Distribution of a Limited Sir2 Protein Pool Regulates the Strength of Yeast rDNA Silencing and Is Modulated by Sir4p

Corresponding author: Jef D. Boeke, Dept. of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 617 Hunterian Building, Baltimore, MD 21205, jef_boeke{at}qmail.bs.jhu.edu (E-mail).

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1. Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels. The endogenous level of Sir2p was shown to be limiting for rDNA silencing. Furthermore, small changes in Sir2p levels altered rDNA silencing strength. In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing. We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein.

CLASSICAL transcriptional silencing in the budding yeast Saccharomyces cerevisiae was originally identified at the silent mating-type loci HML and HMR, which are located on chromosome III (for review see LOO and RINE 1995 ). A related form of silencing also occurs at telomeres and is known as telomere position effect (TPE; GOTTSCHLING et al. 1990 ). Foreign genes inserted in these regions are repressed in a reversible manner that resembles the position effect variegation observed in the heterochromatin of more complex eukaryotes (PILLUS and RINE 1989 ; GOTTSCHLING et al. 1990 ; WEILER and WAKIMOTO 1995 ; DOBIE et al. 1997 ). Both of these chromosomal regions display a general inaccessibility to DNA-modifying enzymes in vitro and in vivo (STRATHERN et al. 1982 ; KOSTRIKEN et al. 1983 ; GOTTSCHLING 1992 ; SINGH and KLAR 1992 ; LOO and RINE 1994 ). Genes transcribed by either RNA polymerase II or III (Pol II or Pol III) are generally repressed (SCHNELL and RINE 1986 ; GOTTSCHLING et al. 1990 ; HUANG et al. 1997 ), although at telomeres, the degree of silencing depends on the intrinsic features of each individual promoter (RENAULD et al. 1993 ). These properties, combined with similarities to classically defined eukaryotic heterochromatin, such as DNA replication late in S phase, location near the nuclear envelope, and physical compaction, have led to proposals that the silent mating-type loci and telomeres in yeast are functionally equivalent to heterochromatin (THOMPSON et al. 1994 ; BRAUNSTEIN et al. 1996 ).

A novel form of silencing was described recently in the ribosomal DNA (rDNA) of S. cerevisiae (BRYK et al. 1997 ; SMITH and BOEKE 1997 ). The rDNA consists of 100–200 copies of a 9.1-kb unit organized into a tandem array on chromosome XII (PETES and BOTSTEIN 1977 ; PHILIPPSEN et al. 1978 ). Each 9.1-kb unit contains the gene for 35S ribosomal RNA precursor, transcribed by RNA Pol I, and the gene for 5S rRNA, transcribed by RNA Pol III (Figure 1). These two transcription units are separated by a nontranscribed spacer (NTS) that contains an origin of DNA replication (SZOSTAK and WU 1979 ; SKRYABIN et al. 1984 ). We originally demonstrated that the expression of several different Pol II–transcribed marker genes integrated into various regions of the rDNA was repressed (SMITH and BOEKE 1997 ). Of the Silent Information Regulator (SIR) genes, rDNA silencing depends only on SIR2 (SMITH and BOEKE 1997 ). In contrast, all four SIR genes contribute to silencing the HM loci, whereas TPE only requires SIR2, SIR3, and SIR4 (HABER and GEORGE 1979 ; KLAR et al. 1979 ; RINE et al. 1979 ; RINE and HERSKOWITZ 1987 ; APARICIO et al. 1991 ). TPE and HM silencing also depend on other factors, including Rap1p (reviewed in SHORE 1994 ), as well as histones H3 and H4 (reviewed in LOO and RINE 1995 ). The role of non-SIR silencing factors and cis-acting elements in mediating rDNA silencing is just beginning to be investigated. The UBC2 (RAD6) gene has been recently implicated in both TPE (HUANG et al. 1997 ) and rDNA silencing (BRYK et al. 1997 ). Topoisomerase I (TOP1), not previously implicated in silencing, is also required for silencing Ty1 elements in rDNA, as well as for suppression of mitotic rDNA recombination (CHRISTMAN et al. 1988 ; BRYK et al. 1997 ).

View larger version (21K): In this window In a new window Download PPT slide   Figure 1. Schematic representation of the Saccharomyces cerevisiae rDNA array structure and Ty1 silencing reporters. The general organization of a single 9.1-kb rDNA gene copy within the tandem array is depicted. The initiation sites for 35S rRNA (Pol I) and 5S rRNA (Pol III) transcription are shown as bent arrows. Three autonomously replicating sequence (ARS) near-consensus elements of the origin of DNA replication in NTS2 are shown as filled circles. Integration sites and orientations of Ty1-mURA3 and Ty1-MET15 insertions into the rDNA are shown as vertical arrows. Downward-pointing arrows represent Ty insertions in the same transcriptional orientation as the 5S rRNA gene (right to left). Upward pointing arrows represent Ty insertions in the opposite orientation. Filled triangles represent Ty1 LTRs. The strain number for each reporter strain is shown.

We previously demonstrated that deletion of SIR2 increases the accessibility of rDNA to psoralen cross-linking in vivo (SMITH and BOEKE 1997 ), suggesting that SIR2 is required for the formation or maintenance of a specialized rDNA chromatin structure that is repressive to RNA Pol II. Using in vivo formaldehyde cross-linking, Sir2p has been found to be associated with rDNA chromatin, mostly within the NTS region (GOTTA et al. 1997 ). In addition, loss of SIR2 increases the frequency of mitotic and meiotic recombination within the rDNA (GOTTLIEB and ESPOSITO 1989 ). Taken together, these findings indicate that SIR2 plays a major role in rDNA function, and they are consistent with the nucleolar localization of Sir2 (GOTTA et al. 1997 ).

Although SIR4 is not required for rDNA silencing, its deletion paradoxically enhances the strength of rDNA silencing (SMITH and BOEKE 1997 ), suggesting that SIR4 may regulate such silencing. TPE and HM silencing regulation can occur at the level of Sir protein dosage. For example, increased SIR3 gene dosage enhances the strength of TPE, resulting in the spreading of repressive telomeric chromatin toward the centromere (RENAULD et al. 1993 ). Sir3p is physically associated with this extended telomeric chromatin (HECHT et al. 1996 ). Another example is that diploid strains containing one copy of SIR4 display unstable repression at HMR (SUSSEL et al. 1993 ), and overexpression of either SIR4 or its C-terminal fragment greatly reduces silencing at both HM loci and telomeres (known as the "anti-SIR" effect; MARSHALL et al. 1987 ; RENAULD et al. 1993 ; COCKELL et al. 1995 ). Sir1p levels are limiting for establishment of HM silencing (STONE et al. 1991 ). So far, there have been no reports that SIR2 dosage affects TPE (RENAULD et al. 1993 ) or HM silencing, although high-copy SIR2 expression does enhance rDNA silencing in the rDNA (FRITZE et al. 1997 ). SIR2 overexpression leads to a decrease in histone acetylation at the HM loci (BRAUNSTEIN et al. 1993 ), and high levels of SIR2 overexpression are toxic to cells, provoking chromosome instability (HOLMES et al. 1997 ).

It has been suggested that the local concentration of Sir proteins is critical in determining silencing efficiency, even at internal chromosomal locations (STAVENHAGEN and ZAKIAN 1994 ; LUSTIG et al. 1996 ; MAILLET et al. 1996 ; MARCAND et al. 1996 ). This hypothesis is supported by other studies showing that telomeres, the Sir proteins, and Rap1p colocalize into a limited number of discrete intranuclear foci, predominantly near the nuclear membrane (PALLADINO et al. 1993 ; COCKELL et al. 1995 ; GOTTA et al. 1996 ). Competition for these limiting amounts of silencing factors has been shown to occur between telomeres and the HM loci. The rap1^s mutation, rap1-12, simultaneously reduces HMR-specific silencing and enhances telomeric silencing (BUCK and SHORE 1995 ). This shift in the balance of silencing to telomeres was proposed to result from sequestration of Sir4p at telomeres, thus reducing the amount of Sir4p available for HMR silencing (BUCK and SHORE 1995 ; MARCAND et al. 1996 ).

We demonstrate in this article that SIR4 regulates rDNA silencing strength by controlling the level of Sir2p that is available to the rDNA. We show that rDNA silencing is highly sensitive to even small changes in Sir2 levels, and that there are normally limiting amounts of Sir2p available for rDNA silencing. We also show that SIR4 negatively affects rDNA silencing. SIR4 overexpression severely reduced the strength of rDNA silencing, and sir4 strengthened rDNA silencing through a SIR2-dependent, SIR3-independent mechanism. Finally, manipulations of SIR4, which were previously shown to redistribute Sir proteins (including Sir2p) to the nucleolus and promote longevity, also cause an increase in rDNA silencing strength. We propose a Sir2p competition model in which Sir4p regulates the shuttling of Sir2p between competing rDNA and telomeric compartments.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media and plasmids:
Unless stated otherwise, media were used as described previously (ROSE et al. 1990 ; SMITH and BOEKE 1997 ). Ty1 transposition induction medium (YNB/casamino acids + Trp galactose) consisted of yeast nutrient broth (YNB; Difco, Detroit) supplemented with 2% casamino acids, 2% galactose, 160 µM adenine, and 800 µM tryptophan. SC (FOA) medium contained 5-FOA (PCR Incorporated) at 2 mg/ml and glucose at 2%. Pb²⁺-containing medium (modified lead acetate, MLA) consisted of 0.3% peptone, 0.5% yeast extract, 4% glucose, 0.02% (w/v) ammonium acetate, 0.1% Pb(NO₃)₂, and 2% agar.

The mURA3-marked GAL-Ty1 overexpression plasmid pJSS36-6 and the CEN, SIR2 plasmid pCAR237 were described previously (SMITH and BOEKE 1997 ). pLP304 (2µ, LEU2 SIR3) and pLP305 (2µ, LEU2 SIR4) were also described previously (STONE and PILLUS 1996 ). pLP347 consists of a HST1 SacI fragment ligated into the SacI site of YEp351. pSIR2µ was constructed by inserting the HindIII genomic SIR2 fragment from pCAR237 into the HindIII site of pRS425 (CHRISTIANSON et al. 1992 ). pJSS71-13 (2µ, HIS3, SIR2) was constructed by ligating a XhoI-NotI fragment from pCAR237, which contains SIR2, into the XhoI-NotI sites of pRS423. pLP754 (2µ LEU2 SIR4-42) consists of a NotI-SalI SIR4-42 fragment that was blunted by Klenow treatment and then ligated into the SmaI site of YEp351 (HILL et al. 1986 ). pLP793 (integrating TRP1 sir4-42) was constructed by ligating the SacI-SalI sir4-42 fragment from pLP754 into the SacI-SalI sites of pRS304. pJH3A and pJH5.1A (provided by JAMES BROACH) were previously described (IVY et al. 1986 ; MARSHALL et al. 1987 ). pJSS73-5 was constructed by ligating an EcoRI-SacII fragment containing the GAL1 promoter and a C-terminal fragment of SIR4 from pRO135 into the same sites of pRS313. pRO135 was a plasmid derived from a galactose-inducible overexpression library (LIU et al. 1992 ) and was provided by ROHINTON KAMAKAKA. The kanMX4 gene of pRS400 (BRACHMANN et al. 1998 ) was derived from pFA6a-kanMX4, provided by ACHIM WACH and PETER PHILIPPSEN (WACH et al. 1994 ).

Yeast strains:
All strains used in this study are derived from JB740 or its derivatives (Table 1; SMITH and BOEKE 1997 ). JS50-1 is a MAT/ diploid isolated during transformation of JB740 with the galactose-inducible Ty1-mURA3 plasmid pJSS36-6 (SMITH and BOEKE 1997 ), presumably via polyethylene glycol (PEG)-mediated fusion (J. S. SMITH and J. D. BOEKE, unpublished data). JS249 was derived from JS50-1 by loss of pJSS36-6. To produce JS101 and JS109, transposition was induced in JS50-1 to produce Ty1-mURA3 integration events into the rDNA or non-rDNA locations, as described previously (SMITH and BOEKE 1997 ). SIR gene deletions are complete ORF deletions and were performed by PCR-mediated gene disruption as described elsewhere (BAUDIN et al. 1993 ; LORENZ et al. 1995 ). The HIS3, LEU2, or kanMX4 genes were PCR amplified from pRS403, pRS405, or pRS400, respectively (SIKORSKI and HEITER 1989 ; BRACHMANN et al. 1998 ), using oligodeoxynucleotide primers containing 5' flanking sequences complementary to the SIR genes (SMITH and BOEKE 1997 ). One SIR2 allele in the MAT/ diploid JS101 was replaced with HIS3 (sir2::HIS3) to produce heterozygote JS106, which can mate. The second SIR2 allele of this heterozygote was replaced with LEU2 (sir2::LEU2) to produce a homozygous SIR2 knockout that was a nonmater.

sir3::kanMX4 was similarly introduced into JS308 (MATa) producing JS316. The MAT sir4::HIS3 deletion strain JS219 has been described previously (SMITH and BOEKE 1997 ). JS316 was transformed with pLP304 (2µ LEU2 SIR3), JS219 was transformed with pLP305 (2µ LEU2 SIR4), and the resulting transformants were crossed. After loss of the LEU2 plasmids, tetrads of the resulting diploid XJS3 were dissected to generate congenic SIR⁺(JS333), sir4 (JS337), sir3 (JS335), and sir3 sir4 (JS339) spores. Diploid XJS5 was similarly sporulated, and tetrads were dissected to generate the congenic sir2 and sir2 sir4 spores. To produce a sir4-42 strain, pLP793 was linearized by SacII cleavage within TRP1 and then integrated into the trp163 locus of the sir4 strain JS337, resulting in strain JS347.

Tetrad dissection:
Diploid strains XJS3 and XJS5 were grown as patches on YPD medium for ~20 hr. Cells from these patches were transferred to 2 ml of liquid sporulation medium (1% potassium acetate, 0.002% zinc acetate) and incubated 1 day at 25°, followed by 4 days at 30°. Tetrads were dissected on a YPD plate and incubated for 3 days at 30° to allow spores to grow up as colonies. Dissection plates were replica plated to SC-His, YPD+G418 (200 µg/ml), and MLA to determine the SIR genotype and rDNA silencing strength.

Sir2p-specific antibody:
A Sir2p-specific antiserum was raised using a synthetic peptide (CGVYVVTSDEHPKTL) comprising the 14 C-terminal amino acids plus a nonencoded cysteine residue. The cysteine residue was added to facilitate m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce, Rockford, IL) conjugation of the peptide to carrier keyhole limpet hemocyanin. Rabbits were immunized, and serum was collected using standard protocols (HARLOW and LANE 1988 ). Serum was diluted and used without further purification, as described below.

Protein extraction and immunoblotting:
Strains were grown to saturation in 10 ml of YPD or SC-Leu medium, diluted to an A₆₀₀ of 0.2 in YPD or SC-Leu, and grown to an A₆₀₀ of ~1.0. Pelleted cells were resuspended in 100 µl lysis buffer (50 mM Tris-HCl, pH 7.5, 1% SDS, 5 mM EDTA, 14.3 mM 2-mercaptoethanol, 2 mM PMSF, 1 µg/ml leupeptin, 2 µg/ml antipain, 10 µg/ml benzamidine, 1 µg/ml chymostatin, 1 µg/ml pepstatin A, and 10 U/ml aprotinin). Glass beads (0.45–0.5 mm diameter) were added to the meniscus, and the tubes were vortexed at full speed for five 30-sec pulses and placed on ice in between pulses. Another 100 µl of lysis buffer was added, followed by boiling for 3 min and centrifugation (14,000 x g for 20 min at 4°). An equal volume of 2x loading buffer was added to the supernatant and then boiled again before loading.

Some 300 A₂₈₀ units of each extract were separated on a 10% SDS polyacrylamide gel and then electroblotted to an Immobilon-P filter (Millipore, Bedford, MA) at 300 mA for 1 hr using a mini-Protean II apparatus (Bio-Rad, Richmond, CA). Filters were blocked for 45 min with 10 ml of blocking solution (1x PBS/0.05% Tween 20/5% milk), then incubated with rabbit polyclonal -Sir2 antibody 2916 (1:5000 dilution in blocking solution) or control mouse monoclonal -tubulin antibody B-5-1-2, 6.1 mg/ml (1:5000 dilution; Sigma, St. Louis, MO) for 1 hr at room temperature. The appropriate secondary antibody (anti-rabbit HRP conjugated or anti-mouse HRP conjugated; Amersham Corp., Arlington Heights, IL) was used at a 1:10,000 dilution for 1 hr in blocking solution. Filters were developed on Kodak XAR5 film using the ECL detection system (Amersham).

RNA blot analysis:
Saturated YPD cultures were diluted to an A₆₀₀ of 0.2 in YPD and grown at 30° to midlog phase (A₆₀₀ of 1.4–1.6). Forty micrograms total RNA, isolated as described by CHAPMAN and BOEKE 1991 , was separated on a formaldehyde-containing 1.2% agarose gel and transferred to Genescreen Plus (NEN-Dupont, Boston, MA) in 10x SSC. The filter was probed with an antisense URA3 RNA probe labeled with [-³²P]UTP (800 Ci/mmol). The URA3 RNA probe was transcribed by T7 RNA polymerase from pBS-URA3 linearized with StuI, producing a 363-nt antisense probe. The filter was prehybridized in 10 ml of 5x SSPE, 5x Denhardt's solution, 1% SDS, 50% formamide (w/v), and 100 µg/ml boiled herring sperm DNA at 60° for 3 h, followed by hybridization in the above solution without DNA at 60° for 16 h. The filter was washed twice at room temperature 2x SSC/0.1% SDS, two times at room temperature in 0.2x SSC/0.1% SDS, and twice at 42° in 0.2x SSC/0.1% SDS. The filter was stripped and reprobed with a 1.1-kb BamHI-HindIII fragment of actin, which was derived from p10-AHX3 (CHAPMAN and BOEKE 1991 ), as a loading control. The filter was imaged on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and quantitated using ImageQuant software.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Ribosomal DNA silencing is highly sensitive to changes in SIR2 dosage:
The silencing reporter strains used in this study all carry mURA3- or MET15-marked Ty1 elements integrated within the NTS of rDNA (Figure 1). Both of these markers are partially silenced when integrated within the rDNA tandem array, but are fully expressed when located outside the rDNA or within a single rDNA gene copy that is artificially positioned outside the array (SMITH and BOEKE 1997 ). mURA3 expression is measured by growth on medium lacking uracil, whereas MET15 expression is measured by growth on medium lacking methionine or by colony color on Pb²⁺ containing medium (COST and BOEKE 1996 ; SMITH and BOEKE 1997 ).

We previously demonstrated that silencing of marker genes in the rDNA is completely dependent on the SIR2 gene. Furthermore, deletion of SIR2 increased the accessibility of rDNA to psoralen cross-linking (SMITH and BOEKE 1997 ). These results are consistent with a role for SIR2 in the formation or maintenance of a specialized rDNA chromatin structure. Because the rDNA tandem array makes up 5–10% of yeast chromatin, there are likely to be limiting amounts of Sir2p available to act on the rDNA. To test this hypothesis, we first constructed the diploid reporter strain JS101 (D39), which contains a single Ty1-mURA3 element integrated at the rDNA (Figure 1; see MATERIALS AND METHODS). SIR2 was then deleted one allele at a time, producing heterozygous and homozygous sir2 deletion strains. The strength of rDNA silencing of the mURA3 reporter, as measured by growth on -Ura medium, was tested using a serial dilution growth assay for each of the resulting strains. Interestingly, when a single copy of SIR2 was deleted (JS106), growth on SC-Ura plates increased approximately fivefold over that of the SIR2/SIR2 (+/+) parent (Figure 2A). At the same time, its sensitivity to FOA increased 5- to 25-fold. This result indicates that a single gene dose of SIR2 is insufficient to fully silence mURA3 within a diploid cell. When the second SIR2 allele was deleted from the heterozygote, creating a homozygous knockout strain (JS108), growth on SC-Ura increased another fivefold to the level of positive control non-rDNA insertion JS109 (Figure 2A). This latter result is identical to that previously observed when SIR2 was deleted from a haploid strain (BRYK et al. 1997 ; SMITH and BOEKE 1997 ). The FOA-resistant colonies that grow in the double knockout are completely Ura^- and represent mitotic loss of mURA3 (data not shown) because of an increased rate of rDNA recombination events leading to complete loss of the mURA3-marked repeat (GOTTLIEB and ESPOSITO 1989 ). The SIR2 deletions did not affect overall growth on nonselective media. The steady-state mURA3 RNA level in these strains was determined by quantitative RNA blot analysis using a URA3-specific riboprobe and is shown schematically in Figure 2B. The relative mURA3 RNA levels corresponded to the relative amount of Ura⁺ growth for each strain, indicating that growth assays are a reliable method for determining silencing efficiency in the rDNA.

View larger version (47K): In this window In a new window Download PPT slide   Figure 2. SIR2 dosage controls rDNA silencing of mURA3. (A) Serial dilution growth assays of diploid strains carrying two, one, or zero copies of SIR2. The SIR2 homozygote (JS101, +/+), heterozygous sir2 deletion (JS106, +/-), and homozygous sir2 deletion (JS108, -/-) strains were patched onto a YPD plate and grown for 48 hr at 30°, resuspended in water, and plated in fivefold dilutions onto SC-Ura, which selects for mURA3 expression, SC+Foa, which selects against mURA3 expression, and SC+Ura to monitor growth. The negative control for Ura+ growth is JS249, and the positive control for Ura+ growth of a non-rDNA Ty1-mURA3 insertion is JS109. SC+Ura and SC+Foa photos are after 48-hr growth. SC-Ura photo is after 4 days. (B) Quantitation of steady-state mURA3 RNA levels. These are the same strains described in A. The ~1.2-kb RNA produced from the mURA3 promoter was detected by Northern analysis using an antisense URA3 RNA probe. The mean absolute expression levels of mURA3, as measured by PhosphorImager analysis and normalized against actin RNA for loading, are plotted on a bar graph in arbitrary units. The background level is represented by a dotted horizontal line. Standard deviations are indicated by vertical error bars (n = 3). (C) Quantitation of steady-state Ty1-mURA3 RNA levels. The ~6.7-kb Ty1-mURA3 RNA produced from the Ty1 promoter was quantitated from the same blot as was used for B. (D) Immunoblot analysis of steady-state cellular Sir2p levels in sir2 deletion strains. Strains are as described in A. Lane 1, JS101. Lane 2, JS106. Lane 3, JS108. The SIR2 alleles are represented by a +, and sir2 alleles are represented by a -. The filter was first incubated with -Sir2 antibody and developed with ECL (top). The same filter was then reincubated with -tubulin antibody and developed a second time with ECL (bottom).

For independent confirmation of the intermediate SIR2 dosage effect, Ty1-mURA3 expression driven from the Ty1 promoter was quantitated from the same URA3-probed filter (Figure 2C). Again, the level of the Ty1-mURA3 6.7-kb message directly correlated with the dosage of SIR2 (Figure 2C), indicating that the dosage effect was not specific to the mURA3 promoter, as measured in Figure 2A and Figure B, but it also influenced the Ty1 promoter. The level of the mURA3 message in a sir2 homozygote was equivalent to that in the non-rDNA control JS109 (Figure 2B), but the level of the larger Ty1-mURA3 message was significantly greater when transcribed from a locus outside the rDNA than within the rDNA, even in the sir2/sir2 homozygote (JS108). We attribute this large difference to the sensitivity of the Ty1 promoter to chromosomal position effects (CURCIO and GARFINKEL 1991 ). In summary, by both a growth assay and by directly examining transcription, the SIR2/sir2 heterozygote is phenotypically intermediate between the sir2/sir2 and SIR2/SIR2 homozygotes.

To confirm that Sir2 protein levels had actually changed in these cells, the relative amounts of Sir2p were analyzed by immunoblotting using an -Sir2 antibody (Figure 2D); the steady-state Sir2p levels corresponded to SIR2 gene dosage. These results indicate that silencing of a single Ty1-mURA3 element inserted at the rDNA of a diploid cell is exquisitely sensitive to Sir2p levels. Whereas rDNA silencing was highly sensitive to SIR2 dosage, HMRa silencing, as measured by a sensitive mating assay, was not affected by the twofold difference in SIR2 dosage (data not shown).

Increased dosage of SIR3 was previously shown to enhance telomeric silencing, but increased SIR2 dosage had no effect (RENAULD et al. 1993 ). To determine whether increased SIR2 or SIR3 dosage could strengthen the rDNA silencing observed in a haploid isolate, CEN (low-copy) and 2µ (high-copy) plasmids containing SIR2 were transformed into the SIR2 haploid Ty1-mURA3 isolate JS124 (S2). This haploid silencing reporter strain contains a single Ty1-mURA3 insertion (within the NTS2) upstream of the 5S rRNA gene (Figure 1; SMITH and BOEKE 1997 ). The level of silencing is shown for each strain in Figure 3A; CEN SIR2 caused a small (less than fivefold) increase in repression compared to the empty CEN vector. Furthermore, the 2µ SIR2 plasmid caused between a 5- and 25-fold increase in repression compared to the empty 2µ vector. These results indicate that there are limiting amounts of cellular Sir2p for rDNA silencing, and that silencing can be enhanced beyond wild-type levels by providing additional Sir2p. In contrast, 2µ plasmids containing SIR3 or the closely related SIR2 homolog HST1 (BRACHMANN et al. 1995 ; DERBYSHIRE et al. 1996 ) had no effect on rDNA silencing strength. In addition, high-copy SIR3 did not effect the enhanced silencing caused by high-copy SIR2 when the two genes were coexpressed in the same strain (data not shown). Another SIR2 homolog, HST3, also had no effect on rDNA silencing when overexpressed from the GAL1 promoter (data not shown). Unexpectedly, the level of Ura⁺ growth was modestly greater for the CEN vector set than for the 2µ vector set, including the empty vectors. The reason for this difference is currently unclear, but could be caused by the known effects of CEN plasmids on the cell cycle (SPENCER and HIETER 1992 ).

View larger version (68K): In this window In a new window Download PPT slide   Figure 3. Increased SIR2 enhances silencing strength. (A) Haploid rDNA Ty-mURA3 insertion strain JS124 (S2), which is SIR2, was transformed with CEN plasmids pRS415 (vector) or pCAR237 (SIR2). It was also transformed with the 2µ plasmids pRS425 (vector), pSIR2µ (SIR2), pLP347 (HST1), and pLP304 (SIR3). The strains were patched onto a SC-Leu glucose plate and incubated for 48 hr. Cells were resuspended in water and plated as fivefold serial dilutions on SC-Ura-Leu and SC-Leu and incubated for 4 days to determine their Ura+ growth phenotypes. The SC-Leu plate was incubated for 2 days. (B) Immunoblot analysis of steady-state cellular Sir2p levels in strains containing the following plasmids: empty CEN vector (pRS415, lane 1), CEN SIR2 vector (pCAR237, lane 2), empty 2µ vector (pRS425, lane 3), and 2µ SIR2 vector (pSIR2µ, lane 4). The filter was first incubated with Sir2 antibody and developed with ECL (top). The same filter was then reincubated with -tubulin antibody and developed a second time with ECL (bottom).

The relative steady-state amounts of Sir2p produced by CEN SIR2 or 2µ SIR2 plasmids in strain S2 are shown in Figure 3B. The modest increase (approximately twofold) in Sir2p caused by CEN SIR2 that is associated with enhanced silencing further emphasizes the extreme sensitivity of rDNA silencing to SIR2 dosage. 2µ SIR2 boosted Sir2p protein levels approximately twofold more than CEN SIR2, concomitant with a greater increase in rDNA silencing strength. SIR2 overexpression is toxic to cells (HOLMES et al. 1997 ), which could explain the relatively small increase in Sir2p levels produced by the 2µ SIR2 plasmid. Cells expressing very high levels of Sir2p would be selected against.

SIR4 regulation of rDNA silencing is dependent on SIR2, but not on SIR3:
We previously demonstrated that sir4 causes enhanced silencing of both mURA3 and MET15 genes inserted in the rDNA (SMITH and BOEKE 1997 ). This result suggested that SIR4 negatively regulates rDNA silencing. We were interested in understanding the nature of this regulation. The enhanced silencing phenotype of a sir4 mutant resembled the enhanced silencing caused by increased SIR2 expression. Therefore, the simplest explanation for enhanced silencing would be a sir4-induced increase in cellular Sir2p levels. This initial hypothesis was incorrect because Sir2p levels were unaffected by sir4 (data not shown). In wild-type cells, Sir4p, Sir3p, Rap1p, telomeric DNA, and some Sir2p are normally colocalized within discrete foci at the nuclear periphery (GOTTA et al. 1996 ). The bulk of Sir2p is nucleolar (GOTTA et al. 1997 ). When SIR4 is deleted, all detectable Sir2p and Sir3p redistributes to the nucleolus (GOTTA et al. 1997 ). The redistribution of Sir3p is especially dramatic as it is undetectable in wild-type nucleoli (GOTTA et al. 1997 ). Another possible explanation for the enhanced rDNA silencing in a sir4 strain could therefore be the nucleolar redistribution of Sir3p, Sir2p, or both. To test these possibilities, congenic SIR, sir2, sir3, sir4, sir2 sir4, and sir3 sir4 strains were qualitatively tested for rDNA silencing strength using a MET15-based colony color assay in an epistasis analysis. In this color assay, Met⁺ cells produce white colonies on Pb²⁺ containing medium (MLA), and Met^- cells produce dark brown colonies (COST and BOEKE 1996 ). Strains containing Ty1-MET15 within the rDNA produce colonies with an intermediate tan color, representing partial silencing of MET15 (SMITH and BOEKE 1997 ). Using this assay, enhanced rDNA silencing is visualized as a darker colony color. As shown in Figure 4, the colony colors of a sir4 strain and a sir3 sir4 strain were equivalent at all stages of growth, indicating that SIR3 was not required for the enhanced rDNA silencing phenotype caused by sir4. The sir2 sir4 strain produced highly sectored white colonies that were phenotypically indistinguishable from the sir2 strain and indicative of loss of rDNA silencing (SMITH and BOEKE 1997 ). Therefore, SIR2 is necessary for the sir4 effect of enhanced rDNA silencing, but SIR3 is not. The specificity of these effects suggests that the enhanced rDNA silencing caused by deletion of SIR4 does not require relocalization of Sir3p to the nucleolus but, rather, occurs by a SIR2-dependent mechanism.

View larger version (99K): In this window In a new window Download PPT slide   Figure 4. Epistasis analysis of SIR2, SIR3, and SIR4 deletions for rDNA silencing phenotypes. Silencing reporter strains containing Ty1-MET15 at the rDNA were generated with combinations of SIR2, SIR3, and SIR4 deletions. Each strain was streaked onto a Pb2+-containing (MLA) agar plate. Photos were taken after 5 days growth at 30°. SIR2 and SIR3 were replaced with the kanMX4 gene and SIR4 was replaced with HIS3. Strains used were (WT) JS333, (sir3) JS335, (sir4) JS337, (sir3 sir4) JS339, (sir2) JS343, and (sir2 sir4) JS344. Darker colony color represents enhanced rDNA silencing, whereas colony colors lighter than WT represent weakened rDNA silencing. Dark sectors represent rDNA recombination events that result in complete loss of the Ty1-MET15 reporter. Note that sir4 strains show strongly enhanced silencing; this enhancement completely depends on SIR2. sir3 strains show a subtle but reproducible enhancement of rDNA silencing.

High-copy SIR4 reduces rDNA silencing:
Whereas deletion of SIR4 enhances rDNA silencing, it drastically reduces HM and telomeric silencing. Overexpression of SIR4 or its C terminus reduces telomeric and HM silencing in a dominant negative fashion, which has been termed the "anti-SIR" effect (MARSHALL et al. 1987 ; RENAULD et al. 1993 ; COCKELL et al. 1995 ). The C terminus of Sir4p interacts with the Sir3 and Rap1 proteins, and these interactions are proposed to help recruit a Sir2p/Sir3p/Sir4p complex to telomeres and the silencer elements of the HM loci (MORETTI et al. 1994 ; COCKELL et al. 1995 ; HECHT et al. 1995 ; MOAZED et al. 1997 ). The dominant negative activity of excess SIR4 toward TPE and HM silencing is proposed to be caused by antagonism of these interactions (MARSHALL et al. 1987 ; MORETTI et al. 1994 ; COCKELL et al. 1995 ). For example, SIR4 overexpression could result in the formation of excess incomplete silencing complexes at the expense of complete ones.

To examine the role of SIR4 dosage on rDNA silencing, a high-copy plasmid containing full-length SIR4 was introduced into the haploid Ty1-mURA3 rDNA insertion isolate S6, and the resulting strains were tested for rDNA silencing strength in a serial dilution growth assay on -Ura medium (Figure 5). Empty vectors had no effect on the repression of Ura⁺ growth. However, the SIR4 plasmid increased the Ura⁺ growth of S6 by at least 25-fold, indicating that high-copy SIR4 severely reduces rDNA silencing strength (Figure 5), very similar to the "anti-SIR" effect that occurs at telomeres and HM loci. Silencing was partially restored by co-overexpression of SIR2 with SIR4, suggesting that SIR4 negatively affects rDNA silencing by interfering with SIR2 function. High-copy SIR4 also reduced silencing in sir4 and sir3 mutant versions of S6 (data not shown), indicating that SIR3 was not required for the high-copy SIR4–dependent loss of silencing. The strength of rDNA silencing therefore correlates inversely with SIR4 dosage.

View larger version (39K): In this window In a new window Download PPT slide   Figure 5. SIR4 overexpression effect on rDNA silencing. Strain JS128 (S6) was transformed with HIS3 2µ and LEU2 2µ plasmids in combinations of two. The HIS3 plasmid was the empty vector pRS423 or the SIR2 vector pJSS71-13. The LEU2 plasmid was the empty vector pRS425 or the SIR4 vector pLP305. Leu+ His+ transformants for each combination were patched onto SC-Leu medium and grown for 2 days. Cells were then resuspended in water and plated as fivefold serial dilutions on SC-His-Leu-Ura to select for Ura+ growth and on SC-His-Leu as a nonselective control. The SC-His-Leu photograph was taken after 2 days, and the SC-His-Leu-Ura photograph was taken after 5 days.

To determine which domains of SIR4 antagonize rDNA silencing, 2µ plasmids expressing various domains of SIR4 were tested for their effect on rDNA silencing using the Ura⁺ growth assay. The results are shown in Figure 6A and tabulated in Figure 6B. Overexpression either of the SIR4-42 allele, which truncates the extreme C terminus, or of the C-terminal 40% of SIR4 (pJH5.1A) had a dominant negative effect on rDNA silencing. pJH5.1A was also moderately toxic to these strains for unknown reasons. Surprisingly, the extreme C terminus of SIR4 actually caused an increase in rDNA silencing strength (pJH3A) similar in amplitude to the effect of a 2µ SIR2 plasmid. Increased rDNA silencing was also observed with pJSS73-5, which overexpresses the C-terminal 23% of Sir4p (Figure 6B). The region in common between the constructs that cause derepression (shaded area) overlaps with the region of Sir4p previously shown to interact with Sir2p, but not with Sir3p (see black bar in Figure 6B; MOAZED et al. 1997 ). Based on these results, we propose that the inhibitory effect of Sir4p on rDNA silencing requires its physical interaction with Sir2p.

View larger version (57K): In this window In a new window Download PPT slide   Figure 6. Differential rDNA-silencing phenotypes caused by overexpression of different SIR4 fragments. (A) High-copy 2µ plasmids expressing N- or C-terminal domains of Sir4 were transformed into the rDNA silencing reporter strain S6 (JS128), and the resulting strains were tested for the effects on silencing by measuring changes in Ura+ growth. The empty vectors are pRS425 and YEp13. Full-length SIR4 is pLP305, whereas SIR4-42 is pLP754. pJH5.1A expresses the C-terminal 40% of SIR4, and pJH3A expresses the C-terminal 12% of SIR4. Cells were grown on SC-Leu medium for 2 days, resuspended in water, and then plated as fivefold serial dilutions on SC-Leu -Ura to select for Ura+ growth and on SC-Leu as a nonselective control. The SC-Leu plate was incubated for 2 days, and the SC-Leu -Ura plate was incubated for 4 days. t, N-terminal–truncated Sir4 protein. (B) Schematic representation of results incorporated from A. The amino acids of Sir4p or Sir2p expressed from each plasmid is indicated. The wild-type level of rDNA silencing is represented as a + for each representative empty vector. The relative silencing phenotype of high-copy SIR2 is included for comparison. Vector sequences are indicated by a thin line. Enhanced rDNA silencing is indicated by ++. Loss of rDNA silencing is indicated by a -. The shaded area of SIR4 is deduced to be required for the negative effect on rDNA silencing. The horizontal black bar represents the region of Sir4p shown to previously interact with Sir2p. The region of Sir4p that interacts with Rap1p and Sir3p is striped. The SIR2 plasmid is indicated by an asterisk.

How could overexpression of the extreme C terminus of Sir4p cause increased rDNA silencing? In addition to the SIR genes, UTH4 and YGL014W are also required for a long life span in yeast (KENNEDY et al. 1997 ). Overexpression of the extreme C-terminal Sir4p domain restores a long life span to specific uth4 mutant strains, a phenotype shared by the same uth4 strains bearing the SIR4-42 mutation (KENNEDY et al. 1995 , KENNEDY et al. 1997 ). Interestingly, the SIR4-42 allele in single copy also results in redistribution of the Sir complex from telomere foci to the nucleolus, regardless of UTH4 genotype (KENNEDY et al. 1997 ). The enhanced rDNA silencing produced by overexpression of the SIR4 C terminus (pJH3A) in Figure 6 could therefore be caused by redistribution of telomeric Sir2p to the nucleolus (rDNA). If this is true, then an integrated SIR4-42 allele would be predicted to also cause enhanced rDNA silencing. To test this hypothesis, we integrated the SIR4-42 allele into a strain deleted for SIR4 and tested rDNA silencing of MET15 (Figure 7). In this case, SIR4-42 caused enhanced rDNA silencing (dark colony color) similar to the sir4 strain (Figure 7). A similar phenotype was also produced by introducing a stop codon after amino acid 1237 of the endogenous SIR4 gene (data not shown). These results suggest that mutations which cause redistribution of the Sir complex to the nucleolus, especially redistribution of Sir2p, also result in enhanced rDNA silencing. Remarkably, pJH3A further strengthened the enhanced rDNA silencing phenotype of a sir4 strain (data not shown), suggesting that overexpression of the SIR4 C terminus may release even more Sir2p for rDNA silencing than does sir4. Alternatively, the SIR4 C terminus could release an additional separate factor that acts at the rDNA.

View larger version (71K): In this window In a new window Download PPT slide   Figure 7. rDNA silencing phenotype of a strain carrying the integrated sir4-42 mutant allele. Cells were plated onto Pb2+ containing MLA plates and incubated for 5 days. Strains used were (SIR4+) JS333, (sir4) JS337, and (sir4-42) JS347. The sir4-42 plasmid pLP793 was integrated into JS337 to produce JS347. Darker colony color represents stronger rDNA silencing.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The importance of SIR2 to cellular function is emphasized by the identification of a family of SIR2 homologs that exist in many diverse organisms, including bacteria, yeast, plants, and mammals (BRACHMANN et al. 1995 ; DERBYSHIRE et al. 1996 ). We and others previously showed that SIR2 was required for rDNA silencing in S. cerevisiae (BRYK et al. 1997 ; SMITH and BOEKE 1997 ). The data presented here demonstrate that rDNA silencing is highly sensitive to the dosage of SIR2 and SIR4, but is independent of SIR3 dosage. The strength of rDNA silencing is proportional to the amount of Sir2p produced, suggesting that modulation of cellular Sir2p levels or the redistribution of the normal Sir2p pool could regulate rDNA silencing levels. Such regulation of silencing by modulation of Sir2p has not been previously observed for the other known forms of silencing and may be specific to the regulation of rDNA silencing.

Model of rDNA silencing regulation by SIR2 and SIR4:
Immunolocalization studies have demonstrated that the Sir2, Sir3, Sir4, and Rap1 proteins colocalize along with telomeric DNA to several subnuclear foci that normally associate with the nuclear periphery (PALLADINO et al. 1993 ; GOTTA et al. 1996 ). This accumulation of Sir proteins at the telomeric foci may act to produce a critical concentration necessary for efficient silencing and chromosomal integrity (PALLADINO et al. 1993 ; COCKELL et al. 1995 ; GOTTA et al. 1996 ). The silencing complex is also required for silencing at the HM loci, resulting in competition between telomeres and the HM loci for limiting amounts of Sir3 and Sir4 proteins (BUCK and SHORE 1995 ; MARCAND et al. 1996 ). Mutations that lengthen telomeres, such as rap1^s, cause the balance of silencing to be tipped toward strengthened TPE (BUCK and SHORE 1995 ). This is proposed to result from titration of Sir4p away from the HM loci. Furthermore, it has been proposed that the sequestration of Sir3p and Sir4p by Rap1p at telomeres controls silencing at artificially created, internal, nontelomeric silencing sites (MAILLET et al. 1996 ; MARCAND et al. 1996 ).

Whereas some Sir2p is localized to the telomeric foci, the bulk is in the nucleolus (GOTTA et al. 1997 ). We have now demonstrated that the wild-type level of Sir2p is limiting for rDNA silencing and that silencing strength is proportional to SIR2 dosage. In a recent independent study, high-copy SIR2 was also shown to decrease the expression of ADE2 and CAN1 markers inserted in the rDNA (FRITZE et al. 1997 ). In contrast to the effect of SIR2 dosage, the strength of rDNA silencing is inversely proportional to SIR4 dosage. We propose a model for the regulation of rDNA silencing in which the amount of Sir2p available in the nucleolus for silencing is controlled by the sequestration of Sir2p at telomeres through an interaction with Sir4p (Figure 8).

View larger version (38K): In this window In a new window Download PPT slide   Figure 8. Model of rDNA silencing regulation by telomeres. In this model, the strength of rDNA silencing is proportional to the amount of Sir2p localized at the nucleolus. Sir4p is proposed to limit rDNA silencing by titrating Sir2p out of the nucleolus through direct interaction with Sir2p. The localization patterns of Sir2p and Sir4p are represented for a wild-type strain (WT), and for sir4, sir2, and sir3 mutants. The normal localization for Sir2p is nucleolar and telomeric (GOTTA et al. 1997 ). sir4 causes redistribution of telomeric Sir2p to the nucleolus (GOTTA et al. 1997 ), which increases the local concentration of Sir2p and enhances rDNA silencing. sir3 causes diffuse nuclear Sir4p localization, which would prevent most of the released telomeric Sir2p from redistributing to the nucleolus. Consistent with this, a very slight but reproducible enhancement of rDNA silencing is observed in sir3 strains (Figure 4). sir2 completely eliminates rDNA silencing and telomeric silencing. Filled circles, Sir2p; open squares, Sir4p.

Several lines of evidence support this model.

1. When SIR4 is deleted, rDNA silencing becomes enhanced (SMITH and BOEKE 1997 ). This is the opposite effect of what happens at telomeres and HM loci, where SIR4 is absolutely required for silencing (IVY et al. 1986 ; RINE and HERSKOWITZ 1987 ; APARICIO et al. 1991 ). Deletion of SIR4 also results in the redistribution of the telomere-localized Sir3p and Sir2p to the nucleolus (GOTTA et al. 1997 ), which is the subnuclear location of the rDNA. Epistasis analysis revealed that SIR2, but not SIR3, is required for the enhanced rDNA silencing caused by sir4. Therefore, the enhancement in silencing cannot be caused simply by relocalization of Sir3p to the nucleolus. It is more likely that the redistribution of telomeric Sir2p to the nucleolus increases the local concentration of Sir2p at the rDNA, resulting in stronger rDNA silencing (see Figure 8, model). In the case of sir3, Sir4p displays a diffuse nuclear localization pattern. Sir2p and Sir4p have been shown to interact independently of Sir3p when not associated with chromatin (MOAZED et al. 1997 ). Therefore, this diffuse nuclear Sir4p would prevent most of the telomeric pool of Sir2p from redistributing to the rDNA (Figure 8, model), thus causing only the minor enhancement in rDNA silencing that we observe.

2. Overexpression of SIR4 results in loss of rDNA silencing (Figure 5) similar to the dominant negative "anti-Sir" effect of SIR4 overexpression on TPE and HM silencing (MARSHALL et al. 1987 ; RENAULD et al. 1993 ; COCKELL et al. 1995 ). The negative effect of SIR4 on HM silencing can be compensated for by co-overexpression of SIR3 (MARSHALL et al. 1987 ). Strikingly, co-overexpression of SIR2 partially alleviates the overexpression effect of SIR4, resulting in restoration of rDNA silencing (Figure 6A). These results suggest that Sir4p disrupts the normal nucleolar function of Sir2p in a titratable manner.

3. We found that a specific domain of Sir4p is responsible for the negative effect on rDNA silencing. This region of Sir4p (amino acids 731–1049) overlaps with a domain previously shown to physically interact with Sir2p (MOAZED and JOHNSON 1996 ; MOAZED et al. 1997 ; STRAHL-BOLSINGER et al. 1997 ). SIR4 overexpression results in delocalization of the telomeric Sir4p staining to a diffuse nuclear pattern (MAILLET et al. 1996 ). This excess nuclear Sir4p could potentially titrate Sir2p away from the rDNA, therefore causing derepression of rDNA silencing (Figure 8, model).

4. A specific mutant allele of SIR4, called SIR4-42, results in redistribution of the Sir silencing complex from telomeres to the nucleolus and suppresses the allele-specific decrease in life span caused by a specific mutation in UTH4, a gene required for long life span (KENNEDY et al. 1995 , KENNEDY et al. 1997 ). Overexpression of the extreme C terminus of SIR4 also increases life span, presumably by redistributing the Sir complex to the nucleolus (KENNEDY et al. 1995 ). Nucleolar relocalization of the Sir complex would increase the intranucleolar Sir2p concentration. Strikingly, both of these SIR4 manipulations cause an increase in rDNA silencing, as predicted by our model. In summary, release of rDNA silencing-competent Sir2p from telomeres should enhance rDNA silencing, whereas increased sequestration of Sir2p at telomeres should weaken rDNA silencing. Consistent with this model, mutant alleles of cdc17, rfc1, and rif1, which increase telomere length in yeast (HARDY et al. 1992 ; ADAMS and HOLM 1996 ), also result in weakened rDNA silencing (J. S. SMITH and J. D. BOEKE, unpublished data).

Deregulation of rDNA silencing could have profound metabolic implications for the yeast cell. For example, loss of the repressive SIR2-dependent rDNA chromatin structure causes an increase in the percentage of actively transcribed rDNA gene copies (SMITH and BOEKE 1997 ). Increased rDNA silencing is therefore predicted to cause a reduction in the percentage of actively transcribed rDNA gene copies.

Structural differences between rDNA and other forms of silencing:
Sir3p is a proposed structural component of yeast telomeric heterochromatin (HECHT et al. 1995 , HECHT et al. 1996 ). Therefore, the lack of SIR3 involvement in rDNA silencing emphasizes the fundamental difference in silencing complexes that act at the rDNA vs. the telomeres/HM loci. Sir3p exclusion from the putative rDNA silencing complex may allow for efficient chromatin remodeling that may be necessary in a genomic region, such as the rDNA, which is efficiently transcribed by Pol I and Pol III, but may need to be constrained in terms of other transactions, such as rDNA recombination (CHRISTMAN et al. 1988 ; GOTTLIEB and ESPOSITO 1989 ; BRYK et al. 1997 ; SMITH and BOEKE 1997 ).

What is the role of SIR2 in rDNA silencing?
Not only is Sir2p localized in the nucleolus (GOTTA et al. 1997 ), but Sir2p preferentially associates with rDNA (GOTTA et al. 1997 ). We have previously demonstrated that in sir2 strains, transcriptionally inactive rDNA regions become more accessible to psoralen cross-linking in vivo, indicating that Sir2p is involved in either establishment or maintenance of a specialized repressive chromatin structure in the rDNA (SMITH and BOEKE 1997 ). Furthermore, a recent study showed that SIR2 modulates the accessibility of rDNA chromatin to micrococcal nuclease digestion in vitro and dam methyltransferase activity in vivo in a dosage-dependent manner (FRITZE et al. 1997 ). These results, taken together, strongly support the hypothesis that Sir2p contributes to the formation of chromatin structure in the rDNA. Therefore, the modulation of rDNA silencing strength by changes in SIR2 dosage is likely to result from direct changes in the Pol II-repressive chromatin in the rDNA. It is still unclear whether Sir2p is a direct structural component of the rDNA chromatin, or whether it is present in a regulatory capacity. Sir2p does not have a demonstrated DNA-binding activity (BUCHMAN et al. 1988 ), suggesting that it might associate with rDNA through interactions with other proteins.

The transcriptional activity of eukaryotic genes correlates with the level of nucleosome acetylation of lysine residues on the N terminus of the core histones (for review see STERNGLANZ 1996 ). Accordingly, histones of the HM loci-associated chromatin have been shown to be hypoacetylated compared to other nonsilenced chromosomal regions (BRAUNSTEIN et al. 1993 , BRAUNSTEIN et al. 1996 ). Overexpression of SIR2 correlates with decreased histone acetylation levels in S. cerevisiae (BRAUNSTEIN et al. 1993 ). It is therefore possible that inactive rDNA chromatin is associated with hypoacetylated histones. The rDNA histone acetylation level might be similarly influenced by SIR2. Interestingly, it has been suggested that deacetylated histones are associated with transcriptionally inactive rRNA genes in rat tumor cells (MUTSKOV et al. 1996 ). However, this has not yet been directly examined in yeast. SIR2 could be directly or indirectly involved in histone deacetylation, or it could prevent acetylation by blocking the access or activity of acetyltransferase on rDNA histones. Whether the histone acetylation state of rDNA histones controls rDNA silencing remains to be determined.

A large family of genes homologous to SIR2 have been identified in other species ranging from bacteria to human (BRACHMANN et al. 1995 ; DERBYSHIRE et al. 1996 ). Included in this family are four homologs in S. cerevisiae known as HST1, HST2, HST3, and HST4. HST1 overexpression has previously been shown to partially complement the mating defect of a sir2 mutant (BRACHMANN et al. 1995 ). However, overexpression of HST1 or HST3 had no effect on rDNA silencing strength. These results suggest that individual members of the HST family of genes do not have identical functions. It will be interesting to test the effects of other hst mutants on rDNA silencing. So far, the only SIR2 homolog in other species that has been characterized is from the yeast Kluyveromyces lactis (CHEN and CLARK-WALKER 1994 ). Mutation of this SIR2 homolog causes an increase in ethidium bromide sensitivity and was reported to cause an increase in rDNA recombination (CHEN and CLARK-WALKER 1994 ). These results suggest that SIR2 homologs may function broadly in regulation of chromatin, including rDNA chromatin.

rDNA silencing and aging:
The life span of S. cerevisiae cells is defined by the relatively fixed number of asymmetric cell divisions that a mother cell can achieve (MORTIMER and JOHNSTON 1959 ). A link between silencing and life span determination was identified through the isolation of a dominant gain of function mutant of SIR4 (SIR4-42), which restored a long life span to a short-lived uth4-14c strain (KENNEDY et al. 1995 ). Because the Sir protein complex is redistributed from telomeric foci to the nucleolus in SIR4-42 cells, the rDNA has been proposed to be the site of this SIR4-42 gain of function and perhaps to be associated with aging (KENNEDY et al. 1997 ). Interestingly, phenotypes associated with the SIR4-42 mutation, including sterility and Sir protein redistribution to the nucleolus, also occur naturally in old mother cells (SMEAL et al. 1996 ; KENNEDY et al. 1997 ), suggesting that the nucleolar redistribution of the Sir protein complex may somehow compensate for or protect against the cumulative effects of aging on the nucleolus (GUARENTE 1997 ; KENNEDY et al. 1997 ).

We have demonstrated that the SIR4-42 mutation or overexpression of the extreme C terminus of SIR4 causes an increase in the strength of rDNA silencing. Does this mean that increased rDNA silencing causes increased life span? As stated above, both of these SIR4 manipulations can restore longevity to specific, short-lived uth4 mutant strains in a SIR3-dependent manner (KENNEDY et al. 1995 ). However, deletion of SIR4 modestly shortens life span (KENNEDY et al. 1995 ) yet enhances rDNA silencing in a SIR2-dependent, SIR3-independent manner. Moreover, SIR3 and SIR4 are required for longevity (KENNEDY et al. 1995 ) but not for rDNA silencing (SMITH and BOEKE 1997 ). Taken together, these findings indicate that increased rDNA silencing is not sufficient to lengthen life span.

Enhanced rDNA silencing may, however, be an important component of the cellular response to aging. Our results suggest that increased rDNA silencing strength is a consequence of mutations or conditions that cause redistribution of the Sir complex (especially Sir2p) to the nucleolus, which could be a phenocopy of what happens in old cells. Deletion of the SGS1 gene was recently demonstrated to accelerate the nucleolar fragmentation phenotype that occurs normally in very old wild-type yeast cells (SINCLAIR et al. 1997 ). SGS1 is a member of the RecQ helicase family, which includes the Werner's syndrome gene WRN that is implicated in premature aging in humans (YU et al. 1996 ). Sgs1p is localized in the nucleolus, and deletion of the gene shortens life span (SINCLAIR et al. 1997 ) and increases the frequency of rDNA recombination (GANGLOFF et al. 1994 ). It was therefore proposed that nucleolar fragmentation represents a cause of yeast aging, and that redistribution of the Sir silencing complex to the nucleolus delays these changes (GUARENTE 1997 ; SINCLAIR et al. 1997 ). More recently, this nucleolar fragmentation has been shown to result from accumulation of extrachromosomal rDNA circles, which form multiple nucleoli (SINCLAIR and GUARENTE 1997 ). These circles also accumulate in strains with a subthreshold level of DNA topoisomerases I and II (KIM and WANG 1989 ). Given that TOP1 and SIR2 are both required for rDNA silencing (BRYK et al. 1997 ; SMITH and BOEKE 1997 ), it is possible that the Sir2p-dependent chromatin structure associated with rDNA silencing could contribute to the counteraction of aging through suppression of rDNA circle excision.

	ACKNOWLEDGMENTS

We thank JAMES BROACH, LEONARD GUARENTE, ROHINTON KAMAKAKA, JOANNA LOWELL, PETER PHILIPPSEN, and ACHIM WACH for supplying plasmids, DANESH MOAZED and SIYUAN LE for comments on the manuscript, and members of the Boeke lab for helpful advice and discussions. L.P. thanks JASPER RINE for his early support (GM54778) of this work, which has been continued with National Insititues of Health (NIH) grant GM54778 (L.P.) J.S.S. is a postdoctoral fellow of the Leukemia Society of America. This work was supported in part by NIH grant CA16519 to J.D.B.

Manuscript received December 24, 1997; Accepted for publication March 23, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ADAMS, A. K. and C. HOLM, 1996  Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.. Mol. Cell. Biol. 16:4614-4620[Abstract/Free Full Text].

APARICIO, O. M., B. L. BILLINGTON, and D. E. GOTTSCHLING, 1991  Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae.. Cell 66:1279-1287[Medline].

BAUDIN, A., O. OZIER-KALOGEROPOULOS, A. DENOUEL, F. LACROUTE, and C. CULLIN, 1993  A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae.. Nucleic Acids Res. 21:3329-3330[Free Full Text].

BOEKE, J. D., D. J. GARFINKEL, C. A. STYLES, and G. R. FINK, 1985  Ty elements transpose through an RNA intermediate. Cell 40:491-500[Medline].

BRACHMANN, C. B., J. M. SHERMAN, S. E. DEVINE, E. E. CAMERON, and L. PILLUS et al., 1995  The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev. 9:2888-2902[Abstract/Free Full Text].

BRACHMANN, C. B., A. DAVIES, G. J. COST, E. CAPUTO, and J. LI et al., 1998  Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115-132[Medline].

BRAUNSTEIN, M., A. B. ROSE, S. G. HOLMES, C. D. ALLIS, and J. R. BROACH, 1993  Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 7:592-604[Abstract/Free Full Text].

BRAUNSTEIN, M., R. E. SOBEL, C. D. ALLIS, B. M. TURNER, and J. R. BROACH, 1996  Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern. Mol. Cell. Biol. 16:4349-4356[Abstract/Free Full Text].

BRYK, M., M. BANERJEE, M. MURPHY, K. E. KNUDSEN, and D. J. GARFINKEL et al., 1997  Transcriptional silencing of Ty1 elements in the RDN1 locus of yeast. Genes Dev. 11:255-269[Abstract/Free Full Text].

BUCHMAN, A. R., W. J. KIMMERLY, J. RINE, and R. D. KORNBERG, 1988  Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae.. Mol. Cell. Biol. 8:210-225[Abstract/Free Full Text].

BUCK, S. W. and D. SHORE, 1995  Action of a RAP1 carboxy-terminal silencing domain reveals an underlying competition between HMR and telomeres in yeast. Genes Dev. 9:370-384[Abstract/Free Full Text].

CHAPMAN, K. B. and J. D. BOEKE, 1991  Isolation and characterization of the gene encoding yeast debranching enzyme. Cell 65:483-492[Medline].

CHEN, X.-J. and G. D. CLARK-WALKER, 1994  sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs. Mol. Cell. Biol. 14:4501-4508[Abstract/Free Full Text].

CHRISTIANSON, T. W., R. S. SIKORSKI, M. DANTE, J. H. SHERO, and P. HIETER, 1992  Multifunctional yeast high-copy number shuttle vectors. Gene 110:119-122[Medline].

CHRISTMAN, M. F., F. S. DIETRICH, and G. R. FINK, 1988  Mitotic recombination in the rDNA of S. cerevisiae is suppressed by the combined action of DNA topoisomerases I and II. Cell 55:413-425[Medline].

COCKELL, M., F. PALLADINO, T. LAROCHE, G. KYRION, and C. LIU et al., 1995  The carboxy termini of Sir4 and Rap1 affect Sir3 localization: evidence for a multicomponent complex required for telomeric silencing. J. Cell Biol. 129:909-924[Abstract/Free Full Text].

COST, G. J. and J. D. BOEKE, 1996  A useful colony colour phenotype associated with the yeast selectable/counterselectable marker MET15.. Yeast 12:939-941[Medline].

CURCIO, M. J. and D. J. GARFINKEL, 1991  Single-step selection for Ty1 element retrotransposition. Proc. Natl. Acad. Sci. USA 88:936-940[Abstract/Free Full Text].

DERBYSHIRE, M. K., K. G. WEINSTOCK, and J. N. STRATHERN, 1996  HST1, a new member of the SIR2 family of genes. Yeast 12:631-640[Medline].

DOBIE, K., M. MEHTALI, M. MCLENAGHAN, and R. LATHE, 1997  Variegated gene expression in mice. Trends Genet. 13:127-130[Medline].

FRITZE, C. E., K. VERSCHUEREN, R. STRICH, and R. E. ESPOSITO, 1997  Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA. EMBO J. 16:6495-6509[Medline].

GANGLOFF, S., J. P. MCDONALD, C. BENDIXEN, L. ARTHUR, and R. ROTHSTEIN, 1994  The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Mol. Cell. Biol. 14:8391-8398[Abstract/Free Full Text].

GOTTA, M., T. LAROCHE, A. FORMENTON, L. MAILLET, and H. SCHERTHAN et al., 1996  The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae.. J. Cell Biol. 134:1349-1363[Abstract/Free Full Text].

GOTTA, M., S. STRAHL-BOLSINGER, H. RENAULD, T. LAROCHE, and B. K. KENNEDY et al., 1997  Localization of Sir2p: the nucleolus as a compartment for silent information regulators. EMBO J. 16:3243-3255[Medline].

GOTTLIEB, S. and R. E. ESPOSITO, 1989  A new role for a yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA. Cell 56:771-776[Medline].

GOTTSCHLING, D. E., 1992  Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo. Proc. Natl. Acad. Sci. USA 89:4062-4065[Abstract/Free Full Text].

GOTTSCHLING, D. E., O. M. APARICIO, B. L. BILLINGTON, and V. A. ZAKIAN, 1990  Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63:751-762[Medline].

GUARENTE, L., 1997  Link between aging and the nucleolus. Genes Dev. 11:2449-2455[Free Full Text].

HABER, J. E. and J. P. GEORGE, 1979  A mutation that permits the expression of normally silent copies of mating-type information in Saccharomyces cerevisiae.. Genetics 93:13-35[Abstract/Free Full Text].

HARDY, C. F., L. SUSSEL, and D. SHORE, 1992  A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation. Genes Dev. 6:801-814[Abstract/Free Full Text].

HARLOW, E., and D. LANE, 1988 Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

HECHT, A., T. LAROCHE, S. STRAHL-BOLSINGER, S. M. GASSER, and M. GRUNSTEIN, 1995  Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast. Cell 80:583-592[Medline].

HECHT, A., S. STRAHL-BOLSINGER, and M. GRUNSTEIN, 1996  Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature 383:92-96[Medline].

HILL, J. E., A. M. MYERS, T. J. KOERNER, and A. TZAGOLOFF, 1986  Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163-167[Medline].

HOLMES, S. G., A. B. ROSE, K. STEUERLE, E. SAEZ, and S. SAYEGH et al., 1997  Hyperactivation of the silencing proteins, Sir2p and Sir3p, causes chromosome loss. Genetics 145:605-614[Abstract].

HUANG, H., A. KAHANA, D. E. GOTTSCHLING, L. PRAKASH, and S. W. LIEBMAN, 1997  The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae.. Mol. Cell. Biol. 17:6693-6699[Abstract/Free Full Text].

IVY, J. M., A. J. KLAR, and J. B. HICKS, 1986  Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.. Mol. Cell. Biol. 6:688-702[Abstract/Free Full Text].

KENNEDY, B. K., J. NICANOR, R. AUSTRIACO, J. ZHANG, and L. GUARENTE, 1995  Mutation in the silencing gene SIR4 can delay aging in S. cerevisiae.. Cell 80:485-496[Medline].

KENNEDY, B. K., M. GOTTA, D. A. SINCLAIR, K. MILLS, and D. S. MCNABB et al., 1997  Redistribution of silencing proteins from telomeres to the nucleolus is associated with extension of life span in S. cerevisiae.. Cell 89:381-391[Medline].

KIM, R. A. and J. C. WANG, 1989  A subthreshold level of DNA topoisomerases leads to the excision of yeast rDNA as extrachromosomal rings. Cell 57:975-985[Medline].

KLAR, A. J. S., S. FOGEL, and K. MACLEOD, 1979  MAR1—a regulator of HMa and HM loci in Saccharomyces cerevisiae.. Genetics 93:37-50[Abstract/Free Full Text].

KOSTRIKEN, R., J. N. STRATHERN, A. J. S. KLAR, J. B. HICKS, and F. HEFFRON, 1983  A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae.. Cell 35:167-174[Medline].

LIU, H., J. KRIZEK, and A. BRETSHER, 1992  Construction of a GAL1-regulated yeast cDNA library and its application to the identification of genes whose overexpression causes lethality in yeast. Genetics 132:665-673[Abstract].

LOO, S. and J. RINE, 1994  Silencers and domains of generalized repression. Science 264:1768-1771[Abstract/Free Full Text].

LOO, S. and J. RINE, 1995  Silencing and heritable domains of gene expression. Annu. Rev. Biol. Dev. 11:519-548[Medline].

LORENZ, M. C., R. S. MUIR, E. LIM, J. MCELVER, and S. C. WEBER et al., 1995  Gene disruption with PCR products in Saccharomyces cerevisiae.. Gene 158:113-117[Medline].

LUSTIG, A. J., C. LIU, C. ZHANG, and J. P. HANISH, 1996  Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae.. Mol. Cell. Biol. 16:2483-2495[Abstract/Free Full Text].

MAILLET, L., C. BOSCHERON, M. GOTTA, S. MARCAND, and E. GILSON et al., 1996  Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression. Genes Dev. 10:1796-1811[Abstract/Free Full Text].

MARCAND, S., S. W. BUCK, P. MORETTI, E. GILSON, and D. SHORE, 1996  Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap1 protein. Genes Dev. 10:1297-1309[Abstract/Free Full Text].

MARSHALL, M., D. MAHONEY, A. ROSE, J. B. HICKS, and J. R. BROACH, 1987  Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae.. Mol. Cell. Biol. 7:4441-4452[Abstract/Free Full Text].

MOAZED, D. and A. D. JOHNSON, 1996  A deubiquitinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae.. Cell 86:667-677[Medline].

MOAZED, D., A. KISTLER, A. AXELROD, J. RINE, and A. D. JOHNSON, 1997  Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3. Proc. Natl. Acad. Sci. USA 94:2186-2191[Abstract/Free Full Text].

MORETTI, P., K. FREEMAN, L. COODLY, and D. SHORE, 1994  Evidence that a complex of SIR proteins interacts with the silencer and telomere binding protein RAP1. Genes Dev. 8:2257-2269[Abstract/Free Full Text].

MORTIMER, R. K. and J. R. JOHNSTON, 1959  Life span of individual yeast cells. Nature 183:1751-1752[Medline].

MUTSKOV, V. J., V. R. RUSSANOVA, S. I. DIMITROV, and I. G. PASHEV, 1996  Histones associated with non-nucleosomal rat ribosomal genes are acetylated while those bound to nucleosome-organized gene copies are not. J. Biol. Chem. 271:11852-11857[Abstract/Free Full Text].

PALLADINO, F., T. LAROCHE, E. GILSON, A. AXELROD, and L. PILLUS et al., 1993  SIR3 and SIR4 proteins are required for the positioning and integrity of yeast telomeres. Cell 75:543-555[Medline].

PETES, T. D. and D. BOTSTEIN, 1977  Simple Mendelian inheritance of the reiterated ribosomal DNA of yeast. Proc. Natl. Acad. Sci. USA 74:5091-5095[Abstract/Free Full Text].

PHILIPPSEN, P., M. THOMAS, R. A. KRAMER, and R. W. DAVIS, 1978  Unique arrangement of coding sequences for 5S, 5.8S, 18S, and 25S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis. J. Mol. Biol. 123:387-404[Medline].

PILLUS, L. and J. RINE, 1989  Epigenetic inheritance of transcriptional states in S. cerevisiae.. Cell 59:637-647[Medline].

RENAULD, H., O. M. APARICIO, P. D. ZIERATH, B. L. BILLINGTON, and S. K. CHHABLANI et al., 1993  Silent domains are assembled continuously from the telomere and are defined by promoter distance and strength, and by SIR3 dosage. Genes Dev. 7:1133-1145[Abstract/Free Full Text].

RINE, J. and I. HERSKOWITZ, 1987  Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae.. Genetics 116:9-22[Abstract/Free Full Text].

RINE, J., J. N. STRATHERN, J. B. HICKS, and I. HERSKOWITZ, 1979  A suppressor of mating-type locus mutations in Saccharomyces cerevisiae: evidence for and identification of cryptic mating-type loci. Genetics 93:877-901[Abstract/Free Full Text].

ROSE, M. D., F. WINSTON and P. HEITER, 1990 Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SCHNELL, R. and J. RINE, 1986  A position effect on the expression of a tRNA gene mediated by the SIR genes of Saccharomyces cerevisiae.. Mol. Cell. Biol. 6:494-501[Abstract/Free Full Text].

SHORE, D., 1994  RAP1: a protean regulator in yeast. Trends Genet. 10:408-412[Medline].

SIKORSKI, R. S. and P. HEITER, 1989  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.. Genetics 122:19-27[Abstract/Free Full Text].

SINCLAIR, D. A. and L. GUARENTE, 1997  Extrachromosomal rDNA circles—a cause of aging in yeast. Cell 91:1033-1042[Medline].

SINCLAIR, D. A., K. MILLS, and L. GUARENTE, 1997  Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants. Science 277:1313-1316[Abstract/Free Full Text].

SINGH, J. and A. J. S. KLAR, 1992  Active genes in budding yeast display enhanced in vivo accessibility to foreign DNA methylases: a novel in vivo probe for chromatin structure of yeast. Genes Dev. 6:186-196[Abstract/Free Full Text].

SKRYABIN, K. G., M. A. ELDAROV, V. L. LARIONOV, A. A. BAYEV, and J. KLOOTWIJK et al., 1984  Structure and function of the nontranscribed spacer regions of yeast rDNA. Nucleic Acids Res. 12:2955-2968[Abstract/Free Full Text].

SMEAL, T., J. CLAUS, B. KENNEDY, F. COLE, and L. GUARENTE, 1996  Loss of transcriptional silencing causes sterility in old mother cells of S. cerevisiae.. Cell 84:633-642[Medline].

SMITH, J. S. and J. D. BOEKE, 1997  An unusual form of transcriptional silencing in yeast ribosomal DNA. Genes Dev. 11:241-254[Abstract/Free Full Text].

SPENCER, F. and P. HIETER, 1992  Centromere DNA mutations induce a mitotic delay in Saccharomyces cerevisiae.. Proc. Natl. Acad. Sci. USA 89:8908-8912[Abstract/Free Full Text].

STAVENHAGEN, J. B. and V. A. ZAKIAN, 1994  Internal tracts of telomeric DNA act as silencers in Saccharomyces cerevisiae.. Genes Dev. 8:1411-1422[Abstract/Free Full Text].

STERNGLANZ, R., 1996  Histone acetylation: a gateway to transcriptional activation. Trends Biochem. Sci. 21:357-358[Medline].

STONE, E. M. and L. PILLUS, 1996  Activation of an MAP kinase cascade leads to Sir3p hyperphoshorylation and strengthens transcriptional silencing. J. Cell Biol. 135:571-583[Abstract/Free Full Text].

STONE, E. M., M. J. SWANSON, A. M. ROMEO, J. B. HICKS, and R. STERNGLANZ, 1991  The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants. Mol. Cell. Biol. 11:2253-2262[Abstract/Free Full Text].

STRAHL-BOLSINGER, S., A. HECHT, K. LUO, and M. GRUNSTEIN, 1997  SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev. 11:83-93[Abstract/Free Full Text].

STRATHERN, J. N., A. J. KLAR, J. B. HICKS, J. A. ABRAHAM, and J. M. IVY et al., 1982  Homothallic switching of yeast mating-type cassettes is initiated by a double-stranded cut in the MAT locus. Cell 31:183-192[Medline].

SUSSEL, L., D. VANNIER, and D. SHORE, 1993  Epigenetic switching of transcriptional states: cis- and trans-acting factors affecting establishment of silencing at the HMR locus in Saccharomyces cerevisiae.. Mol. Cell. Biol. 13:3919-3928[Abstract/Free Full Text].

SZOSTAK, J. W. and R. WU, 1979  Insertion of a genetic marker into the ribosomal DNA of yeast. Plasmid 2:536-554[Medline].

THOMPSON, J. S., A. HECHT, and M. GRUNSTEIN, 1994  Histones and the regulation of heterochromatin in yeast. Cold Spring Harbor Symp. Quant. Biol. 58:247-256.

WACH, A., A. BRACHAT, R. POHLMANN, and P. PHILIPPSEN, 1994  New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae.. Yeast 10:1793-1808[Medline].

WEILER, K. S. and B. T. WAKIMOTO, 1995  Heterochromatin and gene expression in Drosophila.. Annu. Rev. Genet. 29:577-605[Medline].

YU, C.-E., J. OSHIMA, Y.-H. FU, E. M. WIJSMAN, and F. HISAMA et al., 1996  Positional cloning of the Werner's syndrome gene. Science 272:258-262[Abstract].

This article has been cited by other articles:

				C. K. Tsang and X. F. S. Zheng Opposing Role of Condensin and Radiation-sensitive Gene RAD52 in Ribosomal DNA Stability Regulation J. Biol. Chem., August 14, 2009; 284(33): 21908 - 21919. [Abstract] [Full Text] [PDF]

				Y. Mazor and M. Kupiec Developmentally regulated MAPK pathways modulate heterochromatin in Saccharomyces cerevisiae Nucleic Acids Res., August 1, 2009; 37(14): 4839 - 4849. [Abstract] [Full Text] [PDF]

				M. R. Koch and L. Pillus From the Cover: The glucanosyltransferase Gas1 functions in transcriptional silencing PNAS, July 7, 2009; 106(27): 11224 - 11229. [Abstract] [Full Text] [PDF]

				J. Zhou, B. O. Zhou, B. A. Lenzmeier, and J.-Q. Zhou Histone deacetylase Rpd3 antagonizes Sir2-dependent silent chromatin propagation Nucleic Acids Res., June 1, 2009; 37(11): 3699 - 3713. [Abstract] [Full Text] [PDF]

				M. Biswas, N. Maqani, R. Rai, S. P. Kumaran, K. R. Iyer, E. Sendinc, J. S. Smith, and S. Laloraya Limiting the Extent of the RDN1 Heterochromatin Domain by a Silencing Barrier and Sir2 Protein Levels in Saccharomyces cerevisiae Mol. Cell. Biol., May 15, 2009; 29(10): 2889 - 2898. [Abstract] [Full Text] [PDF]

				R. D. Hontz, R. O. Niederer, J. M. Johnson, and J. S. Smith Genetic Identification of Factors That Modulate Ribosomal DNA Transcription in Saccharomyces cerevisiae Genetics, May 1, 2009; 182(1): 105 - 119. [Abstract] [Full Text] [PDF]

				S. Qin, Q. Wang, A. Ray, G. Wani, Q. Zhao, S. R. Bhaumik, and A. A. Wani Sem1p and Ubp6p orchestrate telomeric silencing by modulating histone H2B ubiquitination and H3 acetylation Nucleic Acids Res., April 1, 2009; 37(6): 1843 - 1853. [Abstract] [Full Text] [PDF]

				J. M. McClure, C. M. Gallo, D. L. Smith Jr., M. Matecic, R. D. Hontz, S. W. Buck, F. G. Racette, and J. S. Smith Pnc1p-Mediated Nicotinamide Clearance Modifies the Epigenetic Properties of rDNA Silencing in Saccharomyces cerevisiae Genetics, October 1, 2008; 180(2): 797 - 810. [Abstract] [Full Text] [PDF]

				R. P. Darst, S. N. Garcia, M. R. Koch, and L. Pillus Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 Mol. Cell. Biol., February 15, 2008; 28(4): 1361 - 1372. [Abstract] [Full Text] [PDF]

				S. Venkatasubrahmanyam, W. W. Hwang, M. D. Meneghini, A. H. Y. Tong, and H. D. Madhani Genome-wide, as opposed to local, antisilencing is mediated redundantly by the euchromatic factors Set1 and H2A.Z PNAS, October 16, 2007; 104(42): 16609 - 16614. [Abstract] [Full Text] [PDF]

				C. J. Fry, A. Norris, M. Cosgrove, J. D. Boeke, and C. L. Peterson The LRS and SIN Domains: Two Structurally Equivalent but Functionally Distinct Nucleosomal Surfaces Required for Transcriptional Silencing Mol. Cell. Biol., December 1, 2006; 26(23): 9045 - 9059. [Abstract] [Full Text] [PDF]

				L. Calzari, I. Orlandi, L. Alberghina, and M. Vai The Histone Deubiquitinating Enzyme Ubp10 Is Involved in rDNA Locus Control in Saccharomyces cerevisiae by Affecting Sir2p Association Genetics, December 1, 2006; 174(4): 2249 - 2254. [Abstract] [Full Text] [PDF]

				M. Veron, Y. Zou, Q. Yu, X. Bi, A. Selmi, E. Gilson, and P.-A. Defossez Histone H1 of Saccharomyces cerevisiae Inhibits Transcriptional Silencing Genetics, June 1, 2006; 173(2): 579 - 587. [Abstract] [Full Text] [PDF]

				B. A. Tamburini, J. J. Carson, J. G. Linger, and J. K. Tyler Dominant Mutants of the Saccharomyces cerevisiae ASF1 Histone Chaperone Bypass the Need for CAF-1 in Transcriptional Silencing by Altering Histone and Sir Protein Recruitment Genetics, June 1, 2006; 173(2): 599 - 610. [Abstract] [Full Text] [PDF]

				Y.-H. Kim, D. Ishikawa, H. P. Ha, M. Sugiyama, Y. Kaneko, and S. Harashima Chromosome XII context is important for rDNA function in yeast Nucleic Acids Res., May 31, 2006; 34(10): 2914 - 2924. [Abstract] [Full Text] [PDF]

				A. S. Clarke, E. Samal, and L. Pillus Distinct Roles for the Essential MYST Family HAT Esa1p in Transcriptional Silencing Mol. Biol. Cell, April 1, 2006; 17(4): 1744 - 1757. [Abstract] [Full Text] [PDF]

				R. G. Gardner, Z. W. Nelson, and D. E. Gottschling Ubp10/Dot4p Regulates the Persistence of Ubiquitinated Histone H2B: Distinct Roles in Telomeric Silencing and General Chromatin Mol. Cell. Biol., July 15, 2005; 25(14): 6123 - 6139. [Abstract] [Full Text] [PDF]

				A. H. Michel, B. Kornmann, K. Dubrana, and D. Shore Spontaneous rDNA copy number variation modulates Sir2 levels and epigenetic gene silencing Genes & Dev., May 15, 2005; 19(10): 1199 - 1210. [Abstract] [Full Text] [PDF]

				S. E. Porter, K. L. Penheiter, and J. A. Jaehning Separation of the Saccharomyces cerevisiae Paf1 Complex from RNA Polymerase II Results in Changes in Its Subnuclear Localization Eukaryot. Cell, January 1, 2005; 4(1): 209 - 220. [Abstract] [Full Text] [PDF]

				J. C. Tanny, D. S. Kirkpatrick, S. A. Gerber, S. P. Gygi, and D. Moazed Budding Yeast Silencing Complexes and Regulation of Sir2 Activity by Protein-Protein Interactions Mol. Cell. Biol., August 15, 2004; 24(16): 6931 - 6946. [Abstract] [Full Text] [PDF]

				A. Dasgupta, K. L. Ramsey, J. S. Smith, and D. T. Auble Sir Antagonist 1 (San1) Is a Ubiquitin Ligase J. Biol. Chem., June 25, 2004; 279(26): 26830 - 26838. [Abstract] [Full Text] [PDF]

				N. S. Bae, M. J. Swanson, A. Vassilev, and B. H. Howard Human Histone Deacetylase SIRT2 Interacts with the Homeobox Transcription Factor HOXA10 J. Biochem., June 1, 2004; 135(6): 695 - 700. [Abstract] [Full Text] [PDF]

				I. Orlandi, M. Bettiga, L. Alberghina, and M. Vai Transcriptional Profiling of ubp10 Null Mutant Reveals Altered Subtelomeric Gene Expression and Insurgence of Oxidative Stress Response J. Biol. Chem., February 20, 2004; 279(8): 6414 - 6425. [Abstract] [Full Text] [PDF]

				A. A. Falcon and J. P. Aris Plasmid Accumulation Reduces Life Span in Saccharomyces cerevisiae J. Biol. Chem., October 24, 2003; 278(43): 41607 - 41617. [Abstract] [Full Text] [PDF]

				K. J. Bitterman, O. Medvedik, and D. A. Sinclair Longevity Regulation in Saccharomyces cerevisiae: Linking Metabolism, Genome Stability, and Heterochromatin Microbiol. Mol. Biol. Rev., September 1, 2003; 67(3): 376 - 399. [Abstract] [Full Text] [PDF]

				A. Benguria, P. Hernandez, D. B. Krimer, and J. B. Schvartzman Sir2p suppresses recombination of replication forks stalled at the replication fork barrier of ribosomal DNA in Saccharomyces cerevisiae Nucleic Acids Res., February 1, 2003; 31(3): 893 - 898. [Abstract] [Full Text] [PDF]

				C.D. Smith, D.L. Smith, J.L. DeRisi, and E.H. Blackburn Telomeric Protein Distributions and Remodeling Through the Cell Cycle in Saccharomyces cerevisiae Mol. Biol. Cell, February 1, 2003; 14(2): 556 - 570. [Abstract] [Full Text] [PDF]

				S. N. Garcia and L. Pillus A Unique Class of Conditional sir2 Mutants Displays Distinct Silencing Defects in Saccharomyces cerevisiae Genetics, October 1, 2002; 162(2): 721 - 736. [Abstract] [Full Text] [PDF]

				D. Kasulke, S. Seitz, and A. E. Ehrenhofer-Murray A Role for the Saccharomyces cerevisiae RENT Complex Protein Net1 in HMR Silencing Genetics, August 1, 2002; 161(4): 1411 - 1423. [Abstract] [Full Text] [PDF]

				Y. Huang Transcriptional silencing in Saccharomyces cerevisiae and Schizosaccharomyces pombe Nucleic Acids Res., April 1, 2002; 30(7): 1465 - 1482. [Abstract] [Full Text] [PDF]

				J. J. Sandmeier, I. Celic, J. D. Boeke, and J. S. Smith Telomeric and rDNA Silencing in Saccharomyces cerevisiae Are Dependent on a Nuclear NAD+ Salvage Pathway Genetics, March 1, 2002; 160(3): 877 - 889. [Abstract] [Full Text] [PDF]

				S. U. Åström, A. Kegel, J. O. O. Sjöstrand, and J. Rine Kluyveromyces lactis Sir2p Regulates Cation Sensitivity and Maintains a Specialized Chromatin Structure at the Cryptic {alpha}-Locus Genetics, September 1, 2000; 156(1): 81 - 91. [Abstract] [Full Text]

				J. S. Smith, C. B. Brachmann, I. Celic, M. A. Kenna, S. Muhammad, V. J. Starai, J. L. Avalos, J. C. Escalante-Semerena, C. Grubmeyer, C. Wolberger, et al. A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family PNAS, June 6, 2000; 97(12): 6658 - 6663. [Abstract] [Full Text] [PDF]

				S. Enomoto, S. D. Johnston, and J. Berman Identification of a Novel Allele of SIR3 Defective in the Maintenance, but Not the Establishment, of Silencing in Saccharomyces cerevisiae Genetics, June 1, 2000; 155(2): 523 - 538. [Abstract] [Full Text]

				M. M. Cockell, S. Perrod, and S. M. Gasser Analysis of Sir2p Domains Required for rDNA and Telomeric Silencing in Saccharomyces cerevisiae Genetics, March 1, 2000; 154(3): 1069 - 1083. [Abstract] [Full Text]

				T.-H. Cheng and M. R. Gartenberg Yeast heterochromatin is a dynamic structure that requires silencers continuously Genes & Dev., February 15, 2000; 14(4): 452 - 463. [Abstract] [Full Text]

				A. Kahana and D. E. Gottschling DOT4 Links Silencing and Cell Growth in Saccharomyces cerevisiae Mol. Cell. Biol., October 1, 1999; 19(10): 6608 - 6620. [Abstract] [Full Text] [PDF]

				M. Kaeberlein, M. McVey, and L. Guarente The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms Genes & Dev., October 1, 1999; 13(19): 2570 - 2580. [Abstract] [Full Text]

				S. Kim, A. Benguria, C.-Y. Lai, and S. M. Jazwinski Modulation of Life-span by Histone Deacetylase Genes in Saccharomyces cerevisiae Mol. Biol. Cell, October 1, 1999; 10(10): 3125 - 3136. [Abstract] [Full Text]

				E. Y. Xu, S. Kim, and D. H. Rivier SAS4 and SAS5 Are Locus-Specific Regulators of Silencing in Saccharomyces cerevisiae Genetics, September 1, 1999; 153(1): 25 - 33. [Abstract] [Full Text]

				J. M. Sherman, E. M. Stone, L. L. Freeman-Cook, C. B. Brachmann, J. D. Boeke, and L. Pillus The Conserved Core of a Human SIR2 Homologue Functions in Yeast Silencing Mol. Biol. Cell, September 1, 1999; 10(9): 3045 - 3059. [Abstract] [Full Text]

				Z.-W. Sun and M. Hampsey A General Requirement for the Sin3-Rpd3 Histone Deacetylase Complex in Regulating Silencing in Saccharomyces cerevisiae Genetics, July 1, 1999; 152(3): 921 - 932. [Abstract] [Full Text]

				J. S. Smith, E. Caputo, and J. D. Boeke A Genetic Screen for Ribosomal DNA Silencing Defects Identifies Multiple DNA Replication and Chromatin-Modulating Factors Mol. Cell. Biol., April 1, 1999; 19(4): 3184 - 3197. [Abstract] [Full Text] [PDF]

				T. M. Sandrock, S. M. Brower, K. A. Toenjes, and A. E. M. Adams Suppressor Analysis of Fimbrin (Sac6p) Overexpression in Yeast Genetics, April 1, 1999; 151(4): 1287 - 1297. [Abstract] [Full Text]

				A. Ansari and M. R. Gartenberg Persistence of an alternate chromatin structure at silenced loci in vitro PNAS, January 19, 1999; 96(2): 343 - 348. [Abstract] [Full Text] [PDF]

				M. COCKELL, M. GOTTA, F. PALLADINO, S.G. MARTIN, and S.M. GASSER Targeting Sir Proteins to Sites of Action: A General Mechanism for Regulated Repression Cold Spring Harb Symp Quant Biol, January 1, 1998; 63(0): 401 - 412. [Abstract] [PDF]