distribution of a limited sir2 protein pool regulates the strength of

Copyright 1998 by the Genetics Society of America

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

Jeffrey S. Smith,* Carrie Baker Brachmann,* Lorraine Pillus† and Jef D. Boeke**Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and

†Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347

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

ABSTRACTTranscriptional 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 novelmechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential forother silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanismfor rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2protein 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 mutationswere shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediatedthrough SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reducedby overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effectof SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibitsthe rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promoteextended life span also resulted in enhanced rDNA silencing. We propose a simple model in whichtelomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein.

CLASSICAL transcriptional silencing in the bud- late in S phase, location near the nuclear envelope,and physical compaction, have led to proposals thatding yeast Saccharomyces cerevisiae was originallythe silent mating-type loci and telomeres in yeast areidentified at the silent mating-type loci HML and HMR,functionally equivalent to heterochromatin (Thompsonwhich are located on chromosome III (for review seeet al. 1994; Braunstein et al. 1996).Loo and Rine 1995). A related form of silencing also

A novel form of silencing was described recently inoccurs at telomeres and is known as telomere positionthe ribosomal DNA (rDNA) of S. cerevisiae (Bryk et al.effect (TPE; Gottschling et al. 1990). Foreign genes1997; Smith and Boeke 1997). The rDNA consists ofinserted in these regions are repressed in a reversible100–200 copies of a 9.1-kb unit organized into a tandemmanner that resembles the position effect variegationarray on chromosome XII (Petes and Botstein 1977;observed in the heterochromatin of more complex eu-Philippsen et al. 1978). Each 9.1-kb unit contains thekaryotes (Pillus and Rine 1989; Gottschling et al.gene for 35S ribosomal RNA precursor, transcribed by1990; Weiler and Wakimoto 1995; Dobie et al. 1997).RNA Pol I, and the gene for 5S rRNA, transcribed byBoth of these chromosomal regions display a generalRNA Pol III (Figure 1). These two transcription unitsinaccessibility to DNA-modifying enzymes in vitro andare separated by a nontranscribed spacer (NTS) thatin vivo (Strathern et al. 1982; Kostriken et al. 1983;contains an origin of DNA replication (Szostak andGottschling 1992; Singh and Klar 1992; Loo andWu 1979; Skryabin et al. 1984). We originally demon-Rine 1994). Genes transcribed by either RNA polymer-strated that the expression of several different Pol II–ase II or III (Pol II or Pol III) are generally repressedtranscribed marker genes integrated into various re-(Schnell and Rine 1986; Gottschling et al. 1990;gions of the rDNA was repressed (Smith and BoekeHuang et al. 1997), although at telomeres, the degree1997). Of the S ilent Information Regulator (SIR) genes,of silencing depends on the intrinsic features of eachrDNA silencing depends only on SIR2 (Smith andindividual promoter (Renauld et al. 1993). These prop-Boeke 1997). In contrast, all four SIR genes contributeerties, combined with similarities to classically definedto silencing the HM loci, whereas TPE only requireseukaryotic heterochromatin, such as DNA replicationSIR2, 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 alsoCorresponding author: Jef D. Boeke, Dept. of Molecular Biology and depend on other factors, including Rap1p (reviewed in

Genetics, Johns Hopkins University School of Medicine, 725 N. WolfeShore 1994), as well as histones H3 and H4 (reviewedStreet, 617 Hunterian Building, Baltimore, MD 21205.

E-mail: [email protected] in Loo and Rine 1995). The role of non-SIR silencing

Genetics 149: 1205–1219 ( July 1998)

1206 J. S. Smith et al.

factors and cis -acting elements in mediating rDNA si- brane (Palladino et al. 1993; Cockell et al. 1995;Gotta et al. 1996). Competition for these limitinglencing is just beginning to be investigated. The UBC2

(RAD6) gene has been recently implicated in both TPE amounts of silencing factors has been shown to occurbetween telomeres and the HM loci. The rap1s mutation,(Huang et al. 1997) and rDNA silencing (Bryk et al.

1997). Topoisomerase I (TOP1), not previously impli- rap1-12, simultaneously reduces HMR-specific silencingand enhances telomeric silencing (Buck and Shorecated in silencing, is also required for silencing Ty1

elements in rDNA, as well as for suppression of mitotic 1995). This shift in the balance of silencing to telomereswas proposed to result from sequestration of Sir4p atrDNA recombination (Christman et al. 1988; Bryk et

al. 1997). telomeres, thus reducing the amount of Sir4p availablefor HMR silencing (Buck and Shore 1995; MarcandWe previously demonstrated that deletion of SIR2 in-

creases the accessibility of rDNA to psoralen cross-link- et al. 1996).We demonstrate in this article that SIR4 regulatesing in vivo (Smith and Boeke 1997), suggesting that

SIR2 is required for the formation or maintenance of rDNA silencing strength by controlling the level ofSir2p that is available to the rDNA. We show that rDNAa specialized rDNA chromatin structure that is repres-

sive to RNA Pol II. Using in vivo formaldehyde cross- silencing is highly sensitive to even small changes inSir2 levels, and that there are normally limiting amountslinking, Sir2p has been found to be associated with

rDNA chromatin, mostly within the NTS region (Gotta of Sir2p available for rDNA silencing. We also show thatSIR4 negatively affects rDNA silencing. SIR4 overexpres-et al. 1997). In addition, loss of SIR2 increases the fre-

quency of mitotic and meiotic recombination within the sion severely reduced the strength of rDNA silencing,and sir4D strengthened rDNA silencing through a SIR2-rDNA (Gottlieb and Esposito 1989). Taken together,

these findings indicate that SIR2 plays a major role in dependent, SIR3 -independent mechanism. Finally, ma-nipulations of SIR4, which were previously shown torDNA function, and they are consistent with the nucleo-

lar localization of Sir2 (Gotta et al. 1997). redistribute Sir proteins (including Sir2p) to the nucleo-lus and promote longevity, also cause an increase inAlthough SIR4 is not required for rDNA silencing, its

deletion paradoxically enhances the strength of rDNA rDNA silencing strength. We propose a Sir2p competi-tion model in which Sir4p regulates the shuttling ofsilencing (Smith and Boeke 1997), suggesting that SIR4

may regulate such silencing. TPE and HM silencing Sir2p between competing rDNA and telomeric compart-ments.regulation can occur at the level of Sir protein dosage.

For example, increased SIR3 gene dosage enhances thestrength of TPE, resulting in the spreading of repressive

MATERIALS AND METHODStelomeric chromatin toward the centromere (Renauld

et al. 1993). Sir3p is physically associated with this ex- Media and plasmids: Unless stated otherwise, media wereused as described previously (Rose et al. 1990; Smith andtended telomeric chromatin (Hecht et al. 1996). An-Boeke 1997). Ty1 transposition induction medium (YNB/other example is that diploid strains containing onecasamino acids 1 Trp galactose) consisted of yeast nutrient

copy of SIR4 display unstable repression at HMR (Sussel broth (YNB; Difco, Detroit) supplemented with 2% casaminoet al. 1993), and overexpression of either SIR4 or its acids, 2% galactose, 160 mm adenine, and 800 mm tryptophan.

SC (FOA) medium contained 5-FOA (PCR Incorporated) atC-terminal fragment greatly reduces silencing at both2 mg/ml and glucose at 2%. Pb21-containing medium (modi-HM loci and telomeres (known as the “anti-SIR” effect;fied lead acetate, MLA) consisted of 0.3% peptone, 0.5% yeast

Marshall et al. 1987; Renauld et al. 1993; Cockell etextract, 4% glucose, 0.02% (w/v) ammonium acetate, 0.1%

al. 1995). Sir1p levels are limiting for establishment of Pb(NO3)2, and 2% agar.HM silencing (Stone et al. 1991). So far, there have The mURA3-marked GAL-Ty1 overexpression plasmid

pJSS36-6 and the CEN, SIR2 plasmid pCAR237 were describedbeen no reports that SIR2 dosage affects TPE (Renauld

previously (Smith and Boeke 1997). pLP304 (2m, LEU2 SIR3)et al. 1993) or HM silencing, although high-copy SIR2and pLP305 (2m, LEU2 SIR4) were also described previouslyexpression does enhance rDNA silencing in the rDNA(Stone and Pillus 1996). pLP347 consists of a HST1 SacI

(Fritze et al. 1997). SIR2 overexpression leads to a de- fragment ligated into the SacI site of YEp351. pSIR2m wascrease in histone acetylation at the HM loci (Braun- constructed by inserting the HindIII genomic SIR2 fragment

from pCAR237 into the HindIII site of pRS425 (Christiansonstein et al. 1993), and high levels of SIR2 overexpressionet al. 1992). pJSS71-13 (2m, HIS3, SIR2) was constructed byare toxic to cells, provoking chromosome instabilityligating a XhoI-NotI fragment from pCAR237, which contains(Holmes et al. 1997).SIR2, into the XhoI-NotI sites of pRS423. pLP754 (2m LEU2

It has been suggested that the local concentration of Sir SIR4-42) consists of a NotI-SalI SIR4-42 fragment that wasproteins is critical in determining silencing efficiency, even blunted by Klenow treatment and then ligated into the SmaI

site of YEp351 (Hill et al. 1986). pLP793 (integrating TRP1at internal chromosomal locations (Stavenhagen andsir4-42) was constructed by ligating the SacI-Sal I sir4-42 frag-Zakian 1994; Lustig et al. 1996; Maillet et al. 1996;ment from pLP754 into the SacI-Sal I sites of pRS304. pJH3A

Marcand et al. 1996). This hypothesis is supported byand pJH5.1A (provided by James Broach) were previously

other studies showing that telomeres, the Sir proteins, described (Ivy et al. 1986; Marshall et al. 1987). pJSS73-5and Rap1p colocalize into a limited number of discrete was constructed by ligating an EcoRI-SacII fragment containing

the GAL1 promoter and a C-terminal fragment of SIR4 fromintranuclear foci, predominantly near the nuclear mem-

1207Regulation of rDNA Silencing

TABLE 1

Yeast strains

Strain Genotype

JB740a MATa his3D200 leu2D1 ura3-167JS50-1 MATa/MATa his3D200/his3D200 leu2D1/leu2D1 ura3-167/ura3-167 pJSS36-6JS249 MATa/MATa his3D200/his3D200 leu2D1/leu2D1 ura3-167/ura3-167JS101 MATa/MATa his3D200/his3D200 leu2D1/leu2D1 ura3-167/ura3-167 RDN1/RDN1::Ty1-mURA3JS106 JS101 sir2D::HIS3/SIR2JS108 JS101 sir2D::HIS3/sir2D::LEU2JS109b MATa/MATa his3D200/his3D200 leu2D1/leu2D1 ura3-167/ura3-167 ???/???::Ty1-mURA3JS124 (S2) JB740 RDN1::Ty1-mURA3JS167 JS124 pRS415JS168 JS124 pCAR237JS266 JS124 pRS425JS267 JS124 pLP304JS362 JS124 pLP347JS128 (S6) JB740 RDN1::Ty1-mURA3JS277 JS128 pLP305JS358 JS128 pRS423 pRS425JS359 JS128 pJSS71-13 pRS425JS360 JS128 pRS423 pLP305JS361 JS128 pJSS71-13 pLP305JS366 JS128 pJSS72-1JS367 JS128 pJSS73-5JS375 JS128 pLP754JS384 JS128 YEp13JS385 JS128 pJH3AJS386 JS128 pJH5.1AJS323 (XJS3) MATa/MATa his3D200/his3D200 leu2D1/leu2D1 met15D0/met15D0 trp1D63/trp1D63

ura3-167/ura3-167 RDN1/RDN1::Ty1/MET15 SIR3/sir3D::kanMX4 SIR4/sir4D::HIS3JS333c MATa his3D200 leu2D1 met15D0 trp1D63 ura3-167 RDN1::Ty1-MET15JS335c MATa his3D200 leu2D1 met15D0 trp1D63 ura3-167 RDN1::Ty1-MET15 sir3D::kanMX4JS337c MATa his3D200 leu2D1 met15D0 trp1D63 ura3-167 RDN1::Ty1-MET15 sir4D::HIS3JS339c MATa his3D200 leu2D1 met15D0 trp1D63 ura3-167 RDN1::Ty1-MET15 sir3D::kanMX4

sir4D::HIS3JS347 JS337 trp1D63::pLP793JS341 (XJS5) MATa/MATa his3D200/his3D200 leu2D1/leu2D1 MET15/met15D0 trp1D63/trp1D63

ura3-167/ura3-167 RDN1/RDN1::Ty1-MET15 SIR2/sir2D::kanMX4 SIR4/sir4D::HIS3JS343d MATahis3D200 leu2D1 met15D0 trp1D63 ura3-167 RDN1::Ty1-MET15 sir2D::kanMX4JS344d MATahis3D200 leu2D1 met15D0 trp1D63 ura3-167 RDN1::Ty1-MET15 sir2D::kanMX4 sir4D::HIS3

a Derived from GRF167 (Boeke et al. 1985; Smith and Boeke 1997).b Ty1mURA3 insertion at an unknown non-rDNA locus.c Spores dissected from JS323.d Spores dissected from JS341.

pRO135 into the same sites of pRS313. pRO135 was a plasmid complete ORF deletions and were performed by PCR-mediated gene disruption as described elsewhere (Baudin etderived from a galactose-inducible overexpression library (Liu

et al. 1992) and was provided by Rohinton Kamakaka. The al. 1993; Lorenz et al. 1995). The HIS3, LEU2, or kanMX4 genes were PCR amplified from pRS403, pRS405, orkanMX4 gene of pRS400 (Brachmann et al. 1998) was derived

from pFA6a-kanMX4, provided by Achim Wach and Peter pRS400, respectively (Sikorski and Heiter 1989; Brach-

mann et al. 1998), using oligodeoxynucleotide primers con-Philippsen (Wach et al. 1994).Yeast strains: All strains used in this study are derived from taining 59 flanking sequences complementary to the SIR genes

(Smith and Boeke 1997). One SIR2 allele in the MATa/aJB740 or its derivatives (Table 1; Smith and Boeke 1997).JS50-1 is a MATa/a diploid isolated during transformation diploid JS101 was replaced with HIS3 (sir2D::HIS3) to produce

heterozygote JS106, which can mate. The second SIR2 alleleof JB740 with the galactose-inducible Ty1-mURA3 plasmidpJSS36-6 (Smith and Boeke 1997), presumably via polyethyl- of this heterozygote was replaced with LEU2 (sir2D::LEU2) to

produce a homozygous SIR2 knockout that was a nonmater.ene glycol (PEG)-mediated fusion ( J. S. Smith and J. D.

Boeke, unpublished data). JS249 was derived from JS50-1 by sir3D::kanMX4 was similarly introduced into JS308 (MATa)producing JS316. The MATa sir4D::HIS3 deletion strain JS219loss of pJSS36-6. To produce JS101 and JS109, transposition

was induced in JS50-1 to produce Ty1-mURA3 integration has been described previously (Smith and Boeke 1997). JS316was transformed with pLP304 (2m LEU2 SIR3), JS219 was trans-events into the rDNA or non-rDNA locations, as described

previously (Smith and Boeke 1997). SIR gene deletions are formed with pLP305 (2m LEU2 SIR4), and the resulting trans-


formants were crossed. After loss of the LEU2 plasmids, tetrads prehybridized in 10 ml of 53 SSPE, 53 Denhardt’s solution,1% SDS, 50% formamide (w/v), and 100 mg/ml boiled her-of the resulting diploid XJS3 were dissected to generate con-

genic SIR1( JS333), sir4D ( JS337), sir3D ( JS335), and sir3D ring sperm DNA at 608 for 3 h, followed by hybridization inthe above solution without DNA at 608 for 16 h. The filtersir4D ( JS339) spores. Diploid XJS5 was similarly sporulated,

and tetrads were dissected to generate the congenic sir2D and was washed twice at room temperature 23 SSC/0.1% SDS,two times at room temperature in 0.23 SSC/0.1% SDS, andsir2D sir4D spores. To produce a sir4-42 strain, pLP793 was

linearized by SacII cleavage within TRP1 and then integrated twice at 428 in 0.23 SSC/0.1% SDS. The filter was strippedand reprobed with a 1.1-kb BamHI-HindIII fragment of actin,into the trp1D63 locus of the sir4D strain JS337, resulting in

strain JS347. which was derived from pD10-AHX3 (Chapman and Boeke

1991), as a loading control. The filter was imaged on a Phos-Tetrad dissection: Diploid strains XJS3 and XJS5 weregrown as patches on YPD medium for z20 hr. Cells from phorImager (Molecular Dynamics, Sunnyvale, CA) and quan-

titated using ImageQuant software.these patches were transferred to 2 ml of liquid sporulationmedium (1% potassium acetate, 0.002% zinc acetate) andincubated 1 day at 258, followed by 4 days at 308. Tetrads weredissected on a YPD plate and incubated for 3 days at 308 to RESULTSallow spores to grow up as colonies. Dissection plates were

Ribosomal DNA silencing is highly sensitive toreplica plated to SC-His, YPD1G418 (200 mg/ml), and MLAto determine the SIR genotype and rDNA silencing strength. changes in SIR2 dosage: The silencing reporter strains

Sir2p-specific antibody: A Sir2p-specific antiserum was used in this study all carry mURA3- or MET15-markedraised using a synthetic peptide (CGVYVVTSDEHPKTL) com- Ty1 elements integrated within the NTS of rDNA (Fig-prising the 14 C-terminal amino acids plus a nonencoded

ure 1). Both of these markers are partially silenced whencysteine residue. The cysteine residue was added to facilitateintegrated within the rDNA tandem array, but are fullym-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce, Rock-

ford, IL) conjugation of the peptide to carrier keyhole limpet expressed when located outside the rDNA or within ahemocyanin. Rabbits were immunized, and serum was col- single rDNA gene copy that is artificially positionedlected using standard protocols (Harlow and Lane 1988). outside the array (Smith and Boeke 1997). mURA3Serum was diluted and used without further purification, as

expression is measured by growth on medium lack-described below.ing uracil, whereas MET15 expression is measured byProtein extraction and immunoblotting: Strains were growngrowth on medium lacking methionine or by colonyto 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 z1.0. color on Pb21 containing medium (Cost and Boeke

Pelleted cells were resuspended in 100 ml lysis buffer (50 mm 1996; Smith and Boeke 1997).Tris-HCl, pH 7.5, 1% SDS, 5 mm EDTA, 14.3 mm 2-mer- We previously demonstrated that silencing of markercaptoethanol, 2 mm PMSF, 1 mg/ml leupeptin, 2 mg/ml anti-

genes in the rDNA is completely dependent on thepain, 10 mg/ml benzamidine, 1 mg/ml chymostatin, 1 mg/mlSIR2 gene. Furthermore, deletion of SIR2 increased thepepstatin A, and 10 U/ml aprotinin). Glass beads (0.45–0.5

mm diameter) were added to the meniscus, and the tubes accessibility of rDNA to psoralen cross-linking (Smith

were vortexed at full speed for five 30-sec pulses and placed and Boeke 1997). These results are consistent with aon ice in between pulses. Another 100 ml of lysis buffer was role for SIR2 in the formation or maintenance of aadded, followed by boiling for 3 min and centrifugation

specialized rDNA chromatin structure. Because the(14,000 3 g for 20 min at 48). An equal volume of 23 loadingrDNA tandem array makes up 5–10% of yeast chroma-buffer was added to the supernatant and then boiled again

before loading. tin, there are likely to be limiting amounts of Sir2pSome 300 A280 units of each extract were separated on a available to act on the rDNA. To test this hypothesis,

10% SDS polyacrylamide gel and then electroblotted to an we first constructed the diploid reporter strain JS101Immobilon-P filter (Millipore, Bedford, MA) at 300 mA for

(D39), which contains a single Ty1-mURA3 element inte-1 hr using a mini-Protean II apparatus (Bio-Rad, Richmond,grated at the rDNA (Figure 1; see materials and meth-CA). Filters were blocked for 45 min with 10 ml of blockingods). SIR2 was then deleted one allele at a time, produc-solution (13 PBS/0.05% Tween 20/5% milk), then incubated

with rabbit polyclonal a-Sir2 antibody 2916 (1:5000 dilution ing heterozygous and homozygous sir2 deletion strains.in blocking solution) or control mouse monoclonal a-tubulin The strength of rDNA silencing of the mURA3 reporter,antibody B-5-1-2, 6.1 mg/ml (1:5000 dilution; Sigma, St. Louis, as measured by growth on -Ura medium, was testedMO) for 1 hr at room temperature. The appropriate secondary

using a serial dilution growth assay for each of theantibody (anti-rabbit HRP conjugated or anti-mouse HRP con-resulting strains. Interestingly, when a single copy ofjugated; Amersham Corp., Arlington Heights, IL) was used at

a 1:10,000 dilution for 1 hr in blocking solution. Filters were SIR2 was deleted (JS106), growth on SC-Ura plates in-developed on Kodak XAR5 film using the ECL detection sys- creased approximately fivefold over that of the SIR2/tem (Amersham). SIR2 (1/1) parent (Figure 2A). At the same time, itsRNA blot analysis: Saturated YPD cultures were diluted to

sensitivity to FOA increased 5- to 25-fold. This resultan A600 of 0.2 in YPD and grown at 308 to midlog phase (A600indicates that a single gene dose of SIR2 is insufficientof 1.4–1.6). Forty micrograms total RNA, isolated as described

by Chapman and Boeke (1991), was separated on a formalde- to fully silence mURA3 within a diploid cell. When thehyde-containing 1.2% agarose gel and transferred to Gene- second SIR2 allele was deleted from the heterozygote,screen Plus (NEN-Dupont, Boston, MA) in 103 SSC. The creating a homozygous knockout strain (JS108), growthfilter was probed with an antisense URA3 RNA probe labeled

on SC-Ura increased another fivefold to the level ofwith [a-32P]UTP (800 Ci/mmol). The URA3 RNA probe waspositive control non-rDNA insertion JS109 (Figure 2A).transcribed by T7 RNA polymerase from pBS-URA3 linearized

with StuI, producing a 363-nt antisense probe. The filter was This latter result is identical to that previously observed


Figure 1.—Schematic rep-resentation of the Saccharo-myces cerevisiae rDNA arraystructure and Ty1 silencingreporters. The general or-ganization of a single 9.1-kbrDNA gene copy within thetandem array is depicted.The initiation sites for 35SrRNA (Pol I) and 5S rRNA(Pol III) transcription areshown as bent arrows. Threeautonomously replicatingsequence (ARS) near-con-sensus elements of the ori-gin of DNA replication inNTS2 are shown as filled cir-cles. Integration sites andorientations of Ty1-mURA3and Ty1-MET15 insertionsinto the rDNA are shown asvertical arrows. Downward-pointing arrows representTy insertions in the sametranscriptional orientationas the 5S rRNA gene (rightto left). Upward pointingarrows represent Ty inser-tions in the opposite orien-tation. Filled triangles rep-resent Ty1 LTRs. The strainnumber for each reporterstrain is shown.

when SIR2 was deleted from a haploid strain (Bryk et scribed from a locus outside the rDNA than within therDNA, even in the sir2/sir2 homozygote (JS108). Weal. 1997; Smith and Boeke 1997). The FOA-resistant

colonies that grow in the double knockout are com- attribute this large difference to the sensitivity of theTy1 promoter to chromosomal position effects (Curciopletely Ura2 and represent mitotic loss of mURA3 (data

not shown) because of an increased rate of rDNA recom- and Garfinkel 1991). In summary, by both a growthassay and by directly examining transcription, the SIR2/bination events leading to complete loss of the mURA3-

marked repeat (Gottlieb and Esposito 1989). The sir2 heterozygote is phenotypically intermediate be-tween the sir2/sir2 and SIR2/SIR2 homozygotes.SIR2 deletions did not affect overall growth on nonselec-

tive media. The steady-state mURA3 RNA level in these To confirm that Sir2 protein levels had actuallychanged in these cells, the relative amounts of Sir2pstrains was determined by quantitative RNA blot analysis

using a URA3-specific riboprobe and is shown schemati- were analyzed by immunoblotting using an a-Sir2 anti-body (Figure 2D); the steady-state Sir2p levels corre-cally in Figure 2B. The relative mURA3 RNA levels corre-

sponded to the relative amount of Ura1 growth for sponded to SIR2 gene dosage. These results indicatethat silencing of a single Ty1-mURA3 element insertedeach strain, indicating that growth assays are a reliable

method for determining silencing efficiency in the rDNA. at the rDNA of a diploid cell is exquisitely sensitive toSir2p levels. Whereas rDNA silencing was highly sensi-For independent confirmation of the intermediate

SIR2 dosage effect, Ty1-mURA3 expression driven from tive to SIR2 dosage, HMRa silencing, as measured by asensitive mating assay, was not affected by the twofoldthe Ty1 promoter was quantitated from the same URA3-

probed filter (Figure 2C). Again, the level of the Ty1- difference in SIR2 dosage (data not shown).Increased dosage of SIR3 was previously shown tomURA3 6.7-kb message directly correlated with the dos-

age of SIR2 (Figure 2C), indicating that the dosage enhance telomeric silencing, but increased SIR2 dos-age had no effect (Renauld et al. 1993). To deter-effect was not specific to the mURA3 promoter, as mea-

sured in Figure 2, A and B, but it also influenced the mine whether increased SIR2 or SIR3 dosage couldstrengthen the rDNA silencing observed in a haploidTy1 promoter. The level of the mURA3 message in a

sir2 homozygote was equivalent to that in the non-rDNA isolate, CEN (low-copy) and 2m (high-copy) plasmidscontaining SIR2 were transformed into the SIR2 haploidcontrol JS109 (Figure 2B), but the level of the larger

Ty1-mURA3 message was significantly greater when tran- Ty1-mURA3 isolate JS124 (S2). This haploid silencing


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, 1/1), heterozygous sir2 deletion (JS106, 1/2), and homozygoussir2 deletion (JS108, 2/2) strains were patched onto a YPD plate and grown for 48 hr at 308, resuspended in water, and platedin fivefold dilutions onto SC-Ura, which selects for mURA3 expression, SC1Foa, which selects against mURA3 expression, andSC1Ura to monitor growth. The negative control for Ura1 growth is JS249, and the positive control for Ura1 growth of a non-rDNA Ty1-mURA3 insertion is JS109. SC1Ura and SC1Foa 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 z1.2-kb RNA produced fromthe mURA3 promoter was detected by Northern analysis using an antisense URA3 RNA probe. The mean absolute expressionlevels of mURA3, as measured by PhosphorImager analysis and normalized against actin RNA for loading, are plotted on a bargraph in arbitrary units. The background level is represented by a dotted horizontal line. Standard deviations are indicated byvertical error bars (n 5 3). (C) Quantitation of steady-state Ty1-mURA3 RNA levels. The z6.7-kb Ty1-mURA3 RNA producedfrom the Ty1 promoter was quantitated from the same blot as was used for B. (D) Immunoblot analysis of steady-state cellularSir2p levels in sir2 deletion strains. Strains are as described in A. Lane 1, JS101. Lane 2, JS106. Lane 3, JS108. The SIR2 allelesare represented by a 1, and sir2D alleles are represented by a 2. The filter was first incubated with a-Sir2 antibody and developedwith ECL (top). The same filter was then reincubated with a-tubulin antibody and developed a second time with ECL (bottom).

reporter strain contains a single Ty1-mURA3 insertion et al. 1995; Derbyshire et al. 1996) had no effect onrDNA silencing strength. In addition, high-copy SIR3(within the NTS2) upstream of the 5S rRNA gene (Fig-

ure 1; Smith and Boeke 1997). The level of silencing did not effect the enhanced silencing caused by high-copy SIR2 when the two genes were coexpressed in theis shown for each strain in Figure 3A; CEN SIR2 caused

a small (less than fivefold) increase in repression com- same strain (data not shown). Another SIR2 homolog,HST3, also had no effect on rDNA silencing when over-pared to the empty CEN vector. Furthermore, the 2m

SIR2 plasmid caused between a 5- and 25-fold increase expressed from the GAL1 promoter (data not shown).Unexpectedly, the level of Ura1 growth was modestlyin repression compared to the empty 2m vector. These

results indicate that there are limiting amounts of cellu- greater for the CEN vector set than for the 2m vectorset, including the empty vectors. The reason for thislar Sir2p for rDNA silencing, and that silencing can be

enhanced beyond wild-type levels by providing addi- difference is currently unclear, but could be caused bythe known effects of CEN plasmids on the cell cycletional Sir2p. In contrast, 2m plasmids containing SIR3

or the closely related SIR2 homolog HST1 (Brachmann (Spencer and Hieter 1992).


Figure 3.—Increased SIR2 enhances si-lencing strength. (A) Haploid rDNA Ty-mURA3 insertion strain JS124 (S2), whichis SIR2, was transformed with CEN plasmidspRS415 (vector) or pCAR237 (SIR2). It wasalso transformed with the 2m plasmidspRS425 (vector), pSIR2m (SIR2), pLP347(HST1), and pLP304 (SIR3). The strainswere patched onto a SC-Leu glucose plateand incubated for 48 hr. Cells were resus-pended in water andplated as fivefold serialdilutions on SC-Ura-Leu and SC-Leu andincubated for 4 days to determine theirUra1 growth phenotypes. The SC-Leu platewas incubated for 2 days. (B) Immunoblotanalysis of steady-state cellular Sir2p levelsin strains containing the following plas-mids: empty CEN vector (pRS415, lane 1),CEN SIR2 vector (pCAR237, lane 2), empty2m vector (pRS425, lane 3), and 2m SIR2vector (pSIR2m, lane 4). The filter was firstincubated with Sir2 antibody and devel-oped with ECL (top). The same filter wasthen reincubated with a-tubulin antibodyand developed a second time with ECL(bottom).

The relative steady-state amounts of Sir2p produced bulk of Sir2p is nucleolar (Gotta et al. 1997). WhenSIR4 is deleted, all detectable Sir2p and Sir3p redistrib-by CEN SIR2 or 2m SIR2 plasmids in strain S2 are shown

in Figure 3B. The modest increase (approximately two- utes to the nucleolus (Gotta et al. 1997). The redistribu-tion of Sir3p is especially dramatic as it is undetectable infold) in Sir2p caused by CEN SIR2 that is associated with

enhanced silencing further emphasizes the extreme sen- wild-type nucleoli (Gotta et al. 1997). Another possibleexplanation for the enhanced rDNA silencing in a sir4Dsitivity of rDNA silencing to SIR2 dosage. 2m SIR2

boosted Sir2p protein levels approximately twofold strain could therefore be the nucleolar redistribution ofSir3p, Sir2p, or both. To test these possibilities, congenicmore than CEN SIR2, concomitant with a greater in-

crease in rDNA silencing strength. SIR2 overexpression SIR, sir2, sir3, sir4, sir2 sir4, and sir3 sir4 strains werequalitatively tested for rDNA silencing strength using ais toxic to cells (Holmes et al. 1997), which could explain

the relatively small increase in Sir2p levels produced by MET15-based colony color assay in an epistasis analysis.In this color assay, Met1 cells produce white coloniesthe 2m SIR2 plasmid. Cells expressing very high levels

of Sir2p would be selected against. on Pb21 containing medium (MLA), and Met2 cellsproduce dark brown colonies (Cost and Boeke 1996).SIR4 regulation of rDNA silencing is dependent on

SIR2, but not on SIR3: We previously demonstrated that Strains containing Ty1-MET15 within the rDNA producecolonies with an intermediate tan color, representingsir4D causes enhanced silencing of both mURA3 and

MET15 genes inserted in the rDNA (Smith and Boeke partial silencing of MET15 (Smith and Boeke 1997).Using this assay, enhanced rDNA silencing is visualized1997). This result suggested that SIR4 negatively regu-

lates rDNA silencing. We were interested in understand- as a darker colony color. As shown in Figure 4, thecolony colors of a sir4D strain and a sir3D sir4D straining the nature of this regulation. The enhanced si-

lencing phenotype of a sir4D mutant resembled the were equivalent at all stages of growth, indicating thatSIR3 was not required for the enhanced rDNA silencingenhanced silencing caused by increased SIR2 expres-

sion. Therefore, the simplest explanation for enhanced phenotype caused by sir4D. The sir2D sir4D strain pro-duced highly sectored white colonies that were pheno-silencing would be a sir4D-induced increase in cellular

Sir2p levels. This initial hypothesis was incorrect because typically indistinguishable from the sir2D strain and in-dicative of loss of rDNA silencing (Smith and BoekeSir2p levels were unaffected by sir4D (data not shown).

In wild-type cells, Sir4p, Sir3p, Rap1p, telomeric DNA, 1997). Therefore, SIR2 is necessary for the sir4D effectof enhanced rDNA silencing, but SIR3 is not. The speci-and some Sir2p are normally colocalized within discrete

foci at the nuclear periphery (Gotta et al. 1996). The ficity of these effects suggests that the enhanced rDNA


Sir2p/Sir3p/Sir4p complex to telomeres and the si-lencer 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 SIR4toward TPE and HM silencing is proposed to be causedby antagonism of these interactions (Marshall et al.1987; Moretti et al. 1994; Cockell et al. 1995). Forexample, SIR4 overexpression could result in the forma-tion of excess incomplete silencing complexes at theexpense of complete ones.

To examine the role of SIR4 dosage on rDNA silenc-ing, a high-copy plasmid containing full-length SIR4 wasintroduced into the haploid Ty1-mURA3 rDNA insertionisolate S6, and the resulting strains were tested for rDNAsilencing strength in a serial dilution growth assay on-Ura medium (Figure 5). Empty vectors had no effecton the repression of Ura1 growth. However, the SIR4plasmid increased the Ura1 growth of S6 by at least 25-fold, indicating that high-copy SIR4 severely reducesrDNA 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 ofSIR2 with SIR4, suggesting that SIR4 negatively affectsrDNA silencing by interfering with SIR2 function. High-copy SIR4 also reduced silencing in sir4 and sir3 mutantversions of S6 (data not shown), indicating that SIR3was not required for the high-copy SIR4–dependent lossof silencing. The strength of rDNA silencing thereforecorrelates inversely with SIR4 dosage.

To determine which domains of SIR4 antagonizeFigure 4.—Epistasis analysis of SIR2, SIR3, and SIR4 dele-rDNA silencing, 2m plasmids expressing various do-tions for rDNA silencing phenotypes. Silencing reporter

strains containing Ty1-MET15 at the rDNA were generated mains of SIR4 were tested for their effect on rDNAwith combinations of SIR2, SIR3, and SIR4 deletions. Each silencing using the Ura1 growth assay. The results arestrain was streaked onto a Pb21-containing (MLA) agar plate. shown in Figure 6A and tabulated in Figure 6B. Over-Photos were taken after 5 days growth at 308. SIR2 and SIR3

expression either of the SIR4-42 allele, which truncateswere replaced with the kanMX4 gene and SIR4 was replacedthe extreme C terminus, or of the C-terminal 40% ofwith HIS3. Strains used were (WT)JS333, (sir3D) JS335, (sir4D)

JS337, (sir3D sir4D) JS339, (sir2D) JS343, and (sir2D sir4D) SIR4 (pJH5.1A) had a dominant negative effect onJS344. Darker colony color represents enhanced rDNA silenc- rDNA silencing. pJH5.1A was also moderately toxic toing, whereas colony colors lighter than WT represent weak-

these strains for unknown reasons. Surprisingly, the ex-ened rDNA silencing. Dark sectors represent rDNA recombi-treme C terminus of SIR4 actually caused an increasenation events that result in complete loss of the Ty1-MET15in rDNA silencing strength (pJH3A) similar in ampli-reporter. Note that sir4D strains show strongly enhanced si-

lencing; this enhancement completely depends on SIR2. sir3D tude to the effect of a 2m SIR2 plasmid. Increased rDNAstrains show a subtle but reproducible enhancement of rDNA silencing was also observed with pJSS73-5, which over-silencing. expresses the C-terminal 23% of Sir4p (Figure 6B). The

region in common between the constructs that causederepression (shaded area) overlaps with the region ofsilencing caused by deletion of SIR4 does not requireSir4p previously shown to interact with Sir2p, but notrelocalization of Sir3p to the nucleolus but, rather, oc-with Sir3p (see black bar in Figure 6B; Moazed et al.curs by a SIR2-dependent mechanism.1997). Based on these results, we propose that the inhib-High-copy SIR4 reduces rDNA silencing: Whereas de-itory effect of Sir4p on rDNA silencing requires its physi-letion of SIR4 enhances rDNA silencing, it drasticallycal interaction with Sir2p.reduces HM and telomeric silencing. Overexpression

How could overexpression of the extreme C terminusof SIR4 or its C terminus reduces telomeric and HMof Sir4p cause increased rDNA silencing? In additionsilencing in a dominant negative fashion, which hasto the SIR genes, UTH4 and YGL014W are also requiredbeen termed the “anti-SIR” effect (Marshall et al. 1987;for a long life span in yeast (Kennedy et al. 1997).Renauld et al. 1993; Cockell et al. 1995). The C termi-Overexpression of the extreme C-terminal Sir4p do-nus of Sir4p interacts with the Sir3 and Rap1 proteins,

and these interactions are proposed to help recruit a main restores a long life span to specific uth4 mutant


Figure 5.—SIR4 overexpression effecton rDNA silencing. Strain JS128 (S6) wastransformed with HIS3 2m and LEU2 2mplasmids in combinations of two. The HIS3plasmid was the empty vector pRS423 orthe SIR2 vector pJSS71-13. The LEU2 plas-mid was the empty vector pRS425 or theSIR4 vector pLP305. Leu1 His1 trans-formants for each combination werepatched onto SC-Leu medium and grownfor 2 days. Cells were then resuspended inwater and plated as fivefold serial dilutionson SC-His-Leu-Ura to select for Ura1

growth and on SC-His-Leu as a nonselectivecontrol. The SC-His-Leu photograph wastaken after 2 days, and the SC-His-Leu-Uraphotograph was taken after 5 days.

strains, a phenotype shared by the same uth4 strains rDNA silencing is proportional to the amount of Sir2pproduced, suggesting that modulation of cellular Sir2pbearing the SIR4-42 mutation (Kennedy et al. 1995,

1997). Interestingly, the SIR4-42 allele in single copy levels or the redistribution of the normal Sir2p poolcould regulate rDNA silencing levels. Such regulation ofalso results in redistribution of the Sir complex from

telomere foci to the nucleolus, regardless of UTH4 geno- silencing by modulation of Sir2p has notbeen previouslyobserved for the other known forms of silencing andtype (Kennedy et al. 1997). The enhanced rDNA silenc-

ing produced by overexpression of the SIR4 C terminus may be specific to the regulation of rDNA silencing.Model of rDNA silencing regulation by SIR2 and SIR4:(pJH3A) in Figure 6 could therefore be caused by redis-

tribution of telomeric Sir2p to the nucleolus (rDNA). Immunolocalization studies have demonstrated that theSir2, Sir3, Sir4, and Rap1 proteins colocalize along withIf this is true, then an integrated SIR4-42 allele would

be predicted to also cause enhanced rDNA silencing. telomeric DNA to several subnuclear foci that normallyassociate with the nuclear periphery (Palladino et al.To test this hypothesis, we integrated the SIR4-42 allele

into a strain deleted for SIR4 and tested rDNA silencing 1993; Gotta et al. 1996). This accumulation of Sir pro-teins at the telomeric foci may act to produce a criticalof MET15 (Figure 7). In this case, SIR4-42 caused en-

hanced rDNA silencing (dark colony color) similar to concentration necessary for efficient silencing and chro-mosomal integrity (Palladino et al. 1993; Cockell etthe sir4D strain (Figure 7). A similar phenotype was also

produced by introducing a stop codon after amino acid al. 1995; Gotta et al. 1996). The silencing complex isalso required for silencing at the HM loci, resulting in1237 of the endogenous SIR4 gene (data not shown).

These results suggest that mutations which cause redis- competition between telomeres and the HM loci forlimiting amounts of Sir3 and Sir4 proteins (Buck andtribution of the Sir complex to the nucleolus, especially

redistribution of Sir2p, also result in enhanced rDNA Shore 1995; Marcand et al. 1996). Mutations thatlengthen telomeres, such as rap1s, cause the balance ofsilencing. Remarkably, pJH3A further strengthened the

enhanced rDNA silencing phenotype of a sir4D strain silencing to be tipped toward strengthened TPE (Buck

and Shore 1995). This is proposed to result from titra-(data not shown), suggesting that overexpression of theSIR4 C terminus may release even more Sir2p for rDNA tion of Sir4p away from the HM loci. Furthermore, it

has been proposed that the sequestration of Sir3p andsilencing than does sir4D. Alternatively, the SIR4 C ter-minus could release an additional separate factor that Sir4p by Rap1p at telomeres controls silencing at artifi-

cially created, internal, nontelomeric silencing sitesacts at the rDNA.(Maillet et al. 1996; Marcand et al. 1996).

Whereas some Sir2p is localized to the telomeric foci,DISCUSSION

the bulk is in the nucleolus (Gotta et al. 1997). Wehave now demonstrated that the wild-type level ofThe importance of SIR2 to cellular function is empha-

sized by the identification of a family of SIR2 homologs Sir2p is limiting for rDNA silencing and that silencingstrength is proportional to SIR2 dosage. In a recentthat exist in many diverse organisms, including bacteria,

yeast, plants, and mammals (Brachmann et al. 1995; independent study, high-copy SIR2 was also shown todecrease the expression of ADE2 and CAN1 markersDerbyshire et al. 1996). We and others previously

showed that SIR2 was required for rDNA silencing in inserted in the rDNA (Fritze et al. 1997). In contrastto the effect of SIR2 dosage, the strength of rDNA silenc-S. cerevisiae (Bryk et al. 1997; Smith and Boeke 1997).

The data presented here demonstrate that rDNA silencing is inversely proportional to SIR4 dosage. We proposea model for the regulation of rDNA silencing in whiching is highly sensitive to the dosage of SIR2 and SIR4,

but is independent of SIR3 dosage. The strength of the amount of Sir2p available in the nucleolus for silenc-


Figure 6.—Differential rDNA-silencing phenotypes caused byoverexpression of different SIR4fragments. (A) High-copy 2mplasmids expressing N- or C-ter-minal domains of Sir4 weretransformed into the rDNA si-lencing reporter strain S6(JS128), and the resulting strainswere tested for the effects on si-lencing by measuring changes inUra1 growth. The empty vectorsare pRS425 and YEp13. Full-length SIR4 is pLP305, whereasSIR4-42 is pLP754. pJH5.1A ex-presses the C-terminal 40% ofSIR4, and pJH3A expresses theC-terminal 12% of SIR4. Cellswere grown on SC-Leu mediumfor 2 days, resuspended in water,and then plated as fivefold serialdilutions on SC-Leu -Ura to se-lect for Ura1 growth and on SC-Leu as a nonselective control.The SC-Leu plate was incubatedfor 2 days, and the SC-Leu -Uraplate was incubated for 4 days. t,N-terminal–truncated Sir4 pro-tein. (B) Schematic representa-tion of results incorporated fromA. The amino acids of Sir4p orSir2p expressed from each plas-mid is indicated. The wild-typelevel of rDNA silencing is repre-sented as a 1 for each represen-tative empty vector. The relativesilencing phenotype of high-copy SIR2 is included for com-parison. Vector sequences are in-dicated by a thin line. Enhanced

rDNA silencing is indicated by 11. Loss of rDNA silencing is indicated by a 2. The shaded area of SIR4 is deduced to be requiredfor the negative effect on rDNA silencing. The horizontal black bar represents the region of Sir4p shown to previously interact withSir2p. The region of Sir4p that interacts with Rap1p and Sir3p is striped. The SIR2 plasmid is indicated by an asterisk.

ing is controlled by the sequestration of Sir2p at telo- tion of Sir2p at the rDNA, resulting in stronger rDNAsilencing (see Figure 8, model). In the case of sir3D,meres through an interaction with Sir4p (Figure 8).

Several lines of evidence support this model. Sir4p displays a diffuse nuclear localization pattern.Sir2p and Sir4p have been shown to interact indepen-dently of Sir3p when not associated with chromatin1. When SIR4 is deleted, rDNA silencing becomes en-(Moazed et al. 1997). Therefore, this diffuse nuclearhanced (Smith and Boeke 1997). This is the oppo-Sir4p would prevent most of the telomeric pool ofsite effect of what happens at telomeres and HM loci,Sir2p from redistributing to the rDNA (Figure 8,where SIR4 is absolutely required for silencing (Ivy

model), thus causing only the minor enhancementet al. 1986; Rine and Herskowitz 1987; Aparicio etin rDNA silencing that we observe.al. 1991). Deletion of SIR4 also results in the redistri-

2. Overexpression of SIR4 results in loss of rDNA silenc-bution of the telomere-localized Sir3p and Sir2p toing (Figure 5) similar to the dominant negative “anti-the nucleolus (Gotta et al. 1997), which is the sub-Sir” effect of SIR4 overexpression on TPE and HMnuclear location of the rDNA. Epistasis analysis re-silencing (Marshall et al. 1987; Renauld et al. 1993;vealed that SIR2, but not SIR3, is required for theCockell et al. 1995). The negative effect of SIR4 onenhanced rDNA silencing caused by sir4D. There-HM silencing can be compensated for by co-overex-fore, the enhancement in silencing cannot be causedpression of SIR3 (Marshall et al. 1987). Strikingly,simply by relocalization of Sir3p to the nucleolus. Itco-overexpression of SIR2 partially alleviates the over-is more likely that the redistribution of telomeric

Sir2p to the nucleolus increases the local concentra- expression effect of SIR4, resulting in restoration of


Figure 8.—Model of rDNA silencing regulation by telo-meres. In this model, the strength of rDNA silencing is propor-tional to the amount of Sir2p localized at the nucleolus. Sir4pis proposed to limit rDNA silencing by titrating Sir2p outof the nucleolus through direct interaction with Sir2p. Thelocalization patterns of Sir2p and Sir4p are represented for awild-type strain (WT), and for sir4D, sir2D, and sir3D mutants.The normal localization for Sir2p is nucleolar and telomeric(Gotta et al. 1997). sir4D causes redistribution of telomericSir2p to the nucleolus (Gotta et al. 1997), which increasesthe local concentration of Sir2p andenhances rDNA silencing.sir3D causes diffuse nuclear Sir4p localization, which wouldprevent most of the released telomeric Sir2p from redistribut-ing to the nucleolus. Consistent with this, a very slight butreproducible enhancement of rDNA silencing is observed insir3D strains (Figure 4). sir2D completely eliminates rDNA

Figure 7.—rDNA silencing phenotype of a strain carrying silencing and telomeric silencing. Filled circles, Sir2p; openthe integrated sir4-42 mutant allele. Cells were plated onto squares, Sir4p.Pb21 containing MLA plates and incubated for 5 days. Strainsused were (SIR41) JS333, (sir4D) JS337, and (sir4-42) JS347.The sir4-42 plasmid pLP793 was integrated into JS337 to pro- causing derepression of rDNA silencing (Figure 8,duce JS347. Darker colony color represents stronger rDNA model).silencing.

4. A specific mutant allele of SIR4, called SIR4-42, re-sults in redistribution of the Sir silencing complexfrom telomeres to the nucleolus and suppresses therDNA silencing (Figure 6A). These results suggest

that Sir4p disrupts the normal nucleolar function of allele-specific decrease in life span caused by a spe-cific mutation in UTH4, a gene required for long lifeSir2p in a titratable manner.

3. We found that a specific domain of Sir4p is responsi- span (Kennedy et al. 1995, 1997). Overexpression ofthe extreme C terminus of SIR4 also increases lifeble for the negative effect on rDNA silencing. This

region of Sir4p (amino acids 731–1049) overlaps with span, presumably by redistributing the Sir complexto the nucleolus (Kennedy et al. 1995). Nucleolara domain previously shown to physically interact with

Sir2p (Moazed and Johnson 1996; Moazed et al. relocalization of the Sir complex would increase theintranucleolar Sir2p concentration. Strikingly, both1997; Strahl-Bolsinger et al. 1997). SIR4 overex-

pression results in delocalization of the telomeric of these SIR4 manipulations cause an increase inrDNA silencing, as predicted by our model. In sum-Sir4p staining to a diffuse nuclear pattern (Maillet

et al. 1996). This excess nuclear Sir4p could poten- mary, release of rDNA silencing-competent Sir2pfrom telomeres should enhance rDNA silencing,tially titrate Sir2p away from the rDNA, therefore


whereas increased sequestration of Sir2p at telo- residues on the N terminus of the core histones (forreview see Sternglanz 1996). Accordingly, histones ofmeres should weaken rDNA silencing. Consistent

with this model, mutant alleles of cdc17, rfc1, and the HM loci-associated chromatin have been shown tobe hypoacetylated compared to other nonsilenced chro-rif1, which increase telomere length in yeast (Hardy

et al. 1992; Adams and Holm 1996), also result in mosomal regions (Braunstein et al. 1993, 1996). Over-expression of SIR2 correlates with decreased histoneweakened rDNA silencing ( J. S. Smith and J. D.

Boeke, unpublished data). acetylation levels in S. cerevisiae (Braunstein et al. 1993).It is therefore possible that inactive rDNA chromatin

Deregulation of rDNA silencing could have profound is associated with hypoacetylated histones. The rDNAmetabolic implications for the yeast cell. For example, histone acetylation level might be similarly influencedloss of the repressive SIR2-dependent rDNA chromatin by SIR2. Interestingly, it has been suggested that deacety-structure causes an increase in the percentage of actively lated histones are associated with transcriptionally inac-transcribed rDNA gene copies (Smith and Boeke 1997). tive rRNA genes in rat tumor cells (Mutskov et al. 1996).Increased rDNA silencing is therefore predicted to However, this has not yet been directly examined incause a reduction in the percentage of actively tran- yeast. SIR2 could be directly or indirectly involved inscribed rDNA gene copies. histone deacetylation, or it could prevent acetylation by

Structural differences between rDNA and other blocking the access or activity of acetyltransferase onforms of silencing: Sir3p is a proposed structural compo- rDNA histones. Whether the histone acetylation statenent of yeast telomeric heterochromatin (Hecht et al. of rDNA histones controls rDNA silencing remains to1995, 1996). Therefore, the lack of SIR3 involvement be determined.in rDNA silencing emphasizes the fundamental differ- A large family of genes homologous to SIR2 haveence in silencing complexes that act at the rDNA vs. the been identified in other species ranging from bacteriatelomeres/HM loci. Sir3p exclusion from the putative to human (Brachmann et al. 1995; Derbyshire et al.rDNA silencing complex may allow for efficient chroma- 1996). Included in this family are four homologs intin remodeling that may be necessary in a genomic S. cerevisiae known as HST1, HST2, HST3, and HST4.region, such as the rDNA, which is efficiently transcribed HST1 overexpression has previously been shown toby Pol I and Pol III, but may need to be constrained in partially complement the mating defect of a sir2D mu-terms of other transactions, such as rDNA recombina- tant (Brachmann et al. 1995). However, overexpressiontion (Christman et al. 1988; Gottlieb and Esposito of HST1 or HST3 had no effect on rDNA silencing1989; Bryk et al. 1997; Smith and Boeke 1997). strength. These results suggest that individual members

What is the role of SIR2 in rDNA silencing? Not only of the HST family of genes do not have identical func-is Sir2p localized in the nucleolus (Gotta et al. 1997), tions. It will be interesting to test the effects of otherbut Sir2p preferentially associates with rDNA (Gotta hst mutants on rDNA silencing. So far, the only SIR2et al. 1997). We have previously demonstrated that in homolog in other species that has been characterizedsir2D strains, transcriptionally inactive rDNA regions be- is from the yeast Kluyveromyces lactis (Chen and Clark-

come more accessible to psoralen cross-linking in vivo, Walker 1994). Mutation of this SIR2 homolog causesindicating that Sir2p is involved in either establishment an increase in ethidium bromide sensitivity and wasor maintenance of a specialized repressive chromatin reported to cause an increase in rDNA recombinationstructure in the rDNA (Smith and Boeke 1997). Fur- (Chen and Clark-Walker 1994). These results suggestthermore, a recent study showed that SIR2 modulates that SIR2 homologs may function broadly in regulationthe accessibility of rDNA chromatin to micrococcal of chromatin, including rDNA chromatin.nuclease digestion in vitro and dam methyltransferase rDNA silencing and aging: The life span of S. cerevisiaeactivity in vivo in a dosage-dependent manner (Fritze et cells is defined by the relatively fixed number of asym-al. 1997). These results, taken together, strongly support metric cell divisions that a mother cell can achievethe hypothesis that Sir2p contributes to the formation (Mortimer and Johnston 1959). A link between silenc-of chromatin structure in the rDNA. Therefore, the ing and life span determination was identified throughmodulation of rDNA silencing strength by changes in the isolation of a dominant gain of function mutant ofSIR2 dosage is likely to result from direct changes in SIR4 (SIR4-42), which restored a long life span to a short-the Pol II-repressive chromatin in the rDNA. It is still lived uth4-14c strain (Kennedy et al. 1995). Because theunclear whether Sir2p is a direct structural component Sir protein complex is redistributed from telomeric fociof the rDNA chromatin, or whether it is present in a to the nucleolus in SIR4-42 cells, the rDNA has beenregulatory capacity. Sir2p does not have a demonstrated proposed to be the site of this SIR4-42 gain of functionDNA-binding activity (Buchman et al. 1988), suggesting and perhaps to be associated with aging (Kennedy etthat it might associate with rDNA through interactions al. 1997). Interestingly, phenotypes associated with thewith other proteins. SIR4-42 mutation, including sterility and Sir protein re-

The transcriptional activity of eukaryotic genes corre- distribution to the nucleolus, also occur naturally in oldmother cells (Smeal et al. 1996; Kennedy et al. 1997),lates with the level of nucleosome acetylation of lysine


Insititues of Health (NIH) grant GM54778 (L.P.) J.S.S. is a postdoc-suggesting that the nucleolar redistribution of the Sirtoral fellow of the Leukemia Society of America. This work was sup-protein complex may somehow compensate for or pro-ported in part by NIH grant CA16519 to J.D.B.

tect against the cumulative effects of aging on the nucle-olus (Guarente 1997; Kennedy et al. 1997).

We have demonstrated that the SIR4-42 mutation orLITERATURE CITED

overexpression of the extreme C terminus of SIR4 causesAdams, A. K., and C. Holm, 1996 Specific DNA replication muta-an increase in the strength of rDNA silencing. Does this

tions affect telomere length in Saccharomyces cerevisiae. Mol. Cell.mean that increased rDNA silencing causes increased Biol. 16: 4614–4620.

Aparicio, O. M., B. L. Billington and D. E. Gottschling, 1991life span? As stated above, both of these SIR4 manipula-Modifiers of position effect are shared between telomeric andtions can restore longevity to specific, short-lived uth4silent mating-type loci in S. cerevisiae. Cell 66: 1279–1287.

mutant strains in a SIR3-dependent manner (KennedyBaudin, A., O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute

and C. Cullin, 1993 A simple and efficient method for directet al. 1995). However, deletion of SIR4 modestly shortensgene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21:life span (Kennedy et al. 1995) yet enhances rDNA si-3329–3330.

lencing in a SIR2-dependent, SIR3-independent man-Boeke, J. D., D. J. Garfinkel, C. A. Styles and G. R. Fink, 1985

Ty elements transpose through an RNA intermediate. Cell 40:ner. Moreover, SIR3 and SIR4 are required for longevity491–500.(Kennedy et al. 1995) but not for rDNA silencing

Brachmann, C. B., J. M. Sherman, S. E. Devine, E. E. Cameron, L.

(Smith and Boeke 1997). Taken together, these find- Pillus et al., 1995 The SIR2 gene family, conserved from bacte-ria to humans, functions in silencing, cell cycle progression, andings indicate that increased rDNA silencing is not suffi-chromosome stability. Genes Dev. 9: 2888–2902.cient to lengthen life span.

Brachmann, C. B., A. Davies, G. J. Cost, E. Caputo, J. Li et al., 1998Enhanced rDNA silencing may, however, be an im- Designer deletion strains derived from Saccharomyces cerevisiae

S288C: a useful set of strains and plasmids for PCR-mediatedportant component of the cellular response to aging.gene disruption and other applications. Yeast 14: 115–132.Our results suggest that increased rDNA silencing

Braunstein, M., A. B. Rose, S. G. Holmes, C. D. Allis and J. R.

strength is a consequence of mutations or conditions Broach, 1993 Transcriptional silencing in yeast is associatedwith reduced nucleosome acetylation. Genes Dev. 7: 592–604.that cause redistribution of the Sir complex (especially

Braunstein, M., R. E. Sobel, C. D. Allis, B. M. Turner and J. R.Sir2p) to the nucleolus, which could be a phenocopyBroach, 1996 Efficient transcriptional silencing in Saccharo-

of what happens in old cells. Deletion of the SGS1 gene myces cerevisiae requires a heterochromatin histone acetylationpattern. Mol. Cell. Biol. 16: 4349–4356.was recently demonstrated to accelerate the nucleolar

Bryk, M., M. Banerjee, M. Murphy, K. E. Knudsen, D. J. Garfinkelfragmentation phenotype that occurs normally in very et al., 1997 Transcriptional silencing of Ty1 elements in theold wild-type yeast cells (Sinclair et al. 1997). SGS1 is RDN1 locus of yeast. Genes Dev. 11: 255–269.

Buchman, A. R., W. J. Kimmerly, J. Rine and R. D. Kornberg, 1988a member of the RecQ helicase family, which includesTwo DNA-binding factors recognize specific sequences at silenc-

the Werner’s syndrome gene WRN that is implicated in ers, upstream activating sequences, autonomously replicating se-quences, and telomeres in Saccharomyces cerevisiae. Mol. Cell. Biol.premature aging in humans (Yu et al. 1996). Sgs1p is8: 210–225.localized in the nucleolus, and deletion of the gene

Buck, S. W., and D. Shore, 1995 Action of a RAP1 carboxy-terminalshortens life span (Sinclair et al. 1997) and increases silencing domain reveals an underlying competition between

HMR and telomeres in yeast. Genes Dev. 9: 370–384.the frequency of rDNA recombination (Gangloff et al.Chapman, K. B., and J. D. Boeke, 1991 Isolation and characterization1994). It was therefore proposed that nucleolar frag-

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Chen, X.-J., and G. D. Clark-Walker, 1994 sir2 mutants of Kluyvero-redistribution of the Sir silencing complex to the nucle-myces lactis are hypersensitive to DNA-targeting drugs. Mol. Cell.olus delays these changes (Guarente 1997; SinclairBiol. 14: 4501–4508.

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