A Role for the Saccharomyces cerevisiae RENT Complex Protein Net1 in HMR Silencing

Corresponding author: Ann E. Ehrenhofer-Murray, Otto-Warburg-Laboratories, Ihnestrasse 73, D-14195 Berlin, Germany., ehrenhof{at}molgen.mpg.de (E-mail)

Communicating editor: J. RINE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Silencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD⁺-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2µ-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR.

SILENCING is a form of transcriptional repression that converts regions of eukaryotic chromosomes into an inaccessible chromatin state, which in higher eukaryotes is referred to as heterochromatin. Silenced chromatin is generally refractory to transcription and recombination, replicates late in the S phase of the cell cycle, and is usually located in discrete subnuclear domains (LOO and RINE 1995 ; LUSTIG 1998 ). On a molecular level, silenced chromatin consists of specialized heterochromatin proteins and of nucleosomes carrying deacetylated histones. In the budding yeast Saccharomyces cerevisiae, three classes of loci are known to be silenced: the silent mating-type loci HML and HMR, the telomeres, and the rDNA locus.

The rDNA cluster is the most abundant repetitive sequence in the yeast genome and consists of 100–200 copies of a 9-kb rDNA gene unit. However, only about half of these repeats are active at any given time, whereas the other half is transcriptionally repressed (WARNER 1989 ). Likewise, marker genes inserted in the rDNA locus become metastably repressed (SMITH and BOEKE 1997 ). rDNA silencing is promoted by a protein complex termed RENT (for regulator of nucleolar silencing and telophase; SHOU et al. 1999 ), which contains the proteins Net1, Sir2, Cdc14, and at least one more, yet uncharacterized component. Net1, a 128.5-kD protein with similarity to the topoisomerase interacting factor Tof2, plays a central role in RENT: Net1 is required for nucleolar integrity and for the localization of both Cdc14 and Sir2 to the rDNA (STRAIGHT et al. 1999 ; VISINTIN et al. 1999 ). Furthermore, a mutation in NET1 (net1-1) was identified in a genetic screen for mutants that bypassed the anaphase arrest of cdc15 cells, showing that Net1 is required for the control of exit from mitosis (SHOU et al. 1999 ). The current model posits that the protein phosphatase Cdc14 is tethered to the rDNA via its interaction with Net1 during G1, S, and early M phase. Net1 thereby acts as a competitive inhibitor of Cdc14, thus preventing it from executing its enzymatic activity (TRAVERSO et al. 2001 ). Through a Tem1-dependent signal, Cdc14 is released from the nucleolus during anaphase and thus becomes able to dephosphorylate its targets Sic1, Cdh1, and Swi5 in the nucleus (VISINTIN et al. 1998 ; ZACHARIAE et al. 1998 ). Significantly, the two functions of Net1 in cell cycle regulation and in sustaining the general nucleolar integrity are independent of each other (SHOU et al. 2001 ). In addition, Net1 also recruits RNA polymerase I (PolI) to the rDNA and thereby stimulates the synthesis of rRNA (SHOU et al. 2001 ). The roles of the Net1 protein emphasize its functional complexity and suggest a widespread ability to participate in different cellular processes. However, as Net1 is not likely to bind DNA directly, the mechanism by which the rDNA silencing complex RENT is tethered to the nucleolar DNA still remains to be resolved.

Like Cdc14 and PolI, the silent information regulator protein Sir2 is tethered to the rDNA via Net1 (STRAIGHT et al. 1999 ). Sir2 is a NAD⁺-dependent histone deacetylase specific for lysines 9 and 14 of histone H3 and lysine 16 of H4, and it requires this deacetylase activity for its silencing function (IMAI et al. 2000 ; LANDRY et al. 2000 ). Sir2 is the only Sir protein required for rDNA silencing, suggesting a distinct mechanism of silencing at this locus. The first indication for a role of Sir2 in the rDNA came from the observation that Sir2 represses rDNA recombination (GOTTLIEB and ESPOSITO 1989 ). Recombination between the repetitive rDNA units produces extrachromosomal rDNA circles, which are a cause of cellular senescence in yeast (SINCLAIR and GUARENTE 1997 ). Hence, Sir2 is an anti-aging factor that may provide a link between caloric intake and aging through its NAD⁺ dependence (LIN et al. 2000 ).

Apart from its participation in RENT, nuclear Sir2 is also found at the HM loci, where it forms a complex with the silencing proteins Sir3 and Sir4 (MOAZED et al. 1997 ). The complex is recruited to the target region by an interaction of Sir3/4 with the DNA-binding factor Rap1 (MORETTI et al. 1994 ) and with the deacetylated tails of histone H3 and H4 (HECHT et al. 1995 ), presumably enabling a spreading of the complex along chromatin. Sir1 also takes part in forming the repressed chromatin, but becomes essential only for silencing in compromised silencer mutants. Sir1 is anchored at the silencer by its interaction with the origin recognition complex (ORC; TRIOLO and STERNGLANZ 1996 ; GARDNER et al. 1999 ). Binding sites for ORC, Rap1, and for a third DNA-binding protein, Abf1, are found in the HM silencers. These silencers flank the mating-type genes at HMR and result in their repression, whereas the identical genes at MAT are expressed and determine the cell type. The silencer binding sites will here be referred to as ORC, RAP, and ABF. The factors binding these sites are a prerequisite for the formation of repressed chromatin. A comparable spreading of the Sir complex takes place at the telomeres, where the interaction with telomeric Rap1 enables the anchoring of the complex (MORETTI et al. 1994 ) and where a subsequent arrangement of a telomeric loop structure may drive the formation of a repressed state (PRYDE and LOUIS 1999 ). The Sir2, Sir3, and Sir4 proteins are limiting within the cell, leading to an internal competition between the telomeres and the HM loci (BUCK and SHORE 1995 ) as well as between the telomeres and the rDNA (SMITH et al. 1998 ) for these proteins.

In this study, we investigated the role of the RENT factor Net1 in silencing the HMR locus. We found that a mutation in NET1 (net1-1) as well as NET1 overexpression restored repression at silencing-defective HMR alleles. When overexpressed, Net1 was physically associated with the HMR-E silencer, suggesting that in this scenario, Net1 had a direct role in silencing. Net1 was also capable of providing silencing when artificially tethered to the HMR-E silencer. Interestingly, this repression was dependent upon ORC, but independent of Sir1. Furthermore, we characterized the effect of the net1-1 mutation on silencing. Our data suggested that net1-1 acted indirectly by relieving the competition between the rDNA locus and HMR for the silencing factor Sir2.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plasmid constructions:
The NET1 gene was cloned into a 2µ-plasmid by PCR amplifying a 3925-bp fragment from genomic DNA and ligating it into the SmaI-site of pRS 426 (SIKORSKI and HIETER 1989 ), resulting in pAE 567. To obtain a 2µ-plasmid with a LEU2 marker, the insert was released from pAE 567 with XhoI/SpeI and was inserted into XhoI/SpeI-treated pRS 425 (SIKORSKI and HIETER 1989 ), resulting in pAE 548. To construct the Gal4 DNA-binding domain fusion to Net1, the C terminus of NET1 (amino acids 566–1189) was amplified by PCR from genomic DNA with flanking BamHI and SalI sites and introduced into BamHI/SalI-treated pAE 107 behind the Gal4 DNA-binding domain, yielding pAE 610. pAE 107 is identical to pJR1639 (FOX et al. 1997 ) and is a CEN/URA3 vector in which GAL4 (amino acids 1–147) is cloned between the GPD promoter and the PGK terminator. To fuse NET1 with an epitope tag (pAE 622), we made use of 6xc-myc containing pRS 426 (pAE 569). In a first step, the promoter of NET1 was inserted upstream of the tag sequence as a PCR fragment with flanking KpnI and XhoI sites. In a second step, the NET1 open reading frame and terminator region were inserted downstream of the tag as a SpeI/NotI PCR fragment. The complete insert of pAE 622 was cloned as a KpnI/NotI fragment into the CEN-based plasmid pRS 316 to yield pAE 711. All fusions were verified by DNA sequence analysis.

Yeast strains and methods:
The relevant genotypes of the yeast strains used in this study are listed in Table 1. Strains were constructed by standard techniques of crossing, subsequent sporulation, and tetrad analysis. Genomic tagging by 3xHA was performed as described (KNOP et al. 1999 ). Growth and manipulation of yeast were carried out according to standard procedures (SHERMAN 1991 ). Patch-mating assays and quantitative mating assays were performed as described (EHRENHOFER-MURRAY et al. 1997 ), using AEY 264 (MATa his4) as the a mating tester strain. All quantitative mating efficiencies are the average of at least two independent determinations and were normalized to the wild-type strain AEY 1.

Chromatin crosslinking and immunoprecipitation:
Formaldehyde fixation as well as subsequent chromatin preparation and immunoprecipitation reactions were performed essentially as described (HECHT et al. 1996 ). Fixation was performed with 2% formaldehyde for 1 hr and quenched with 250 mM glycine. The crude lysate was precleared with protein A sepharose beads (Pharmacia, Piscataway, NJ) for 30 min and the resulting supernatant was subsequently incubated with -myc antibody (Invitrogen, San Diego) overnight at 4°. Immune complexes were isolated by incubating the extracts for 1 hr with protein A sepharose beads. Elution was performed at room temperature for 15 min, using 1% SDS/0.1 M NaHCO₃. To analyze the presence of specific DNA loci, 1/15 of the purified material was amplified by PCR for 28 cycles (HMR: HMR-1 5'-gctgatgcatgccaaacaaaaccc-3' and HMR-2 5'-ccctctcctcagacactactaag-3'; NTS: NTS-up 5'-tcgcatgaagtacctcccaactac-3' and NTS-down 5'-tccgcttccgcttccgcagtaaaa-3'; 25S: 25S-up 5'-aggacgtcatagagggtgagaatc-3' and 25S-down 5'-ttgacttacgtcgcagtcctcagt-3'; actin: act-up 5'-cggtagaccaagacaccaagg-3' and act-down 5'-gtcagtcaaatctctaccggcc-3'). PCR products were separated on 1% agarose gels and visualized with ethidium bromide.

Co-immunoprecipitations:
The preparation of extracts and co-immunoprecipitations (co-IP) were performed as described (SHOU et al. 1999 ) except that extracts were incubated with -myc or with -hemagglutinin (HA) overnight at 4°, followed by a 1-hr incubation with protein A sepharose beads.

Cell extract preparation and Western blotting:
Whole cell extracts were prepared by glass bead lysis (HAMPTON and RINE 1994 ), and proteins were separated on 8% SDS-polyacrylamide gels and immunoblotted with the following antibodies: Sir2, Sir3, and Sir4 (Santa Cruz Biotechnology; Santa Cruz, CA); -myc (Invitrogen); and -HA (Covance).

DNA preparation and Southern blotting:
Genomic DNA was prepared as described (HOFFMAN and WINSTON 1987 ), digested with HaeII, and separated on a 0.8% agarose gel, followed by DNA blot hybridization and detection of DNA using a URA3 probe.

Immunostaining:
Fixation, spheroblasting, and spreading of yeast nuclei were performed as described (TRELLES-STICKEN et al. 1999 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The net1-1 mutation improved silencing at a defective HMR allele:
The protein complex RENT is required for rDNA silencing, and mutations in NET1 cause derepression of marker genes inserted at the rDNA locus. Furthermore, the deletion of NET1 causes a slight increase in telomeric silencing (STRAIGHT et al. 1999 ). In this study, we sought to investigate in detail if Net1 also exerted an influence on HM silencing. Derepression of the HMR locus results in the simultaneous expression of a and information in MAT cells, which abolishes the mating ability of the cells. Thus, a loss of HMR silencing in MAT strains can be measured as a loss of mating ability.

In a first attempt, we determined the influence of a mutation in NET1, net1-1 (SHOU et al. 1999 ), on silencing of the HMR locus. We chose to use the net1-1 allele instead of a NET1 deletion, because the poor growth of net1 cells would have complicated the analysis (VISINTIN et al. 1999 ). net1-1 cells display a temperature-sensitive phenotype and have an abnormal, elongated shape. The capacity of Net1 to interact with Cdc14 is abolished in net1-1 cells as shown by co-immunoprecipitation experiments (SHOU et al. 1999 ). Introducing the mutation net1-1 did not derepress the wild-type HMR and HML loci, as MATa or MAT net1-1 strains showed no mating defect in a patch-mating assay (data not shown). However, in silencing, genes can act as both positive or negative regulators. Thus, to have an experimental setup with the possibility of observing both increases or decreases in silencing, we made use of a version of the synthetic HMR-E silencer in which the Abf1-binding site is mutated and where HMR-I is deleted (HMR-SS abf1^- I; MCNALLY and RINE 1991 ). The synthetic silencer is a minimal version of HMR-E that lacks most of the functional redundancies present at natural HMR-E. The introduction of the additional mutation in the Abf1-binding site causes a moderate 50-fold derepression.

A strain that was MAT HMR-SS abf1^- I and carried net1-1 was constructed, and its mating ability was compared to that of an isogenic NET1 strain (Fig 1A). The NET1 strain displayed a reduced -mating ability due to the expression of a1 information from the mutant HMR-SS abf1^- I allele. However, introducing net1-1 improved the mating ability, suggesting that silencing at the defective HMR locus was restored.

View larger version (50K): In this window In a new window Download PPT slide   Figure 1. The net1-1 mutation and the overexpression of NET1 from a 2µ-plasmid increased the silencing capacity of mutated HMR alleles without affecting the Sir2 protein level. (A) The mating ability of MAT HMR-SS abf1- I cells that were either NET1 (AEY 726) or net1-1 (AEY 1885) was tested in a patch-mating assay. (B) Expression of NET1 from a 2µ-plasmid increased the silencing ability of a MAT HMR-SS abf1- I (AEY 726), of a MAT HMRACSABF (AEY 70), and of a MAT HMRa-e** (AEY 403) strain, as measured by quantitative mating analysis. lc, low copy; hc, high copy. (C) Expression of NET1 from a 2µ-plasmid did not increase the silencing ability of a MAT ACS hmr::TRP1 strain with mutations in the RAP1 gene (AEY 760, AEY 72) as measured by growth on medium lacking tryptophane. (D) Expression of NET1 from a 2µ-plasmid was approximately sixfold higher than that from a CEN-based vector, as tested by Western blotting analysis of 6xmyc-tagged NET1 (pAE 711, pAE 622) in AEY 1558. (E) Sir2 protein levels were unaffected by additional NET1 expression. Protein extracts of AEY 726, transformed with pRS 426, pAE 711, or pAE 622, were analyzed by Western blotting.

NET1 overexpression enhanced silencing at defective HMR alleles:
The observation that net1-1 improved mating suggested that Net1 had a negative effect on HMR silencing. We next determined the effect of NET1 overexpression on silencing of the defective HMR-SS abf1^- I locus. For this purpose, a 2µ-plasmid containing NET1 under the control of its own promoter was introduced into a MAT strain carrying HMR-SS abf1^- I, and the mating ability of the strain was determined. Surprisingly, expression of NET1 from this plasmid improved the mating ability of the strain by 130-fold compared to the strain transformed with an empty vector (Fig 1B), suggesting that the overexpression of NET1 increased silencing at HMR. To determine whether a lower amount of Net1 might already lead to an increase in silencing capacity, we introduced NET1 on a low-copy CEN-based plasmid into the MAT HMR-SS abf1^- I strain and measured the mating ability. Indeed, the slightly overexpressed NET1 could improve the mating ability, albeit to a lesser extent than the 2µ-overexpression (Fig 1B). These observations suggested that upon increase of the Net1 protein amount, HMR silencing in this yeast strain could be restored, depending upon the amount of additional Net1. The extent of NET1 expression was determined by measuring the amount of a 6xmyc-epitope-tagged version of NET1 when present on the high-copy 2µ-plasmid or on the low-copy CEN-based vector. The epitope-tagged Net1 was functional in that it complemented the temperature sensitivity of the net1-1 mutation and suppressed HMR silencing defects when overexpressed (data not shown). 2µ-expressed 6xmyc-NET1 was expressed at least sixfold higher than that from the CEN-based vector as determined by Western blotting analysis (Fig 1D).

The fact that NET1 overexpression improved repression at an HMR allele mutated in the Abf1-binding site of HMR-E (HMR-SS abf1^- I) suggested that Abf1 was not required for Net1-mediated silencing. We next determined the cis requirements for this silencing. Mutations in the ORC- and Rap1-binding sites of the synthetic HMR-E silencer cause complete derepression and therefore are not suitable for this analysis. In contrast, the deletion of a combination of binding sites in the natural HMR-E silencer in the presence of HMR-I causes a moderate derepression (BRAND et al. 1987 ), comparable to HMR-SS abf1^- I, and thus was used here. High-copy expression of NET1 improved the mating ability of a MAT strain carrying deletions in the ORC- and Abf1-binding sites of the wild-type HMR-E silencer (HMR ACSABF) by 10-fold, whereas HMR ACSRAP was only slightly affected and HMR RAPABF was not affected by NET1 overexpression (Fig 1B). These results suggested that overexpressed NET1 required an intact Rap1-binding site at HMR-E to improve silencing. Moreover, rap1-12 and rap1-13 mutants, carrying a substitution of the HMRa information by the TRP1 gene, also did not show an enhanced silencing of the TRP1 reporter upon NET1 overexpression, as measured by growth on medium lacking tryptophane (Fig 1C), suggesting that a functional Rap1 protein was also required. However, silencing at an HMR allele with subtle point mutations in the Rap1- and Abf1-binding sites (HMRa-e**; KIMMERLY et al. 1988 ) was also affected by NET1 overexpression. Perhaps the point mutation in the Rap1-binding site in this context has a less severe effect than the deletion of the Rap1 site and thus could be suppressed more easily.

Silencing by NET1 overexpression depended on the Sir proteins:
In principle, the suppression of mating defects by high-copy NET1 could occur by mechanisms other than those that improve classical Sir-mediated silencing. To determine whether the restoration of mating was caused by a restoration of this type of silencing, we tested whether the presumptive silencing by NET1 overexpression depended upon the known structural components of silenced chromatin, the Sir proteins. MAT strains were constructed that carried a defective HMR allele (HMR-SS abf1^- I or HMR ACSABF) and a deletion of SIR1, SIR2, SIR3, or SIR4, and the mating ability of these strains in the presence or absence of overexpressed NET1 was determined. All strains were complete non-maters (data not shown), showing that HMR was derepressed in these strains. This was similarly the case in the sir2, sir3, and sir4 strains carrying the wild-type HMR allele and for a HMR-SS I sir1 strain (data not shown). Thus, the effect of NET1 overexpression required the SIR genes, suggesting that it established bona fide silencing at the mutant HMR alleles and that it functioned there via the Sir proteins.

NET1 overexpression did not influence expression of the Sir proteins:
How does Net1 increase silencing at HMR? One possibility is that Net1 would do so by increasing the cellular levels of silencing proteins, since higher expression of Sir proteins has been shown to lead to improved silencing (XU et al. 1999B ). To test whether NET1 overexpression affected Sir protein levels, we measured the amount of Sir2, Sir3, and Sir4 in a MAT HMR-SS abf1^- I strain that carried NET1 or 6xmyc-tagged NET1 on a 2µ-plasmid. As shown in Fig 1E, the amount of Sir2 as well as of Sir3 and Sir4 (data not shown) did not change upon additional expression of NET1. This showed that Net1 did not function in silencing by changing Sir protein levels and suggested a more direct role for Net1 at HMR.

Overexpressed Net1 was physically associated with the HMR-E silencer in chromatin:
One hypothesis for Net1's role in HMR silencing is that Net1, when overexpressed, interacts with the silencer binding proteins and helps to attract other silencing factors to establish silenced chromatin, perhaps through its ability to interact with Sir2. If Net1 is associated with the HMR-E silencer in chromatin, then HMR-E DNA should be enriched in a protein-DNA fraction prepared by immunoprecipitation of Net1. Yeast cells harboring the HMR-SS abf1^- I or the HMR-SS rap1^- I allele and overexpressing 6xmyc-NET1 were crosslinked with formaldehyde to generate covalent linkages between closely associated proteins and between proteins and DNA. Chromatin was isolated and sonicated to an average size of 0.5–1.0 kb, and 6xmyc-Net1 was immunoprecipitated from this mixture by using an -myc antibody. Net1-associated DNA was subsequently analyzed by PCR using specific primers. The overexpressed, myc-tagged Net1 protein could be found associated with rDNA loci (NTS, 25S), as has been shown previously (STRAIGHT et al. 1999 ; data not shown). Similarly, HMR-E was enriched in the immunoprecipitates of the HMR-SS abf1^- I strain but not of the HMR-SS rap1^- I strain, and the enrichment required both 6xmyc-NET1 and the -myc antibody (Fig 2A and data not shown). Conversely, ACT1 sequences were not enriched in the immunoprecipitates, arguing against unspecific binding of Net1 to chromatin. We further tested the possibility that overexpressed Net1 was bound on non-rDNA chromatin by performing immunostaining on chromatin of spread nuclei with 6xmyc-Net1. Even at high levels of NET1 expression, Net1 was located specifically on rDNA, but not on other chromatin (Fig 2B). HMR staining was not detected in this assay, presumably because the Net1 concentration at this locus was too low for detection. These results demonstrated that overexpressed NET1 was physically present at HMR-SS abf1^- I, but did not unspecifically coat chromatin, and thus likely had a direct role in HMR silencing, which depended upon the architecture of the HMR allele.

View larger version (61K): In this window In a new window Download PPT slide   Figure 2. Overexpressed Net1 protein was specifically associated with the HMR-E silencer. (A) Chromatin immunoprecipitates from formaldehyde crosslinked cells (MAT HMR-SS abf1- I, AEY 726) were analyzed by PCR using primers specific to the HMR-E silencer and to the ACT1 gene simultaneously. Identical immunoprecipitations were carried out without addition of antibody (--myc) and with cells lacking the overexpressed, tagged NET1 (no tag). The inverse image of ethidium-bromide-stained gels with representative amplifications is shown. (B) Net1-immunostaining experiments showed the nucleolar Net1 staining pattern that was independent of the extent of NET1 expression. The images show spread nuclei of fixed cells of AEY 726, transformed with pAE 426, pAE 711, or pAE 622.

Tethered Gal4-Net1 promoted ORC-dependent, Sir1-independent repression of HMR:
Since overexpressed Net1 was physically associated with the HMR-E silencer, we reasoned that it might have the capacity to recruit silencing proteins and to nucleate a repressive chromatin structure. To test this possibility, we asked whether Net1 could provide silencing when artificially tethered to the silencer in a so-called targeted silencing assay (CHIEN et al. 1993 ). In this assay, one of the protein binding sites in the HMR silencer is replaced by a varying number of Gal4-binding sites, which on their own have no silencing capacity. Silencing is then achieved by expressing fusions of silencing proteins with the Gal4 DNA-binding domain (amino acids 1–147, referred to as Gal4). The prototype silencing protein used in this assay is Gal4-Sir1 (CHIEN et al. 1993 ).

To test tethered silencing by Net1, a Gal4-Net1 hybrid was constructed by fusing the NET1 C terminus (amino acids 566–1189) with the Gal4-binding domain. We chose this part of Net1 because it shows an interaction with Sir2 in the yeast two-hybrid assay (CUPERUS et al. 2000 ). HMR silencing by Gal4-Net1 was tested in MAT strains in which either the ORC- or the Rap1-binding site was replaced by a single Gal4-binding site (HMR-Gal4-RAP-ABF I and HMR-ACS-Gal4-ABF I, respectively). As expected, in the absence of a fusion protein, these strains were non-maters due to derepression of HMR, but became efficient maters upon introduction of Gal4-Sir1 (Fig 3). When Gal4-Net1 was expressed in these strains, efficient mating was observed in the HMR-ACS-Gal4-ABF strain, showing that tethered Net1 could provide silencing. However, no silencing was found when Net1 was tethered to the ORC-binding site (data not shown). Since the number of Gal4-binding sites for some silencing proteins has previously been shown to be limiting (FOX et al. 1997 ), we also tested Gal4-Net1-mediated silencing when three or five Gal4-binding sites replaced the ACS. Only with five binding sites did we observe weak tethered silencing by Gal4-Net1 (Fig 3). These results showed that tethered Net1 had the capability of establishing repressive chromatin at HMR and suggested that Net1 required the presence of specific silencer binding proteins to mediate silencing.

View larger version (98K): In this window In a new window Download PPT slide   Figure 3. Net1 induced silencing when tethered to the Rap1-binding site of the HMR-E silencer. Repression at HMR was measured by patch-mating assays in MAT HMR-5xGal4-RAP-ABF I (AEY 500) and MAT HMR-ACS-Gal4-ABF I (AEY 454) cells. Gal4-Sir1 served as a positive control for targeted silencing. The dependence of Gal4-Net1-mediated silencing on Sir1, Sir2, and ORC was investigated in strains carrying a SIR1 or SIR2 deletion (AEY 1934 and AEY 1968, respectively) or the orc2-1 mutation (AEY 2132).

The observation that Gal4-Net1 silencing was efficient only when the single Gal4-binding site replaced the Rap1-binding site suggested that tethered silencing mediated by Net1 required a functional ORC-binding site. We therefore tested whether Gal4-Net1-mediated silencing also required one of the ORC proteins, Orc2, by introducing orc2-1 into the MAT HMR-ACS-Gal4-ABF strain. Silencing by Gal4-Net1 was abolished in this strain (Fig 3), showing that ORC was required for tethered Net1 silencing. ORC is thought to act in silencing by recruiting Sir1 to the silencer via an interaction between Sir1 and Orc1 (TRIOLO and STERNGLANZ 1996 ). If this recruitment were the major task of ORC in silencing, then we would also expect the Net1-driven silencing to be dependent on Sir1. We therefore tested the tethered Gal4-Net1 silencing in a sir1 strain. Interestingly, tethered Net1 was still able to establish silencing in the absence of Sir1 (Fig 3), suggesting that Sir1 function in silencing became dispensable in this scenario.

If the silencing property of tethered Net1 was based on the interaction between Net1 and Sir2 (CUPERUS et al. 2000 ), then Gal4-Net1-mediated silencing was expected to depend upon Sir2. Indeed, silencing by tethered Net1 could not be detected in the absence of Sir2 (Fig 3), suggesting that tethered Net1 established a repressive chromatin structure recruiting Sir2, and potentially other silencing proteins, to the silencer.

NET1 overexpression did not affect rDNA silencing and telomeric silencing:
Since increased cellular levels of Net1 were capable of improving HMR silencing, we next asked whether silencing at the other known silenced loci was influenced by NET1 overexpression. For this purpose, silencing of the URA3 reporter gene was monitored when inserted in the rDNA locus (SMITH and BOEKE 1997 ) or at an artificial telomere (GOTTSCHLING et al. 1990 ) by testing growth on medium lacking uracil or on medium containing 5-fluoroorotic acid (FOA), which inhibits growth of URA3-expressing cells due to the synthesis of the toxic compound 5-fluorouracil. However, NET1 overexpression had no effect on telomeric URA3 silencing, even when telomeric silencing was compromised by an orc2-1 mutation (Fig 4A). Also, expression of URA3 in the rDNA locus was unaffected by high-copy NET1 (Fig 4B). We also sought to investigate the effect of NET1 on HML silencing. Since no suitable HML silencer mutants are available, we tested whether NET1 overexpression could suppress the intermediate silencing defect at HML in a sir1sas2 strain (EHRENHOFER-MURRAY et al. 1997 ). However, NET1 overexpression was unable to suppress, probably because NET1-mediated silencing required Sir1 (Fig 4C).

View larger version (74K): In this window In a new window Download PPT slide   Figure 4. NET1 overexpression did not affect telomeric, rDNA, or HML silencing. (A) Telomeric silencing was determined by measuring the expression of URA3, when inserted in the telomeric region of chromosome VII (AEY 1017), by testing growth on medium lacking uracil and on FOA-containing medium. Experiments were also performed with a telomeric silencing-deficient orc2-1 strain (AEY 2143). (B) rDNA silencing was determined by measuring the expression of the URA3 gene inserted in the nontranscribed region of an rDNA unit (AEY 1778). (C) HML silencing was measured in a silencing-deficient MATa sir1 sas2 strain (AEY 346), using a patch-mating assay. Introduction of a SAS2-containing plasmid but not a NET1 overexpressing plasmid restored its mating deficiency.

Net1-based HMR silencing did not involve a stable interaction with Rap1:
The above data led us to further investigate the functional importance of Rap1 in Net1-based silencing mechanisms. Rap1- and ORC-binding sites also exist inside the repeated rDNA locus, where Net1 is present in the RENT complex. As it is not yet clear how RENT is tethered to the DNA, we sought to elucidate whether Rap1 or ORC was important for rDNA silencing.

For this purpose, we integrated Ty1-mURA3 into the 25S region of the rDNA locus (RDN1) in strains that either were wild type or carried mutations in RAP1 (rap1-12, rap1-13) or ORC (orc2-1, orc5-1). To estimate the number of tandem integrations, the band intensity of URA3 in RDN1::Ty1-mURA3 was compared to that of the native URA3 locus in a Southern blot. Transformants were chosen that carried one or two integrated copies of the URA3 gene. The level of URA3 expression was measured by growth on medium lacking uracil as well as on FOA-containing medium. While mutations in the RAP1 gene had only a minor influence on the cells' ability to grow in the presence of FOA, orc mutants were FOA sensitive, indicating a higher level of URA3 expression and, thus, less Ty1-mURA3 silencing in these strains (Fig 5A). However, neither orc nor rap mutations repressed URA3 at its native locus, since such strains were completely FOA sensitive (Fig 5A and data not shown). Taken together, these results suggested that ORC, but not Rap1, was required for rDNA silencing.

View larger version (43K): In this window In a new window Download PPT slide   Figure 5. orc mutations reduced rDNA silencing. (A) RDN1::Ty1-mURA3 expression was tested by plating serial dilutions of the respective strains on complete medium, on medium lacking uracil, or on FOA-containing medium. The number of Ty1-mURA3 integrations in each strain was measured by Southern blotting analysis. The strains used were AEY 2741 (wt URA3+), AEY 2742 (wt), AEY 2744 (rap1-13), AEY 2743 (rap1-12), AEY 2745 (orc5-1), and AEY 2746 (orc2-1). (B) Net1 and Rap1 did not interact in co-immunoprecipitation experiments. The interaction of Sir2 and Net1 served as a positive control. The respective strains were obtained by transforming AEY 2548 (Rap-3xHA) and AEY 2551 (Sir2-3xHA) with a 6xmyc-Net1-containing plasmid (pAE 622).

We further tested whether Net1 interacted physically with Rap1 by co-IP. For this purpose, the 2µ-plasmid carrying the 6xmyc-tagged version of NET1 was introduced into a yeast strain that expressed an HA-tagged Rap1 protein (Rap1-3xHA) from its native promoter. As a positive control, a co-IP was also performed between 6xmyc-tagged Net1 and Sir2-3xHA, since Sir2 and Net1 have previously been shown to interact (GHIDELLI et al. 2001 ; SHOU et al. 2001 ). Whereas Sir2 co-immunoprecipitated with Net1, we could not detect coprecipitated Rap1 (Fig 5B), suggesting that Rap1 and Net1 were not or were only weakly associated with each other.

Increased dosage of Sir2 recapitulated the net1-1 silencing phenotype:
The experiments above supported the view that the Net1 protein, when overexpressed, interacted with silencer binding proteins at the HMR-E silencer and recruited the Sir proteins to form repressed chromatin. Thus, one might propose that, under normal expression, Net1 would be present at HMR and that the mutation of NET1 would therefore decrease silencing. However, contrary to this view, we had observed that net1-1 improved the mating of a MAT HMR-SS abf1^- I strain (see above), thus arguing that the influence of net1-1 in silencing was distinct from that of NET1 overexpression. We therefore sought to determine by what mechanism net1-1 was restoring silencing.

In a first set of experiments, we further characterized the net1-1 effect on defective silencer variants. net1-1 was able to improve the mating ability of strains carrying all the tested HMR alleles (Fig 6), confirming the hypothesis that the net1-1 mutation and the NET1 overexpression nucleated silencing in two distinct ways. The increase in the mating capacity most likely reflected an increase in silencing at HMR, because the net1-1 strains became complete non-maters upon a deletion of SIR1, SIR2, or SIR3 (data not shown).

View larger version (118K): In this window In a new window Download PPT slide   Figure 6. Increased HMR silencing in net1-1 cells was recapitulated by high-copy expression of Sir2. The effect of net1-1 and SIR2 overexpression was tested in MAT strains carrying various mutations in the HMR-E silencer. Strains AEY 70, 69, 98, 403, and 726 (from top to bottom) were NET1, and strains AEY 1793, 1889, 1891, 1888, and 1885 were net1-1. SIR2 overexpression was tested in the NET1 series of strains by introducing SIR2 on the dLEU2 plasmid pJDB 207 (BUCHMAN et al. 1988 ).

Net1 interacts with Sir2 in the RENT protein complex at the rDNA locus and is required for Sir2's association with the rDNA (STRAIGHT et al. 1999 ). We therefore reasoned that in the net1-1 strain, the interaction between Net1 and Sir2 might be disturbed such that Sir2 failed to localize to the nucleolus. Perhaps Sir2 would then be released into the nucleus, thus increasing the level of Sir2 available for HMR silencing. If the effect of net1-1 on HMR was indirect through the release of nucleolar Sir2, one prediction would be that the third class of transcriptional repression, telomeric silencing, would also improve in the absence of Net1. In agreement with this, a slightly increased level of telomeric silencing has been detected in net1 cells (STRAIGHT et al. 1999 ). Also supporting this theory, SIR2 overexpression has previously been shown to suppress the silencing defect of the HMRa-e** allele (XU et al. 1999A ). Therefore, we tested the effect of increased Sir2 dosage on the other defective HMR alleles. All HMR alleles tested were suppressed by high-copy Sir2 to a similar extent as by the net1-1 mutation (Fig 6), indicating that the enhanced availability of nuclear Sir2 in net1-1 cells might be responsible for the improved HMR silencing.

Sir2 was distributed throughout the nucleus in net1-1 cells:
Our hypothesis posits that net1-1 liberates Sir2 from the nucleolus and increases the amount of Sir2 in the whole nucleus. The Sir2 association with rDNA has previously been shown to be lost upon deletion of NET1 (STRAIGHT et al. 1999 ), but it remained unclear what happened to Sir2 in net1-1 mutant cells. To follow the fate of Sir2 in net1-1 cells, we introduced a green fluorescent protein (GFP)-tagged Sir2 (CUPERUS et al. 2000 ) into a wild-type NET1 and into a mutant net1-1 strain and observed its cellular distribution by fluorescence microscopy. In a wild-type strain, as well as in a NET1 overexpressing strain, nucleolar Sir2 was present as a half-moon-shaped structure at the edge of the nucleus. In contrast, Sir2-GFP showed a strikingly different staining pattern in net1-1 cells, in that the nucleolar half-moon structure was completely absent (Fig 7). Instead, the whole nucleus appeared weakly stained by Sir2-GFP. We next measured the amount of Sir2-GFP protein in wild-type and net1-1 strains by Western blotting analysis. Both strains displayed equal levels of Sir2-GFP (data not shown), showing that net1-1 did not decrease the cellular level of Sir2. Therefore, the nuclear staining pattern of Sir2-GFP demonstrated that Sir2 became distributed throughout the nucleus in net1-1 strains, which supported the notion that net1-1 acted indirectly in HMR silencing by liberating Sir2 from the nucleolus. Thus, these experiments suggested that the rDNA locus and HMR competed for limiting amounts of Sir2.

View larger version (37K): In this window In a new window Download PPT slide   Figure 7. Delocalization of Sir2 from the nucleolus in a net1-1 mutant. Sir2-GFP was visualized in living cells by fluorescence microscopy under a FITC filter. Hoechst was used to stain the cellular DNA. Sir2-GFP appeared as a distinct nucleolar shape at the nuclear periphery in wild type (wt) and NET1 overexpressing cells, but was spread over the whole nucleus in net1-1 cells. The strains used were HMR-SS abf1-I NET1 (AEY 726) and HMR-SS abf1-I net1-1 (AEY 1885), transformed with GLC462 (CUPERUS et al. 2000 ), and, for overexpression of NET1, with pAE 622.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Net1 protein is a central component of the nucleolar protein complex RENT that promotes rDNA silencing and nucleolar integrity and regulates the exit from mitosis in yeast. In this study, we have identified a role for Net1 in repression of the silent mating-type locus HMR of S. cerevisiae. Significantly, both high-copy expression and mutation of NET1 suppressed mutations in the HMR-E silencer. Both effects were dependent upon the function of the Sir proteins, suggesting that bona fide silencing was established under these conditions. Thus, NET1 joins genes such as the cell cycle genes CDC7 (AXELROD and RINE 1991 ), CDC45, and POL30 (EHRENHOFER-MURRAY et al. 1999 ) as well as the SAS genes SAS2, SAS3, SAS4, and SAS5 as genes that, when mutated, improve silencing (REIFSNYDER et al. 1996 ; EHRENHOFER-MURRAY et al. 1997 ; XU et al. 1999B ). However, the mechanisms of how these genes act in silencing are likely to be quite distinct. We invoke two models for the effect of Net1 on HM silencing: (1) a direct role in silencing for overexpressed Net1, since it was physically associated with HMR-E sequences, and (2) an indirect role for net1-1, namely, by increasing the amount of Sir2 available for HMR silencing by releasing Sir2 from the nucleolus.

There are several possibilities for how elevated levels of Net1 could improve silencing. Overexpressed Net1 could have indirect effects on silencing, for instance, by disrupting the RENT complex in a dominant-negative fashion and releasing Sir2 or other components from the nucleolus or by influencing cell cycle progression, the deceleration of which has been proposed to increase the likelihood of establishing repressed chromatin (LAMAN et al. 1995 ). However, we do not favor these interpretations, because Sir2 was still nucleolar in cells overexpressing NET1 (Fig 7). Additionally, these cells did not show any detectable growth defect or differences in doubling time, arguing against an effect on cell cycle progression. Importantly, we found that Net1 was physically associated with HMR-E DNA, suggesting a direct role for Net1 in HMR silencing. Since Net1 has no recognizable DNA-binding capacity (STRAIGHT et al. 1999 ), this interaction is likely to be mediated by silencer binding proteins. The observation that Net1-mediated HMR silencing required a functional Rap1-binding site at HMR-E and that NET1 overexpression could not overcome the silencing defect of a rap1-12 or a rap1-13 strain suggests a direct or indirect recruitment of Net1 to HMR-E through Rap1. However, we were unable to detect an interaction between Rap1 and Net1 by co-immunoprecipitation, which supports the argument that a potential association, if present, may be unstable or bridged by other proteins. Alternatively, Net1 may localize to HMR via its interaction with Sir2, which in turn may require an intact Rap1-binding site. Notably, silencing at an artificial telomere, which depends upon tandem Rap1-binding sites, was not strengthened by NET1 overexpression (Fig 4A), indicating that other cis-acting elements at HMR-E were required to mediate the NET1 silencing effect. In agreement with this, rDNA silencing was not weakened by mutations in Rap1 (rap1-12, rap1-13). However, the possibility remains that these mutations alter only rDNA-independent Rap1 functions. Interestingly, in tethered silencing experiments, Net1 was able to promote silencing only when Gal4-Net1 substituted for Rap1, further suggesting that the position of Net1 within the silencer was important for its silencing effect. Notably, Net1 established Sir1-independent silencing in the artificial tethering experiments, whereas silencing upon NET1 overexpression required Sir1. Perhaps Sir1 is required to recruit Net1 to the silencer and hence is dispensable in the tethered silencing experiments. Alternatively, tethered Net1 might be associated with the silencer in a more stable fashion than overexpressed Net1. Tethered Net1 may therefore recruit the Sir2/3/4 complex more efficiently, thus circumventing the need for Sir1. Interestingly, the Net1-tethered silencing was abolished in an orc2-1 mutant, showing its dependence upon ORC. Also, rDNA silencing, which is independent of Sir1, was compromised in an orc2-1 strain. Thus, ORC displayed functions in silencing that went beyond the recruitment of Sir1. In light of this, it is interesting that the association of Sir1 at HMR requires not just ORC, but also Sir2 (GARDNER and FOX 2001 ), suggesting that the roles of both ORC and Sir1 are more complex than the simple Sir1 recruitment by ORC. With respect to rDNA silencing, our data suggest the possibility that ORC is involved in RENT binding to rDNA.

Interestingly, overexpressed NET1 yielded silencing at the HMR ACSABF allele, suggesting that this silencing was ORC independent due to the deletion of the ACS site. This seems at odds with the observation that tethered Net1 silencing required ORC. Thus, a possible connection between ORC and Net1 may depend upon the way Net1 is recruited to the silenced region. Alternatively, silencing at the HMR ACS allele may still require ORC, because several origins close to HMR-E become active upon mutation of the silencer origin (PALACIOS DEBEER and FOX 1999 ).

How does Net1 promote silencing once it is recruited to the silencer? Since Net1 interacts with Sir2 in the nucleolus, it may also primarily attract Sir2 (and potentially other unidentified RENT components) to the HMR-E silencer. Sir2 may then deacetylate histones in the HMR chromatin domain, which would lead to a more efficient recruitment of Sir3 and Sir4 through their interaction with unacetylated histone tails (HECHT et al. 1995 ). The Net1-driven anchoring of the Sir2/3/4 complex to DNA may be related to the attraction of this complex to the nucleolar rDNA that takes place in aging mother cells and in sir4-42 cells (KENNEDY et al. 1997 ). In this respect, it is interesting to note that cells coexpressing a and information display accelerated aging (KAEBERLEIN et al. 1999 ). Thus, the prevention of HMR expression by excess Net1 may be interpreted as an additional mechanism to promote longevity in yeast.

An alternative possibility as to how silencer-bound Net1 promotes silencing is that Net1 may locally stabilize the Sir2 protein or the whole Sir complex at HMR, similar to Cdc14, whose degradation is prevented through its nucleolar association with Net1 (SHOU et al. 1999 ). This may lead to increased local concentrations of Sir2, which would then improve silencing at HMR. Net1 may also act at HMR by directly modifying the deacetylase activity of Sir2, much as it regulates the phosphatase activity of Cdc14 (TRAVERSO et al. 2001 ), for instance, by activating Sir2 through physical contact or by deactivating a yet unidentified inhibitor of Sir2.

Formally, the silencing improvement in a net1-1 mutant invokes a negative effect of Net1 on HMR silencing, since the lack of Net1 function leads to enhanced HMR silencing. For instance, Net1 might act in silencing by inhibiting a silencing factor, or net1-1 might indirectly improve silencing by changing cell cycle progression. However, we favor the model that the availability of Sir2 for HMR silencing is increased in the net1-1 mutant, because we observed a Sir2 delocalization from the nucleolus in net1-1 cells and because SIR2 overexpression phenocopied the net1-1 effect at HMR (Fig 6 and Fig 7). Formally, we cannot exclude the possibility that the net1-1 mutation releases other nucleolar factors that might participate in HMR silencing. In summary, our data suggest a competition for Sir2 (and potentially other silencing proteins) between the rDNA and HMR, similar to the competition that has previously been observed between rDNA and telomeric silencing (SMITH et al. 1998 ) as well as between the telomeres and HM silencing (BUCK and SHORE 1995 ).

As in yeast, rDNA gene units are arranged in multiple tandem repeats in higher eukaryotes. For instance, rDNA arrays in Drosophila lie on the sex chromosomes, where they act as pairing sites between the X and Y chromosomes during male meiosis (BRISCOE and TOMKIEL 2000 ). Net1 homologs have been identified in Drosophila as well as in other eukaryotes (COSTANZO et al. 2000 ). Thus, in analogy to Net1's role in yeast, these homologs may likewise be involved in nucleolar functions and in epigenetic gene regulation in these organisms. Furthermore, our experiments shed light on the dynamics between the different silenced regions in the genome of S. cerevisiae. Perhaps a similar intranuclear competition for limiting silencing factors also is in play in higher organisms.

	ACKNOWLEDGMENTS

We thank D. Shore, R. H. Deshaies, J. Boeke, and J. Rine for strains and plasmids and M. Grunstein for the ChIP protocol. We also thank A. Geissenhöner, A. Grünweller, and S. Meijsing for comments on the manuscript, A. Barduhn and K. Vogel for excellent technical assistance, H. Scherthan and E. Trelles-Sticken for support with immunostainings, and all members of our laboratory for many stimulating discussions.

Manuscript received December 13, 2001; Accepted for publication May 7, 2002.

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