The CCR4-NOT Complex Maintains Stability and Transcription of rRNA Genes by Repressing Antisense Transcripts

The rRNA genes (rDNA) in eukaryotes are organized into highly repetitive gene clusters. Each organism maintains a particular number of copies, suggesting that the rDNA is actively stabilized. We previously identified about 700 Saccharomyces cerevisiae genes that could contribute to rDNA maintenance. Here, we further analyzed these deletion mutants with unstable rDNA by measuring the amounts of extrachromosomal rDNA circles (ERCs) that are released as by-products of intrachromosomal recombination.

evolution, cellular demand for ribosomes has grown due to the increase in cell size and the enhanced number of cellular functions. Ribosomes consist of ribosomal proteins and RNA that occupy, in a cell of budding yeast Saccharomyces cerevisiae, ϳ70% of the total protein and ϳ60% of the total RNA (1). To sustain the synthesis of large numbers of ribosomes, the yeast cell has ϳ150 tandemly repeated copies of rRNA genes (rDNA) on chromosome XII (chr. XII).
Repetitive sequences form unstable regions in a genome, as spontaneous recombination between repeats can lead to loss or gain of these sequences. The rDNA is one of the most abundant repetitive regions in eukaryotic genomes in which the copy number can vary, as has been observed in several organisms (2,3). In spite of this unstable nature, each organism maintains its rDNA copy number at a particular, stable level, thereby suggesting that there are mechanisms at play that keep the overall repeat structure intact (4).
Some of the mechanisms that help to maintain the rDNA copy number are well studied in budding yeast (3). A major mechanism is gene amplification (5) (see Fig. S1 in the supplemental material). When rDNA copies are lost by intrachromosomal recombination, gene amplification occurs to compensate for the lost copies. The main players in the process of gene amplification are the replication fork barrier (RFB) site and the examined the rDNA stability in ϳ4,800 gene deletion mutants and classified them by rDNA stability into four ranks, rank 1 (more stable than the wild type [wt]), rank 2 (as stable as the wt), rank 3 (more unstable than the wt), and rank 4 (extremely unstable) (13). Around 700 mutants with unstable rDNA (of ranks 3 and 4) were identified, and some of these have been analyzed in more detail (13)(14)(15). The possibility that among these~700 mutants, identified in a first step to select genes important for rDNA stability from ϳ4,800 candidates, there will be false positives cannot be excluded (16). Therefore, any follow-up analysis requires confirmation of the rDNA instability in these mutants, which we have done for about one-third of the mutants categorized in rank 3. We reexamined 242 of these strains by pulsed-field gel electrophoresis (PFGE), and 73 strains were subjected to more quantitative assays that assessed the level of extrachromosomal rDNA circles (ERCs) that are produced by recombination in the rDNA and, thus, are indicative of rDNA instability (17,18) (Fig. S1). Among the mutants examined, a mutant lacking one of the components of the CCR4-NOT complex (19)(20)(21)(22)(23), Pop2 (Caf1), produced an extremely high level of ERCs. The rDNA was also unstable in mutants lacking other members of the complex (ccr4, dhh1, not3, and not4). The CCR4-NOT complex is a multifunctional protein complex that is involved in the regulation of gene expression of various classes of RNA: mRNA, rRNA, and other nonprotein-coding RNA (24)(25)(26)(27). The deadenylase activity of Pop2 and Ccr4 can trigger mRNA degradation by exonucleases such as Rrp44 (28).
Here, we report that in the pop2 and ccr4 mutants, levels of noncoding RNA transcribed from E-pro are highly increased, and the extent of association to rDNA by cohesin and condensin was reduced. Moreover, the amount of rRNA in the pop2 mutant is reduced to about half of the wild-type level. These mutant phenotypes depended on the presence of the E-pro promoter. We conclude that the CCR4-NOT complex mediates the degradation of noncoding RNA transcribed from the E-pro promoter and in this manner contributes to maintaining rDNA stability and rRNA synthesis.

RESULTS
Screening of mutants with unstable rDNA. To confirm rDNA instability and identify mutants with highly unstable rDNA, we conducted a secondary screen with 242 mutants out of ϳ660 categorized as rank 3 in our previous screen that lack genes whose products function in DNA replication, recombination, repair, and transcription according to the gene ontology annotation in the Saccharomyces Genome Database (29,30). First, genomic DNA prepared from these mutants was separated by PFGE, and rDNA stability was analyzed by comparing the degree of smearing of the chr. XII band in a mutant to that of wild-type chr. XII. Of the 242 strains examined, for 73 strains the rDNA instability phenotype was reproduced and deletion of the genes of interest was confirmed (data not shown). These strains were then subjected to more quantitative ERC assays. Genomic DNA, prepared from mutant strains that were grown from two independent single colonies, was separated by conventional agarose gel electrophoresis, and ERCs were detected by Southern blotting (see Fig. S2 in the supplemental material). As controls, we included DNA isolated from the wild-type strain and also from the sir2 mutant that is known to produce a high level of ERCs (31). The amount of ERCs was normalized to the amount of genomic rDNA to correct for differences in rDNA copy number. More ERCs accumulated in 62 mutants than in wild-type cells (Fig. S2L). Among them, the pop2 mutant contained the highest level of ERCs, which was ϳ50-fold higher than that of the wild type and ϳ4-fold higher than that of the sir2 mutant (Fig. S2). Pop2 is a component of the CCR4-NOT complex (20,21). Interestingly, two other members of this complex, Ccr4 and Dhh1, had originally also been classified as rank 3 (13). Unfortunately, both genes were omitted from the ERC assay, as DHH1 did not pass the selection by gene ontology and a deletion of CCR4 was not in the library used for the secondary screen. Overall, these findings indicate that the CCR4-NOT complex plays an important role in maintaining rDNA stability.
The CCR4-NOT complex is required for the maintenance of rDNA stability. To examine the contribution of the CCR4-NOT complex to the maintenance of rDNA stability, we constructed mutants lacking components of this complex in the W303 genetic background, which is different from the BY4741 background used for the yeast knockout library, and analyzed rDNA stability in these mutants by PFGE. Of the ten subunits in the CCR4-NOT complex (27), we could not establish deletion mutants for NOT1, NOT2 or NOT5, suggesting that these genes are essential in the W303 background. The mutants lacking Pop2, Ccr4, Dhh1, Not3, or Not4, but not Caf40 or Caf130, showed smeared bands of chr. XII, as seen in the sir2 mutant, after staining with ethidium bromide (EtBr), indicating that the rDNA copy number frequently changes in these strains and that their rDNA is unstable ( Fig. 1B; see also Fig. S3 in the supplemental material). Consistent with these results, these mutants produced a high level of ERCs ( Fig. 1C and D). In addition, compared to the wild-type strain or the sir2 mutant, the size of chr. XII, and therefore the rDNA copy number, were increased in all these strains except the not3 mutant ( Fig. 1B and Fig. S3).
To examine the relationship between rDNA recombination and the CCR4-NOT complex, we established double mutants with fob1. In the absence of Fob1, replication fork arrest and DSB formation that can induce recombination do not occur and rDNA becomes stable (10,12,15). The chr. XII band in each of the double mutants was as sharp as that in the fob1 single mutant ( Fig. 1B and Fig. S3), suggesting that the CCR4-NOT complex functions downstream of Fob1. In other words, the CCR4-NOT complex maintains rDNA stability after replication fork arrest and DSB formation.
Pop2 and Ccr4 are generally considered to have deadenylase activities that can promote cytoplasmic mRNA degradation (32,33). However, whether Pop2 has a catalytic function is still unclear. In yeast, the catalytic activity of Pop2 is not required for mRNA poly(A) removal (34), while Not4 is a ubiquitin protein ligase (35,36). Dhh1 interacts with the CCR4-NOT complex and has helicase activity (37,38). Thus, the absence of CCR4-NOT-associated enzymatic activity, by deleting Dhh1 or one of the subunits Ccr4, Pop2, and Not4, results in a severely unstable rDNA phenotype, namely, a large but variable increase in rDNA copy number and extensive ERC formation.
Deadenylase activity of CCR4-NOT is required to maintain rDNA stability. Defects in deadenylation activity in the pop2 and ccr4 mutants may cause the observed rDNA instability. To test this possibility, we examined the effect of point mutations in POP2 or CCR4 that would abolish enzymatic activity on rDNA stability (39)(40)(41)(42). We introduced an empty plasmid, a plasmid carrying wild-type POP2 or CCR4, or plasmids carrying catalytically dead alleles of pop2 (S44A, E46A) or ccr4 (E556A) into the diploid strain heterozygous for pop2 or ccr4 gene deletion, followed by tetrad dissection to isolate the haploid clones of interest. As seen before (Fig. 1B), in the pop2 mutant carrying the empty plasmid, large, smeared chr. XII bands were observed ( Fig. 2A). In contrast, expression of wild-type or catalytically dead Pop2 proteins in pop2 mutant cells did not exhibit the rDNA instability seen in the pop2 mutant ( Fig. 2A), suggesting that the presence, rather than the catalytic activity, of Pop2 is important for maintaining rDNA stability. Expression of wild-type Ccr4 in the ccr4 mutant suppressed the rDNA instability ( Fig. 2A). The rDNA in the ccr4 mutant expressing the catalytically inactive Ccr4-E556A protein, however, was unstable and similar to the rDNA in the mutant carrying an empty plasmid. Thus, Ccr4 activity is essential for rDNA maintenance, while Pop2 has a mediating role. Indeed, the presence of Pop2, not its activity, is found to be required for tethering Ccr4 to the complex (21,43,44). Thus, in the pop2 deletion mutant, the complex loses both Pop2 and Ccr4, and thereby all deadenylase activity, leading to rDNA instability.
After complementation of the deletion strains (pop2 and ccr4) by reintroducing wild-type or catalytically inactive alleles, growth is restored in the case of Pop2 but only barely so in the case of Ccr4 (Fig. 2B). This result confirms the rDNA analysis and points to a correlation of a functional CCR4-NOT complex with the recovery of rDNA stability ( Fig. 2A). Interestingly, rDNA instability by itself does not affect growth (see below).
Noncoding transcripts accumulate in CCR4-NOT mutants. According to the current model for the mechanism of rDNA recombination (Fig. S1), rDNA stability is mainly affected by three events: (i) replication fork arrest, (ii) DSB formation, and (iii) noncoding transcription from E-pro. To identify which event is linked to a malfunctioning CCR4-NOT complex, we first determined the levels of arrested forks at the RFB site in the pop2, ccr4, and dhh1 mutants, in which the unstable rDNA phenotype was most prominent. To this end, replication intermediates were analyzed by twodimensional (2D) gel electrophoresis (6,45) in which arrested replication forks at the RFB site appear as a strong signal along the arc of Y-shaped replication intermediates. The levels of arrested forks in the CCR4-NOT mutants were similar to that in the wild type ( Fig. S4A to C). DSBs at the RFB site were detected by one-dimensional gel electrophoresis (15), which provided an indication for a partial correlation between the DSB levels and rDNA instability ( Fig. S4D and E), that is, levels of DSBs were ϳ1.6-fold higher in the pop2 and ccr4 mutants than in the wild type, but no difference was observed for the dhh1, not3, and not4 mutants. Although these increases in DSB levels may be a cause of rDNA instability in these mutants, the difference seems too small to explain, for example, the large increase of ERCs in the pop2 mutant.
The noncoding transcripts from E-pro were detected by Northern blot analysis ( Fig.  3A and B). Transcripts in either direction accumulated in the sir2 and pop2 mutants relative to the levels in wild-type cells, in which they were barely detectable. Quantitation of the amount of E-pro-derived transcripts showed that these markedly increased in the pop2 mutant compared to those in the wild type (Fig. 3C). A strong accumulation of transcripts initiated at E-pro was also observed in the absence of other subunits, in particular the enzymatic subunits of the CCR4-NOT complex ( Fig. S5A to F). In addition, in the pop2 sir2 double mutant more E-pro-derived transcripts were formed than produced in each single mutant (Fig. S6), which was also observed for the sir2 double mutant with dhh1 ( Fig. S5C and D). These results indicate that on top of reduced E-pro suppression in the absence of Sir2, the synthesized transcripts that are derived from E-pro are not degraded in the CCR4-NOT mutants when the enzymatic activity of the complex is compromised or are more efficiently produced. The accumulation of these noncoding transcripts is linked to a dramatically reduced rDNA stability.
E-pro transcripts increase rDNA instability in CCR4-NOT complex mutants. To find out whether reduced rDNA stability in the pop2 mutant is directly related to the noncoding transcription from E-pro itself, we established the pop2 deletion in a mutant lacking the E-pro sequence from its rDNA repeats (11). In this strain, E-pro was replaced with the GAL1/10 promoter that is not induced in medium containing raffinose as the carbon source and not regulated by Sir2. In raffinose-containing medium, the rDNA in this strain is stable with fewer ERCs, but in galactose-containing medium the opposite results are observed (46). The absence of Pop2 in this E-pro-less (E-proΔ) strain, when grown in raffinose-containing medium, did not affect rDNA stability, as shown by PFGE (Fig. 4). The band of chr. XII in the E-pro⌬ pop2 mutant was as sharp as that in wild-type and fob1 cells. The presence of Ccr4 or Dhh1, other CCR4-NOT complex subunits required for maintaining stable rDNA, also is not essential for rDNA maintenance in the absence of E-pro (Fig. 4). In contrast, in galactose-containing medium, where transcription of the E-pro locus is activated in E-proΔ cells, the absence of CCR4-NOT complex subunits Pop2 and Ccr4 caused smearing and lengthening of chr. XII bands (Fig. S7). In the dhh1 mutant, however, the chr. XII band is more comparable to that in wild-type and sir2 cells. These results indicate that transcripts from E-pro that accumulate in the absence of the deadenylase subunits of the CCR4-NOT complex increase rDNA instability.
Increased E-pro transcription in the absence of Pop2 leads to reduced rDNA association of cohesin and condensin. Transcription from E-pro, which is increased in the sir2 mutant, prevents cohesin from associating with rDNA, resulting in decreased rDNA stability (12). We tested whether a similar scenario occurs in the pop2 mutant by chromatin immunoprecipitation (ChIP) (47). In this assay, protein associated with DNA is precipitated and the DNA is detected by real-time PCR. To analyze rDNA association of cohesin (Mcd1), we used a FLAG-tagged version of the protein and four PCR primer sets that amplify various regions in the rDNA (Fig. 5A). In the pop2 mutant, the rDNA  association of Mcd1 is comparable to that in the sir2 mutant and reduced compared to that of the wild type (Fig. 5B). We also tested condensin (Smc2), the dissociation of which from rDNA is linked to rDNA instability (48). Using FLAG-tagged Smc2, the association with rDNA was, compared to that of the wild-type strain, reduced by more than 2-fold in the pop2 and sir2 mutants (Fig. 5C). These results indicate that increased levels of E-pro transcription inhibit cohesin and condensin association with rDNA and reduce rDNA stability.
Accumulation of E-pro-derived transcripts interferes with rRNA synthesis. Enhanced E-pro transcription in CCR4-NOT mutants could interfere with the synthesis and processing of the 35S rRNA precursor. To see whether the RNA polymerase I and II machineries collide, we measured rDNA transcripts. As shown in Fig. 6A to C, the amount of 25S and 18S rRNA in the pop2 and ccr4 mutants was reduced about 2-fold compared to the wild-type level or that of the sir2 mutant. Moreover, the amount of preprocessed 35S rRNA was also reduced (Fig. S8B), suggesting that the absence of enzymatic active CCR4-NOT interfered with rDNA transcription and/or processing. To confirm whether this reduction of rRNA transcripts is related to the activity of E-pro, we measured the levels of rDNA transcripts in the E-pro⌬ pop2 mutant that was grown in  (Fig. 1A), transcripts from E-pro were detected. As the GAL promoter is not activated in raffinose-containing medium, the transcript was undetectable in the E-proΔ strain. The same gel as that used in panel A was hybridized. (C) The quantitation of rRNA transcripts in panel A. Each signal intensity was normalized to total RNA. The values are relative to that of the wt. Error bars show the range from two independent experiments. (D) Growth of the pop2 and sir2 mutants was determined by a spot assay on raffinose-containing medium. noninducing medium. This replacement resulted in more wild-type-like amounts of 18S and 25S rRNA ( Fig. 6A and C), while accumulation of IGS1-R transcripts was no longer detectable (Fig. 6B). Moreover, the poor growth in the pop2 mutant partially recovered in the E-pro⌬ pop2 mutant ( Fig. 6D; also see below).
Apart from colliding transcription machines, it is possible that the transcripts from E-pro form R-loops (DNA/RNA hybrids) that inhibit 35S transcription. To test this possibility, we precipitated DNA/RNA hybrids with S9.6 antibody from wild-type and pop2 mutant cells and checked by quantitative PCR (qPCR) for hybrid formation at various sites on the rDNA. As shown in Fig. S9, the amount of DNA/RNA hybrids detected in the pop2 mutant was 2-fold lower than that for the wild type at any site. Therefore, R-loop formation seems not to be related to reduced rDNA transcription and, while often associated with genome instability, not instrumental in the rDNA instability phenotypes of the pop2 mutant.
Accumulation of E-pro-derived transcripts is related to growth in CCR4-NOT complex mutants. In the pop2 mutant, E-pro transcripts increase and rDNA becomes unstable, like that in the sir2 mutant ( Fig. 1B and 3). The major difference between the pop2 and sir2 mutants is that the pop2 mutant grows poorly compared to the wild-type strain and the sir2 mutant (Fig. 6D). The effect on cell growth by accumulation of noncoding RNA transcribed from E-pro could be compared to a situation where less noncoding RNA was formed from an Sir2-independent, noninduced GAL promoter that replaced the E-pro (E-proΔ) (Fig. 6D). This experiment revealed that the growth defect observed in the absence of Pop2 was alleviated in the E-pro⌬ strain, suggesting that accumulation of E-pro transcripts is related to a cause of the observed growth defect.
Concomitant with an accumulation of E-pro transcripts, the pop2 mutant has about 3-fold more rDNA (Fig. S5G) and 8-to ϳ50-fold more ERCs (Fig. 1D and Fig. S2L) than the wild type. Although not observed for the sir2 mutant, such an increase in unstable rDNA may be a burden and could affect cell growth. Therefore, we tested the growth of the pop2 fob1 double mutant, which has rDNA that is stably kept at the normal rDNA copy level (Fig. 1B). In the double mutant, ERCs did not accumulate ( Fig. S10A and B), but despite this decrease in rDNA stress, cell growth in the absence of Pop2 and Fob1 was still poor (Fig. S10C). In contrast to the absence of Sir2, CCR4-NOT mutations could affect a variety of processes that support removal of unwanted compounds (possibly E-pro-derived transcripts). Therefore, our results suggest that increases in rDNA copy and ERCs are not a major cause of poor growth of CCR4-NOT mutants. Most likely, the accumulation of products that normally are cleared by processes controlled by this complex reduces the production of rRNA and ribosomes, causing poor growth.

DISCUSSION
rRNA is one of the most abundant RNAs in organisms. During the course of evolution, cells have developed systems to produce large amounts of rRNA transcripts. One of them is gene amplification of rDNA. In the budding yeast, the noncoding promoter E-pro regulates the type of recombination that supports rDNA amplification (11) (see Fig. S1 in the supplemental material). We found that in the pop2 mutant rDNA becomes unstable and that the E-pro transcripts accumulate, which interferes with rDNA association of cohesin and condensin. As a result, unequal sister chromatid recombination is expected to increase and to induce rDNA instability (12,48) (Fig. 7A). Moreover, an accumulation of E-pro transcripts was observed to lead to a reduction of rRNA production only in the absence of Pop2 or Ccr4, not Sir2 (Fig. 6). Whether this reduction would have caused a growth defect, as seen upon destabilizing RNA polymerase I (49), or other processes are affected by CCR4-NOT complex mutations remains to be seen. We did observe, however, an improvement in cell growth when E-pro transcription was reduced, indicating that accumulation of E-pro transcripts is part of the problem these mutant cells face.
The CCR4-NOT complex functions to mediate degradation of mRNA through its deadenylation activity (22, 50-52), which in essence is a 3=-5=-exoribonuclease activity. Here, we found that the complex may also be required for the degradation of noncoding RNAs that are transcribed from E-pro in the rDNA. It has been shown that transcripts from E-pro are degraded by the exosome and the TRAMP complex containing Trf4 (53). Trf4 is a poly(A) polymerase component of the TRAMP complex that mediates nuclear RNA degradation by the exosome. Additionally, it has been shown that the 3= end of IGS1-R is deadenylated by Pap1 (53). The CCR4-NOT complex can interact with components of TRAMP and the nuclear exosome during the degradation of noncoding nuclear RNAs, such as misprocessed and polyadenylated snoRNAs (24). Moreover, it has been shown that CCR4-NOT subunits are physically enriched in the rDNA (26). Therefore, it is feasible that the CCR4-NOT complex also functions, together with TRAMP and the nuclear exosome, in the degradation of noncoding RNA transcribed from E-pro in the rDNA. However, in the trf4 mutant, only the transcripts made in the direction of a replication fork that would be colliding with 35S transcription (IGS1-R) accumulated (53). In the CCR4-NOT mutants (pop2, ccr4, dhh1, and not4), we observed the accumulation of transcripts produced in both directions from the E-pro ( Fig. 3 and Fig. S5). In fact, for recombination that leads to rDNA amplification, bidirectional transcription from the E-pro is required (11). This may be the reason that rDNA stability is not obviously reduced in the trf4 mutant (53). Our analysis of the yeast deletion collection also showed that rDNA in the trf4 mutant and in exosome mutants was as stable as that in the wild-type strain (13). Therefore, we speculate that one of the reasons the rDNA integrity is so severely affected in CCR4-NOT mutants is that the removal of E-pro transcripts is more seriously impaired in these strains than in the trf4 or exosome mutants.
Accumulation of E-pro transcripts is also observed in the sir2 mutant, in which rDNA is unstable (Fig. 3) (11). In contrast to the CCR4-NOT mutants (pop2 and ccr4), in the case of the sir2 mutant, cell growth and the amount of rDNA transcripts were normal ( Fig.  6 and Fig. S8). In the sir2 mutant, E-pro transcription is not repressed and more RNA is transcribed (11) (Fig. 3 and Fig. S5 and S6) but can be cleared out by CCR4-NOTdependent processes. In the pop2 mutant the transcripts accumulate, presumably because they are not degraded (Fig. 7B). E-pro transcripts may anneal with 35S rRNA and interfere with its transcription and/or promote its degradation. The exact length of E-pro transcripts that run against the direction of 35S transcription (IGS1-R) remains unclear, because the transcription does not appear to terminate at a specific site while a huge amount of 18S rRNA appears to comigrate and could interfere with Northern analyses (Fig. 6A and B). Still, the noncoding transcripts could be long enough to anneal to large sections of 35S rRNA. In the pop2 mutant but not in the sir2 mutant (where the E-pro transcripts can be degraded), the accumulated antisense RNA may interfere with rRNA production when such hybrids are formed and not sufficiently untangled (Fig. 7B, panel c). Further study is required to elucidate how the CCR4-NOT complex controls the degradation of E-pro transcripts. The deadenylase-defective allele of pop2 (S44A, E46A) (41) had a small effect on rDNA stability and growth compared to that of ccr4 (E556A) (40) (Fig. 2). This suggests that the deadenylase activity of Pop2 is less important for CCR4-NOT function than that of Ccr4 (32). There is structural evidence that Pop2 has a role to connect Ccr4 to the complex (23), so that in the pop2 deletion mutant not only Pop2 but also Ccr4 is absent from the complex. In the case of the deadenylase-defective Pop2 (S44A, E46A), however, it is possible that the protein can hold on to Ccr4 and keeps the main deadenylase activity linked to the complex. In the deletion-or deadenylase-defective mutants of Ccr4, only the enzymatic activity of Pop2 resides in the complex, which is not sufficient to maintain rDNA stability and normal growth (Fig. 2), as is supported by other reports (44,54).
The rDNA transcriptional interference by antisense transcription from E-pro, which leads to reduced rRNA synthesis, could explain another phenotype caused by a defective CCR4-NOT complex in rDNA maintenance. In addition to unstable rDNA in the pop2 and ccr4 mutants, their rDNA copy number is significantly increased (Fig. 1B and  Fig. S5G). This suggests that cells have a means to sense the amount of rRNA, which feeds back to stimulate rDNA amplification when rRNA production is down. Consistent with this hypothesis, it has been shown that the levels of nascent rRNA are increased in CCR4-NOT complex-defective mutants by PolI ChIP analysis (26). If more copy numbers produce more E-pro transcripts, then these transcripts could interfere with rDNA transcription to the extent that amplification recombination remains induced. This ongoing rDNA amplification would result in a huge amount of rDNA copies on the chromosome and, upon recombination, as ERCs. Further analysis is required to unravel these possibilities.

MATERIALS AND METHODS
Yeast strains, oligonucleotides, and growth conditions. Mutant yeast strains used for ERC assays, shown in Fig. S2 in the supplemental material, are derived from the Yeast Knockout Collection (YSC1053; Open Biosystems). The wild-type strain shown in Fig. S2 is BY4741 (MATa his3-1 leu2-0 met15-0 ura3-1). Other yeast strains used in this study are derivatives of NOY408-1b, which is derived from W303, and are listed in Table S1. Gene deletion was performed by one-step gene replacement. Oligonucleotides used in this study are listed in Table S2.
For PFGE and ERC assays, cells were grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) at 30°C overnight. For PFGE shown in Fig. 4 and Fig. S7, cells were grown in YPRaffinose (1% yeast extract, 2% peptone, and 2% raffinose) or YPGalactose (1% yeast extract, 2% peptone, and 2% galactose). To prepare RNA, cells were grown overnight at 30°C in YPAD medium (YPD containing 1.67% adenine). To perform 2D and DSB analyses, cells were first grown in 5 ml of YPD at 30°C overnight until saturation. Cells were then inoculated into 30 to 50 ml fresh YPD medium at a density of 0.5 ϫ 10 6 to 1.0 ϫ 10 6 cells/ml and grown until the culture reached a density of 2 ϫ 10 6 to 3 ϫ 10 6 cells/ml. To perform ChIP analysis, cells were first grown in 5 ml of YPAD at 30°C overnight until saturation. Cells were then inoculated into 50 ml of fresh YPAD at an optical density at 600 nm (OD 600 ) of 0.1. Cells were incubated until the culture reached an OD 600 of 0.8 to 1.0 (log phase).
Plasmid construction. YCplac33-POP2 and YCplac33-CCR4 were constructed by the In-Fusion cloning method as follows. The genomic regions containing either the POP2 or the CCR4 open reading frame plus ϳ500 bp upstream and downstream were PCR amplified from S. cerevisiae genomic DNA using primers HS405 and HS406 (in the case of POP2) or HS409 and HS410 (for CCR4). Linearized YCplac33 vector fragments were generated by PCR amplification of YCplac33 using primers HS411 and HS412 (POP2 specific) or HS413 and HS414 (CCR4 specific). The gene and vector fragments with 18 bases of homology at each end were fused using the In-Fusion HD cloning kit (TaKaRa) according to the manufacturer's instructions. The reaction mixes were used for transformation of Escherichia coli DH5␣, and plasmid DNA from positive clones was analyzed by Sanger sequencing.
Plasmids YCplac33-pop2 (S44A, E46A) and YCplac33-ccr4 (E556A) were constructed using the Prime-STAR mutagenesis basal kit (TaKaRa) according to the manufacturer's instructions. Briefly, primers were designed to have 15 nucleotides of overlap at their 5= ends and to contain the mutations that change Pop2 serine 44 into alanine and glutamate 46 into alanine (primers HS533 and HS534) or that changes Ccr4 glutamate 556 into alanine (primers HS535 and HS536). These primers were used to perform PCR using YCplac33-POP2 or YCplac33-CCR4 as a template. The resulting PCR products were transformed into E. coli DH5␣. Plasmid DNA was sequenced to confirm that the mutations had been introduced.
Genomic DNA preparation. For PFGE, 2D gel analysis, DSB analysis, and ERC assays, genomic DNA was prepared in low-melting-temperature agarose plugs as described previously (15). For 2D gel analysis and DSB analysis, cells were collected and treated with 0.1% sodium azide prior to genomic DNA preparation. To assess rDNA copy numbers, genomic DNA was prepared by the conventional method as described previously (15).
ERC assays. One-half of an agarose plug was cut and electrophoresed on 0.4% agarose gel in 1ϫ Tris-acetate-EDTA (40 mM Tris base, 20 mM acetic acid, and 1 mM EDTA, pH 8.0) at 1.0 V/cm for 48 h at 4°C with buffer circulation. The buffer was changed after ϳ24 h of electrophoresis. DNA was transferred to Hybond-N ＋ (GE Healthcare), followed by Southern blot hybridization as described previously (15). The probe to detect genomic rDNA and ERCs was PCR amplified from S. cerevisiae genomic DNA using primers HS204 and HS205. Quantification of signals was performed by Fujifilm MultiGauge, version 2.0, software (Fujifilm). The membranes were first exposed to phosphor screens so that signals did not become saturated. Scanned images were used to quantify genomic rDNA. The membranes were then exposed for several days, and scanned images were used to quantify supercoiled monomers, relaxed monomers, supercoiled dimers, and relaxed dimers. The sum of ERC signals was normalized to signals of genomic rDNA. For each panel (blot) in Fig. S2A to K, the mean value for the wt was set to 1. For Fig. S2L, ERC levels in all the mutants examined were combined. As a representative ERC score for sir2, the value shown in Fig. S2B was used.
PFGE. One-third of an agarose plug and Hansenula wingei chromosomal DNA markers (Bio-Rad) were electrophoresed on 1.0% agarose gel (pulsed-field certified agarose; Bio-Rad) in 0.5ϫ Tris-borate-EDTA (TBE) buffer (44.5 mM Tris base, 44.5 mM boric acid, and 1 mM EDTA, pH 8.0) on a Bio-Rad contourclamped homogeneous electric field DR-III system under the following conditions: 68 h at 3.0 V/cm at 14°C, 120°included angle, initial switch time of 300 s, and final switch time of 900 s. DNA was transferred to Hybond XL, followed by Southern blot hybridization as described previously (15). The probe to detect chr. XII was PCR amplified from S. cerevisiae genomic DNA using primers HS210 and HS211.
2D gel electrophoresis. To detect replication intermediates, 2D neutral/neutral gel electrophoresis was performed as described previously (45). Briefly, one-third of an agarose plug was equilibrated four times in 1 ml Tris-EDTA (TE; pH 9.0) for 15 min and then equilibrated twice with 1 ml of 1ϫ H buffer (TaKaRa) for 20 min and digested in 200 l of 1ϫ H buffer containing 150 U of BglII (TaKaRa) for 8 to 16 h at 37°C. DNA was separated on 0.4% agarose gel (Star Agarose; Rikaken) in 1ϫ TBE at 1.32 V/cm for 14 h at room temperature. DNA was stained with 0.3 g/ml of EtBr. Gel slices containing replication intermediates in rDNA were excised, rotated 90°, and separated on 1.2% agarose gel in 1ϫ TBE containing 0.3 g/ml of EtBr at 6.0 V/cm for 6 h at 4°C. DNA was transferred to Hybond-N ϩ (GE Healthcare), followed by Southern blot hybridization as described previously (15). The probe to detect replication intermediates was prepared by PCR amplification of S. cerevisiae genomic DNA using primers HS266 and HS275. Quantification was performed with ImageJ (NIH). We determined the signal for the RFB spot (a) and the intensity of the replication intermediate with the spots for 1N and 2N (b). The relative replication-intermediate levels were calculated as a divided by b. The score was normalized to the wt value of 1. DSB analysis. To assess DSB frequency, physical assays were performed as described previously (15). Briefly, one-third of an agarose plug was equilibrated four times in 1 ml TE for 15 min, equilibrated twice with 1 ml of 1ϫ NEBuffer 3.1 for 30 min, and digested in 150 l of 1ϫ NEBuffer 3.1 containing 150 U of BglII (New England Biolabs) for 12 to 16 h at 37°C. DNA was separated on 0.7% agarose gel in 1ϫ TBE at 2.0 V/cm for 20 to 22 h at room temperature with buffer circulation. DNA was transferred to Hybond XL (GE Healthcare), followed by Southern blot hybridization as described previously (15). The probe used for DSB analysis was PCR amplified from S. cerevisiae genomic DNA using primers HS206 and HS207. Quantification was performed by Fujifilm MultiGauge, version 2.0, software. Signals of DSB fragments and replication intermediates containing arrested forks were quantified. The level of DSBs was assessed by normalizing DSB signals to signals of replication intermediates containing arrested forks. The values for the wt were set to 1.
rDNA copy number assays. Approximately 5 g of genomic DNA was digested with 50 U of BglII (TaKaRa). DNA was separated on 1% agarose gel at 10 V/cm for 30 to 40 min. DNA was transferred to Hybond N ϩ (GE Healthcare), followed by Southern blot hybridization as described previously (15). Quantification was performed by ImageJ (NIH). The membranes were first hybridized by probes to detect MCM2, which was PCR amplified using primers HS542 and HS543. Probes were stripped and the membranes were hybridized with an rDNA probe that was PCR amplified using primers HS393 and HS394. Signals of the rDNA fragment were normalized to that of the MCM2 fragment. The mean values for the wt were set to 1.
Yeast RNA preparation. Cells were collected and washed with ice-cold water treated with 0.1% diethylpyrocarbonate. Cells were resuspended in 400 l of TES (10 mM Tris-HCl [pH 7.5], 10 mM EDTA at pH 7.5, 0.5% SDS). An equal volume of acidic phenol, a mixture consisting of equal volumes of phenol and 50 mM sodium acetate at pH 4.0, was added to the cell suspension, followed by vigorous vortexing for about 10 s. Cells were incubated at 65°C for 1 h, incubated for 5 min on ice, and centrifuged at 17,400 ϫ g for 10 min at 4°C. The aqueous phase was reextracted with 200 l of acidic phenol and 200 l of chloroform, after which the RNA was isolated by ethanol precipitation. RNA was quantified by measuring the OD 260 using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
Northern blotting and hybridization. Either 10 g of total RNA (to detect noncoding RNA from IGS1) or 1 to 5 g of total RNA (to detect rRNA) and, as a size standard, DynaMarker RNA High (BioDynamics Laboratory Inc.) were separated on 1% agarose gel containing 6% formaldehyde and 1ϫ morpholinepropanesulfonic acid (MOPS; 20 mM MOPS, 2 mM sodium acetate, 1 mM EDTA, 0.1% diethylpyrocarbonate, adjusted to pH 7.0 with NaOH) in 1ϫ MOPS running buffer at 10 V/cm for 30 to 40 min. RNA was transferred to Hybond N ϩ (GE Healthcare).
To prepare strand-specific probes used for Northern blot hybridization, 10 ng of PCR product was mixed with the primer at a final concentration of 0.1 M in a reaction volume of 17.25 l. The PCR product was heat denatured by boiling for 3 min at 95°C, followed by gradual cooling to 50°C, and then 2.5 l of 10ϫ Ex Taq buffer, 0.25 l of Ex Taq (1.25 U; TaKaRa), 2.5 l of 0.2 mM deoxynucleoside triphosphate mixture, and 2.5 l of [␣-32 P]dCTP (3,000 Ci/mmol; Perkin Elmer) was added and the mixture was incubated at 72°C for 15 to 20 min. Unincorporated labeled nucleotides were removed using ProbeQuant G-50 microcolumns (GE Healthcare). Noncoding RNA from IGS1 was detected by hybridization overnight at 65°C using Rapid-hyb buffer (GE Healthcare), and rRNA was detected by hybridization overnight at 65°C using ULTRAhyb ultrasensitive hybridization buffer (Thermo Fisher Scientific). Quantification was performed by Fujifilm MultiGauge, version 2.0, software to quantify results of Northern blotting and ImageJ (NIH) to quantify rRNA levels in gels stained with EtBr. Each signal intensity of rRNA was normalized to that of total RNA. The transcript levels are relative to those of the wt (set to 1). Primers to obtain PCR products used as a template for linear PCR yielding the labeled probes were the following: for the 25S rRNA probe, HS391 and HS392; for 18S rRNA, HS387 and HS388; for 35S rRNA, HS397 and HS398; for IGS1-F transcripts, HS274 and HS275; and for IGS1-R transcripts, HS266 and HS296. Primers used for probe synthesis by linear PCR are the following: for the 25S rRNA probe, HS392; for 18S rRNA, HS388; for 35S rRNA, HS398; to detect IGS1-F transcripts, HS275; and to detect IGS1-R transcripts, HS266.
ChIP. ChIP was performed as described previously, with some modifications (47). Cells at an OD 600 equivalent to 50 to 60 were collected and cross-linked with 1% formaldehyde at room temperature for 15 min, and formaldehyde was quenched for 5 min after adding glycine to a final concentration of 125 mM. Cells were washed twice with cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) and resuspended in 600 l lysis buffer (50 mM HEPES-KOH at pH 7.5, 500 mM NaCl, 1 mM EDTA at pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and cOmplete, EDTA free) with 0.5-mm-diameter zirconia beads (YZB05; Yasui Kikai) in 2-ml screwcap tubes. Cells were disrupted by vigorous shaking for 20 cycles of 30 s at 2,700 rpm followed by 30 s at rest at 4°C in a Multibeads shocker (Yasui Kikai). Disrupted cells were collected and centrifuged at 700 ϫ g for 10 min at 4°C. The pellet with the chromatin fraction was resuspended in 300 l of lysis buffer. Chromatin was sheared by sonication at 4°C for 10 cycles of 30 s on and 30 s off on the high setting on a Bioruptor (Cosmo Bio). Samples were centrifuged at 3,800 ϫ g for 2 min at 4°C. Twenty microliters of lysate was set aside as input DNA.
Ten microliters of Dynabeads protein G (no. 10004D; Thermo Fisher) was prewashed with PBS containing 5 mg/ml bovine serum albumin (BSA). Beads were incubated with 2.0 g of antibody in 200 l of PBS containing 5 mg/ml BSA at 4°C for 2 h, followed by washing the beads with lysis buffer. RNA-DNA hybrids were precipitated using S9.6 antibody (MABE1095; Merck). Mcd1-Flag and Smc2-Flag were precipitated using anti-FLAG antibody (F3165; Sigma). Beads were incubated with a mixture of 120 l of lysate and 120 l of lysis buffer at 4°C overnight. Beads were washed twice with lysis buffer at 4°C for 5 min, once with wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% Nonidet P40 [IGEPAL-CA630; Sigma], 0.5% Na-deoxycholate, 1 mM EDTA [pH 8.0], and cOmplete mini EDTA-free proteinase inhibitor cocktail [Sigma]) at 4°C for 10 min and once with 1ϫ TE (pH 8.0) at 4°C for 1 min. Immunoprecipitates were eluted by incubating beads with 120 l of TE containing 1% SDS at 65°C for 10 min. The supernatants were saved. Beads were washed with 130 l of TE containing 1% SDS, and the supernatants were combined with the first eluates. Cross-links were reversed from input and IP samples by incubating samples at 65°C overnight. Samples were incubated with 10 l of 20 mg/ml proteinase K at 50°C for 2 h. Finally, DNA was isolated by phenol-chloroform extraction and ethanol precipitation. Isolated DNA was resuspended in 100 l of TE.