (Fig. 4B), due to the failure of chromatids to separate from one another. The majority of these cells did arrest in the subsequent G1 phase, as indicated by the transition from elongated (mitotic) to short (G1) spindles (Fig. 4C).

Figure 4.

Figure 4

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Silencing is not established in the presence of noncleavable Scc1/Mcd1p. (A) Experimental outline. The scheme for this experiment is similar to that described in Figure 1, except that sir3-8 MET-SCC1/MCD1 GAL-SCC1-RRDD (yAL230) cells were initially grown in raffinose media without methionine to express cleavable Scc1/Mcd1p and repress noncleavable Scc1-RRDDp. Subsequently, these cells were switched to raffinose/galactose media with methionine to repress cleavable Scc1/Mcd1p and express noncleavable Scc1-RRDDp. (B) DNA content. Samples were taken at the indicated times throughout the experiment and treated as described in Figure 1. (C) Long spindle count. Cells expressing either wild-type (□) or noncleavable (▪) Scc1/Mcd1p were taken at the indicated times throughout the experiment, fixed, stained, and counted as described in Figure 2. (D) HMRa1 expression. RNA was isolated from samples at the indicated times throughout the experiment, and analyzed as in Figure 1.

In the presence of noncleavable Scc1-RRDDp, HMRa1 expression remained elevated throughout the experiment (Fig. 4D). At most, only a moderate reduction in HMRa1 expression was observed at the last timepoint in cells allowed to exit mitosis (HMRa1/ACT1 = 0.30, Fig. 4D). Thus noncleavable Scc1-RRDDp severely reduced the rate and the extent of the establishment of silencing, suggesting that cleavage of Scc1p is necessary for the establishment of silencing.

Discussion

The findings presented here provide an in vivo analysis of the cell-cycle requirements for the establishment of mating-type silencing in S. cerevisiae and suggest roles for both S- and M-phase events. We demonstrate that full establishment of silencing does not occur when cell-cycle progression is blocked in S or G2/M phase. In contrast, silencing is largely restored when cells reach the next G1 phase. We find that Sir protein recruitment to a silencer is impaired in cells arrested in S phase. Interestingly, even when normal levels of Sir protein association are achieved in G2/M, robust silencing is not observed. The incomplete silencing observed prior to passage through mitosis requires Scc1/Mcd1p. In addition, the establishment of silencing is inhibited by a noncleavable allele of Scc1/Mcd1p. These findings support a model in which the full establishment of silencing is permitted only after sufficient Sir protein association with a silencer and Scc1/Mcd1p cleavage by Esp1p.

An M-phase requirement for the establishment of silencing

Our observations indicate only a moderate level of repression in cells blocked in G2/M phase and more robust silencing in cells that reach at least telophase of the cell cycle. Compared to fully derepressed cells, G2/M-blocked cells show a two- to fourfold decrease in HMRa1 expression, whereas G1-blocked cells show more substantial HMRa1 repression of 9- to 17-fold. These levels may reflect a mixed population of cells that have and have not completely established silencing or an intermediate level of silencing in all cells. In either case, there is a significant population of cells whose levels of repression most closely approach the fully silenced state only after cells have reached G1 (or in G2/M-arrested cells lacking Scc1/Mcd1p). Despite increased repression, HMRa1 expression in G1 cells does not reach zero. This discrepancy is most likely due to a small fraction of cells that fail to reenter the cell cycle after the initial αF arrest (Fig. 1B). Alternatively, our findings may reflect the inability of all cells to establish silencing “from scratch” in a single cell cycle.

The moderate decrease in HMRa1 expression that we observe in G2/M-blocked cells is likely related to the findings of earlier studies in which levels of repression between three- and ninefold were recorded for G2/M-arrested cells (Miller and Nasmyth 1984; Fox et al. 1995, 1997; Kirchmaier and Rine 2001; Li et al. 2001). Although the majority of these previous studies did not report an increase in silencing after passage through mitosis, this discrepancy may be due to the different methods of detection of the HMRa1 transcript. The RT–PCR and real-time sequence detection assays used here are more sensitive and quantitative than either Northern blot or S1 nuclease assays and may have allowed the detection of transcripts not observed previously. Alternatively, the different cell-cycle requirements may be the result of the different mechanisms used to control silencing in other studies.

All but one of the previous studies of the cell-cycle control of the establishment of silencing used a modified HMRE silencer that depended on the expression of a Gal4-Sir1p or LexA-Sir1p fusion protein to establish silencing (Fox et al. 1995, 1997; Kirchmaier and Rine 2001; Li et al. 2001). The modified silencers used in those studies have multiple binding sites for the Sir1 fusion protein, and these proteins bind DNA as dimers (Carey et al. 1989; Thliveris et al. 1991), implying that the Sir1 fusion protein is present on the modified HMRE at quantities higher than expected at a wild-type silencer. Recent studies regarding the order of Sir protein recruitment to a silencer indicate that Sir1p association with a silencer is a critical, early step in the efficient recruitment of the remaining Sir proteins (Rusche et al. 2002). Thus, the unusual abundance of Sir1p at a modified silencer may enhance Sir protein recruitment during the cell cycle, allowing the bypass of one or more cell-cycle requirements. It is worth noting, however, that one study using the modified silencer reported increased silencing when cells where allowed to proceed beyond the G2/M arrest point (Li et al. 2001). This raises the possibility that the same increase in silencing occurs in these experiments as well. Only a detailed comparison of these different approaches to control silencing will fully address these potential differences.

In the present study, as in the original study of the cell-cycle requirements for the establishment of silencing (Miller and Nasmyth 1984), a temperature-sensitive allele of SIR3 (sir3-8) was used to inactivate silencing. This approach allows the use of native silencers and wild-type levels of Sir protein expression. Both our own and previous studies support the wild-type function of Sir3-8p at 23°C. For example, wild-type- and Sir3-8p-dependent silencing of HMRa1 are indistinguishable at 23°C (data not shown), and sir3-8 cells mate as well as wild-type cells in a SIR1-dependent manner (Stone et al. 2000). The cell-cycle requirements for the establishment of silencing that we observe are not due to a temporal requirement for Sir3-8p resynthesis or folding. Even if cells are given additional time at 23°C either prior to or after release from αF, they still must pass G2/M before silencing is fully established (Supplementary Fig. 1). In contrast, silencing can be established rapidly (by 2 h) in G2/M-arrested cells when Scc1/Mcd1p is eliminated or in cells that are allowed to reach the next G1. Thus, the ability of Sir3-8p to assemble at HMR is not facilitated by the passage of time per se, but by passage through the cell cycle. Importantly, in the experiments described here, the HMR locus and all of the other Sir proteins are wild-type, arguing that previously described synthetic effects with sir3-8 (Stone et al. 2000) are unlikely to affect the outcomes of these experiments. Although it remains possible that Sir3-8p is particularly susceptible to a negative effect of Scc1/Mcd1p (e.g., competition for binding sites at HMR), we saw no additional Sir protein association after the loss of Scc1/Mcd1p as measured by ChIP. Whether cells were in G2/M phase (poorly silenced with Scc1/Mcd1p) or the next G1 phase (robustly silenced without Scc1/Mcd1p), Sir3-8p, Sir2p, and Sir4p were equivalently associated with HMR (Fig. 1C). Thus, the defect responsible for the M-phase requirement was not the bulk association of Sir proteins with HMR.

Sister-chromatid cohesion and silencing

We have demonstrated that the establishment of silencing depends on the loss of Scc1/Mcd1p and that this explains the requirement for passage through M phase. Three lines of evidence support this conclusion. First, silencing was established by telophase (Fig. 2D), a time in the cell cycle when Scc1/Mcd1p is lost. Second, when Scc1/Mcd1p was absent, silencing was established after passage through S phase (Fig. 3D). Finally, when Scc1/Mcd1p cannot be degraded, silencing was substantially delayed (Fig. 4D). The simplest model to explain these findings is that the role of Scc1/Mcd1p is mediated through its participation in the cohesin complex. From our data, however, it is not possible to rule out a more specific negative effect of Scc1/Mcd1p, independent of its role in cohesion. It is also possible that the key event controlling the establishment is not sister-chromatid cohesion, but instead is a different event that depends on cohesion (e.g., chromosome condensation; Guacci et al. 1997). Future experiments addressing the effect of other elements of the sister-chromatid cohesion process will help to distinguish between these different models.

Previous studies of HMR found both Smc1p and Smc3p to be important for the function of a boundary element that defined the extent of the silenced domain (Donze et al. 1999). Supporting a more direct role for cohesin components in boundary element function, Scc1/Mcd1p has been shown to bind to these elements in vivo (Laloraya et al. 2000). Our findings identify an inhibitory role for Scc1/Mcd1p in the establishment of silencing in M phase. The involvement of cohesin components in promoting boundary element function and inhibiting the establishment of silencing could help to coordinate these two events. Such a dual role of Scc1/Mcd1p in silencing could ensure the formation of functional boundary elements prior to full establishment of silencing.

The S-phase requirement

Our studies clearly distinguish a requirement for passage through S phase from a requirement for passage through M phase during the establishment of silencing. Under conditions that eliminate the M-phase requirement (the absence of Scc1/Mcd1p), S-phase-arrested cells are still unable to establish silencing (Fig. 3D). Thus, the S-phase requirement is independent of Scc1/Mcd1p. We observe reduced association of Sir proteins with the HMR silenced domain during S phase. Although this observation offers a molecular explanation for the S-phase requirement in the establishment of silencing, it does not define the mechanism(s) that regulates Sir protein recruitment to a silencer at this stage of the cell cycle. Perhaps Sir proteins freed from HMR upon Sir3-8p inactivation preferentially reassociate with telomeres and/or the rDNA locus, since these sites represent the majority of Sir protein targets. If so, the transient diffusion of Sir proteins observed during G2/M phase (Straight et al. 1999; Laroche et al. 2000) may provide a mechanism for redistributing Sir proteins to the silencers after S phase. Alternatively, the reduced association of Sir proteins with HMR in S-phase-arrested cells may be due to a lack of one or more chromatin assembly factors at the silencer. The HMR region is unreplicated in HU-arrested cells (Santocanale et al. 1999), and this may reduce or eliminate the recruitment of chromatin assembly factors in this region. It is unlikely, however, that the S-phase requirement is due to the passage of the replication fork through the silenced region, as plasmids lacking an origin of replication can still establish silencing (Kirchmaier and Rine 2001; Li et al. 2001).

Possible mechanisms for the control of silencing by Scc1/Mcd1p

Scc1/Mcd1p has been shown to bind to the HMR locus in vivo, but whether a direct interaction with silencing proteins is necessary for its function in inhibiting establishment is unknown. If so, then removing Scc1/Mcd1p binding sites from the HMR locus could abrogate the effects of Scc1/Mcd1p on the establishment of silencing. In S. pombe, physical and genetic interactions have been shown between a heterochromatin component, Swi6, and the cohesin complex (Bernard et al. 2001; Nonaka et al. 2002). Although Scc1/Mcd1p does not appear to inhibit Sir protein association with HMR, a similar interaction between Scc1/Mcd1p and the Sir proteins could inhibit Sir protein function after they are assembled at the silenced region.

The requirement for passage through M phase to establish silencing at HMR may be a consequence of a chromatin maturation process. As chromosomes are duplicated, the chromatin is largely dismantled and reassembled. Concurrently, the chromosomes are prepared for segregation into daughter cells. Coordinating these two events may necessitate a delay in the assembly of complex chromatin domains such as those at HMR. Our studies of Sir protein association argue against a model in which the chromosome segregation machinery inhibits Sir protein recruitment to HMR. Instead, we propose that there is a process beyond HMR association that is required for the Sir proteins to perform their function in silencing. The activation of the HMR-associated Sir proteins could require the direct participation of the segregation machinery, perhaps involving both its assembly and disassembly. Such a process may affect silencing at locations other than HMR. Studies of telomeric heterochromatin indicate that it is particularly susceptible to activator-induced gene expression in G2/M (Aparicio and Gottschling 1994), indicating that silencing at newly replicated telomeres is weak. Future studies examining the differences between the derepressed G2/M chromatin and repressed G1 chromatin at HMR during the establishment of silencing should provide insights into the nature of the development of heterochromatic structures during the cell cycle.

Materials and Methods

Yeast strains

All strains used were haploid, bar1∷hisG derivatives of W303a (W303Ba, Table 1). To construct yAL170, a SalI/HinDIII fragment containing the 5′ portion of sir3-8 (Stone et al. 2000), its upstream region, and TRP1 inserted at the HpaI site was integrated into the SIR3 locus; plasmid pSB1598 was integrated at HML to delete this locus; and plasmid pSB1597 was integrated to delete the z1 region of MATa1. To construct strain yAL208, the MET3 promoter was amplified by PCR with ends homologous to the SCC1/MCD1 locus and transformed into yAL170 to replace the SCC1/MCD1 promoter. To construct strain yAL218, plasmid pWS103 (Shou et al. 1999) was modified to carry URA3 as its selectable marker, and integrated at the TEM1 locus. Strain yAL230 was constructed by integration of plasmid pAA355, containing GAL-SCC1 (R180D,R268D) (Uhlmann et al. 1999), at the LEU2 gene of yAL208. yAL227 and yAL231 are segregants of crosses between AAy1289 and yAL208 or yAL170, respectively.

Table 1.

Strains used

Strain
Genotype
Source
W303a MATa ura3-1 trp1-1 leu2-3,  112 his3-11, 15 ade2-1  can1-100 R. Rothstein
W303Ba W303a bar1::hisG This laboratory
yAL170 W303Ba sir3-8::TRP1  Δhml::HIS3 Δmatal::hisG This study
yAL208 yAL170 MET-SCCI1::URA3 This study
yAL218 yAL170 GAL-UPL-TEM1::URA3 This study
yAL230 yAL208 leu2::GAL-SCC1-RRDD  LEU2 This study
AAy1289 W303α ura3::3x tetO112 URA3  leu2::tetR-GFP LEU2 A. Amon
yAL227 Segregant of yAL208xAAy1289  cross W303a BAR1 sir3-8::TRP1  Δhml::HIS3 Δmata1::hisG  MET-SCC1::URA3 ura3::3x  tetO112 URA3 leu2::tetR-GFP  LEU2 This study
yAL231 Segregant of yAL170xAAy1289  cross W303a bar1::hisG sir3-8::TRP1  Δhml::HIS3 Δmata1::hisG  ura3::3x tetO112 URA3  leu2::tetR-GFP LEU2 This study
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In a typical experiment, cells were treated with 50 ng/mL α-factor (26 μg/mL for BAR1 strains) at 23°C for 2.5 h, raised to 37°C for 2 h, filtered and washed with 1.5 volumes of water, and resuspended in fresh media at 23°C containing 200 mM hydroxyurea (Sigma), 10 μg/mL nocodazole (Sigma), or 50ng/mL α-factor added after the appearance of buds. To deplete GAL-UPL-Tem1p, cells were switched at the time of the shift to 37°C to media containing 2% glucose. To repress MET-SCC1, methionine was added at a final concentration of 0.03% at the time of the shift to 37°C. To induce GAL-SCC1-RRDD, galactose was added at a final concentration of 2% at the time of the shift to 37°C.

Flow cytometric DNA quantitation

Measurement of DNA content by flow cytometry was performed as described (Bell et al. 1993).

Western blot analysis

To monitor SIR3 protein in the sir3-8 strain, samples from culture were withdrawn and proteins were isolated by TCA precipitation. TCA-precipitated protein was run on a 10% SDS gel, blotted to nitrocellulose, and probed with α-Sir3p antibodies.

Chromatin immunoprecipitation assay

ChIP assays were performed as described previously (Aparicio et al. 1997) with polyclonal antibodies to Sir3p (Mills et al. 1999), Sir2p (Imai et al. 2000), and Sir4p with the exception that RNase-treatment was omitted. PCR analysis (26 cycles) was performed on 2% of the precipitated DNA and 0.2% of the input DNA. PCR products were resolved on 6% PAG. Primer sequences to HMRE, HMRa1, or URA3 are available upon request.

RT–PCR

Total RNA was isolated by bead mill lysis and column purification (QIAGEN). Concentrations were adjusted to 0.5 μg/μL, and 0.5 μg of total RNA was used in 20 μL reaction with RT (Stratagene) to generate cDNA. PCR analysis (26 cycles) was performed using 5% of total cDNA. PCR products were resolved on 6% PAG. Primer sequences to HMRa1 or ACT1 are available upon request.

Real-time sequence detection

One-step RT–PCR was performed using the ABI Prism 7000 Sequence Detection System, and results were analyzed with accompanying software. We used 0.5 μg of total RNA in reactions with HMRa1 primers and probe, and 0.05 μg of total RNA was used in reactions with ACT1 primers and probe. A standard curve for each primer set was generated using 10-fold serial dilutions of yeast genomic DNA, ranging from 108 to 105 targets for primer annealing. Primer and TaqMan probe sequences for HMRa1 or ACT1 were selected using the Primer Express 2.0 software, and sequences are available upon request.

Indirect immunofluorescence

Immunofluorescence was performed as described (Visintin et al. 1999).

Acknowledgments

We thank F. Stegmeier, B. Lee, C. Armstrong, M. Kaeberlein, L. Pillus, F. Uhlmann, and J. Haber for their generous gifts of strains, plasmids, antibodies, and protocols; A. Amon, F. Solomon, and D. Sinclair for helpful discussions and comments on the manuscript; and J. Lindow and D. Rines for microscopy assistance. This work was supported by the Howard Hughes Medical Institute and by an award from the Searle/Chicago Community Trust. S.P.B. is an employee of the Howard Hughes Medical Institute, A.L. was supported by a predoctoral training grant from the N.I.H., and H.B. was supported by a predoctoral fellowship from the Howard Hughes Medical Institute.

 The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Footnotes

Supplemental material is available at http://www.genesdev.org.

E-MAIL spbell@mit.edu; FAX (617) 253-4043.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.764102.

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