Copyright 0 1997 by the Genetics Society of America

Hyperactivation of the Silencing Proteins, Sir2p and SirSp, Causes Chromosome Loss

Scott G. Holmes, Alan B. Rose,' Kristin Steuerle, Enrique Saez,' Sandra Sayegh,' Young Mi Lee and James R. Broach Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 Manuscript received March 28, 1996 Accepted for publication November 14, 1996

ABSTRACT The SIRgene products maintain transcriptional repression at the silent mating type loci and telomeres in Saccharomyces cerevisiae, although no enzymatic or structural activity has been assigned to any of the Sir proteins nor has the role of any of these proteins in transcriptional silencing been clearly defined. We have investigated the functions and interactions of the Sir2, Sir3,and Sir4 proteins by overexpressing them in yeast cells. We find that Sir2p and Sir3p are toxic when overexpressed,while high Sir4p levels have no toxic effect. Epistasis experiments indicate that Sir2pinduced toxicity is diminished in strains lacking the SIR3 gene, while both Sir2p and Sir4p are required for Sir3p to manifest its full toxic effect. In addition, the effects of Sir2 or Sir3 overexpression are exacerbated by specific mutations in the N- terminus of the histone H4 gene. These results are consistent with a model in which Sir2p, Sir3p and Sir4p function as a complex and interact with histones to modify chromatin structure. We find no evidence that toxicity from high levels of the Sir proteins results from widespread repressionof transcrig tion. Instead, we find that high levels of Sir2p and/or Sir3p cause a profound decrease in chromosome stability. These results can be appreciated in the context of the effects of Sir2p in histone acetylation and of chromatin structure on chromosome stability.

APLOID Saccharomyces cereuisim contains three loci ucts of the four SIR genes. Each of the SIR genes was H that code for mating-type information. Mating initially identified by genetic screens for loss of repres- type is determined by genes present at the expressed sion at the silent mating type loci (WERand GEORGE MAT locus, while similar or identical genes present at 1979; KLAR et al. 1979; RINE et al. 1979; &NE and HER- the HML and HMR are expressed only when transposed SKOWITZ 1987). None of the SIR genes is essential for to MAT following mating type switching. Repression at growth, but null mutations in SIR2, SIB, or SIR4 lead HML and HMR is achieved by a position effect mecha- to complete expression of the silent mating type loci. nism known as silencing, which extends to other yeast Deletion of the SIRl gene leads to an intermediate phe- genes placed at HML or HMR (for review see HERSKOW- notype in which some cells are repressed and others ITZ et al. 1992; LAURENSON and RINE 1992; HOLMESet derepressed (PILLUSand RINE 1989). An understanding al. 1996). A similar position effect is exerted on genes of the function of the SIR genes is central to determin- artificially placed at yeast telomeres (&ARICIO et al. ing the mechanism of silencing in yeast. 1991; GOTTSCHLINGet al. 1990). Silencing is likely to Each of the fourSIR genes has been cloned, but their involve the formation of the yeast equivalent of hetero- sequences have not suggested clear functions for their chromatin, a repressive chromatin structure that under- protein products. SIRl codes for a novel protein (STONE lies the phenomena of X chromosome inactivation in et al. 1991) while SIR2 codes for a zinc finger protein mammals and position effect variegation in Drosophila that is a member of a gene family withfour othermem- (THOMPSONet al. 1993; BRAUNSTEINet al. 1996a,b). bers in yeast and at least one similar member in mam- Silencing at HML and HMR depends on sequences mals (SHOREet al. 1984; BRACHMANNet al. 1995). The flanking each locus, known as the E and I silencers, as SIR3 gene codes for a protein of unknown function well as several transacting factors, including the prod- that has similarity to Orclp, part of the six subunit complex that recognizes DNA replication origins in Corresponding author: James Broach, Department of Molecular R. yeast (SHORE et al. 1984; BELLet al. 1995). Finally, SIR4 Biology, Princeton University, Princeton, NJ 08540. E-mail: [email protected] codes for a large protein thatshows similarityto nuclear 'Present address: University of California, Davis, College of Agricul- lamins (MARSHALLet al. 1987; DI~EYand STILLMAN ture and Environmental Sciences, Department of Vegetable Crops, 1989). None of the Sir proteins have been shown to Davis, CA 9561G8746. interact directly with the silencer sequences. If individ- 'Present address: Dana Farber Cancer Institute, 44 Binney St., Boston, MA 02114. ual Sir proteins are fused to the Gal4p DNA binding 'Present nddress: School of Public Health, Columbia University, New domain and recruited to the silent mating type loci York, NY 10032. using a GAL4 UAS sequence, Sirlp can promote weak

Genetics 145: 605-614 (March, 1997) 606 S. G. Holmes et al.

silencing; no silencing is observed by recruitment of treated with exonuclease BAL31, and reclosed in thepresence SirSp, SiAp, or Sir4p (CHIENet al. 1993). of SalI linkers. The 3.5-kb Sall-BamHI fragment from one of the recovered plasmids, in whichthe SulI site is 7 bp upstream Several independent approacheshave suggested that of the initial ATG codon of the SZR3 open reading frame, was the Sir proteins interact with silencer-binding factors inserted into YEp51 and YEp54 to create pAR16 and pAR82, and histone proteins to promote transcriptional repres- respectively. The GALJO::SZR4 plasmid, pSIR4.7, was derived sion. MORETTI et al. (1994) used a two hybrid screen by insertion of a KpnI-BamHI fragment spanning the 2-p circle to find proteins that interact with the silencer-binding replication and partitioning sequences of plasmid pSIR4.3 into the equivalent sites of plasmid pSIR4.6, each of which factors and identified an association of Rap1 with Sir3p has been previously described (MARSHALL et al. 1987). pSAS2 and Sir4p. The Raplp-Sir3p association may be direct, was constructed by isolating a 5.0-kb EcoRV fragment from as these proteins physically interact in vitro. MORETTIet pAR82, containing the GALIO::SIR3 fusion, and subcloning al. (1994) also used the two hybrid assay to show that it into pAR14, cut with Hind111 and made blunt with Klenow the Sir3 and Sir4 proteins interact in vivo. In an inde- enzyme. pYML2 was made by first subcloning a ChI-KpnI frag- ment containing the SZR3-R3 mutation from pLJ90 (JOHNSON al. pendent study HECHTet (1995) investigated the abil- et al. 1990) into pAR12, forming pYMLl. A SalI-BamHI frag- ity of Sir2p, SirSp, and Sir4p to interact with the N- ment containing the SZm-R3 mutation was then isolated from terminal tails of histone H3 and H4in vitro.While they pYMLl and subcloned into pAR34 cut with SUR and BamHI, found no evidence for a Sir2p-histone association, they forming pYML2. pYML2 is identical to pAR16, except for the showed that both Sir3p and Sir4p bind the tails of H3 SZR3-R? mutation. pSH105, used to delete the SZR3 gene, was made by replacing the large BglII fragment within the SIR3 and H4 (HECHTet al. 1995). Using protein affinity chro- open reading frame with a BglII fragment containing the matography, MOAZEDand JOHNSON (1996) found that URA3 gene. Sir2p and Sir3p associate with Sir4p. Finally, immuno- Strains: Yeast strains used in this study are listed in Table fluorescence experiments using antibodies to Sir3p, 1. Plasmid C369 (SHOREet al. 1984) was used to make the Sir4p, and Raplpsuggest that these proteins colocalize sir2::TRpl deletion. Plasmid pSH105 was used to make the sir3:: URA3 deletion. Plasmid pAR59 (MARSHALL et al. 1987) in discrete foci associated with the nuclear periphery was used to make the sir4::URA3 deletion. Strain Y1191 was (PALLADIN0 et al. 1993; COCKELL et al. 1995). a segregant from a cross between strains PKY499 (KAYNE et Genetic approaches to determining the function of al. 1988) and lab strainAB8-16C. Isogenic derivatives ofY1191 the Sir proteins have been limited by the identical null that differed only in the plasmid-borne HHF2 allele were de- phenotype of the SIR2, SIR3, or SIR4 genes and the rived from strain Y1191 by a plasmid shuffle protocol. Strain Y1191 was transformed to Trp+ with plasmid pMH3lO CyCp absence of alleles with reduced or novel functions. The TRPHHF2) and a Ura- segregant of one such transformant SIR genes, particularly SIRl, SZE, and SIR4, are tran- was recovered. This segregant was transformed to Ura+ with scribed at low levels, which may reflect their highly spe- plasmids pPK617, pPK618, or pPK606, hearing HHF2 alleles cific role in the cell (IVYet al. 1986). We have examined A414, A420, and A423, respectively (KAYNE et al. 1988). the effects of overexpressing the SIR genes in hopes of Trp segregants of selected transformants were recovered to observing phenotypes that would help us understand yield strains Yl186, Y1181, and Y1176, respectively. SIR2 or SZR3 overexpressing versions of these strains were obtained their normal function. We have previously shown that by transforming each of them to Trp+ with plasmids pAR44 overexpression of SIR4 leads to a dominant disruption or pAR82. of silencing, the "anti-SIR"effect (MARSHALL et al. 1987), Immunological procedures: Preparation of antibodies to and that overexpression of SZRZleads to a global deacety- Sir2p and Sir3p was described previously (BRAUNSTEINet al. lation of histone molecules in the cell (BRAUNSTEINet 1993). Immunoprecipitation experiments were performed in the proteasedeficient strain BJ2169. Cultures of this strain al. 1993). Here we report that overexpression of SIR2 harboring the indicated plasmid or plasmids were grown at or SIR3 is toxic to yeast cells. Using the overexpression 30" in SC raffinose medium to a density of lo' cells/ml, at phenotypes in epistasis analysis,we find evidence for the which point galactose was added to 2% and incubation con- functional interaction of the Sir proteins in a complex tinued for 3 hr. Extractswere prepared as described that interacts with histones and, athigh levels,interferes (BRAUNSTEINet al. 1993) and incubated with the indicated antibody (15 pl/ml extract) at 0" for 3 hr. Formalin-fixed with the mitotic transmission of chromosomes. Staph A cells (ImmunoPrecipin, BRL, 30 pl/ml extract) were added and incubation continued for20 min. Immunoprecipi- MATERIALS AND METHODS tates were then harvested by centrifugation and washed twice with RIPA buffer (150 mM NaCI, 1% sodium deoxycholate, Plasmids: Plasmids in which SZRZ or SZR3 expression was 1% Triton X-100, 0.1% SDS, 10 mM Tris-HC1 pH 7.2). Samples placed under control of the GAL10 promoter were con- were boiled in PAGE sample buffer and the presence of Sir structe,d from the high copy expression vectors YEp51 and proteins was determined by Western blot analysis asdescribed YEp54 (BROACHet al. 1983; ARMSTRONG et al. 1990). The SIR2 (BRAUNSTEINet al. 1993). and SZR3 genes were subcloned using plasmids pJHZO.1 and Chromosome loss: StrainWH1015 (provided by P. HIETER) pJSAN59, respectively (Iw et al. 1986). A SalI linker was in- contains a HZS3marked 125-kb linear chromosome fragment serted into the AccI site of plasmid pJH20.1, which lies 45 bp derived from chromosome ZZZ (SHEROet al. 1991). A LEU2- upstream of the initial ATG codon of SZRZ. The 3.5-kb SulI- marked 73-kb circular derivative of chromosome 111 was intro- Hind111 fragment from theresulting plasmid was inserted into duced as described (RUNGEand ZAK~AN1993) into an isolate eitherYEp51, forming pAR14, orYEp54, forming pAR44. The of strainYPHlO15 lackingthe linear chromosome Illfragment. 3.7-kb HpuI fragment from pKAN63 was ligated into the SmaI Each strain was then transformed with appropriate SZR-overex- site of pUCl2. This plasmid (pAR3) was linearized with SacI, pressing plasmids and controls. Chromosome loss was deter- SIR Gene Overexpression 607

TABLE 1 Description of yeast strains used in this study

Strain Genotype Source Y2155 HMLaMATa HMh ade2 leu2 lys2 trpl-289 ura3-52 BRAUNSTEINet al. (1993) Y2156Y2155; Asir2::TRPl This study Y2157Y2155; Asir3::URA3 This study E158 Y2155; Asir4::URA3 This study BJ2169 MATa leu2 trpl urn3 prb1-1122,407p@4-3 E. JONES Y1191 MATa ade2 his3Al leu2-3,112 trplA901 hhfl::HIS3 hhf2::LEUZ LYS2::GALl-lacZ This study lyCp URA3-HHF21 Y1186 MATa ade2 his3Al leu2-3,112 trplA901 hhfl::HIS3 hhf2::LEUZ LYS2::GALl-lacZ Thisstudy lyCp-URA3-hhf2A4-14] Y1181 MATa ade2 his3Al leu2-3,112 trplA901 hhfl::HIS3 hhP::LEU2 LYS2::GALl-lacZ Thisstudy lyCp-URA3-hhjL?A419] Y1176 MATa ade2 his3Al leu2-3,112 trplA901 hhfl::HIS3 hhJ2::LEUZLYS2::GALl-GacZ Thisstudy PCP-URA3-hhf2A4-231 E215 MATcv leu2-?,112 ura3-52 ade2 trpl-289 can1 Ahisl::URA3 This study WH1015 MATa ade2-101 his3A200 leu2Al lys2-801 trplA63 urd-52 [CEN 3L YPH985.his.suplll SHEROet al. (1991) Y2279 Y2215 X YPH1015 This study mined by fluctuation analysis: for the linear chromosome III shown to result in a large increase in the levels of each fragment individual colonies grown on glucose medium lack- Sir protein (MARSHALLet al. 1987; BRAUNSTEINet al. ing leucine were suspended in SC raffinose medium lacking leucine, induced by theaddition ofgalactose to 2%, then 1993). grown at 30". Cells were plated on -Leu and -Leu -His plates High levels of Sir4p have previously been shown to at the time of induction and at various times following induc- cause a dominant disruptionof silencing, known as the tion to determine the initial and final percentageof cells bear- anti-SIR effect (IVYet al. 1986; MARSHALL et al. 1987). ing the nonessential chromosonle fragment.An identical prc- We find that increasing the abundance ofSir2p or Sir3p cedure was usedto determine the lossrate of the circular chromosome Illderivative, excepta TRP-marked SIR3 plasmid does not affect mating efficiency (datanot shown). was used, and the test chromosome was followed by the LEU2 However, increased levels of Sir2p or Sir3p lead to a marker. The values shown are the means of at least two inde- significant decrease in cell viability. Cultures carrying pendent determinations; trials deviatedfrom the means by different SIR plasmids were grown to log phase in raffi- <20%. Chromosome V lossrate was determinedin strain nose medium, which neither induces nor represses the Y2279 as described (HARTWFLL and SMITH1985), except that GAL1 0 promoter. Serial dilutions of these cultures were a disruption of the HIS1 locus was used in place of the recessive horn3 mutation. Recombination was not sigificantly altered in then plated on media containing galactose, which in- strains overexpressing SIR genes by this assay. Loss rates re- duces high expression of the SIR genes. Figure 1 shows ported are the meansof at least three independent determina- that the SIR4 overexpressing plasmid did not reduce tions; trials varied from the means by <25%. the ability of the culture to form colonies when com- pared to cells containing a vector control. However, RESULTS high-level expression of Sir2p or Sir3p is toxic to yeast cells, leading to a 10%-lo4 decrease in plating efficiency. Overexpression of SIR2 or SZR.3 is toxic: Deletion of Decreased colony number and size are also observed if SIR2, SIR.3, or SIR4 leads to an identical phenotype in cultures are plated on galactose medium containing which the silent mating type loci are fully derepressed, leucine (not shown),indicating that toxicity is not solely but none of the deletions yields any additional pheno- due to a SIRZ- or SIR3-induced decrease inplasmid types*that might provide insights into the role of the stability. SIR proteins in transcriptional silencing. Since the SIR Interactions among the SIR proteins: The complete genes are transcribed at low levels, we reasoned that loss of silencing due to null mutations in any one of high levels of the SIR proteins might induce an activity the SIR2, SIR?, or SIR4 genes initially led to the pro- normally constrained to silenced loci to act at other posal that they acted as a complex. In support of this places on the chromosome and result in novel pheno- proposal, MORETTIet al. showed that the Sir3 and Sir4 types. To test this hypothesis we constructed a set of proteins associate with each other, and with Raplp, in plasmids allowing inducible, high level expression of viuo ( MORETTI et al. 1994), while Sir2p and Sir3p associ- the SIR genes. Overexpression was achieved by placing ate with Sir4p in vitro (MOAZEDand JOHNSON 1996). the SIR genes under thecontrol of the galactose-induc- The ability to induce agrowth phenotype by expressing ible GALlOpromoter on high copy number vectors and individual SIRgenes allowed 11s to explore furtherpossi- adding galactose to the growth medium of strains con- ble functional interactions among the Sir proteins in taining these plasmids. Theseconditions have been vivo. We first overexpressed combinations of the SIR 608 S. G. Holmes ~t nl. FIGURE1 .-Toxic effects of SIR gene overexpression. Strain Y2155 was trans formed with plasmids con- taining galactose-inducible SIR genes, then grown over- night in raffinose medium lacking leucine. Sets of 10- fold serial dilutions from these cultures were spotted on glucose medium lacking leucine (-LEU) or on ga- lactose medium lacking leu- cine (-LEU GAL). Photo- graphs were taken after 2- 4 day growth at 30". genes to assay their ability to cooperate in inducing of the same strain expressing either SIR2 or SIR3alone. toxic effects. We foundthat overexpressing SIR4 in This synergistic effect in reducing cell viability suggests combination with SIR2, or in combination with SlR3, that Sir2p and Sir3p share a common or related func- neither increased nor reducedthe level of toxicity tion, and is consistent with a physical association. caused by SIR2 or SIR3overexpression alone (Table 2). We furtherexplored the functional interactions However, cells bearing a plasmid that expresses both among the Sir proteins by overexpressing specific SIR SIR2 and SIR3 at high levels exhibited a decrease in genes in backgrounds containing SIR gene deletions. viability that was substantially greater than for cells ex- If the Sir proteins exert toxic effects by acting as a pressing either SIR2 or SIR3 alone (Figure 1). complex, then the absence of one of the SIR genes We performed a time course experimentto examine might abrogate the growth defect resulting from over- the consequences of SIR2 and SIR3 coexpression fb- expression. However, if Sir2p or Sir3p act alone to in- ther. Strains carrying plasmids expressing a single SIR duce toxicity, the absence of the otherSIR genes would gene, or both SIR2 and SIR3 were grown to log phase not affect the phenotype. Accordingly, we overex- in raffinose medium,induced with galactose, and pressed SIR2 or SIR3 in strains lacking the SIR2 gene. plated for viability. For convenience these results are As shown in Figure 2A, we found that the toxic effects presented in Table 2as the ratio of viable cells following growth in galactose for 24 hr us. the viable cells present following growth of the same strain for 24 hr in the absence of galactose. The values presented accurately reflect the behavior of the strains throughoutthe growth curve. The viabilityof strains overexpressing both SIR2 and SIR3 was at least 300 times less than that

TABLE 2 Viability of cultures overexpressing SIR genes

PlasmidSIR gene overexpressed Relativeviability

YEp.5 1mp.54 None 1.1 PAR1 4m.p.54 SIR2 0.07 pAR82/YEp.51 SIR3 0.067 pSIR4.5/Yt.,p54 SIR4 0.78 pAR14/pAR82 SIR2/ SIR3 0.00028 pAR14/pSIR4.7 SIR2/SIR4 0.048 pAR82/pSIR4.7 SII<3//sIR4 0.14 CMtrlres of strain Y21.55 carrying the indicated plasmids were grown at 30" to log phase in raffinose medium lacking leucine and tryptophan, when the cultures were divided and galactose was added to half to a concentration of 2%. Cultures FIGL~RE2.-SIIi gcnc-s coopcmtr to CAIISC toxic cllims. The were incubated for an additional 24 hr. The number of viable, effects of SIR gene owrexprrssion was assayed in strains lack- plasmid-bearing cells was determined at this time by plating ing individual SIR genes. SIR gene deletions were created in appropriate culture aliquots on selective glucose media. Rela- strain Y215.5,which was then transformed with a set of plas- tiveviability represents the ratio of the number of viable,mids containing galactose-inducibleSIRgenes. Serial dilution plasmid-bearing cells in the induced culture at the end of 24 analysis was performed as described in the legend to Figure hr to that in the uninduced culture at the end of 24 hr. 1. (A) Asir2 strain. (R) Asir3 strain. (C) Asir4 strain. SIR Gene Overexpression 609 of SIR3 overexpression were diminished in this strain, SIR2 SIR2/SIR3 SIR3 suggesting that Sir2pis required to manifest SIWsover- expression phenotype. Similarly, we assayed Sir-induced 1234 1234 1234 toxicity in a strain deleted for the SIR3 gene (Figure 2B). In thisstrain SIR2's effects werenearly absent, 200 - indicating thatSIR3 is required forSIR2 toxicity. Finally, the effects of SIR2 or SIR3 overexpression were assayed 97 - in a strain deleted for theSIR4 gene (Figure 2C).SIR3 induced toxicity was reduced in this strain, suggesting that in addition to SIR2, SIR3 requires SIR4 to exert a 68 - ' .I growth defect. The toxic effect of SIR2 overexpression i: was only slightly diminished in this strain, indicating 43 that Sir2p is less dependenton SIR4.SIR4 overex- -

pression was not toxic inany of these backgrounds, FIGLXI;.3.-Sir!?p ;Ind Sir311 ctrimmullol~r~~il,it~~t~~.(.III- indicating that a potential for SIR4induced growth de- tures of strain I3J2169 carrying phR14 (G41A-.SIIU), phR82 fects is not held in check by the presence of the other (GU,-SIR3) or both plasmids were grown in SC raffinose me- Sir proteins (not shown). dium to log phase then induced by the addition of galactose to 2%. Extracts of the induced strains were prepared as de- Sir2p and Sir3p are physically associatedin vivo: Our scribed in SIATI:.RIAI.SASD Sit:TIIOI)S and 0.5-ml aliquots were functional assays are consistent with physical binding immunoprecipitated with anti-Sir2p serum, anti-Sir8p serum, studies that indicate that SIR3 and SIR4 interact with or no prima? antibody. Immllnoprecipitates and samples of each other, and suggest a previously uncharacterized the total cell extracts were fractionated by SDSPAGE, trans- interaction behveen SIR2 and SIR?. To examine this ferred to nitrocellulose and probed with a mixture of anti- Sir2p and anti-Sir3p sera. The filter was developed using an interaction further we determined whether Sir2p and alkaline phosphatase-conillgated secondary antibody and Sir3p are physically associated in vim. Extracts of strains photographed after staining for alkaline phosphatase activity. containing high levels of SiRp, SirSp, or both were The I;hel a1m.e each set 01' live lanes indicates the SIR gene incubated with antibodiesdirected against Sir2p or ovcrc*xpressedin those strains. L~ne1, whole cell extract; lane Sir3p. The immunoprecipitates were fractionated by 2, no prilnay antil,otly; lane 3, immrlnoprccipitated with anti- Sir'Lp sera; lane 4, immunoprecipitated with antiSir3p sera. SDSPAGE, transferredto nitrocellulose, andthen probed with a mixture of Sir2p and Sir3p antibodies. The results of this experiment are shown in Figure 3. strains with the A420 or A423alleles are defective for Anti-Sir2p antibody did not immunoprecipitate Sir3p repression ( IGU'NE P/ nl. 1988). M7e examined the effects from extracts of a strain expressing Sir3p alone. Simi- of overexpressing the SIR2 gene in combination with larly, anti-Sir3p antibody failed to immunoprecipitate these alleles (Table 3). Although Sir2p does not bind significant amounts of Sir2p from a strain expressing to this region of histone H4 in vi/ro (HIKHTPI nl. 1995), high levels of Sir2p alone. In contrast, each of the anti- we have previously shown that high levels of Sir2p lead bodies precipitates significantamounts of both proteins to a decrease of acetylation on the four lysine residues from an extract of a strain containing high levels of in the N-terminus of H4 (BRALWSTEISP/ 01. 1993). SIR2 both. Thus,Sir2p and Sir3p forma complex whencoex- overexpression in strainscarrying each of the HHF2 pressed, indicating that the two proteins are physically deletion alleles showed essentially the same small but associated in vivo. significant decrease in viability, compared to that for Mutational alterations of histone H4 enhance SIR2 SIR2 overexpression in strainscarrying the wild-type and SIIU-induced lethality: Silencing likely involves an HHF2 allele. interaction between the Sir proteins and histones. To We also examined the effects of overexpressing SIR3 investigate Sir-histoneinteractions we examinedthe in the HHF2 mutant backgrounds. Overexpression of consequences of overexpressing SIR2 or SIR3 in back- SIR3 in a backgroundcontaining the A414 allele, grounds containing mutantforms of histone H4. Muta- which deletes a region that is not required for binding tions in histone H4 that lead to a silencing defect map of Sir3p to H4 in vitro (HECHTPI nl. 1995), resulted in to the N-terminus of the protein (KAYNE PI nl. 1988; a substantial decrease inviability compared to thatfrom MEGEEet nl. 1990; PARKand SZOSTAK1990); this region overexpression of SIR3 in the context of a wild-type H4 of the protein is also required for anin vitro interaction allele (Table 3). More extensive deletions in H4, which with SlR3and SIR4 (HECHTet al. 1995). M'e constructed would be predicted not to bind Sir3p based on in vitro strains carrying different deletion alleles of HHF2 as studies, suppress the increased lethality, producing a well as either thehigh expression SIR2 or SlR3plasmids. level of toxicity equivalent to a wild-type histone back- Three histone H4 deletion alleles were assayed, dif- ground. These results show that SIR2 and SIR3 geneti- fering in the extent of the N-terminaldeletion they cally interact with histone H4 and highlight a specific contain. Wild-type cells containing the smaller A414 interaction with SIR3 and regions in the X-terminal tail. deletion do not exhibit a defect in silencing; however, Although noother function besides silencing has 61 0 S. G. Holmes ct /I/.

TABLE 3 Viability of histone deletion strains overexpressing SIR genes

Relative viahility in thc presence of the IfIfI~Zallclc SIR gene ovcrcxpresscd M'ild typc A414 A4-19 A423 Sonc SIR2 S111'3

been established for the SIR genes, the overexpression ing at telomeres and the silent-mating type loci, but effects we observed might have been due to an increase insteadcause :I decrease in transcriptionthrottgho1~t in an activity that was unrelated to silencing. To cxam- the genome and repress expression of essential genes. ine this possibility we tested the effects of overex- To investigatethis possibility we examined the influ- pressing the SIRFR3 allele. This allele was isolated in a ence of SIR overexpression on the steady-state levels of screen for suppressorsof a histone H4 N-terminal point a variety of mRNXs. We grew caltures tolog ph:lse, mutation that was defective in silencing (JOIISSOS r/ intl~~cctl theSIR genes, then prepared RNA tronl in- NI. 1990). Thesesuppressors werenot allele specific, dt~cctl and rminduced cdturcs4 111- later. At this time and their mutations did not mapto the region of SIR3 cells carrying the SI113 and SIR2/SIR3 ovcrcxpressing that is requiredfor an in vifro association with H4 plasmids had substantially reduced plating efficiency, (HIX:III r/ crl. 199.5). An independent screenfor in- while cells can7ing the vector control. S1112, or SIR4 creased silencing at telomeres in the background of a overexpressing plasmids exhibited no decrease in pht- hypomorphic mutation in the I&\l'l gene also yielded ing efficiency. In inducing or noninducing conditions the SIIU-IU allele (LIVand LVSTK;1996). Therefore, an equal numbcrof cells yielded an equivalent amount the SIR" allele likely encodes a form of SirSp that of RNA. Therefore, Sir protein overexpression did not promotes more efficient silencing. If the toxic effects qlobally affect steadystate levels of RSA or cell integrity. of SIIU overexpression were due to a function related M'e examined the effects of SIR gene overexpression to silencing, then overexpression of the SIR3-R3 allele on the levels of several specific messages by Northern might be particularly toxic. To test this prediction we analysis (Fig1u-e 5 and data not shown). We observed introduced the SIIc3-IU mutation into ourSIR3 overex- no effectof' SIR overexpression on the levels of AC7'1, pressing plasmid and compared the effects of overex- GAI,I, or 77U'l mRNA, or on 18s rRNA. This indicates pressing the SIIU-IU to that of overexpressing wild-type that SIR overexpression is not inducing a general shut SIR3. The results of this experiment are presented in down of pol1 or pol11 transcription. Figure 4. Overexpressionof the SIR3-R3 allele was R~:s,\v~npt 01. (1993) have shown that SIR3 overex- clearly more toxic than overexpression of the SIR3wild- pression causes an increase in spreading of telomere type allele, suggesting that the effectsof overexpression positioneffect. Therefore, SIR overexpressionmight are related to SirSp'srole in silencing, and not to a not establish silencing in new locations, but rather ex- novel or uncharacterized function. tend silencing from the known foci of establishment SIR overexpression and transcription: A straightfor- into contiguousessential genes. To test if silencing nu- ward hypothesis for Sir-induced lethality is that the SIR cleated at IfMI- or HMR were spreading into essential proteins are no longer constrained to establish silenc- genes, we overexpressed SIR genes in a strainthat

-LEU -LEU GAL FIGCRE 4."Increasrd le- thality is induced by overex- pression of the SIRjr-IL? al- vector lele. A plasmid containing the SIFU-FU allele under control of the galactose SIR3 promoter was introduced into strain Y213.i and tested SIR3-R3 for galactose-induced ef- fects. Serial dilution analy- sis was performed as de- SIR2BIR3 scribed in the legend to Figure 1. SIR Gene Overexpression 61 1 TABLE 4 SIR gene overexpression increases chromosomeloss

Loss rate SIR gene 12.5-kb 73-kb overexpressed linear circular Chromosome V None 0.022 3.0 x l0-3 3.0 x lo-" SIR2 0.12 ND 3.9 x lo-.' SIR3 0.31 1.1 x 10" 3.8 x 10P SIR4 0.016 ND 1.3 x lo-" SIR2/SIR3 0.92 ND 5.2 X 10" Strain YPH1015 contains a nonessential chromosome frag- ment bearing the HIS3 marker. SIR overexpressing plasmids were transformed into strain YPH1015, grown to log phase in raffinose medium lacking leucine, then divided in two, when half was induced by the addition of galactose to 2%. The FIGURE.i.-Transcription in strains overexpressing SIR number of plasmid-bearing cells that were His' was deter- genes. Strain Y2155 containing IJW2-marked SIR overex- mined at the time of induction and at later times; chrome pressing plasmids was grown to log phasein raffinose medium some loss rates were determined by fluctuation analysis (see lacking leucine. The culture was divided, and galactose was MATERIAIS AND METHODS). ND, not determined. added to half to a final concentration of 2%. Following 4 hr of galactose induction, growth was suspended and RNA of chromosomes, we introduced our plasmids into a collected from the cells, fractionated on formaldehyde/agar- ose gels, blotted to nitrocellulose, and probed sequentiallv haploid yeast strain bearing a marked, nonessential with the indicated gene fragments. Lane 1, YEp51 (vector); chromosome fragment. Thisstrain background was also lane 2, pAR14 (SIR.?); lane 3, pAR16 (SIR3);lane 4, pSIR4.7 highly sensitive to SIR gene overexpression, with SIR3 (SIR4); lane 5, pSAS2 (SIR2 and SIR3). expression in this case showing increased toxicity com- pared to SIR2. We measured the stability of the nones- lacked HML and HMR. These cells remained sensitive sential chromosome by fluctuation analysis in inducing to Sir-induced toxicity (not shown). We also tested the and noninducing conditions. These data are shown in possibility that high levels of SIR protein could extend Table 4. We observed a profound effect on the mitotic telomere position effect into essential genes. RNA levels stability of the test chromosome, observing loss rates of of PKCl and SUC.2, genes tightly linked to the ends of 15-30% in SIR2 or SIR3 overexpressing strains, and a their chromosomes,were not influenced by SIR overex- loss rate of 90% in the strain expressing both SIR2 and pression (Figure 5 and data not shown).Therefore, SIR.?. we consider the possibility unlikely that chromosomal To determine if this loss of stability extended to au- genes become subject to telomere position effect as a thentic yeast chromosomes, we measured the loss rate result of SIR overexpression. of a markedchromosome V (HARTWELLand SMITH Finally, we examined the expression of RPLI6, a gene 1985) in diploid strain Y2279. In this strain SIR3in- that is positively regulated by binding of the Rap1 pro- duced toxicity was substantially greater than that for tein in wild-type cells. Since Sir3p and Sir4p interact cells overexpressing SR!?. Once again we observed a with Raplp, SIRgene overexpression might exerta spe- significant decrease in the stability of the test chromo- cific effect on genes regulated by Raplp. In this case, some. The SIR genes mediate telomere position effect we observed a twofold reduction in RNA levels regard- (APARICIO et dl. 1991), and the Sir3p and Sir4p proteins less of the SIR gene overexpressed. Since the same ef- may localize to the endsof chromosomes (GOLTAet nl. fect is observed in the SIR4 overexpressing strain, we 1996). As alterations in telomere metabolism are known conclude that this difference is not responsible for the to affect chromosome stability in yeast, it is possible that decrease in viability observed when SIR2 or SIR3 is over- the decrease in chromosome stability we observed was expressed. Therefore, we find no evidence for the pro- due to aberrant telomere function. To examine this posal that a reduction in transcription is responsible possibility we measured the stability of a circular chro- for the toxic effects of SIR gene overexpression. mosome derivative in a strain overexpressing SIR3. SIR overexpression decreases chromosome stability: Once again, we observed an increase in the loss rate of Sir proteins most likely manifest their effects through the test chromosome, indicating that theeffects of SIR an alteration of chromatin structure. Failing to observe overexpression are not solely due to an affect on te- an effect ofSIR overexpression on transcription, we lomeres. The increase in chromosome loss is correlated examined the effects of SIR overexpression on other with the reduction in plating efficiency in each case, fundamental processes of chromosome dynamics that and this reduction in mitotic stability islikely to be are influenced by chromatin. To investigate the effect sufficient to account for the lethaleffects ofSIR overex- of high levels of Sir proteins on the mitotic stability pression. 612 S. G. Holmes et al.

Eukaryotic cells have checkpoints that monitor the First, we observed that SZBinduced toxicity is sup- integrity of chromosomes and their fitness for segrega- pressed by deleting the SIR4 gene. These results are tion (HARTWELL and WEINERT1989; MURRAY1992). consistent with the observation that the two proteins Given the high rates of chromosome loss we investi- physically interact, and suggest that SIR3's function is gated whether SIR overexpression leads to a delay or dependent on the SIR4 protein. In contrast, the toxic lethality at a particular point of the cell cycle, which effects of SIR2 overexpression were not affected to the would indicate the activation of a checkpoint mecha- same degree in the Dsir4 strain, indicating that SIR2's nism. To look for activation of a checkpoint, we grew function is less dependent on Sir4p. cultures to log phase, induced high expression of the Our experiments revealed a previously uncharacter- SIR genes, then monitored cell morphology in the cul- ized interaction between SIR2 and SZR?. First, the two tures over a 24hr period. For each gene tested, the proteins act in synergy to promote inviability; second, proportion of cells in each phase of the cell cycle was the toxic effects induced by expressing SIR2 or SIR3 not changed in the induced culturewhen compared to individually are dependent on thepresence of the other uninduced controls (not shown),This suggests that the protein. Finally, we have shown that the two proteins defectin chromosome stability does not activate a can be co-immunoprecipitated. Although these experi- checkpoint mechanism. ments do not indicate whether SZR2 and SZR? associate with each other directly, they provide a framework for DISCUSSION bringing each of the Sir proteins to the silent mating Protein-protein interactionsand silencing: The inter- type loci. actions among thevarious Sir proteins, silencer binding Sir2p and Sir3p show an interesting interaction with factors, and histones have been explored extensively by the histone H4 N-terminus. High levelsof Sir3p are genetic and biochemical means. Recessive alleles of the synthetically lethal with the A414 H4 allele. Since this SZR? or SIR4 genes show unlinked noncomplementa- deletion does not interfere with the in vitro interaction tion with recessive alleles of SIRl and SIR2 (&NE and of Sir3p with H4, one interpretation of this result is HERSKOWITZ1987). We previously showed that high ex- that this deletion increases the ability of Sir3p to bind pression of SZR? can suppress the disruption of silenc- H4. This model is consistent with in vitro experiments: ing caused by overexpressing the SIR4 gene (-HALL binding of Sir3p is inhibited in vitro by specific point et al. 1987), which suggested an interaction between mutations at position 16; theability of Sir3p to bind H4 the two proteins that has been confirmed by assay in containing this point mutation in vitro is restored by the two hybrid system (MORETTI et al. 1994). The two- deleting positions 4- 14 ( HECHTet al. 1995). hybrid assay also demonstrated that Sir3p and Sir4p can Based on in vitro binding studies, Sir3p would not be associate with the Rap1 protein (MORETTIet al. 1994). expected to bind H4 containing the A419 or A423 Alleles of SZR? can suppress silencing defects caused by deletions. Consistent with this, the synthetic lethal phe- mutations in theRAP1 and histone H4 gene (JOHNSON notype caused by the A414 mutation is suppressed by et al. 1990; LIU and LUSTIG1996), while recombinant theadditional deletions; in these backgrounds, SZR3 Sir3p and Sir4p can bind to the N-terminal tails of his- overexpression is no more toxic than in wild-type cells. tones H3 and H4 in vitro (HECHTet al. 1995). Sir2p This suggests that the distal portion of the histone H4 and Sir3p bind to an affinity column containing the C- N-terminus may restrict the binding of proteins such as terminal half of Sir4p (MOAZEDand JOHNSON 1996). SZR3 and SIR4 and strengthens the model thatsilencing Finally, increasing the copy number of the SIRl gene involves a specific interaction between and SZR? and can suppress a variety of silencing defects, including histone H4. temperature-sensitive alleles of the SIR3 and SIR4 genes High levels of Sir2p also exhibit a synthetic pheno- (STONEet al. 1991), while Sirlp exhibits a two-hybrid type with deletions in the H4 N-terminus, but in this interaction with Orclp, a subunit of a silencer-binding case the interactions are not specific to a single H4 complex of proteins (ORC) that also binds yeast ARS allele. Sir2p fails to bind this region of H4 in vitro. This elements (TRIOLOand STERNCLANZ1996). suggests that SZR2 interacts with histones in aless direct We have investigated the functionsof the Sir proteins manner. Similar to the interaction between Sir3p and by determiningthe consequences of overexpressing the H4 A414 allele, overexpression of both SZR2 and them in yeast cells.We report here thatoverexpression SZR3leads to a synthetic lethality. One model to account of SZR2 or SIR? is toxic to yeast cells.The identification for this relationship is that SZR2 functions to increase of specific growth phenotypes as a consequenceof over- the accessibility of the histone H4 N-terminal tail to expressing the SIR2 or SZR3 gene has allowed us to SIW, and thus mimics the effect of the shorter (a4 describe a set of genetic interactions linking the Sir2, 14) H4 N-terminal deletion. Sir3, and Sir4 proteins and histone H4. Our results How might Sir2p act to increase access to histones? complement and extend the previously identified ge- One possibility is that Sir2p affects the acetylation levels netic and physical interactions among the SZR genes of histone H4 and the other histone proteins. We have and histones. previously shown that overexpression of the SIR2 gene Gene Overexpression SIR Gene 61 3 leads to a global decrease in the acetylation of histone and indicated the need for a dynamic equilibrium in molecules in the cell, and that the silent mating type the acetylation status of the lysine residues. We have loci are bound by chromatin that is underacetylated shown that SZR overexpression alters the acetylation of compared to the rest of the genome. Consistent with histones and might be predicted to lead to similar de- this model, we have shown that SIR3 overexpression, fects in genome integrity. Additional processes that may either alone orin combination with SZm, had no effect be sensitive to alterations in chromatin would include on histone acetylation (BRAUNSTEINet al. 1993). How- kinetochore function. The formation of an inappropri- ever, as we have shown here, overexpressing SIR3 in ate chromatin structure across centromeres could im- addition to SIR2 has a profound effect on cell viability. pair chromosome segregation and lead to nondisjunc- There are several ways in which a change in acetyla- tion. A third possibilitywould involve telomere tion may be involved in silencing. First, Sir2p may in- metabolism. Sir3p and Sir4p likely interact withte- duce an decrease in acetylation that is sufficient to in- lomeres through their association with Raplp, which ducetranscriptional repression; the Sir3 and Sir4 has multiple binding sites on each telomere. Deletions proteins may then be involved in preserving the unace- of SIR2 or SZR3 delocalize Raplp in the nucleus and tylated state. Second, a lack of acetylation due to Sir2p lead to small decreases in chromosome stability and activity may increase the accessibility of histone tails to telomere length (PALLADINOet al. 1993). Finally, Sir3p Sir3p and Sir4p, and these proteins may induce the has similarity to Orclp, part of the origin recognition formation of a repressive complex. A change in the complex (ORC) that is essential for DNA replication in acetylation state of a nucleosome has been shown to yeast cells. It has been proposed that Sir3p may substi- alter the binding of a protein factor to DNA in vitro tute for Orclp in ORC specifically at silencers (BELLPt (LEEet al. 1993). This model would predict that highly al. 1995). At artificially high levels Sir3p may compete acetylated histones may have reduced affinity for inter- Orclp out of ORC and interfere with the initiation of actions with SirSp and Sir4p. The in vitro experiments DNA replication. in which Sir3p and Sir4p were shown to bind the N- High levels of Sir2p and Sir3p induce a toxic effect terminal tails of histone H3 and H4 were performed without inducing transcriptional silencing. In addition, on unacetylated histones (HECHTet al. 1995); it is not these effects are observed in strains deleted for theSIR4 known what the influence of acetylation is on this in gene. This suggests that the Sir proteins may not func- vitro interaction.A final model would proposethat tion as a complex to accomplish a single task, but have Sir2p may increase access of histones to Sir3p by an independent functions of whichsilencing is the cumula- unknown mechanism, and a lack of acetylation may be tive result. Consistent with this, SZR2 and SZR3 overex- a consequence of silencing, rather than a cause. pression is more pronouncedin combination, butsome The nature of the SIR-induced toxic effect: We find toxicity remains in the absence of the other. In addi- no evidence to support the hypothesis that SIR overex- tion, the SIR2 gene has been shown to participate in pression leads to widespread transcriptional silencing. suppressing recombination at therDNA repeats, a func- First, SIR2 or SIR3 overexpression remains toxic in tion that does not involve any of the other SZR genes strains lacking SZR4, or in strains in which the histone (GOTTLIEBand ESPOSITO1989). Our results are consis- H4 N-terminal domain is absent, backgrounds that do tent with a model in which the Sir proteins have inde- not support repression at the silent mating type loci or pendent activities yet cooperate in a complex to modify telomeres. Second, the steady-state levels ofa variety of chromatin structure. transcripts is unchanged upon induction of high levels of the SIR proteins. This is consistent with the high LITERATURE CITED specificity of silencing in wild-type cells, and suggests that the silencer sequences provide strict controls on APARIC:IO, 0.M., B. 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