INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Heterochromatin in eukaryotic chromosomes is usually associated with transcriptional silencing of nearby genes and also the suppression of recombination. In Saccharomyces cerevisiae, silencing occurs at several different genetic loci, including the cryptic mating-type loci (HML and HMR) (for a review see reference 50), and telomeres (34), both of which are generally recognized as the yeast heterochromatin equivalent. Remarkably, silencing of several RNA polymerase II (Pol II)-transcribed reporter genes also occurs within the rDNA locus (11, 70), even though this region of the yeast genome is very actively transcribed by Pol I and III. The ribosomal DNA (rDNA) of S. cerevisiae consists of 100 to 200 copies of a 9.1-kb unit organized into a perinuclear tandem array (59, 60), an arrangement reminiscent of the heterochromatin of higher eukaryotes.

Silencing in yeast is mediated by a specialized heterochromatin-like structure that is dependent on a series of trans-acting factors, including the proteins encoded by the four silent information regulator (SIR) genes. Efficient silencing at the HM loci requires all four SIR genes, while telomere position effect (TPE) requires SIR2, SIR3, and SIR4 (2). SIR1 contributes to the efficient establishment of silencing only at the HM loci (61). TPE and HM silencing also share requirements for Rap1, histones H3 and H4, and several other factors (50). As a result of this overlap in required silencing factors, the underlying mechanism of repression is thought to be similar between the HM loci and telomeres, although the mechanism is not yet well understood. Current models indicate that Sir2p, Sir3p, and Sir4p form a multimeric complex which interacts with the hypoacetylated N-terminal tails of histones H3 and H4 within nucleosomes, leading to the formation of a silenced chromatin domain at telomeres and the HM loci (35). The Sir proteins may therefore be structural components of yeast heterochromatin, although their exact functions have not yet been identified.

rDNA silencing is distinct from TPE and HM silencing in that SIR2 is the only absolutely required SIR gene (11, 70), implying that there are underlying differences in the mechanism of repression. Furthermore, unlike the HM and telomere loci, the rDNA is possibly the most transcriptionally active region of the entire genome, making rDNA silencing paradoxical. It is currently unknown whether the Pol I or Pol III transcription of rDNA plays any role in silencing. rDNA silencing is also exquisitely sensitive to alterations in SIR2 dosage (31, 71), suggesting that Sir2p is a structural component of rDNA chromatin; indeed, Sir2p specifically associates with rDNA by chromatin immunoprecipitation analysis (32). Although SIR4 function is not directly required for efficient rDNA silencing, it plays a regulatory role, mediating competition between telomeres and the rDNA for limiting amounts of Sir2 protein (71). This is consistent with the cellular localization of Sir2p, which is mostly nucleolar; smaller amounts of Sir2p also localize to perinuclear telomeric foci (32).

There are several potential functions of rDNA silencing in the yeast cell. The first is suppression of mitotic and meiotic recombination within the tandemly repeated rDNA. Deletion of SIR2 not only causes a loss of silencing in the rDNA (11, 70) but also increases the rate of rDNA recombination (33). The second is suppression of a cryptic Pol II promoter in the rDNA that overlaps with the well-characterized Pol I promoter (20). The third is modulation of rRNA transcription by Pol I. Deletion of SIR2 increases the percentage of rDNA repeats that are actively transcribed by Pol I (70). Fourth, silencing has been linked to the regulation of life span in yeast cells (47, 69). Certain mutations of SIR4 which promote longevity (47) also strengthen rDNA silencing (71), suggesting that there may be a link between the counteraction of aging and rDNA silencing. However, this link is complex, as aging is associated with redistribution of Sir3p and Sir4p to the nucleolus (46), yet neither of these proteins participates directly in rDNA silencing, as operationally defined by the silencing of Pol II reporter genes placed in the rDNA (70).

Thus far, a few genes other than SIR2 and SIR4 have been implicated as rDNA silencing factors. These genes encode topoisomerase I (TOP1), the ubiquitin-conjugating enzyme (UBC2/RAD6), histones H2A and H2B (11), and most recently Sas10p (42). Using multiple rDNA silencing reporter genes, we have performed a genetic screen that identified numerous non-SIR genes with rDNA silencing functions. Interestingly, multiple genes with known roles in DNA replication and/or chromatin modulation were identified. Several of the rDNA silencing genes identified in this screen have similar functions in TPE and HM silencing, implying that the rDNA silencing mechanism is distinct yet has some features in common with the other forms of silencing in yeast. We propose a model for rDNA silencing in which multiple cellular processes collaboratively lead to an rDNA chromatin structure that is repressive to Pol II reporter gene expression and recombination.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Media, plasmids, and yeast strains. Unless stated otherwise, media used were as previously described (63, 70). Pb²⁺-containing medium (MLA) consisted of 0.3% peptone, 0.5% yeast extract, 4% glucose, 0.02% (wt/vol) ammonium acetate, 0.1% Pb(NO₃)₂, and 2% agar. Glucose was the sole carbon source in all media. All yeast strains used (Table 1) were congenic to GRF167 (6, 70). All liquid and plate incubations of yeast strains were performed at 30°C. pJSS70-9 (2µm TRP1 SIR2) was constructed by ligating a XhoI-NotI SIR2 fragment from pCAR237 (70) into the XhoI-NotI sites of pRS424 (17).

Mutagenesis and identification of affected genes. Haploid strains JS306 and JS311 were mutagenized using transposon Tn3::lacZ::LEU2 as previously described by Burns et al. (13). A yeast genomic DNA library was obtained from Mike Snyder's lab (via Susan Michaelis). This library had been mutagenized in Escherichia coli by random Tn3::lacZ::LEU2 integration events. The mutated genomic DNA inserts were removed from the vector backbone by digestion with NotI and transformed into JS306 and JS311 by using a high-efficiency lithium acetate-polyethylene glycol-dithiothreitol procedure. Cells were plated onto leucine-deficient synthetic complete (SCLeu) medium (approximately 200 to 250 transformants/plate) to select for Leu⁺ mutant colonies in which a transposon-disrupted DNA fragment had integrated into the genome by homologous recombination.

Dominance tests. Each mutant was mated to a strain of the opposite mating type which did not contain reporter genes in the rDNA and was Trp⁺. Briefly, mutant strains were streaked out for single colonies on SCLeu, grown for 3 days, and replica plated onto a YPD plate, along with a lawn of either JS314 (MAT) or JS315 (MATa). The two strains were allowed to mate for 5 h at 30°C and the YPD mating plate was then replica plated to SCLeuTrp and grown overnight to select for diploid formation. The resulting diploids were then replica plated to the silencing indicator medium SCLeuTrpUra or to MLA to test for dominance. The heterozygous lrs diploids were also restreaked for single colonies onto MLA to test for dominance by the rDNA sectoring assay. Backcross analysis was used to confirm cosegregation of the mutant phenotype with the Tn3::lacZ::LEU2 transposon insertion for mutants that were represented by a single isolate in the screen.

PCR-mediated gene disruption. Complete open reading frame (ORF) deletions were made for several genes identified in the screen to confirm the Lrs or Irs silencing phenotype of that particular mutant. PCR-mediated gene disruption was performed as described elsewhere (5, 51). The dominant drug resistance marker, kanMX4 (79), was PCR amplified from pRS400 (8, 71), using oligonucleotide primers containing 39 nucleotides complementary to the 5' and 3' ends of the targeted ORF. The resulting PCR fragments were transformed into JS306 or JS311, and gene replacement with kanMX4 was selected for by growth on YPD medium containing G418 (200 µg/ml).

Silencing growth assays. Strains to be tested were patched onto YPD, or selective medium if they contained a plasmid, and grown overnight. Cells were scraped from the plates and resuspended in 1 ml of sterile water. The cell suspension was normalized to an A₆₀₀ reading of 0.5 and then serially diluted in fivefold increments; 5 µl of each dilution was spotted onto either nonselective or selective SC agar plates, using an eight-channel pipette. Plates were incubated for 2 to 5 days. Polaroid photographs were taken of all plates except SCUra after 2 days. Photographs of SCUra plates were taken after 4 days unless specified otherwise in the figure legends.

Colony color silencing assays. Strains to be tested were patched onto YPD and grown overnight. Cells were then streaked onto MLA plates with four or six sectors per plate. The plates were wrapped with Parafilm to prevent dehydration and then allowed to grow 5 days. Photographs were taken at day 5, using a Leica stereoscopic microscope equipped with a 35-mm color camera.

Telomere length analysis. Two independent colonies for each mutant were lightly inoculated into 10 ml of YPD cultures and incubated for approximately 16 h. Cells were pelleted, and genomic DNA was isolated as previously described, using a Teeny prep spheroplasting method (6). Nucleic acid pellets were resuspended in 100 µl of Tris-EDTA (TE), and 15 µl (2 to 4 µg of DNA) was digested in a 30-µl reaction with XhoI and separated on a 0.7% agarose gel. The DNA was transferred to Genescreen Plus (NEN-Dupont), UV cross-linked, and hybridized with a telomere specific probe in Church and Gilbert hybridization solution (1 mM EDTA, 0.5 M Na₂HPO₄ [pH 7.2], 7% sodium dodecyl sulfate, and 1% bovine serum albumin) at 60°C overnight. The blot was washed once at room temperature and three times (10 min) at 60°C with washing solution (2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% sodium dodecyl sulfate). The probe was a 350-bp EcoRI fragment from plasmid pYLPV, which contained a 280-bp TG_1-3 telomeric repeat (81).

Psoralen cross-linking analysis. In vivo psoralen cross-linking assays were performed as previously described (14, 22, 70), with several minor modifications. Fresh 50-ml YPD cultures were inoculated from saturated YPD cultures to an A₆₀₀ of 0.3 and grown for 6.5 h into log phase. Approximately 2.5 × 10⁸ cells were washed with ice-cold H₂O and resuspended in 0.7 ml of cold TE in a 24-well tissue culture plate; 40 µl of a 200-µg/ml solution of 4,5',8-trimethylpsoralen (Sigma) in 100% ethanol was added to each well, and the plate was UV irradiated for 5 min on ice at a distance of 6 cm five times. The light source was a long-wave UV lamp (model B-100A; Ultraviolet Products, Inc.). Cells were washed, spheroplasted with zymolyase at 37°C, lysed, proteinase K treated, phenol-chloroform extracted, and ethanol precipitated. Total nucleic acid was resuspended in 40 µl of TE and normalized to an A₂₆₀ of 0.04. DNA (4 µl) was digested for 5 h at 37°C with EcoRI in a 30-µl reaction containing RNase A (80 ng/µl). DNA was separated on 1.3% agarose gel (14.5 by 24 cm) at 80 V for 17 h. Cross-linking was reversed in a Stratagene Stratalinker at 0.6 J/cm². DNA was transferred to Genescreen Plus in 10× SSC and hybridized with probe A, which is a 2.2-kb EcoRI-SmaI fragment of the rDNA nontranscribed spacer (NTS).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of rDNA silencing mutants. As a first step toward understanding the molecular mechanism of rDNA silencing, we carried out a genetic screen designed to identify mutations in genes which contribute either positively or negatively to silencing. Strains JS306 (MATa) and JS311 (MAT) were constructed to contain three different Pol II-transcribed reporter genes in the rDNA, namely, a single MET15 reporter gene (embedded in a Ty1 element) located within NTS2 of one rDNA repeat and a mURA3/HIS3 expression cassette within the 18S rRNA-coding region of a second repeat (Fig. 1A). Met⁺ strains produce white colonies on Pb²⁺-containing (MLA) medium, whereas Met strains produce dark brown colonies (21, 58). rDNA silencing of MET15 results in a characteristic intermediate tan colony color (70). Mutants which weaken rDNA silencing were predicted to produce a lighter colony color than wild type (WT), and mutants which strengthen rDNA silencing were predicted to produce darker colonies. The mURA3 reporter was also repressed by the rDNA and resulted in a very weak Ura⁺ phenotype (70). HIS3 was not observed as silenced in previous studies in a replica plating test (11, 70), and so it was used as a marker for the presence of the tightly linked mURA3 reporter. The relevant WT phenotypes of these reporter strains were therefore a tan colony color on MLA plates, weakly Ura⁺, and fully His⁺. During this study we found that HIS3 is in fact partially silenced when assayed by a more quantitative colony spotting assay.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Genetic screen for rDNA silencing mutants. (A) Schematic representation of the rDNA structure of strains JS306 and JS311, showing the positions of each reporter gene. (B) Schematic drawing showing the transposon mutagenesis strategy used in the screen. NotI yeast genomic DNA fragments disrupted with the transposon mTn3::lacZ::LEU2 (13), were transformed into strains JS306 and JS311. Homologous recombination resulted in the replacement of a specific segment of yeast chromosome (gene X), with an identical DNA fragment which was disrupted with the transposon. Transposon-disruption mutants are selected for the presence of LEU2 by growth on SCLeu medium. (C) Flow chart describing the screening procedure and number of isolates at each stage. Sequences for 20 of the 65 of the 2° irs isolates were not recovered.

sir2

Description of lrs mutants and their silencing phenotypes. The LRS class of genes were predicted to encode proteins which contributed structurally or were positive regulators of rDNA silencing. In this paper we describe LRS1 (RFC1), LRS4 (YDR439W), LRS5 (TOP1), LRS7 (RIF1), LRS8 (CAC1), and LRS9 (CDC17) (Table 2). The remaining LRS genes will be described elsewhere. Figure 2 shows the effects of a subset of these mutants on mURA3, HIS3, and MET15 silencing in the rDNA as measured by quantitative growth assays, which were previously shown to correlate with reporter gene expression levels (71). Figure 3 shows the effect of the mutations on MET15 expression as measured by a qualitative colony color assay. Each lrs mutant was more Ura⁺ and His⁺ than WT (Fig. 2A) and produced white colonies with a hyperrecombination sectoring phenotype on Pb²⁺ medium (Fig. 3). The lrs5 insertions were in the topoisomerase I gene (TOP1), a known rDNA silencing factor (11, 18), and therefore validated the specificity of the lrs screen.

View larger version (98K): [in this window] [in a new window]   FIG. 2.   rDNA silencing phenotypes of selected mutants. (A) Quantitative growth assays measuring the silencing of mURA3 and HIS3 within the rDNA. Fivefold serial dilutions of freshly grown cells were plated onto either SC (Complete), SCUra (Ura), or SCHis (His) medium. Strains shown are WT (JS306 or JS311), top1 (M122), cac1 (M179), cac1 (JS400), rif1 (M158), rif1 (JS418), lrs4 (JS574), sir2 (JS576), rpd3 (M480), rpd3 (JS490), sin3 (JS493), sap30 (M475), hir3 (M489), tup1 (M419 and M432), and irs4 (M469). The photographs were taken for the SCUra plates at day 3 for the lrs mutant series (top of panel) and day 4 for the irs series (bottom of panel). (B) Quantitative growth assay measuring silencing of MET15 in NTS2. Fivefold serial dilutions of cells were plated onto SC and SCMet Cys medium. Strains were the same as for panel A.

View larger version (95K): [in this window] [in a new window]   FIG. 3.   Qualitative colony color assay showing the lrs and irs phenotypes of selected mutants. Freshly grown cells were scraped from YPD medium and streaked onto MLA medium for single colonies. Cells were grown 5 days before photographs were taken. lrs mutants produce white colonies and often display a hypersectoring phenotype; irs mutants produce a darker colony color, indicative of reduced MET15 expression compared to WT. lrs strains shown are WT (JS306), top1 (M122), rif1 (M158), cac1 (M179), sir2 (JS576), and lrs4 (JS574); irs strains shown are WT (JS311), hir3 (M487), rpd3 (M480), sin3 (JS493), sap30, M475, irs4 (M469), and tup1 (M419).

Description of irs mutants and their phenotypes. The IRS class of genes was predicted to include negative regulators of rDNA silencing. IRS1 was identified as SIR4, deletion of which was previously shown to increase rDNA silencing (70). Isolation of a sir4 mutant therefore validated the specificity of this screen for irs mutants. Other IRS genes described in this study are IRS2 (RPD3), IRS4 (YKR019C), IRS8 (SAP30), IRS10 (HIR3), and IRS18 (TUP1) (Table 2). Each of these genes (except IRS4) has previously been shown to have a chromatin-related function (26, 39, 44, 64, 73, 84). Other IRS genes will be described elsewhere.

sin3

sin3

cac1

rpd3

rif1

lrs4

sin3

rDNA silencing in lrs and irs mutants is controlled by Sir2p levels. rDNA silencing is exquisitely sensitive to SIR2 dosage (31, 71). We were therefore interested in determining whether the lrs and irs mutants remained susceptible to SIR2 dosage effects. If any of these genes were downstream of SIR2 in a silencing pathway, then their Lrs or Irs phenotypes should become resistant to changes in SIR2 dosage. To test this hypothesis, we overexpressed SIR2 in several mutant backgrounds and tested rDNA silencing strength in Ura⁺ and His⁺ growth assays. SIR2 overexpression increased the silencing strength of the cac1 (lrs8) mutant, as measured by less Ura⁺ growth, compared to a strain containing an empty vector (Fig. 4A). However, less of an effect was observed in the His⁺ assay, consistent with the HIS3 reporter being more resistant to rDNA silencing in this mutant. The effect of SIR2 overexpression was less pronounced for the top1 mutant in the Ura⁺ and His⁺ assays, suggesting that top1 mutants become partially resistant to increased SIR2 dosage. rif1 and lrs4 mutants were fully sensitive to SIR2 dosage (data not shown). SIR2 overexpression also further strengthened rDNA silencing in all irs mutants tested, including tup1, sin3, sap30 (not shown), rpd3, and hir3 (Fig. 4B). These results indicated that most lrs and irs mutants are fully responsive to elevations in SIR2 dosage, and they localized these genes either upstream of SIR2 or in different genetic pathways to rDNA silencing.

View larger version (64K): [in this window] [in a new window]   FIG. 4.   Effect of SIR2 overexpression on rDNA silencing mutants. (A) The high-copy-number empty vector pRS424 or the SIR2 vector pJSS70-9 was transformed into the WT strain JS306, several lrs mutants including cac1 (M179), and the top1 mutant (M154). Fivefold serial dilutions were spotted onto SCTrp, which selects for the plasmid, SCTrpUra to measure silencing of mURA3, and SCTrp His to measure silencing of HIS3. (B) Fivefold serial dilutions of irs mutants containing a high-copy-number empty vector or a high-copy-number SIR2 vector.

Several mutants with long telomeres also weaken rDNA silencing. We previously proposed that rDNA silencing strength could be modulated by telomere length due to a competition between the rDNA and telomeres for a limited pool of Sir2p (71). Similarly, extra telomeres weaken silencing of a telomeric reporter gene by titrating an unidentified silencing factor (82). Certain mutations in the DNA replication genes cdc17 (pol1) and rfc1, and null mutations of rif1, cause telomeres to lengthen compared to WT strains (1, 38). Lrs insertion alleles were isolated for each of these genes (Table 2; Fig. 5A). The Lrs phenotype of these mutants might therefore be due to long telomeres sequestering Sir2p and depleting it from the rDNA. As predicted by this model, the viable rfc1 (lrs1) and cdc17 (lrs9) mutants indeed had significantly longer telomeres than the parent strains (Fig. 5B). The rif1 (lrs7) mutant had longer telomeres which produced a qualitatively different banding pattern than the other mutants (Fig. 5B). This unusual banding pattern was specific to the rif1 mutation because it cosegregated in backcrosses.

View larger version (69K): [in this window] [in a new window]   FIG. 5.   A class of rDNA silencing mutants with abnormally long telomeres. (A) The DNA replication mutants cdc17 and rfc1, and the telomere regulation mutant rif1, were each isolated as lrs mutants. Each mutant was crossed to a sir4::HIS3 mutant of the opposite mating type to produce a heterozygous diploid which was sporulated and dissected for tetrads. The resulting haploid strains were grown on MLA medium and assayed for loss of silencing of MET15 as measured by colony color. Strains shown are WT (JS333), sir4 (JS337), cdc17 (JS432), cdc17 sir4 (JS436), rfc1 (JS443), rfc1 sir4 (JS445), rif1 (JS422), and rif1 sir4 (JS426). (B) Steady-state telomere length of lrs mutants in combination with sir3 and sir4 mutations. Genomic DNA was isolated from two isolates of each mutant listed, digested with XhoI, and separated on a 0.7% agarose gel. The transferred DNA was detected with a C1-3A-specific DNA probe, which on this blot will detect typical Y' telomeres (shown schematically at bottom). The variable-length telomeres are visualized as a smear (brackets). In addition to the mutants described in panel A, there are congenic sir3 (JS335), cdc17 sir3 (JS434), rif1 sir3 (JS424), rif2 (JS495), and rif2 rif1 (JS497) strains.

sir3

sir4

sir4

sir4

sir4

sir3

rif1 sir4

rif1 rif2

lrs and irs mutants alter the structure of rDNA chromatin. To determine whether mutations which weaken rDNA silencing (lrs) also disrupt rDNA chromatin structure, we tested each lrs mutant in an in vivo psoralen cross-linking assay (22). This assay measures the intracellular accessibility of the rDNA to psoralen cross-linking. The Lrs phenotype of sir2 mutants was previously shown to be associated with an increase in psoralen cross-linking at the rDNA, reflecting a more open chromatin structure (70). Increased psoralen cross-linking was detected by the slower mobility of isolated rDNA fragments on a native agarose gel. The accessibility of the NTS in a subset of the lrs mutants is shown in Fig. 6A. The 2.5-kb NTS fragment released by EcoRI digestion displayed a slower gel mobility for each lrs mutant, similar to the sir2 control. This result indicated that the NTS had a more open chromatin structure in most lrs mutants, not just those carrying sir2.

View larger version (35K): [in this window] [in a new window]   FIG. 6.   rDNA chromatin accessibility of mutants as measured by in vivo psoralen cross-linking. (A) Log-phase cultures of lrs mutants were UV cross-linked with psoralen in vivo. Isolated DNA was digested with EcoRI and separated on a 1.3% agarose gel, and the transferred DNA was detected with a probe specific for the NTS of the rDNA (C). The sir2 strain (JS343) acted as a positive control for increased accessibility to psoralen cross-linking. Other strains shown are WT (JS306), top1 (M122), rif1 (M158), and cac1 (JS400). (B) An identical cross-linking procedure using the same NTS-specific probe was carried out on a subset of the irs mutants. The strains tested are WT (JS311), rpd3 (JS490), sin3 (JS493), sap30 (M475), hir3 (M411), and tup1 (M419). The control lanes show the 2.5-kb EcoRI fragment which is observed when the strains are not cross-linked. (C) Schematic drawing of the EcoRI rDNA fragment detected from this assay. ARS, autonomously replicating sequence.

sir4

rpd3

sin3

rpd3

sin3

The Irs phenotype of an rpd3 mutant is partially reversed by cac1, rif1, and sir2 mutations. The opposing silencing and chromatin accessibility phenotypes of rpd3, sin3, and sap30 mutants prompted a more in-depth examination of the genetic interactions between RPD3 and the LRS genes. We asked whether the Irs phenotype caused by rpd3 mutations depends on any of the LRS genes, specifically SIR2, CAC1, or RIF1. Double-mutant combinations were generated and tested in an epistasis analysis for rDNA silencing strength using the Ura⁺ and His⁺ growth assays and the colony color assay (Fig. 7). We obtained different epistasis results that correlated with the location of the silencing reporter gene in the rDNA repeat. For example, with the mURA3/HIS3 cassette readouts, rpd3 mutations fully overrode the effects of the cac1 and rif1 mutations (Fig. 7A). However, for the MET15 reporter in the same strains, the double mutants produced Met⁺ (data not shown) and colony color phenotypes that were intermediate between those of the two single mutants (Fig. 7B). Thus, rpd3 partially overrode the cac1 and rif1 Lrs phenotypes. Therefore, mURA3 and HIS3, both located within the 18S coding region, appeared to be influenced by the rpd3 mutant effect more than MET15, which was located within NTS2. Alternatively, these differences could be caused by simple promoter differences.

View larger version (80K): [in this window] [in a new window]   FIG. 7.   Epistasis analysis between rpd3 and lrs mutations for rDNA silencing phenotypes. (A) Silencing reporter strains were generated with combinations of rpd3 mutations with either sir2, cac1, or rif1 mutations. The resulting strains were assayed for rDNA silencing strength in Ura+ and His+ growth assays. Fivefold serial dilutions of each haploid strain were spotted on indicator plates. A plus sign indicates the strain is WT for a particular gene, and a minus sign indicates a mutation of a particular gene. (B) The same strains were plated onto MLA medium and tested for silencing strength in the colony color assay. Each column represents strains grown on the same plate.

rpd3 sir2

sir2

rpd3 sir2

rpd3 sir3

rpd3 mutations partially restore mating competence to sir3 mutants. In the process of analyzing the interactions of rpd3 and sir3 mutations, we made some surprising observations about effects on HM locus silencing. Previous studies have noted enhancement of silencing effects by rpd3 mutations on a weakened HMRA silencer (77). We observed behavior suggesting that rpd3 mutations can restore silencing to the native HMR silencer in sir3 mutants. All of the 17 MAT rpd3 sir3 spores resulting from a cross of rpd3 and sir3 strains efficiently mated to a MATa tester strain, indicating that the rpd3 mutation partially reversed the silencing defect caused by the sir3 mutation. This effect was specific for MAT cells, suggesting that HMRa was affected but HML was not affected (Fig. 8). In contrast, the rpd3 mutation did not rescue the mating defect of MAT sir2 or MAT sir4 strains (Fig. 8).

View larger version (51K): [in this window] [in a new window]   FIG. 8.   Mating phenotypes of rpd3 sir double-mutation combinations. The rpd3::mTn3 mutation was combined with either sir2, sir3, or sir4 mutation to determine the effect on mating ability. Strains were patched onto YPD, grown for 24 h, and then mated for 6 h with a lawn of MATa or MAT tester strains. Diploids were selected on SD minimal medium by growth for 18 h. The original YPD master plate was replica plated to SC medium as a nonselective growth control. The double-mutation combinations were tested in both MAT and MATa genetic backgrounds. Mating is measured by the efficiency of diploid formation on SD medium.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Multiple genetic pathways to rDNA silencing. An unanticipated outcome from this screen was the absence of genes involved in transcription by RNA Pol I or III, especially since this specialized transcription is intimately associated with the rDNA. It was possible that rDNA silencing of Pol II reporter genes was caused by promoter interference from Pol I or III. Instead, we recovered multiple genes involved with DNA replication or chromatin modulation. The DNA replication genes included POL1, RFC1, and DPB3. The chromatin modulating class included RPD3, SIN3, SAP30, TUP1, HIR3, SIR4, and RIF1. The TOP1 and CAC1 genes could be placed in either DNA replication or chromatin modulating classes, since they participate in both processes. These results, coupled with the intermediate phenotypes of several double-mutation combinations, are strong evidence for several independent pathways which converge on rDNA silencing (Fig. 9). The SIR2 pathway is extremely important, but it appears that all the pathways must be functional to achieve full silencing strength.

View larger version (16K): [in this window] [in a new window]   FIG. 9.   Model of multiple pathways to rDNA silencing. Sir2p is a central factor in rDNA silencing. Cdc17p and Rfc1p inhibit formation of extended telomeres, limiting the impact of telomere-rDNA competition for Sir2p, which is mediated by Sir4p. Cdc17p and Rfc1p may also positively act on rDNA silencing through their DNA replication functions. Silencing in most lrs and irs mutants can be increased by overexpression of Sir2p. CAF-I and Rif1p could be upstream of Sir2p, act in cooperation with Sir2p, or be completely separate inputs. Rpd3p counteracts rDNA silencing mostly through a Sir2p-dependent mechanism but also through a SIR-independent mechanism. Topoisomerase I may influence rDNA silencing by a mechanism that is partially independent of Sir2p.

Telomere length effects on rDNA silencing. Sir4p mediates competition between the rDNA and telomeres for a limiting amount of Sir2 protein (71). Variations in telomere length are therefore predicted to influence the strength of rDNA silencing by changing the balance of Sir2p between the rDNA and telomere compartments. Indeed, three different lrs mutants with significantly longer than WT telomeres were isolated. Also consistent with this competition model, introducing a sir4 mutation into rfc1 or cdc17 mutant strain backgrounds partially reversed their Lrs phenotypes back to at least WT silencing strength and also significantly reduced telomere length. This was an important result because in the absence of Sir4p, all cellular Sir2p is localized in the nucleolus and not sequestered at telomeres (32). Taken together, these two findings suggest that rfc1 and cdc17 mutations weaken the Irs phenotype of a sir4 mutant through a telomere length-independent mechanism, perhaps related to their replication functions.

rif2

sir4

sir3

rif2

rif1

rif1

DNA replication and rDNA silencing. Connections between DNA replication and silencing are not unprecedented. First, establishment of silencing at the HM loci requires progression through S phase (30, 53). Second, the six-subunit origin recognition complex (ORC) is required for silencing at HML and HMR (29, 52). One role of ORC in silencing at the HM loci appears to be recruitment of the Sir-silencing complex through interactions with Sir1p (16, 76). However, ORC is also required for silencing at telomeres in a SIR1-independent manner (30). Interestingly, the silencing function of one ORC subunit (Orc5p) at the HMR-E silencer can be genetically separated from its replication initiation function, implying that replication initiation is not required for silencing (25). Silencing at the rDNA is qualitatively different from HM and telomere silencing, and so it is currently unknown whether ORC functions in rDNA silencing.

dpb3

Histone balancing act. Nucleosomes are formed from two molecules of each of the core histones H2A, H2B, H3, and H4 which tightly associate with 146 bp of DNA. Histones H3 and H4 have been shown to contribute directly to silencing at the HM and telomere loci (35). Furthermore, histones H2A and H2B are important for rDNA silencing. Deletion of HTA1-HTB1, one of two gene pairs producing H2A and H2B, results in a loss of rDNA silencing (11), suggesting that histone stoichiometry within the nucleosome may be critical. We recovered four independent mutations in the HIR3 gene from our screen as irs10 mutants. Mutations in HIR1, HIR2, or HIR3 cause deregulation of a transcriptional feedback mechanism which controls HTA1-HTB1 expression (73). The Irs phenotype of irs10 (hir3) mutants may therefore be due to increased H2A H2B expression levels. Consistent with this model, telomeric silencing is also slightly increased in a hir1 mutant strain (44).

cac1

Histone deacetylation and rDNA silencing. Transcriptionally silenced regions of eukaryotic genomes are generally hypoacetylated. In S. cerevisiae, the HM loci and telomeres are hypoacetylated on histones H3 and H4, with an acetylation pattern similar to that in higher eukaryotes (10). The yeast RPD3 gene product is the proposed catalytic component of a large multiprotein histone deacetylase complex (HDB) which acts in targeted transcriptional repression (40, 64). Sin3p, another subunit of HDB, acts to target the complex to specific Pol II promoters through association with specific DNA binding proteins (41). Sap30p is also a member of this complex (84). Paradoxically, rpd3 and sin3 mutations increase silencing at HM loci (77), telomeres (23, 64), and the rDNA (this study). Similarly, Drosophila TPE is enhanced by null mutations of its RPD3 homolog (23). In another study, sap30 mutants strengthened all types of silencing in yeast, including the rDNA (37). These findings were completely consistent with our results for the irs8 (sap30) mutation using a different strain background and different rDNA silencing reporters. In both studies, these results are paradoxical because loss of histone deacetylase function is expected to result in hyperacetylation of histone N-terminal tails, resulting in derepression of transcription.

rpd3

sir3

rpd3 sir2

rpd3 sir4

sir3