Swapping the Gene-Specific and Regional Silencing Specificities of the Hst1 and Sir2 Histone...

MOLECULAR AND CELLULAR BIOLOGY, Apr. 2007, p. 2466–2475 Vol. 27, No. 70270-7306/07/$08.00�0 doi:10.1128/MCB.01641-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Swapping the Gene-Specific and Regional Silencing Specificities of theHst1 and Sir2 Histone Deacetylases�

Janet Mead,1 Ron McCord,1† Laura Youngster,1 Mandakini Sharma,2‡Marc R. Gartenberg,2 and Andrew K. Vershon1*

Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854,1 andDepartment of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey,

Piscataway, New Jersey 088542

Received 2 September 2006/Returned for modification 9 November 2006/Accepted 8 January 2007

Sir2 and Hst1 are NAD�-dependent histone deacetylases of budding yeast that are related by strongsequence similarity. Nevertheless, the two proteins promote two mechanistically distinct forms of gene repres-sion. Hst1 interacts with Rfm1 and Sum1 to repress the transcription of specific middle-sporulation genes. Sir2interacts with Sir3 and Sir4 to silence genes contained within the silent-mating-type loci and telomerechromosomal regions. To identify the determinants of gene-specific versus regional repression, we created aseries of Hst1::Sir2 hybrids. Our analysis yielded two dual-specificity chimeras that were able to perform bothregional and gene-specific repression. Regional silencing by the chimeras required Sir3 and Sir4, whereasgene-specific repression required Rfm1 and Sum1. Our findings demonstrate that the nonconserved N-terminal region and two amino acids within the enzymatic core domain account for cofactor specificity andproper targeting of these proteins. These results suggest that the differences in the silencing and repressionfunctions of Sir2 and Hst1 may not be due to differences in enzymatic activities of the proteins but rather maybe the result of distinct cofactor specificities.

The Sirtuin family of proteins is a large class of NAD�-dependent deacetylases with a highly conserved enzymatic coredomain (9). These enzymes are found in all kingdoms of life,and many organisms contain multiple Sirtuin family members(13). Although Sirtuin homologs have NAD�-dependentdeacetylase activities, their cellular functions differ as a resultof their subcellular localization and specific protein-proteininteractions (15, 32). For example, although human SIRT2deacetylates histone H4 during mitosis, it has also been foundin the cytoplasm and appears to function as a tubulin deacety-lase required for exit from mitosis (11, 30, 32, 46). The SIRT1protein deacetylates and activates acetyl-coenzyme A syn-thetase, as well as regulating transcription factors, such as p53,FOXO, and human immunodeficiency virus Tat (16, 23, 31, 45,47, 48). Determining how these proteins form complexes withtheir cofactors and recognize their targets is therefore key tounderstanding how these proteins function in the cell.

The yeast Saccharomyces cerevisiae has five members of theSirtuin family, HST1 to HST4 and SIR2, the founding memberof the family (3, 10). Similar to the homologs in higher eu-karyotes, the yeast proteins vary in their cellular localizationsand perhaps substrate specificities (32, 40). Sir2 functions as aNAD�-dependent histone deacetylase and is involved in mod-ifying chromatin structure and functioning as a regional tran-scriptional silencer (19, 21, 43). Silencing in yeast, like hetero-chromatin in higher eukaryotes, renders large regions of the

chromosome transcriptionally inactive in a non-gene-specificmanner. These regions include the silenced mating type loci,the subtelomeric domains, and the ribosomal DNA (rDNA)locus (39). The histone tails within these regions are hy-poacetylated, and the DNA is generally refractory to modifyingenzymes. Although Sir2 is required for silencing at all three ofthese loci, the protein forms two distinct complexes with non-overlapping sets of binding partners (44). One complex is re-quired for silencing the telomeres and the mating type loci andincludes the Sir3 and Sir4 silencing cofactors (17, 28). Thesecond complex is required for silencing the rDNA loci andincludes the Net1 and Cdc14 proteins (18, 38, 41). Mutations inthese cofactors result in mislocalization of Sir2 and loss ofsilencing at the respective loci. These studies indicate that theinteraction of Sir2 with these cofactors is critical to properlocalization and silencing.

Hst1 is the closest homolog of Sir2 and, like Sir2, is aNAD�-dependent deacetylase and a component of at least twodistinct complexes (22, 34). Hst1 is tethered to the DNA-binding protein Sum1 through interactions with Rfm1 to forma complex that represses middle-sporulation genes during veg-etative growth (26, 35, 42, 49). Hst1 is also a component of theSet3c complex, which appears to repress transcription of mei-osis-specific genes during early meiosis (34). The functionalsimilarity between Hst1 and Sir2 is further demonstrated bythe observation that in high copy numbers, the two proteins areable to partially function in place of each other. Overexpres-sion of Sir2 can partially suppress hst1 defects in repression ofmiddle-sporulation genes, and overexpression of Hst1 can par-tially restore silencing at HMR in the absence of Sir2 (3, 10,49). However, under normal levels of expression, neither pro-tein functions in place of the other. These proteins thereforehave distinct regulatory activities: Sir2 functions as a transcrip-

* Corresponding author. Mailing address: Waksman Institute, 190Frelinghuysen Rd., Piscataway, NJ 08854-8020. Phone: (732) 445-2905.Fax: (732) 445-5735. E-mail: [email protected].

† Present address: Department of Medicine, Stanford University,Palo Alto, CA 94305.

‡ Present address: Genentech, South San Francisco, CA 94080.� Published ahead of print on 22 January 2007.

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tional silencer of relatively large regions of the genome, whileHst1 functions as a transcriptional repressor, acting locally at aspecific set of promoters. In this paper, we investigate themechanism through which these highly conserved proteinshave distinct functions in the cell.

Although Sir2 and Hst1 share strong sequence similaritythroughout the enzymatic cores of the proteins, their N terminiare considerably more divergent (Fig. 1). We show that thisdifference accounts, in part, for the specificity of cofactor in-teractions by Hst1 and Sir2, and this, in turn, accounts fordifferences between Sir2-mediated silencing and Hst1-medi-ated gene-specific repression mechanisms. Interestingly, wehave found that relatively subtle differences in two amino acidswithin the catalytic cores of the proteins also contribute tocofactor specificity. These findings provide insight into howother members of the Sirtuin family may discriminate betweendifferent sets of cofactors and have different regulatory roles inthe cell.

MATERIALS AND METHODS

Strains and plasmids. The yeast strains used in this study are shown in Table1. To construct null mutations for each gene of interest in the desired strain

FIG. 1. Sequence similarity of Sir2 and Hst1. Amino acid alignment of the yeast Sir2 and Hst1 proteins. Identical residues are shaded in gray.Endpoints of the internal deletions constructed in Hst1 are shown with arrows above the sequence. Junctions of the Hst1::Sir2 chimeras indicatingthe Hst1 residues that were replaced with the corresponding regions of Sir2 are shown with arrows below the sequence. The asterisks indicatepositions in which residues in Sir2 were replaced with the amino acids at the corresponding positions in Hst1.

TABLE 1. Yeast strains

Strain Genotype Source orreference

JXY5 MATa ade2-1 trp1-1 his3-11,15 can1-100ura3-1 leu2-3,112 hst1�::kanMX4

49

JXY20 JXY5 with sir2::TRP1 49JMY055 JXY20 with sir3�::HIS3 This studyMPY13 W303-1B with rfm1�::kanMX4RMY8 W303-1B with rfm1�::kanMX4

hst1�::kanMX26

LPY2447 MAT� his3�200 leu2�1 ura3-52sir2�2::HIS3 RDN1::Ty1-mURA3 (S6)

37

LPY3923 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 sir2�::HIS3hmr�::TRP1

37

JMY047 LPY3923 with rfm1�::kanMX4 This studyLPY1953 MATa his3�200 leu2�1 lys2�202

trp1�63 ura3-52 sir2�2::TRP1ADH4::URA3-TEL

37

JMY049 LPY1953 with sir3�::kanMX4 This studyJMY053 LPY1953 with sir4�::kanMX4 This studyJMY058 LPY1953 with rfm1�::kanMX4 This studyTHC13 MAT� ade2-1 can1-100 his3-11,15 leu2-

3,112 trp1-1 ura3-1 sir3�::HIS3JMY045 THC13 with hst1�::KanMX This study

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backgrounds, we obtained the diploid KanMX null mutant strains from ResearchGenetics. Primers were designed to generate a fragment via PCR that includedthe KanMX replacement of the gene of interest and 500 bp of homology up-stream and downstream. These fragments were then transformed into the vari-ous strain backgrounds, and the integration of the KanMX null mutation wasverified using primers internal and external to the transformed fragment.

Plasmid pJX43 is a lacZ transcription reporter plasmid containing a promoterthat is repressed by the SMK1 middle sporulation element (MSE) (33). PlasmidpRAM29 contains a V5-epitope-tagged version of HST1 expressed from its ownpromoter on a 2�m LEU2 vector (26). Plasmid pRAM27 contains SIR2 ex-pressed from its own promoter on a 2�m LEU2 vector (26). Amino acid substi-tutions in HST1 and SIR2 were introduced by site-directed mutagenesis using theStratagene QuikChange mutagenesis kit. HST1::SIR2 chimeras were created bygenerating PCR fragments with regions of SIR2 and cloning those regions intothe corresponding homologous regions within HST1 on plasmid pRAM29 usingeither the appropriate restriction sites or gapped plasmid repair (29). The sir2mutants were cloned into pJM491, which contains a V5 epitope tag at the Cterminus of the protein. A complete list of the chimera and point mutantplasmids used in this study is available upon request. The SUM1-myc (pMP208)and RFM1-HA (pDI14) plasmid constructs that were used in the coimmunopre-cipitation (co-IP) experiments were previously described (26).

Transcription and silencing assays. MSE-dependent repression activity wasassayed by measuring �-galactosidase expression from the MSE-lacZ transcrip-tion reporter, pJX43. Quantitative liquid �-galactosidase activity assays wereperformed as described previously (14). To measure the level of silencing by theHST1 or SIR2 constructs at the HM loci, transformants of strain LPY3923 and itsderivatives were grown overnight at 30°C, diluted to a starting A600 of 1.0, seriallyfivefold diluted, and plated on selective media as described previously (37). ForrDNA- and telomeric-silencing assays, transformants of strains LPY2447 andLPY1953 and their derivatives were diluted to a starting A600 of 4.0 for the rDNAassays and 2.5 for the telomeric assays. Cultures were then serially fivefolddiluted and plated on the appropriate selective medium, SD-Trp or 5-fluoro-orotic acid (5-FOA), respectively.

Western blot analysis and co-IP experiments. Yeast lysates for Western blotanalysis were prepared by washing cells once in 1 ml cold water plus 0.2 mMphenylmethylsulfonyl fluoride. The cells were resuspended in 1 ml cold 0.2 mMphenylmethylsulfonyl fluoride in water plus 150 �l cold 2 N NaOH, 8% 2-mer-captoethanol. Following a 10-min incubation on ice, the proteins were precipi-tated with 150 �l cold 50% trichloroacetic acid on ice for 10 min. The proteinswere pelleted by centrifugation at 4°C, washed twice with 1 ml cold acetone, andbriefly dried under vacuum. The pellet was resuspended in 100 �l sample buffer(0.1 M Tris, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 200 mM 2-mer-captoethanol, 25 mM Tris base, 0.1% bromophenol blue). The proteins wereseparated on an 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresisgel; transferred to a nitrocellulose membrane; probed with antibodies specific foreither the Myc epitope (Babco/Covance), the hemagglutinin (HA) epitope(Boehringer Manheim), or the V5 epitope (Invitrogen); and detected using ECLWestern blot detection (Amersham Pharmacia Biotech). Rabbit polyclonal an-tibody to Sir4 was prepared by Cocalico Biologicals with affinity-purified bacte-rially expressed His-tagged full-length Sir4 protein. Co-IP experiments wereperformed as described previously (26).

RESULTS

Deletion analysis of Hst1. Although Hst1 shows significantsequence similarity to regions of Sir2 that correspond to theenzymatic core of the protein, the N-terminal and C-terminalregions are more divergent (Fig. 1). Mutational analysis of Sir2has shown that some of these nonconserved regions are essen-tial for Sir2 function and, in part, specify interactions with itsdifferent cofactors (5, 7, 37). To determine which regions ofHst1 are required for its function, we constructed a series ofdeletions in the protein and assayed for their abilities to re-press transcription of middle-sporulation genes and to interactwith the Sum1-Rfm1 complex (Fig. 2). Many of the deletionswere unable to repress transcription of an MSE-regulated pro-moter in an hst1� strain (Fig. 2B). However, two of the dele-tions repressed transcription almost as well as wild-type Hst1.One of these deletions, Hst1�8-54, removed the nonconserved

N-terminal region of the protein. Despite the ability to repressreporter genes, the truncated protein was less stable, since thelevel of expression was decreased and multiple fragmentswere present, presumably due to partial proteolysis (Fig. 2C).The other deletion in Hst1 that showed partial repression,

FIG. 2. Deletion analysis of Hst1. (A) Schematic of the deletionsthat were constructed in Hst1. The top line shows the conservation ofdifferent domains of Hst1 with Sir2. The numbers above the lineindicate the endpoints of the different regions of the protein, and thepercentages are the sequence identity with Sir2. Each mutant wasnamed for the residues that were deleted. (B) MSE repression assaysof wild-type and Hst1 deletions. The indicated mutants on high-copy-number 2�m plasmids were tested for the ability to repress transcrip-tion of an MSE-regulated promoter driving lacZ expression in JXY5,an hst1� strain. The bars indicate the average �-galactosidase activitiesof five independent isolates, and the standard deviations are shown bythe error bars. (C) (Top) Western blot with antibody to the V5 epitopeto monitor the levels of expression of tagged (lane 1) and untagged(lane 2) wild-type Hst1 or the indicated V5-tagged Hst1 deletion mu-tants (lanes 3 to 9). (Bottom) Western blot with Myc antibody to detectthe presence of Myc-tagged Sum1 protein that coimmunoprecipitatedwith tagged or untagged wild-type Hst1 or the indicated mutants.

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Hst1�350-371, removed a portion of the enzymatic core of theprotein. This region of the core domain is not strongly con-served between Hst1 and Sir2 and is not present in many of theSir2 family members (Fig. 1).

We next performed co-IP experiments to determine whichof the Hst1 deletion proteins were able to interact with theSum1-Rfm1 complex. As expected from the results of the re-pression assays, Sum1 coimmunoprecipitated with the Hst1�8-54

and Hst1�350-371 deletions, indicating that these regions are notessential for interactions with the Sum1-Rfm1 complex (Fig.2C). The Sum1 protein was able to weakly interact with theHst1�54-81, Hst1�164-327, and Hst1�479-502 mutants and failed tointeract with the Hst1�118-158 and Hst1�329-474 mutants, indi-cating that residues within these regions are required for fullinteraction with the Sum1-Rfm1 complex.

Hst1::Sir2 chimeras show the N-terminal region is requiredfor silencing. To distinguish between regions within Hst1 thatare required for gene-specific repression and regions withinSir2 that are required for regional silencing, we created a seriesof Hst1::Sir2 chimeras by swapping regions of Sir2 into thecorresponding regions of Hst1 (Fig. 3A). Each chimera wasthen tested for the ability to complement defects in silencing insir2 strains that contain reporter genes at the rDNA or HMRmating type locus or telomeres. As shown by the strong growthon media lacking uracil, none of the chimeras silence theURA3 marker at the rDNA locus (Fig. 3B). Each of thesechimera proteins may therefore lack a region of Sir2 that isrequired to form a complex with Cdc14 and Net1 and to silencethe rDNA loci.

Most of the Hst1::Sir2 chimeras also failed to silence re-porter genes at HMR and telomeres (Fig. 3C and D). However,the Hst1::Sir212-155 chimera, in which the N-terminal region ofHst1 (residues 12 to 155) is replaced with the correspondingregion of Sir2, showed a significant decrease in growth onmedia lacking tryptophan, indicating that the TRP1 reportergene at HMR was silenced by this chimera (Fig. 3C). We alsofound that Hst1::Sir212-155 was the only chimera that permittedgrowth of a sir2 strain with a telomeric URA3 marker on5-FOA, a drug that kills cells that express URA3 (Fig. 3D).Silencing by this chimera was not dependent upon the genedosage of the chimera, as it silenced equally well when ex-

FIG. 3. Transcriptional silencing by Hst1::Sir2 chimeras. (A) Aschematic representation of the Hst1::Sir2 chimeras is shown. The topline shows a schematic of Hst1 with numbers indicating the percentidentity of the different regions of the protein with Sir2. The rowsbelow show the different Hst1::Sir2 chimeras, in which the numbers

indicate the positions of the residues in Hst1 that were replaced withthe corresponding residues in Sir2. (B) High-copy-number (2�m) plas-mids containing either wild-type SIR2, HST1, or the indicatedHST1::SIR2 chimeras were transformed into strain LPY2447 (sir2rDNA::URA3), and normalized serial fivefold dilutions of the cultureswere spotted on plates with (�Ura) and without (�Ura) uracil. Failureto grow on �Ura medium indicates silencing of the rDNA loci. (C)Wild-type and chimera constructs were transformed in strain LPY3923(sir2 hmr::TRP1), and normalized serial dilutions of the cultures werespotted on media with (�Trp) and without (�Trp) tryptophan. Failureto grow on �Trp medium indicates silencing of the HMR loci. (D)Normalized cultures of the indicated transformants of strain LPY1953(sir2 TEL::URA3) were serially diluted and spotted on media withand without the drug 5-FOA. 5-FOA prevents growth of the cellsthat express URA3. The ability to grow on the 5-FOA plate indicatessilencing of the telomeric loci in the cell. (E) Low-copy-number(CEN) plasmids containing either wild-type SIR2, HST1, or theHST1::SIR212-155 chimera were transformed into strain LPY3923and assayed as for panel C.



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pressed from both high- and low-copy-number vectors (Fig. 3Eand data not shown), which indicates that the Hst1::Sir212-155

chimera is able to silence the telomere and the mating type locialmost as well as Sir2. Since this chimera is able to silenceHMR and telomeres but not the rDNA locus, it supports pre-vious observations that different regions of Sir2 are involved inthe formation of the Sir3-Sir4 and RENT complexes (7, 17).Since Sirtuins may form oligomers, we also assayed silencing inan hst1� sir2� double-mutant strain (6). Our results in thedouble-mutant background were indistinguishable from thoseperformed in the sir2� strain, indicating that the Hst1::Sir212-155

chimera silences through a Sir2-like mechanism rather thanthrough repression involving Hst1.

Recently, it was shown that Sum1 binds at the HML lociand has a role in DNA replication (20). Since theHst1::Sir212-155 chimera contains large regions of Hst1, it ispossible that this construct was able to bypass the require-ment for the Sir3 and Sir4 silencing cofactors through re-cruitment by Sum1 and Rfm1 bound to the silenced regions.To determine if Sir3 and Sir4 were required for silencing bythis chimera, we transformed sir2 sir3 and sir2 sir4 double-mutant strains with SIR2 or the HST1::SIR212-155 chimeraand assayed for silencing. We found that the chimera failedto silence the telomeric reporter gene, indicating that bothSir3 and Sir4 are required for silencing by the chimera (Fig.4). Conversely, the Hst1::Sir212-155 chimera was able to si-

lence as well as Sir2 in a strain lacking both SIR2 and RFM1.Thus, Rfm1 is not required for silencing, strongly suggestingthat the chimera does not silence the HML loci or telomereregions through a pathway that involves Sum1.

The ability of the Hst1::Sir212-155 chimera to complementsir2 strains but not sir2 strains lacking sir3 or sir4 indicatesthat it acts in the normal Sir pathway and may form acomplex with Sir3 and Sir4. To verify this model, co-IPexperiments were performed in which the tagged Hst1::Sir212-155

chimera, Sir2, or Hst1 was immunoprecipitated, and the pelletswere then assayed by Western blotting for the presence of Sir4.As expected, Sir4 was not present in the immunoprecipitatedpellet of the untagged Sir2 lysate but was strongly present inthe immunoprecipitate of the V5-tagged Sir2 lysate (Fig. 5,lanes 3 and 2). Importantly, while wild-type Hst1 was unable toimmunoprecipitate Sir4, the Hst1::Sir212-155 chimera inter-acted quite well with Sir4 (Fig. 5, lanes 1 and 4). This suggeststhat residues within the 12-to-198 region of Sir2 are requiredfor association with the Sir3-Sir4 silencer complex and that thedifferences between Sir2 and Hst1 in this region are importantfor targeting Sir2 to silence the telomeres and mating type loci.

Hst1::Sir2 chimeras define a region in the enzymatic core ofHst1 that is required for MSE-mediated repression. The re-sults described above show that swapping the N-terminal re-gion of Sir2 onto Hst1 enabled the protein to function inregional silencing of chromosomal domains. One appealingmodel is that the nonconserved N-terminal regions of Sir2 andHst1 specify the interactions with the Sir3-Sir4 and Sum1-Rfm1 complexes, respectively. This model would predict thatthe Hst1::Sir212-155 chimera would not strongly complementdefects in MSE-dependent repression in an hst1 mutant. Totest this model, the chimeras were assayed for the ability torepress transcription of an MSE-regulated promoter in an hst1strain. Surprisingly, the Hst1::Sir212-155 N-terminal chimeraand most of the other chimeras functioned as well as wild-typeHst1 in low or high copy numbers and in either hst1� or hst1�sir2� strains (Fig. 6A and data not shown). However, a chimerain which a region of the core domain was replaced by thecorresponding region in Sir2 (Hst1::Sir2266-325) was unable to

FIG. 4. Silencing by the Hat1::Sir212-155 chimera is dependent onSir3 and Sir4 but independent of Rfm1. Plasmids containing wild-typeHST1 or SIR2 or the Hat1::Sir212-155 chimera, along with a blankvector, were transformed in strain (A) LPY1953 (sir2 TEL::URA3),(B) JMY049 (LPY1953 with sir3�::KanMX), (C) JMY053 (LPY1953with sir4�::KanMX), and (D) JMY058 (LPY1953 with rfm1�::KanMX),and normalized fivefold serial dilutions of cultures were spotted on plateswith and without 5-FOA. Growth on 5-FOA indicates silencing of thetelomere loci.

FIG. 5. The Hat1::Sir212-155 chimera coimmunoprecipitates withSir4. Lysates from transformants of strain JXY20 (sir2� hst1�) con-taining plasmids with V5-tagged wild-type Hst1 (lane 1), V5-taggedSir2 (lane 2), untagged Sir2 (lane 3), or V5-tagged Hat1::Sir212-155chimera (lane 4) were immunoprecipitated with antibody to the V5epitope. The top panel shows a Western blot with antibody to the V5epitope to monitor the levels of expression of V5-tagged proteins incrude lysates. The bottom panel shows a Western blot of the immu-noprecipitated pellets using polyclonal antibodies to Sir4.



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complement hst1 defects in repression in either low or highcopy numbers (Fig. 6A and data not shown). This suggests thatresidues 266 to 325 of Hst1 are required for MSE-mediatedrepression.

Since the chimeras contained large portions of Sir2, it waspossible that repression by these chimeras could be indepen-dent of Rfm1 and instead depend on Sir2 cofactors, such asSir3 or Sir4. However, with the exception of the Hst1::Sir2266-325

chimera, all of the constructs were able to fully complementrepression of the reporter in hst1 sir3 and hst1 sir4 strains (Fig.6B and data not shown). Moreover, none of the chimeras wereable to repress transcription of the reporter in an rfm1 strain(Fig. 6B). These data suggest that, with the exception ofHst1::Sir2266-325, all of these chimeras interact with the Sum1-Rfm1 complex to repress MSE-regulated promoters. This re-sult, along with the HM and telomeric-silencing assays, sug-gests that the Hst1::Sir212-155 chimera is a dual-specificityprotein that functions as both a gene-specific repressor and aregional transcriptional silencer.

To test if the chimeras are able to physically interact withthe Sum1-Rfm1 complex, we performed co-IP experimentsin which the V5 epitope-tagged Hst1::Sir2 chimeras wereimmunoprecipitated using a V5 antibody, and the pelletswere then assayed by Western blotting for the presence ofSum1. Western blot analysis of the lysates with V5 antibodyshowed that all of the chimeras were present at levels com-parable to those of the wild-type protein (Fig. 6C, top). Asexpected from the complementation results, most of theHst1::Sir2 chimeras were able to interact with Sum1 (Fig.6C, bottom). However, Sum1 failed to immunoprecipitatewith the Hst1::Sir2266-325 chimera. This suggests that resi-dues within the 266-to-325 region of Hst1 are required forassociation with the Sum1-Rfm1 complex and that the dif-ferences between Hst1 and Sir2 in this region are importantfor targeting Hst1 to repress middle-sporulation genes.

Residues in the Zn ribbon region of Hst1 are important forspecifying interactions with Rfm1. The results describedabove show that a region in the conserved enzymatic core ofthe Hst1 protein (residues 266 to 325) is important fordetermining the specificity of Hst1 interactions with theSum1-Rfm1 complex. The crystal structures of the enzy-matic domains of several members of the Sir2 family ofNAD�-dependent deacetylases have been solved (2, 4, 12,27, 50). When mapped to the human SIRT2 crystal structure(PDB accession no. 1J8F), the 266-to-325 region of Hst1encompasses the first two cysteines of the conserved zincribbon motif, along with a region on the back side of theprotein, away from the NAD� binding pocket and active site(Fig. 7A) (12). Hst1 and Sir2 have strong sequence similarityin this region of the protein, with only eight amino aciddifferences (Fig. 1). The model of the human SIRT2 crystalstructure suggests that several of these residues are likely tobe exposed to solvent in the Hst1 and Sir2 proteins. It ispossible that these residues play important roles in targetingHst1 to Rfm1, instead of to Sir4. Therefore, by swappingthese residues in Sir2 with the amino acids found at thecorresponding positions in Hst1, we hypothesized that itmight be possible to target Sir2 to Rfm1. To test this model,we engineered amino acid substitutions in Sir2 and assayedthe affects of these changes on Sir2-mediated repression of

FIG. 6. MSE-mediated repression by the Hst1::Sir2 chimeras.(A) Plasmids containing either wild-type SIR2, HST1, or the indi-cated HST1::SIR2 chimeras were transformed into strain JXY5(hst1�::KanMX) and assayed for the ability to repress the MSE-regulated promoter on plasmid pJX43. The bars indicate the aver-age levels of �-galactosidase activity of five independent transfor-mants, and the standard deviations are shown by the error bars.(B) Wild-type SIR2, HST1, or the indicated HST1::SIR2 chimeraswere transformed into strain RMY8 (hst1�::KanMX rfm1�::KanMX)(hatched bars) or JMY045 (hst1�::KanMX sir3�::HIS3) (solid bars)and assayed for the ability to repress the MSE-regulated promoter asin panel A. (C) The upper blot shows a Western blot analysis withantibody to the V5 epitope to compare the levels of expression oftagged (lane 1) and untagged (lane 2) wild-type Hst1 and the indicatedHst1::Sir2 chimeras (lanes 3 to 7). Extracts from these strains wereimmunoprecipitated with antibodies specific for the V5 epitope, andthe pellets were assayed by Western blot analysis with Myc antibodiesto detect the presence of Myc epitope-tagged Sum1 in the immuno-precipitate (bottom).



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the MSE-regulated promoter in an hst1 mutant background.Sir2 mutants with amino acid substitutions of residues K320,I321, M334, S356, T357, and T371, alone and in combina-tion (Sir2-6H), were unable to repress the MSE-regulatedpromoter any better than wild type Sir2 (Fig. 7B and datanot shown). However, the Sir2-2H mutant, containing theN378Q and L379I amino acid substitutions, producedroughly the same level of repression of the reporter aswild-type Hst1. This result supports the model in whichdifferences in this region of Hst1 and Sir2 are important fordistinguishing between their different cofactors. Repressionof the MSE-regulated promoter by the Sir2-2H mutant didnot require Sir3 or Sir4 but did require Rfm1 (Fig. 7B anddata not shown). This result suggests that this double aminoacid substitution was sufficient to increase the affinity of Sir2interactions with Rfm1.

Repression of the MSE-regulated promoter by the Sir2-2Hmutant suggests that residues N378 and L379 in Sir2 are im-portant for specifying interactions with Rfm1. Residues at thecorresponding positions in Hst1, Q324 and I325, were there-fore likely to be important for interaction with Rfm1. To test

this model, we swapped the amino acids at these positions inHst1 with those found at the corresponding positions in Sir2and measured the level of Hst1-dependent repression of areporter promoter. The Hst1-2S mutant, containing theQ324N and I325L substitutions, showed decreased levels ofrepression, suggesting that the substitution of these residuesaffects interactions with Rfm1 (Fig. 7B).

To test the model that the Sir2-2H mutant interacts withRfm1, co-IP assays with Rfm1 were performed with wild-type Hst1, Sir2, and the Sir2-2H mutant. The Sir2 proteinwas unable to coimmunoprecipitate with HA-tagged Rfm1in this assay (Fig. 7C). In contrast, the Sir2-2H mutant wasclearly able to interact with Rfm1. This result suggests thatthese relatively conserved differences between Sir2 and Hst1enable these proteins to discriminate in their interactionswith Rfm1.

None of the amino acid substitutions that we constructed inSir2 affected silencing at the HMR, telomere, or rDNA loci(data not shown). This result suggests that these mutants havenot lost the ability to interact with either the Sir3-Sir4 or theNet1-Cdc14 complexes to silence the different loci. It therefore

FIG. 7. Residues in the 266-to-325 region of Hst1 specify interactions with Rfm1. (A) A space-filling model of the human SIRT2 protein thatwas derived from the crystal structure is shown (12). This view is of the back side of the protein, away from the NAD� binding pocket and thepredicted binding site of the histone tail. The region corresponding to Hst1 residues 266 to 325 (Sir2 residues 314 to 379) is shown in yellow. Theresidues in green are the positions in the Sir2-6H mutant that were replaced with the corresponding amino acids in Hst1. The residues in redcorrespond to positions N378 and L379 in Sir2 that were replaced in the Sir2-2H mutant. (B) The level of repression of an MSE-regulatedpromoter by Hst1, Sir2, Sir2-6H (K320N, I321M, M334D, S356D, T357P, and T371S), Sir2-2H (N378Q and L379I), and Hst1-2S (Q324N andI325L) mutants on low-copy-number CEN plasmids were assayed as described in the legend to Fig. 2B. The assays for the six bars on the left wereperformed in strain JXY5 (hst1�), while the assays for the three bars on the right were performed in strain RMY8 (hst1� rfm1�). (C) A co-IP assayshowed that Sir2-2H interacts with Rfm1. Strain JXY5 (hst1�::KanMX) was cotransformed with high-copy-number 2�m plasmids expressingRFM1-HA and HST1 (lanes 1 and 4), SIR2 (lanes 2 and 5), and SIR2-2H (lanes 3 and 6). A Western blot of the crude lysates (lanes 1 to 3) andRfm1-immunoprecipitated samples (lanes 4 to 6) was probed with polyclonal antibody to the V5 epitope.



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appears that the Sir2-2H mutant is able to interact with Sir2and Hst1 cofactors and function as both a regional transcrip-tional silencer and a gene-specific repressor.

DISCUSSION

The hallmark of the Sirtuin family of NAD�-dependentdeacetylase enzymes is a highly conserved enzymatic core ofapproximately 260 amino acids. Interestingly, while the bacte-rial homologs consist only of the conserved enzymatic coredomain, homologs in higher eukaryotes have amino- and car-boxyl-terminal extensions that are highly divergent amongmembers of the family. It is likely that these nonconservedregions contribute to the specific functions of many proteins inthe Sirtuin family. The N-terminal regions of these proteins areof particular interest because of their potential regulatoryroles. For example, both Sir2 and Hst2 form homotrimers insolution, and it has been shown that the amino-terminal me-thionine of one Hst2 subunit interacts with the active site inanother monomer in the homotrimer (6, 50). Similarly, humanSIRT3 is catalytically inactive as a full-length protein and onlybecomes active after cleavage of the first 100 amino acidsfollowing import into the mitochondria (36). Mutational anal-ysis of the yeast Sir2 protein showed the importance of theN-terminal domain in regional transcriptional silencing (5, 8,37). It was therefore likely that the nonconserved N-terminalextensions of Hst1 and Sir2 contribute to the cofactor interac-tions and target specificities of the two proteins. This model issupported by our finding that a Hst1 chimera protein contain-ing the N-terminal region of Sir2, Hst1::Sir212-155, is able tointeract with Sir4 and silence the mating loci and telomeres ina sir2 strain. Interestingly, the Hst1::Sir212-155 chimera is stillable to interact with Rfm1 and to function as a repressor ofmiddle-sporulation genes. This result indicates that althoughamino acid differences in this region of Hst1 and Sir2 areimportant for specifying interactions with Sir2 cofactors, thetwo proteins contain sufficient sequence similarity in this re-gion to enable both of them to productively interact withRfm1.

The analysis of the Hst1::Sir212-155 chimera suggested thatdifferences in other regions of Sir2 and Hst1 must also beimportant for specifying interactions with Rfm1. In support ofthis model, the Hst1::Sir2266-325 chimera failed to interact withRfm1 or to repress an MSE-regulated reporter. This regionlies on the surface of the catalytic domain that is opposite fromthe NAD� binding pocket and likely provides a suitable inter-face for cofactor interactions. This region incorporates the firsttwo cysteines of the Zn� ribbon motif, and the importance ofthis region was demonstrated by mutational analysis of thesecysteine residues in Sir2, which disrupt both the enzymaticactivity and cofactor interactions of the protein (27, 37). Inter-estingly, we found that swapping only two Sir2 residues withthe corresponding Hst1 residues (N378Q and L379I) enabledthe Sir2 protein to interact with Rfm1 and repress MSE-reg-ulated genes. Although the cysteine residues are highly con-served, residues directly surrounding this motif are relativelydivergent among different members of the Sirtuin family. Thecrystal structures of several different Sirtuin proteins showedthat there are significant differences in the positions of theZn�-ribbon motif relative to the Rossmann fold when bound

by ligand (12, 25, 27, 51, 52). The sequence conservation of theCys residues, along with the differences in conformation, sug-gests that there may be an important mechanistic or structuralrole for this region in Sirtuin proteins. Our data suggest thatdifferences within this region may be important for specifyinginteractions with distinct sets of cofactors.

The Hst1::Sir212-155 chimera and the Sir2-2H mutant areable to function as both regional silencers of the HM loci andtelomere- and gene-specific repressors of middle-sporulationgenes. These proteins have both gained the ability to interactwith different cofactor complexes and are therefore dually spe-cific in function. It was possible that these proteins formed asingle complex that was able to both silence and repress. Thismodel was enticing, because studies of the dominant SUM1-1mutant have shown that it is able to bypass the requirement forSir2 for silencing at HMR by directly interacting with the ORCcomplex and recruiting Rfm1 and Hst1 to this region (24, 26,35). However, we found that the Hat1::Sir212-155 chimera andthe Sir2-2H mutant required Sir3 and Sir4 to silence the HMloci and telomeres and Rfm1 and Sum1 to repress the middle-sporulation genes. This result suggests that the Hat1::Sir212-155

chimera and the Sir2-2H mutant form two separate complexesin the cell: one in complex with Sir3 and Sir4 to silence the HMloci and telomeres and another in complex with Sum1 andRfm1 to repress middle-sporulation genes. In support of thismodel, we have been unable to observe by chromatin im-munoprecipitation assays Sir4 binding to promoters re-pressed by the Sum1-Rfm1-Hst1 complex or Rfm1 binding totelomere regions silenced by the Sir2-Sir3-Sir4 complex in theHat1::Sir212-155 chimera and Sir2-2H mutant strains (data notshown). In addition, while we observe that both Sir4 and Sum1or Rfm1 interact with the Hst1::Sir212-155 chimera and theSir2-2H mutant, we were unable to coimmunoprecipitate Sir4and Rfm1 with each other in these same strains, suggestingthat Sir4 and Rfm1 do not simultaneously interact in a complexwith these deacetylases (data not shown). These results suggestthat Rfm1 binding to the Hst1::Sir212-155 chimera or theSir2-2H mutant may exclude binding by Sir4 and that bindingby Sir4 to these proteins may exclude Rfm1. Direct competi-tion between Sir4 and Rfm1 may play an important role in thenormal activities of Hst1 and Sir2. For example, if Rfm1 couldbind Sir2, then the ability of the deacetylase to spread andcreate domains of repression at the HM loci and telomeresmight be blocked. The disruption of regional silencing at theseloci would cause improper expression of the silenced matingtype cassettes, as well as a host of subtelomeric genes that arethought to be activated by external cell stress (1). Moreover, itwould expose the Ho endonuclease recognition sites at the HMloci, permitting DNA cleavages that would disrupt directionalmating type switching in native yeast. Similarly, if Sir4 couldbind Hst1, then the ability of this deacetylase to act locallymight be replaced by nonspecific regional repression. SinceHst1 targets are interspersed throughout the genome and areoften in close proximity to other genes, regional silencing atthese loci would have deleterious effects on the cell (26). Themutual exclusion of one set of cofactors when bound by theother may therefore play an important role in distinguishingwhether these proteins function as regional silencers or gene-specific repressors when targeted to specific loci.

Despite the functional similarities between Hst1 and Sir2,



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each enzyme is involved in mechanistically different methodsof turning off transcription. However, we have found that theHst1::Sir212-155 chimera and the Sir2-2H mutant function asboth regional silencers and gene-specific repressors. These re-sults show that differences between Sir2-mediated silencingand Hst1-mediated repression are not the results of the intrin-sic enzymatic activity or substrate specificity of each enzyme,but rather the results of differences in the interactions with theSir3-Sir4 and Sum1-Rfm1 complexes. The abilities of the en-zymes to discriminate between different cofactor complexestherefore have important roles in specifying the functions ofthese proteins. It is likely that differences in the nonconservedregions, as well as subtle differences within the enzymatic core,contribute to the specificities of other members of the Sirtuinfamily.

ACKNOWLEDGMENTS

We thank Lorraine Pillus for generously providing strains and plas-mids.

This research was supported by grants from the National Institute ofHealth (GM 58762 to A.K.V. and GM51402 to M.G.).

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