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Molecular and Cellular Biology, April 2007, p. 2466-2475, Vol. 27, No. 7
0270-7306/07/$08.00+0     doi:10.1128/MCB.01641-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Swapping the Gene-Specific and Regional Silencing Specificities of the Hst1 and Sir2 Histone Deacetylases{triangledown}

Janet Mead,1 Ron McCord,1,{dagger} Laura Youngster,1 Mandakini Sharma,2,{ddagger} Marc R. Gartenberg,2 and Andrew K. Vershon1*

Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854,1 Department 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


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sir2 and Hst1 are NAD+-dependent histone deacetylases of budding yeast that are related by strong sequence similarity. Nevertheless, the two proteins promote two mechanistically distinct forms of gene repression. Hst1 interacts with Rfm1 and Sum1 to repress the transcription of specific middle-sporulation genes. Sir2 interacts with Sir3 and Sir4 to silence genes contained within the silent-mating-type loci and telomere chromosomal regions. To identify the determinants of gene-specific versus regional repression, we created a series of Hst1::Sir2 hybrids. Our analysis yielded two dual-specificity chimeras that were able to perform both regional and gene-specific repression. Regional silencing by the chimeras required Sir3 and Sir4, whereas gene-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 and proper targeting of these proteins. These results suggest that the differences in the silencing and repression functions of Sir2 and Hst1 may not be due to differences in enzymatic activities of the proteins but rather may be the result of distinct cofactor specificities.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Sirtuin family of proteins is a large class of NAD+-dependent deacetylases with a highly conserved enzymatic core domain (9). These enzymes are found in all kingdoms of life, and many organisms contain multiple Sirtuin family members (13). Although Sirtuin homologs have NAD+-dependent deacetylase activities, their cellular functions differ as a result of their subcellular localization and specific protein-protein interactions (15, 32). For example, although human SIRT2 deacetylates histone H4 during mitosis, it has also been found in the cytoplasm and appears to function as a tubulin deacetylase required for exit from mitosis (11, 30, 32, 46). The SIRT1 protein deacetylates and activates acetyl-coenzyme A synthetase, 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 with their cofactors and recognize their targets is therefore key to understanding how these proteins function in the cell.

The yeast Saccharomyces cerevisiae has five members of the Sirtuin family, HST1 to HST4 and SIR2, the founding member of the family (3, 10). Similar to the homologs in higher eukaryotes, the yeast proteins vary in their cellular localizations and perhaps substrate specificities (32, 40). Sir2 functions as a NAD+-dependent histone deacetylase and is involved in modifying chromatin structure and functioning as a regional transcriptional silencer (19, 21, 43). Silencing in yeast, like heterochromatin in higher eukaryotes, renders large regions of the chromosome transcriptionally inactive in a non-gene-specific manner. 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 hypoacetylated, and the DNA is generally refractory to modifying enzymes. Although Sir2 is required for silencing at all three of these loci, the protein forms two distinct complexes with nonoverlapping sets of binding partners (44). One complex is required for silencing the telomeres and the mating type loci and includes the Sir3 and Sir4 silencing cofactors (17, 28). The second complex is required for silencing the rDNA loci and includes the Net1 and Cdc14 proteins (18, 38, 41). Mutations in these cofactors result in mislocalization of Sir2 and loss of silencing at the respective loci. These studies indicate that the interaction of Sir2 with these cofactors is critical to proper localization and silencing.

Hst1 is the closest homolog of Sir2 and, like Sir2, is a NAD+-dependent deacetylase and a component of at least two distinct complexes (22, 34). Hst1 is tethered to the DNA-binding protein Sum1 through interactions with Rfm1 to form a complex that represses middle-sporulation genes during vegetative growth (26, 35, 42, 49). Hst1 is also a component of the Set3c complex, which appears to repress transcription of meiosis-specific genes during early meiosis (34). The functional similarity between Hst1 and Sir2 is further demonstrated by the observation that in high copy numbers, the two proteins are able to partially function in place of each other. Overexpression of Sir2 can partially suppress hst1 defects in repression of middle-sporulation genes, and overexpression of Hst1 can partially restore silencing at HMR in the absence of Sir2 (3, 10, 49). However, under normal levels of expression, neither protein functions in place of the other. These proteins therefore have distinct regulatory activities: Sir2 functions as a transcriptional silencer of relatively large regions of the genome, while Hst1 functions as a transcriptional repressor, acting locally at a specific set of promoters. In this paper, we investigate the mechanism through which these highly conserved proteins have distinct functions in the cell.

Although Sir2 and Hst1 share strong sequence similarity throughout the enzymatic cores of the proteins, their N termini are considerably more divergent (Fig. 1). We show that this difference accounts, in part, for the specificity of cofactor interactions by Hst1 and Sir2, and this, in turn, accounts for differences between Sir2-mediated silencing and Hst1-mediated gene-specific repression mechanisms. Interestingly, we have found that relatively subtle differences in two amino acids within the catalytic cores of the proteins also contribute to cofactor specificity. These findings provide insight into how other members of the Sirtuin family may discriminate between different sets of cofactors and have different regulatory roles in the cell.


Figure 1
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  		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 indicating the Hst1 residues that were replaced with the corresponding regions of Sir2 are shown with arrows below the sequence. The asterisks indicate positions in which residues in Sir2 were replaced with the amino acids at the corresponding positions in Hst1.

 

   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and plasmids. The yeast strains used in this study are shown in Table 1. To construct null mutations for each gene of interest in the desired strain backgrounds, we obtained the diploid KanMX null mutant strains from Research Genetics. Primers were designed to generate a fragment via PCR that included the KanMX replacement of the gene of interest and 500 bp of homology upstream and downstream. These fragments were then transformed into the various strain backgrounds, and the integration of the KanMX null mutation was verified using primers internal and external to the transformed fragment.


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  		TABLE 1. Yeast strains

 
Plasmid pJX43 is a lacZ transcription reporter plasmid containing a promoter that is repressed by the SMK1 middle sporulation element (MSE) (33). Plasmid pRAM29 contains a V5-epitope-tagged version of HST1 expressed from its own promoter on a 2µm LEU2 vector (26). Plasmid pRAM27 contains SIR2 expressed from its own promoter on a 2µm LEU2 vector (26). Amino acid substitutions in HST1 and SIR2 were introduced by site-directed mutagenesis using the Stratagene QuikChange mutagenesis kit. HST1::SIR2 chimeras were created by generating PCR fragments with regions of SIR2 and cloning those regions into the corresponding homologous regions within HST1 on plasmid pRAM29 using either the appropriate restriction sites or gapped plasmid repair (29). The sir2 mutants were cloned into pJM491, which contains a V5 epitope tag at the C terminus of the protein. A complete list of the chimera and point mutant plasmids used in this study is available upon request. The SUM1-myc (pMP208) and RFM1-HA (pDI14) plasmid constructs that were used in the coimmunoprecipitation (co-IP) experiments were previously described (26).

Transcription and silencing assays. MSE-dependent repression activity was assayed by measuring ß-galactosidase expression from the MSE-lacZ transcription reporter, pJX43. Quantitative liquid ß-galactosidase activity assays were performed as described previously (14). To measure the level of silencing by the HST1 or SIR2 constructs at the HM loci, transformants of strain LPY3923 and its derivatives were grown overnight at 30°C, diluted to a starting A600 of 1.0, serially fivefold diluted, and plated on selective media as described previously (37). For rDNA- and telomeric-silencing assays, transformants of strains LPY2447 and LPY1953 and their derivatives were diluted to a starting A600 of 4.0 for the rDNA assays and 2.5 for the telomeric assays. Cultures were then serially fivefold diluted and plated on the appropriate selective medium, SD-Trp or 5-fluoroorotic acid (5-FOA), respectively.

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


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Deletion analysis of Hst1. Although Hst1 shows significant sequence similarity to regions of Sir2 that correspond to the enzymatic core of the protein, the N-terminal and C-terminal regions are more divergent (Fig. 1). Mutational analysis of Sir2 has shown that some of these nonconserved regions are essential for Sir2 function and, in part, specify interactions with its different cofactors (5, 7, 37). To determine which regions of Hst1 are required for its function, we constructed a series of deletions in the protein and assayed for their abilities to repress transcription of middle-sporulation genes and to interact with the Sum1-Rfm1 complex (Fig. 2). Many of the deletions were unable to repress transcription of an MSE-regulated promoter in an hst1{Delta} strain (Fig. 2B). However, two of the deletions repressed transcription almost as well as wild-type Hst1. One of these deletions, Hst1{Delta}8-54, removed the nonconserved N-terminal region of the protein. Despite the ability to repress reporter genes, the truncated protein was less stable, since the level of expression was decreased and multiple fragments were present, presumably due to partial proteolysis (Fig. 2C). The other deletion in Hst1 that showed partial repression, Hst1{Delta}350-371, removed a portion of the enzymatic core of the protein. This region of the core domain is not strongly conserved between Hst1 and Sir2 and is not present in many of the Sir2 family members (Fig. 1).


Figure 2
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  		FIG. 2. Deletion analysis of Hst1. (A) Schematic of the deletions that were constructed in Hst1. The top line shows the conservation of different domains of Hst1 with Sir2. The numbers above the line indicate the endpoints of the different regions of the protein, and the percentages are the sequence identity with Sir2. Each mutant was named for the residues that were deleted. (B) MSE repression assays of wild-type and Hst1 deletions. The indicated mutants on high-copy-number 2µm plasmids were tested for the ability to repress transcription of an MSE-regulated promoter driving lacZ expression in JXY5, an hst1{Delta} strain. The bars indicate the average ß-galactosidase activities of five independent isolates, and the standard deviations are shown by the error bars. (C) (Top) Western blot with antibody to the V5 epitope to monitor the levels of expression of tagged (lane 1) and untagged (lane 2) wild-type Hst1 or the indicated V5-tagged Hst1 deletion mutants (lanes 3 to 9). (Bottom) Western blot with Myc antibody to detect the presence of Myc-tagged Sum1 protein that coimmunoprecipitated with tagged or untagged wild-type Hst1 or the indicated mutants.

 
We next performed co-IP experiments to determine which of the Hst1 deletion proteins were able to interact with the Sum1-Rfm1 complex. As expected from the results of the repression assays, Sum1 coimmunoprecipitated with the Hst1{Delta}8-54 and Hst1{Delta}350-371 deletions, indicating that these regions are not essential for interactions with the Sum1-Rfm1 complex (Fig. 2C). The Sum1 protein was able to weakly interact with the Hst1{Delta}54-81, Hst1{Delta}164-327, and Hst1{Delta}479-502 mutants and failed to interact with the Hst1{Delta}118-158 and Hst1{Delta}329-474 mutants, indicating that residues within these regions are required for full interaction with the Sum1-Rfm1 complex.

Hst1::Sir2 chimeras show the N-terminal region is required for silencing. To distinguish between regions within Hst1 that are required for gene-specific repression and regions within Sir2 that are required for regional silencing, we created a series of Hst1::Sir2 chimeras by swapping regions of Sir2 into the corresponding regions of Hst1 (Fig. 3A). Each chimera was then tested for the ability to complement defects in silencing in sir2 strains that contain reporter genes at the rDNA or HMR mating type locus or telomeres. As shown by the strong growth on media lacking uracil, none of the chimeras silence the URA3 marker at the rDNA locus (Fig. 3B). Each of these chimera proteins may therefore lack a region of Sir2 that is required to form a complex with Cdc14 and Net1 and to silence the rDNA loci.


Figure 3
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  		FIG. 3. Transcriptional silencing by Hst1::Sir2 chimeras. (A) A schematic representation of the Hst1::Sir2 chimeras is shown. The top line shows a schematic of Hst1 with numbers indicating the percent identity of the different regions of the protein with Sir2. The rows below show the different Hst1::Sir2 chimeras, in which the numbers indicate the positions of the residues in Hst1 that were replaced with the corresponding residues in Sir2. (B) High-copy-number (2µm) plasmids containing either wild-type SIR2, HST1, or the indicated HST1::SIR2 chimeras were transformed into strain LPY2447 (sir2 rDNA::URA3), and normalized serial fivefold dilutions of the cultures were spotted on plates with (+Ura) and without (–Ura) uracil. Failure to 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 were spotted on media with (+Trp) and without (–Trp) tryptophan. Failure to 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 with and without the drug 5-FOA. 5-FOA prevents growth of the cells that express URA3. The ability to grow on the 5-FOA plate indicates silencing of the telomeric loci in the cell. (E) Low-copy-number (CEN) plasmids containing either wild-type SIR2, HST1, or the HST1::SIR212-155 chimera were transformed into strain LPY3923 and assayed as for panel C.

 
Most of the Hst1::Sir2 chimeras also failed to silence reporter genes at HMR and telomeres (Fig. 3C and D). However, the Hst1::Sir212-155 chimera, in which the N-terminal region of Hst1 (residues 12 to 155) is replaced with the corresponding region of Sir2, showed a significant decrease in growth on media lacking tryptophan, indicating that the TRP1 reporter gene at HMR was silenced by this chimera (Fig. 3C). We also found that Hst1::Sir212-155 was the only chimera that permitted growth of a sir2 strain with a telomeric URA3 marker on 5-FOA, a drug that kills cells that express URA3 (Fig. 3D). Silencing by this chimera was not dependent upon the gene dosage of the chimera, as it silenced equally well when expressed from both high- and low-copy-number vectors (Fig. 3E and data not shown), which indicates that the Hst1::Sir212-155 chimera is able to silence the telomere and the mating type loci almost as well as Sir2. Since this chimera is able to silence HMR and telomeres but not the rDNA locus, it supports previous observations that different regions of Sir2 are involved in the formation of the Sir3-Sir4 and RENT complexes (7, 17). Since Sirtuins may form oligomers, we also assayed silencing in an hst1{Delta} sir2{Delta} double-mutant strain (6). Our results in the double-mutant background were indistinguishable from those performed in the sir2{Delta} strain, indicating that the Hst1::Sir212-155 chimera silences through a Sir2-like mechanism rather than through repression involving Hst1.

Recently, it was shown that Sum1 binds at the HML loci and has a role in DNA replication (20). Since the Hst1::Sir212-155 chimera contains large regions of Hst1, it is possible that this construct was able to bypass the requirement for the Sir3 and Sir4 silencing cofactors through recruitment by Sum1 and Rfm1 bound to the silenced regions. To determine if Sir3 and Sir4 were required for silencing by this chimera, we transformed sir2 sir3 and sir2 sir4 double-mutant strains with SIR2 or the HST1::SIR212-155 chimera and assayed for silencing. We found that the chimera failed to silence the telomeric reporter gene, indicating that both Sir3 and Sir4 are required for silencing by the chimera (Fig. 4). Conversely, the Hst1::Sir212-155 chimera was able to silence as well as Sir2 in a strain lacking both SIR2 and RFM1. Thus, Rfm1 is not required for silencing, strongly suggesting that the chimera does not silence the HML loci or telomere regions through a pathway that involves Sum1.


Figure 4
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  		FIG. 4. Silencing by the Hat1::Sir212-155 chimera is dependent on Sir3 and Sir4 but independent of Rfm1. Plasmids containing wild-type HST1 or SIR2 or the Hat1::Sir212-155 chimera, along with a blank vector, were transformed in strain (A) LPY1953 (sir2 TEL::URA3), (B) JMY049 (LPY1953 with sir3{Delta}::KanMX), (C) JMY053 (LPY1953 with sir4{Delta}::KanMX), and (D) JMY058 (LPY1953 with rfm1{Delta}::KanMX), and normalized fivefold serial dilutions of cultures were spotted on plates with and without 5-FOA. Growth on 5-FOA indicates silencing of the telomere loci.

 
The ability of the Hst1::Sir212-155 chimera to complement sir2 strains but not sir2 strains lacking sir3 or sir4 indicates that it acts in the normal Sir pathway and may form a complex with Sir3 and Sir4. To verify this model, co-IP experiments were performed in which the tagged Hst1::Sir212-155 chimera, Sir2, or Hst1 was immunoprecipitated, and the pellets were then assayed by Western blotting for the presence of Sir4. As expected, Sir4 was not present in the immunoprecipitated pellet of the untagged Sir2 lysate but was strongly present in the immunoprecipitate of the V5-tagged Sir2 lysate (Fig. 5, lanes 3 and 2). Importantly, while wild-type Hst1 was unable to immunoprecipitate Sir4, the Hst1::Sir212-155 chimera interacted quite well with Sir4 (Fig. 5, lanes 1 and 4). This suggests that residues within the 12-to-198 region of Sir2 are required for association with the Sir3-Sir4 silencer complex and that the differences between Sir2 and Hst1 in this region are important for targeting Sir2 to silence the telomeres and mating type loci.


Figure 5
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  		FIG. 5. The Hat1::Sir212-155 chimera coimmunoprecipitates with Sir4. Lysates from transformants of strain JXY20 (sir2{Delta} hst1{Delta}) containing plasmids with V5-tagged wild-type Hst1 (lane 1), V5-tagged Sir2 (lane 2), untagged Sir2 (lane 3), or V5-tagged Hat1::Sir212-155 chimera (lane 4) were immunoprecipitated with antibody to the V5 epitope. The top panel shows a Western blot with antibody to the V5 epitope to monitor the levels of expression of V5-tagged proteins in crude lysates. The bottom panel shows a Western blot of the immunoprecipitated pellets using polyclonal antibodies to Sir4.

 
Hst1::Sir2 chimeras define a region in the enzymatic core of Hst1 that is required for MSE-mediated repression. The results described above show that swapping the N-terminal region of Sir2 onto Hst1 enabled the protein to function in regional silencing of chromosomal domains. One appealing model is that the nonconserved N-terminal regions of Sir2 and Hst1 specify the interactions with the Sir3-Sir4 and Sum1-Rfm1 complexes, respectively. This model would predict that the Hst1::Sir212-155 chimera would not strongly complement defects in MSE-dependent repression in an hst1 mutant. To test this model, the chimeras were assayed for the ability to repress transcription of an MSE-regulated promoter in an hst1 strain. Surprisingly, the Hst1::Sir212-155 N-terminal chimera and most of the other chimeras functioned as well as wild-type Hst1 in low or high copy numbers and in either hst1{Delta} or hst1{Delta} sir2{Delta} strains (Fig. 6A and data not shown). However, a chimera in which a region of the core domain was replaced by the corresponding region in Sir2 (Hst1::Sir2266-325) was unable to complement hst1 defects in repression in either low or high copy numbers (Fig. 6A and data not shown). This suggests that residues 266 to 325 of Hst1 are required for MSE-mediated repression.


Figure 6
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  		FIG. 6. MSE-mediated repression by the Hst1::Sir2 chimeras. (A) Plasmids containing either wild-type SIR2, HST1, or the indicated HST1::SIR2 chimeras were transformed into strain JXY5 (hst1{Delta}::KanMX) and assayed for the ability to repress the MSE-regulated promoter on plasmid pJX43. The bars indicate the average levels of ß-galactosidase activity of five independent transformants, and the standard deviations are shown by the error bars. (B) Wild-type SIR2, HST1, or the indicated HST1::SIR2 chimeras were transformed into strain RMY8 (hst1{Delta}::KanMX rfm1{Delta}::KanMX) (hatched bars) or JMY045 (hst1{Delta}::KanMX sir3{Delta}::HIS3) (solid bars) and assayed for the ability to repress the MSE-regulated promoter as in panel A. (C) The upper blot shows a Western blot analysis with antibody to the V5 epitope to compare the levels of expression of tagged (lane 1) and untagged (lane 2) wild-type Hst1 and the indicated Hst1::Sir2 chimeras (lanes 3 to 7). Extracts from these strains were immunoprecipitated with antibodies specific for the V5 epitope, and the pellets were assayed by Western blot analysis with Myc antibodies to detect the presence of Myc epitope-tagged Sum1 in the immunoprecipitate (bottom).

 
Since the chimeras contained large portions of Sir2, it was possible that repression by these chimeras could be independent of Rfm1 and instead depend on Sir2 cofactors, such as Sir3 or Sir4. However, with the exception of the Hst1::Sir2266-325 chimera, all of the constructs were able to fully complement repression of the reporter in hst1 sir3 and hst1 sir4 strains (Fig. 6B and data not shown). Moreover, none of the chimeras were able to repress transcription of the reporter in an rfm1 strain (Fig. 6B). These data suggest that, with the exception of Hst1::Sir2266-325, all of these chimeras interact with the Sum1-Rfm1 complex to repress MSE-regulated promoters. This result, along with the HM and telomeric-silencing assays, suggests that the Hst1::Sir212-155 chimera is a dual-specificity protein that functions as both a gene-specific repressor and a regional transcriptional silencer.

To test if the chimeras are able to physically interact with the Sum1-Rfm1 complex, we performed co-IP experiments in which the V5 epitope-tagged Hst1::Sir2 chimeras were immunoprecipitated using a V5 antibody, and the pellets were then assayed by Western blotting for the presence of Sum1. Western blot analysis of the lysates with V5 antibody showed that all of the chimeras were present at levels comparable to those of the wild-type protein (Fig. 6C, top). As expected from the complementation results, most of the Hst1::Sir2 chimeras were able to interact with Sum1 (Fig. 6C, bottom). However, Sum1 failed to immunoprecipitate with the Hst1::Sir2266-325 chimera. This suggests that residues within the 266-to-325 region of Hst1 are required for association with the Sum1-Rfm1 complex and that the differences between Hst1 and Sir2 in this region are important for targeting Hst1 to repress middle-sporulation genes.

Residues in the Zn ribbon region of Hst1 are important for specifying interactions with Rfm1. The results described above show that a region in the conserved enzymatic core of the Hst1 protein (residues 266 to 325) is important for determining the specificity of Hst1 interactions with the Sum1-Rfm1 complex. The crystal structures of the enzymatic domains of several members of the Sir2 family of NAD+-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 Hst1 encompasses the first two cysteines of the conserved zinc ribbon motif, along with a region on the back side of the protein, away from the NAD+ binding pocket and active site (Fig. 7A) (12). Hst1 and Sir2 have strong sequence similarity in this region of the protein, with only eight amino acid differences (Fig. 1). The model of the human SIRT2 crystal structure suggests that several of these residues are likely to be exposed to solvent in the Hst1 and Sir2 proteins. It is possible that these residues play important roles in targeting Hst1 to Rfm1, instead of to Sir4. Therefore, by swapping these residues in Sir2 with the amino acids found at the corresponding positions in Hst1, we hypothesized that it might be possible to target Sir2 to Rfm1. To test this model, we engineered amino acid substitutions in Sir2 and assayed the affects of these changes on Sir2-mediated repression of 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 combination (Sir2-6H), were unable to repress the MSE-regulated promoter any better than wild type Sir2 (Fig. 7B and data not shown). However, the Sir2-2H mutant, containing the N378Q and L379I amino acid substitutions, produced roughly the same level of repression of the reporter as wild-type Hst1. This result supports the model in which differences in this region of Hst1 and Sir2 are important for distinguishing between their different cofactors. Repression of the MSE-regulated promoter by the Sir2-2H mutant did not require Sir3 or Sir4 but did require Rfm1 (Fig. 7B and data not shown). This result suggests that this double amino acid substitution was sufficient to increase the affinity of Sir2 interactions with Rfm1.


Figure 7
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  		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 that was 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 the predicted binding site of the histone tail. The region corresponding to Hst1 residues 266 to 325 (Sir2 residues 314 to 379) is shown in yellow. The residues in green are the positions in the Sir2-6H mutant that were replaced with the corresponding amino acids in Hst1. The residues in red correspond to positions N378 and L379 in Sir2 that were replaced in the Sir2-2H mutant. (B) The level of repression of an MSE-regulated promoter by Hst1, Sir2, Sir2-6H (K320N, I321M, M334D, S356D, T357P, and T371S), Sir2-2H (N378Q and L379I), and Hst1-2S (Q324N and I325L) 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 were performed in strain JXY5 (hst1{Delta}), while the assays for the three bars on the right were performed in strain RMY8 (hst1{Delta} rfm1{Delta}). (C) A co-IP assay showed that Sir2-2H interacts with Rfm1. Strain JXY5 (hst1{Delta}::KanMX) was cotransformed with high-copy-number 2µm plasmids expressing RFM1-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) and Rfm1-immunoprecipitated samples (lanes 4 to 6) was probed with polyclonal antibody to the V5 epitope.

 
Repression of the MSE-regulated promoter by the Sir2-2H mutant suggests that residues N378 and L379 in Sir2 are important for specifying interactions with Rfm1. Residues at the corresponding positions in Hst1, Q324 and I325, were therefore likely to be important for interaction with Rfm1. To test this model, we swapped the amino acids at these positions in Hst1 with those found at the corresponding positions in Sir2 and measured the level of Hst1-dependent repression of a reporter promoter. The Hst1-2S mutant, containing the Q324N and I325L substitutions, showed decreased levels of repression, suggesting that the substitution of these residues affects interactions with Rfm1 (Fig. 7B).

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

None of the amino acid substitutions that we constructed in Sir2 affected silencing at the HMR, telomere, or rDNA loci (data not shown). This result suggests that these mutants have not lost the ability to interact with either the Sir3-Sir4 or the Net1-Cdc14 complexes to silence the different loci. It therefore appears that the Sir2-2H mutant is able to interact with Sir2 and Hst1 cofactors and function as both a regional transcriptional silencer and a gene-specific repressor.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The hallmark of the Sirtuin family of NAD+-dependent deacetylase enzymes is a highly conserved enzymatic core of approximately 260 amino acids. Interestingly, while the bacterial homologs consist only of the conserved enzymatic core domain, homologs in higher eukaryotes have amino- and carboxyl-terminal extensions that are highly divergent among members of the family. It is likely that these nonconserved regions contribute to the specific functions of many proteins in the Sirtuin family. The N-terminal regions of these proteins are of particular interest because of their potential regulatory roles. For example, both Sir2 and Hst2 form homotrimers in solution, and it has been shown that the amino-terminal methionine of one Hst2 subunit interacts with the active site in another monomer in the homotrimer (6, 50). Similarly, human SIRT3 is catalytically inactive as a full-length protein and only becomes active after cleavage of the first 100 amino acids following import into the mitochondria (36). Mutational analysis of the yeast Sir2 protein showed the importance of the N-terminal domain in regional transcriptional silencing (5, 8, 37). It was therefore likely that the nonconserved N-terminal extensions of Hst1 and Sir2 contribute to the cofactor interactions and target specificities of the two proteins. This model is supported by our finding that a Hst1 chimera protein containing the N-terminal region of Sir2, Hst1::Sir212-155, is able to interact with Sir4 and silence the mating loci and telomeres in a sir2 strain. Interestingly, the Hst1::Sir212-155 chimera is still able to interact with Rfm1 and to function as a repressor of middle-sporulation genes. This result indicates that although amino acid differences in this region of Hst1 and Sir2 are important for specifying interactions with Sir2 cofactors, the two proteins contain sufficient sequence similarity in this region to enable both of them to productively interact with Rfm1.

The analysis of the Hst1::Sir212-155 chimera suggested that differences in other regions of Sir2 and Hst1 must also be important for specifying interactions with Rfm1. In support of this model, the Hst1::Sir2266-325 chimera failed to interact with Rfm1 or to repress an MSE-regulated reporter. This region lies on the surface of the catalytic domain that is opposite from the NAD+ binding pocket and likely provides a suitable interface for cofactor interactions. This region incorporates the first two cysteines of the Zn+ ribbon motif, and the importance of this region was demonstrated by mutational analysis of these cysteine residues in Sir2, which disrupt both the enzymatic activity and cofactor interactions of the protein (27, 37). Interestingly, we found that swapping only two Sir2 residues with the corresponding Hst1 residues (N378Q and L379I) enabled the Sir2 protein to interact with Rfm1 and repress MSE-regulated genes. Although the cysteine residues are highly conserved, residues directly surrounding this motif are relatively divergent among different members of the Sirtuin family. The crystal structures of several different Sirtuin proteins showed that there are significant differences in the positions of the Zn+-ribbon motif relative to the Rossmann fold when bound by ligand (12, 25, 27, 51, 52). The sequence conservation of the Cys residues, along with the differences in conformation, suggests that there may be an important mechanistic or structural role for this region in Sirtuin proteins. Our data suggest that differences within this region may be important for specifying interactions with distinct sets of cofactors.

The Hst1::Sir212-155 chimera and the Sir2-2H mutant are able to function as both regional silencers of the HM loci and telomere- and gene-specific repressors of middle-sporulation genes. These proteins have both gained the ability to interact with different cofactor complexes and are therefore dually specific in function. It was possible that these proteins formed a single complex that was able to both silence and repress. This model was enticing, because studies of the dominant SUM1-1 mutant have shown that it is able to bypass the requirement for Sir2 for silencing at HMR by directly interacting with the ORC complex and recruiting Rfm1 and Hst1 to this region (24, 26, 35). However, we found that the Hat1::Sir212-155 chimera and the Sir2-2H mutant required Sir3 and Sir4 to silence the HM loci 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 complexes in the cell: one in complex with Sir3 and Sir4 to silence the HM loci and telomeres and another in complex with Sum1 and Rfm1 to repress middle-sporulation genes. In support of this model, we have been unable to observe by chromatin immunoprecipitation assays Sir4 binding to promoters repressed by the Sum1-Rfm1-Hst1 complex or Rfm1 binding to telomere regions silenced by the Sir2-Sir3-Sir4 complex in the Hat1::Sir212-155 chimera and Sir2-2H mutant strains (data not shown). In addition, while we observe that both Sir4 and Sum1 or Rfm1 interact with the Hst1::Sir212-155 chimera and the Sir2-2H mutant, we were unable to coimmunoprecipitate Sir4 and Rfm1 with each other in these same strains, suggesting that Sir4 and Rfm1 do not simultaneously interact in a complex with these deacetylases (data not shown). These results suggest that Rfm1 binding to the Hst1::Sir212-155 chimera or the Sir2-2H mutant may exclude binding by Sir4 and that binding by Sir4 to these proteins may exclude Rfm1. Direct competition between Sir4 and Rfm1 may play an important role in the normal activities of Hst1 and Sir2. For example, if Rfm1 could bind Sir2, then the ability of the deacetylase to spread and create domains of repression at the HM loci and telomeres might be blocked. The disruption of regional silencing at these loci would cause improper expression of the silenced mating type cassettes, as well as a host of subtelomeric genes that are thought to be activated by external cell stress (1). Moreover, it would expose the Ho endonuclease recognition sites at the HM loci, permitting DNA cleavages that would disrupt directional mating type switching in native yeast. Similarly, if Sir4 could bind Hst1, then the ability of this deacetylase to act locally might be replaced by nonspecific regional repression. Since Hst1 targets are interspersed throughout the genome and are often in close proximity to other genes, regional silencing at these loci would have deleterious effects on the cell (26). The mutual exclusion of one set of cofactors when bound by the other may therefore play an important role in distinguishing whether these proteins function as regional silencers or gene-specific repressors when targeted to specific loci.

Despite the functional similarities between Hst1 and Sir2, each enzyme is involved in mechanistically different methods of turning off transcription. However, we have found that the Hst1::Sir212-155 chimera and the Sir2-2H mutant function as both regional silencers and gene-specific repressors. These results show that differences between Sir2-mediated silencing and Hst1-mediated repression are not the results of the intrinsic enzymatic activity or substrate specificity of each enzyme, but rather the results of differences in the interactions with the Sir3-Sir4 and Sum1-Rfm1 complexes. The abilities of the enzymes to discriminate between different cofactor complexes therefore have important roles in specifying the functions of these proteins. It is likely that differences in the nonconserved regions, as well as subtle differences within the enzymatic core, contribute to the specificities of other members of the Sirtuin family.


   ACKNOWLEDGMENTS
 
We thank Lorraine Pillus for generously providing strains and plasmids.

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


   FOOTNOTES
 
* Corresponding author. Mailing address: Waksman Institute, 190 Frelinghuysen Rd., Piscataway, NJ 08854-8020. Phone: (732) 445-2905. Fax: (732) 445-5735. E-mail: vershon{at}waksman.rutgers.edu. Back

{triangledown} Published ahead of print on 22 January 2007. Back

{dagger} Present address: Department of Medicine, Stanford University, Palo Alto, CA 94305. Back

{ddagger} Present address: Genentech, South San Francisco, CA 94080. Back


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Molecular and Cellular Biology, April 2007, p. 2466-2475, Vol. 27, No. 7
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