SAS4 and SAS5 Are Locus-Specific Regulators of Silencing in Saccharomyces cerevisiae

Corresponding author: David H. Rivier, Department of Cell and Structural Biology, University of Illinois, 601 S. Goodwin Ave., Urbana, IL 61801., rivier{at}uiuc.edu (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sir2p, Sir3p, Sir4p, and the core histones form a repressive chromatin structure that silences transcription in the regions near telomeres and at the HML and HMR cryptic mating-type loci in Saccharomyces cerevisiae. Null alleles of SAS4 and SAS5 suppress silencing defects at HMR; therefore, SAS4 and SAS5 are negative regulators of silencing at HMR. This study revealed that SAS4 and SAS5 contribute to silencing at HML and the telomeres, indicating that SAS4 and SAS5 are positive regulators of silencing at these loci. These paradoxical locus-specific phenotypes are shared with null alleles of SAS2 and are unique among phenotypes of mutations in other known regulators of silencing. This work also determined that these SAS genes play roles that are redundant with SIR1 at HML, yet distinct from SIR1 at HMR. Furthermore, these SAS genes are not redundant with each other in silencing HML. Collectively, these data suggest that SAS2, SAS4, and SAS5 constitute a novel class of regulators of silencing and reveal fundamental differences in the regulation of silencing at HML and HMR. We provide evidence for a model that accounts for the observation that these SAS genes are both positive and negative regulators of silencing.

THREE regions of the yeast genome, the HML and HMR cryptic mating-type loci and the regions adjacent to the telomeres, are each assembled into a heterochromatic structure that inactivates transcription. Inactivation of transcription at HML and HMR is referred to as silencing, whereas inactivation of transcription in the telomeric regions is typically referred to as the telomeric position effect or TPE. Silencing and TPE depend on histone H3, histone H4, and on Sir2p, Sir3p, and Sir4p, which associate with each other to form heterochromatin in the silent regions (reviewed in GRUNSTEIN 1997 , GRUNSTEIN 1998 ; LUSTIG 1998 ). Furthermore, silencing and TPE are mitotically stable forms of gene inactivation; once a gene is silenced it remains silent through many rounds of cell division (PILLUS and RINE 1989 ; GOTTSCHLING et al. 1990 ). The initial inactivation of the gene, establishment, corresponds to the assembly of heterochromatin, whereas the clonal propagation of silencing, inheritance, presumably results from the duplication of heterochromatin during DNA replication and mitosis. A related form of silencing also occurs at the RDN1 locus, the region of the yeast genome that contains approximately 200 repeated copies of the ribosomal DNA (rDNA; BRYK et al. 1997 ; SMITH and BOEKE 1997 ).

Silencing at HML and HMR requires DNA elements known as silencers (ABRAHAM et al. 1983 ; FELDMAN et al. 1984 ; BRAND et al. 1985 ). The two silencers that flank HML are known as HML-E and HML-I, and the two that flank HMR are known as HMR-E and HMR-I. The silencers bind combinations of three proteins: ORC, the replication initiator protein, and two transcriptional activators, Rap1p and Abf1p (reviewed in LAURENSON and RINE 1992 ; LOO and RINE 1995 ).

The establishment of silencing and assembly of heterochromatin in the silent regions is thought to occur in two steps. The first step, nucleation, involves the initial recruitment of Sir3p and Sir4p to the silent regions. The second step involves the subsequent spreading or polymerization of heterochromatin throughout the region. At least one role of the silencers, telomeres, and their associated proteins is to nucleate the formation of heterochromatin. In particular, Rap1p binds the silencers and telomeres and recruits Sir3p and Sir4p to the loci, and Sir3p and Sir4p, in turn, nucleate the assembly of heterochromatin (MORETTI et al. 1994 ; LUSTIG et al. 1996 ; MARCAND et al. 1996 ). Similarly, Sir1p binds to ORC, recruits Sir4p, and plays a central role in nucleating silencing (CHIEN et al. 1993 ; TRIOLO and STERNGLANZ 1996 ; GARDNER et al. 1999 ).

Each of the silenced regions is differentially sensitive to mutations in the genes that contribute to, but are not required for, silencing. For instance, NAT1 and ARD1 encode subunits of an N-terminal acetyl transferase that positively regulates silencing (WHITEWAY et al. 1987 ; MULLEN et al. 1989 ; APARICIO et al. 1991 ; PARK and SZOSTAK 1992 ). Mutations in NAT1 or ARD1 result in a loss of TPE and a partial loss of silencing at HML but do not result in a loss of silencing at HMR (WHITEWAY et al. 1987 ; MULLEN et al. 1989 ; APARICIO et al. 1991 ). However, mutation of NAT1 or ARD1 results in a substantial loss of silencing at HMR in combination with a mutation in SIR1 (WHITEWAY et al. 1987 ; STONE et al. 1991 ). Consequently, it has been proposed that a hierarchy of silencing exists in which silencing at the telomeres is less efficient than silencing at HML, which is less efficient than silencing at HMR (APARICIO et al. 1991 ).

The differential efficiency of silencing among the silent loci is due, at least in part, to the locus-specific action of Sir1p. Deletion of SIR1 results in a partial loss of silencing at HML and HMR but does not result in a defect in TPE (APARICIO et al. 1991 ). Thus, Sir1p contributes to silencing at HML and HMR but does not contribute to TPE. Consequently, the increased efficiency of silencing at HML and HMR relative to TPE is likely due, at least in part, to the action of Sir1p at HML and HMR but not at the telomeres. The basis for the greater efficiency of silencing at HMR relative to HML is not known.

The efficiency of silencing in a particular region can also be influenced indirectly by perturbations that alter the physical distribution of the protein components of heterochromatin within the nucleus. For instance, deletion of SIR4 results in increased silencing at the RDN1 locus (SMITH and BOEKE 1997 ). In contrast to HM silencing and TPE, SIR4 is not a direct regulator of silencing at RDN1 (J. S. SMITH et al. 1998 ). However, SIR2 is required for rDNA silencing, and furthermore, the endogenous level of Sir2p is limiting for silencing within the rDNA. It has been proposed that deletion of SIR4 results in a loss of TPE and a failure of Sir2p to sequester at the telomeres, thereby increasing the effective concentration of free Sir2p and resulting in increased silencing in the rDNA (J. S. SMITH et al. 1998 ). Therefore, deletion of SIR4 is thought to increase silencing in the rDNA as an indirect consequence of disruption of TPE.

Taken together, these observations suggest that silencing is regulated by three classes of genes: (1) genes that encode components of heterochromatin or direct regulators of silencing at HML, HMR, and the telomeres; (2) genes that encode locus-specific regulators of silencing; and (3) genes that encode proteins that indirectly effect silencing by altering the distribution of components of the silencing machinery.

Deletion of SAS2 causes silencing defects at HML and telomeres but suppresses silencing defects at HMR (REIFSNYDER et al. 1996 ; EHRENHOFER-MURRAY et al. 1997 ). Therefore, SAS2 behaves as a positive regulator of TPE and silencing at HML and a negative regulator of silencing at HMR. These opposite phenotypes at HML and HMR are unique among mutations known to effect silencing, suggesting that an understanding of the basis for these locus-specific phenotypes will likely lead to new insights into the regulation of silencing. We recently identified two genes, SAS4 and SAS5, that, when mutated, are capable of restoring silencing at HMR in the presence of a partially defective HMR-E silencer (XU et al. 1999 ). Thus, SAS4 and SAS5, like SAS2, are formally negative regulators of silencing at HMR. In this report we investigated whether the SAS4 and SAS5 genes had the same set of unique locus-specific regulatory properties as SAS2. Furthermore, we investigated a possible mechanism by which SAS2 acts as a positive regulator of silencing at HML and a negative regulator of silencing at HMR.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strain construction:
The entire coding regions of the SAS4 and SAS5 genes were deleted by PCR-mediated gene disruption (BAUDIN et al. 1993 ) as described previously (XU et al. 1999 ). SAS4 was deleted from the haploid strains UCC1001 and DRY439 to generate DRY1371 and DRY1364, respectively. All gene disruptions were confirmed by DNA blot analysis. All additional W303-derived strains containing the sas4::kanMX4 allele were derived from crosses of DRY1322 to standard laboratory strains, as described below (see Table 1). SAS5 was deleted from haploid strains UCC1001, UCC1003, and JRY5273, resulting in DRY1372, DRY1392, and DRY1314, respectively. All additional W303-derived strains containing the sas5::HIS3 allele were derived from crosses of DRY1314 to standard laboratory strains, as described below. sas2-1::TRP1 strains were similarly derived from crosses with JRY5071 (MAT sas2-1::TRP1; EHRENHOFER-MURRAY et al. 1997 ).

A series of strains (DRY1655–1657, DRY1661–1664, and DRY1697–1699) containing various combinations of null alleles of the SAS genes with wild-type HMR were segregants derived from a diploid formed from a cross between JRY5071 and DRY1345 (W303-1a; hmr::URA3 sas4::kanMX4 sas5::HIS3).

Strains containing combinations of null alleles of the SAS genes together with a null allele of SIR1 were generated from two crosses. DRY1658 and DRY1800 were segregants from a cross between JRY4622 and DRY1805 (W303-1a; MAT sas2-1::TRP1). DRY1659, DRY1660, DRY1801, and DRY1802 were segregants from a cross between JRY4622 and DRY1806 (W303-1a; MAT sas4::kanMX4 sas5::HIS3). DRY1805 and DRY1806 were segregants from the cross between JRY5071 and DRY1345 described above.

DRY1399 (HMRa-e** sir1::LEU2) was a segregant derived from a cross between JRY4622 (sir1::LEU2) and DRY1314 (HMRa-e** sas5::HIS3). DRY1424 (HMR-SS I sas5::HIS3) was a segregant from a cross between DRY439 (HMR-SS I) and DRY1316 (W303-1a; MATa HMR-ssabf1::ADE2 sas5::HIS3).

PCR protocol:
PCR reactions for gene disruption were carried out using the high-fidelity Elongase kit (GIBCO, Grand Island, NY) under the conditions recommended by the manufacturer.

Plasmid construction:
pDR590 (pRS426-SIR3) was constructed by cloning a 4.5-kb SalI fragment containing the SIR3 gene from pJR508 (provided by J. Rine) into SalI cleaved pRS426 (CHRISTIANSON et al. 1992 ). pDR583 (pRS426-SIR4) was constructed in two steps. A 6.8-kb EcoRI-SstII fragment of SIR4 derived from pJR368 (provided by J. Rine) was inserted into pBluescript cleaved with EcoRI and SstII resulting in pDR304. The XhoI-SstII SIR4-containing fragment of pDR304 was inserted into XhoI-SstII-cleaved pRS246 resulting in pDR583.

Quantitative and patch mating assays:
Quantitative matings were performed as described previously (XU et al. 1999 ). For patch mating analysis, test strains were patched onto solid rich medium, grown overnight, replica plated onto a lawn of ~1.2 x 10⁷ MATa cells (JRY2726) or MAT cells (JRY2728) on YM plates supplemented with adenine, and grown for 1–2 days at 30°. Strains containing pRS426-derived plasmids were patched onto solid minimal medium lacking uracil, incubated for 2 days at 30°, and replica plated onto mating lawns as described above.

Assay for TPE:
Silencing of the TEL(VIIL) adh4::URA3 gene (GOTTSCHLING et al. 1990 ) was measured as a function of growth on medium containing 5-fluoroorotic acid (5-FOA; GUTHRIE and FINK 1991 ). Aliquots (5 µl) of 10-fold serial dilutions containing from 10⁶ to 10² cells per aliquot were spotted onto solid minimal medium containing 5-FOA and incubated for 2–3 days at 30°. As a control for cell viability, 5-µl aliquots of the serial dilutions were also spotted onto solid rich medium and onto minimal medium supplemented with uracil.

Media and genetic manipulations:
Rich medium (YPD) and minimal medium (YM) were as described (SHERMAN 1991 ). Medium containing 5-FOA was as described (GUTHRIE and FINK 1991 ). Transformation was by a modified lithium-acetate method (GIETZ and SCHIESTL 1991 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

SAS4 and SAS5 are required for TPE:
The HMR-E silencer is composed of an ARS consensus sequence (ACS) element, which is the binding site for ORC, and one binding site each for Rap1p and Abf1p (BRAND et al. 1987 ; KIMMERLY et al. 1988 ; MCNALLY and RINE 1991 ). SAS2, SAS4, and SAS5 were identified by recessive mutations that restored silencing to an allele of HMR that contained the defective HMRa-e** silencer (AXELROD and RINE 1991 ; EHRENHOFER-MURRAY et al. 1997 ; XU et al. 1999 ). This silencer contains a point mutation in the Rap1 binding site and a 1-bp insertion in the Abf1 binding site and is almost completely defective in silencing. Null mutations in SAS2, SAS4, or SAS5 restore silencing to HMRa-e** (REIFSNYDER et al. 1996 ; EHRENHOFER-MURRAY et al. 1997 ; XU et al. 1999 ).

To further characterize the role of SAS4 and SAS5 in silencing, we tested whether these genes were required for TPE. Yeast strains that transcribe URA3 are sensitive to the drug 5-FOA, whereas strains that do not transcribe URA3 are resistant to 5-FOA (GUTHRIE and FINK 1991 ). Strains that contain URA3 inserted into the ADH4 locus adjacent to an artificial telomere [TEL(VIIL) adh4::URA3] display a variegated phenotype of URA3 expression (GOTTSCHLING et al. 1990 ). In approximately half the cells in the TEL(VIIL) adh4::URA3 population, heterochromatin spreads from the telomere to the URA3 gene and silences it, resulting in 5-FOA resistance. In the other half of the cells in the population, URA3 is not silenced, resulting in 5-FOA sensitivity. To test the possible role of SAS4 and SAS5 in TPE, these genes were individually deleted from a strain carrying the TEL(VIIL) adh4::URA3 allele and silencing was monitored by 5-FOA sensitivity. The proportion of cells sensitive to 5-FOA increased by at least five orders of magnitude as a result of deletion of either SAS4 (DRY1371) or SAS5 (DRY1372; Figure 1). In contrast, deletion of SAS4 or SAS5 did not alter the proportion of cells sensitive to 5-FOA in strains containing a mutant allele of URA3 (DRY1391) or a copy of URA3 that was not adjacent to a telomere (DRY1392) and was not subject to TPE (Figure 1 and data not shown). Thus, SAS4 and SAS5 are required for TPE. Furthermore, these results indicate that Sas4p and Sas5p can play an essential role in silencing independent of Sir1p, since, as described above, Sir1p does not play a role in TPE.

View larger version (43K): In this window In a new window Download PPT slide   Figure 1. SAS4 and SAS5 are required for TPE. (A) Deletion of SAS4 or SAS5 disrupts telomeric silencing and results in sensitivity to 5-FOA. A dilution series of isogenic strains plated on solid medium containing 5-FOA is shown. The number of cells plated per dilution is indicated at the bottom. The strains shown are UCC1001 (WT, TEL-VIIL adh4::URA3), DRY 1372 (sas5, TEL-VIIL adh4::URA3), DRY 1371 (sas4, TEL-VIIL adh4::URA3), DRY 1392 (sas5 URA3), and DRY1391 (sas5 ura3). (B) Viability of strains on solid rich medium. Aliquots of the serial dilutions from A were plated onto rich medium to control for cell viability.

SAS4 and SAS5 are positive regulators of silencing at HML:
To determine whether SAS4 or SAS5 is required for silencing at HML, we used a quantitative mating-type assay to monitor expression of the HML genes. Wild-type MATa strains display the a-mating phenotype, whereas MATa strains in which silencing at HML is disrupted display the nonmating phenotype. Similar to deletion of SIR1 (JRY4622), deletion of either SAS4 (DRY1656) or SAS5 (DRY1657) results in a modest reduction in silencing at HML as indicated by quantitative mating analysis (Figure 2A). Therefore, both SAS4 and SAS5 contribute to the efficient silencing of HML but neither is required for silencing of HML.

View larger version (84K): In this window In a new window Download PPT slide   Figure 2. Contribution of SAS2, SAS4, and SAS5 to silencing at HML. Strains shown are isogenic with W303-1a. Qualitative patch mating assays are shown in the panels with quantitative mating analysis given below and genotypes above. (A) a-Mating phenotype of MATa HML strains mutant in individual genes. Strains shown are JRY4622 (sir1), DRY1655 (sas2), DRY1656 (sas4), and DRY1657 (sas5). (B) a-Mating phenotype of strains mutant in SIR1 and individual SAS genes. Strains shown are W303-1a (WT), DRY1236 (sir4), DRY1658 (sir1 sas2), DRY1659 (sir1 sas4), and DRY1660 (sir1 sas5). (C) a-Mating phenotype of strains mutant in combinations of SAS genes. Strains shown are DRY1661 (sas2 sas5), DRY1662 (sas2 sas4), DRY1663 (sas4 sas5), and DRY1664 (sas2 sas4 sas5).

As described above, SIR1 plays a role in the nucleation of heterochromatin and the establishment of silencing. However, deletion of SIR1 results in only a modest silencing defect at HML (PILLUS and RINE 1989 ). Thus, Sir1p is redundant with other molecules that contribute to the establishment of silencing, or Sir1p acts in collaboration with other molecules, such as Rap1p, to collectively nucleate silencing. In contrast, strains lacking SIR1 do not appear to be defective in the clonal propagation of silencing through mitosis. Thus the role of SIR1 in silencing may be limited to the initial formation of heterochromatin (PILLUS and RINE 1989 ). As described above, deletion of both SIR1 and SAS2 causes a much more severe silencing defect at HML than deletion of either gene alone (REIFSNYDER et al. 1996 ). Thus, SAS2 and SIR1 appear to play redundant roles in silencing HML.

To explore the possibility that either SAS4 or SAS5 plays a role in silencing HML that is redundant with SIR1, we analyzed the mating phenotype of strains harboring a null allele of SIR1 in combination with a null allele of either SAS4 or SAS5. The -mating phenotype of a sas4 sir1 strain (DRY1659) and a sas5 sir1 strain (DRY1660) was four orders of magnitude less than that of the wild-type strain or the singly mutated sir1 (JRY4622), sas4 (DRY1656), or sas5 (DRY1657) strains (Figure 2B). Thus, both SAS4 and SAS5 are required in combination with SIR1 for efficient silencing at HML.

The observation that the role of SAS4 and SAS5 in silencing HML is redundant with that of SIR1 raised the possibility that SAS4 and SAS5 provide redundant functions with each other in silencing HML. Similarly, since the role of SAS2 in silencing HML is redundant with SIR1, it is possible that SAS2, SAS4, and SAS5 provide redundant functions with each other. Alternatively, SAS2, SAS4, and SAS5 may act collectively to provide a single function in silencing. To determine whether SAS2, SAS4, and SAS5 provided silencing functions that were redundant with each other, we quantitated the extent of silencing at HML in strains that contained combinations of null alleles of SAS2, SAS4, and SAS5. Deletion of both SAS4 and SAS5 (DRY1663) resulted in no greater silencing defect at HML than deletion of either gene alone (Figure 2). Similarly, strains containing null alleles of SAS2 and SAS4 (DRY1662), SAS2 and SAS5 (DRY1661), or SAS2, SAS4, and SAS5 (DRY1664) were no more defective for HML silencing than any of the single mutant strains, indicating that the roles of SAS2, SAS4, and SAS5 in silencing of HML were not redundant.

Null alleles of SAS4 and SAS5 have phenotypes at HMR opposite to that of a null allele of SIR1:
The observation that the SAS genes were redundant with SIR1 in silencing at HML suggests that there are fundamental differences in the regulation of HML and HMR. In particular, the SAS genes and SIR1 do not appear to be redundant at HMR as they are at HML, since deletion of SIR1 results in a silencing defect at HMR, whereas deletion of the SAS genes suppresses silencing defects at HMR (REIFSNYDER et al. 1996 ; EHRENHOFER-MURRAY et al. 1997 ; XU et al. 1999 ). However, these previous observations are not directly comparable since the phenotypes of null alleles of SIR1 and null alleles of SAS4 or SAS5 were observed in strains containing different versions of the HMR silencers. To test directly whether SAS4 or SAS5 mutants display HMR phenotypes opposite to those of SIR1 mutants, we compared the phenotypes of null mutations in SAS4, SAS5, and SIR1 in two genetic backgrounds. One background contained the HMR-SS I allele of HMR, which is composed of a synthetically constructed version of the HMR-E silencer in combination with a deletion of the HMR-I silencer. The HMR-SS I (DRY439) allele is partially defective in silencing and mates with an efficiency of 0.265 relative to wild type (Figure 3). In this strain, deletion of SIR1 (JRY4624) dramatically reduced silencing at HMR, whereas deletion of either SAS4 or SAS5 restored silencing to near wild-type levels (Figure 3). The other background contained the defective HMRa-e** allele. Deletion of SAS4 or SAS5 in this background restored silencing, whereas deletion of SIR1 did not (Figure 3). These results confirm and extend the observation that deletion of SAS4 (DRY1322) or SAS5 (DRY1314) suppresses silencing defects at HMR. Furthermore, these results directly demonstrate that null alleles of SAS4 and SAS5 have HMR phenotypes opposite to a null allele of SIR1. Hence, in contrast to the redundant roles of the SAS genes with SIR1 at HML, the SAS genes and SIR1 have opposite roles in silencing at HMR.

View larger version (64K): In this window In a new window Download PPT slide   Figure 3. sas4 and sas5 have opposite phenotypes to sir1 at HMR as revealed by mutant alleles of the HMR-E silencer. Qualitative patch mating assays of isogenic strains are shown in the panels with quantitative mating analysis given below and genotypes above. (A) -Mating phenotype of MAT HMRa-e** strains mutant in SIR1, SAS4, or SAS5. Strains shown are JRY5273 (HMRa-e**), DRY1399 (HMRa-e** sir1), DRY1322 (HMRa-e** sas4), and DRY1314 (HMRa-e** sas5). (B) -Mating phenotype of strains mutant in SIR1, SAS4, or SAS5. Strains shown are DRY439 (HMR-SS I), JRY4624 (HMR-SS I sir1), DRY1364 (HMR-SS I sas4), and DRY1424 (HMR-SS I sas5). (C) -Mating phenotype of wild-type and sir4 control strains. Strains shown are JRY3009 (WT) and DRY1235 (sir4).

Increased dosage of SIR1, SIR2, SIR3, or SIR4 results in a SAS phenotype:
How might mutations in SAS2, SAS4, and SAS5 suppress the silencing defects of the HMRa-e** silencer? In principle, deletion of the SAS genes could suppress silencing defects at HMR as an indirect consequence of disruption of silencing at the telomeres. In particular, as a result of disruption of TPE, Sir2p, Sir3p, and/or Sir4p could be released from the telomeres, effectively increasing the concentration of the pool of these proteins available for silencing at HMR. Since one role of the silencers is to nucleate silencing, it is possible that an increased concentration of the pool of the available Sir proteins could drive nucleation even in the presence of the defective HMRa-e** silencer. A prediction of this model is that increasing the concentration of Sir2p, Sir3p, and/or Sir4p would suppress the defects of the HMRa-e** silencer in an otherwise wild-type cell.

To test whether the HMRa-e** silencing defects could be suppressed by an increased dosage of any of the Sir proteins, high copy number plasmids containing the individual SIR1, SIR2, SIR3, or SIR4 genes were introduced into a strain harboring the HMRa-e** allele. An increased dosage of either SIR1 (DRY2107), SIR2 (DRY2108), SIR3 (DRY1464), or SIR4 (DRY1460) suppressed the silencing defect caused by the HMRa-e** allele (Figure 4). The simplest interpretation of these data is that mutations in SAS2, SAS4, or SAS5 suppress defects in silencing at HMR as an indirect effect of disrupting telomeric silencing. By inference, these results suggest that the primary role of SAS2, SAS4, and SAS5 is to bring about silencing at the telomeres and HML.

View larger version (58K): In this window In a new window Download PPT slide   Figure 4. Increased dosage of SIR1, SIR2, SIR3, or SIR4 suppresses the HMRa-e** silencing defect. (A) -Mating phenotype of control strains transformed with the 2µ-based vector pRS426. Strains shown are DRY1448 [WT (pRS426)] and DRY1456 [MAT HMRa-e** sas5 (pRS426)]. (B) -Mating phenotype of DRY1452 [MAT HMRa-e** (pRS426)], DRY1464 [MAT HMRa-e** (pRS426-SIR3)], and DRY1460 [MAT HMRa-e** (pRS426-SIR4)]. (C) -Mating phenotype of DRY2107 [MAT HMRa-e** (pRS426-SIR1)] and DRY2108 [MAT HMRa-e** (pRS426-SIR2)].

Deletion of SAS4 or SAS5 does not result in a silencing defect at HMR:
The data presented above suggest that SAS2, SAS4, and SAS5 are positive regulators of silencing at the telomeres and HML but not at HMR. However, the positive contribution of the SAS genes to silencing at HML was revealed by analysis of HML flanked by wild-type alleles of the HML-E and HML-I silencers, whereas the negative regulatory effect of the SAS genes on HMR was revealed by analysis of HMR flanked by mutant alleles of the HMR-E silencer. To assess more directly the role of the SAS genes at HMR, we tested whether SAS2, SAS4, or SAS5 contributed to silencing of wild-type HMR. Deletion of SAS2 (DRY1797), SAS4 (DRY1798), or SAS5 (DRY1799) did not result in a detectable reduction in silencing of HMR as measured by a quantitative mating assay (Figure 5). Thus, in contrast to HML, deletion of the SAS genes does not result in a silencing defect at HMR.

View larger version (67K): In this window In a new window Download PPT slide   Figure 5. SAS4 and SAS5 do not contribute to silencing at the wild-type HMR locus. Results of quantitative analysis of the -mating phenotype of MAT HMRa strains are presented. The relevant genotype of each strain analyzed is given below the corresponding result. Strains analyzed were JRY3009 (WT), JRY4621 (sir1), DRY1264 (sir3), DRY1797 (sas2), DRY1800 (sir1 sas2), DRY1798 (sas4), DRY1801 (sir1 sas4), DRY1799 (sas5), and DRY1802 (sir1 sas5).

As described above, a null allele of SIR1 in combination with a null allele in SAS2, SAS4, or SAS5 resulted in a severe defect in silencing at HML, whereas deletion of any of these genes alone resulted in only a modest silencing defect. To explore further the possible role of the SAS genes in silencing wild-type HMR, we determined whether null alleles of the SAS genes caused a substantial defect in silencing at HMR in combination with a null allele of SIR1. Deletion of SIR1 and SAS2 (DRY1800), SIR1 and SAS4 (DRY1801), or SIR1 and SAS5 (DRY1802) did not result in a detectable silencing defect at HMR (Figure 5). In fact, deletion of SAS2, SAS4, or SAS5 appeared to suppress the modest silencing defect that results from deletion of SIR1 (JRY4621) alone (Figure 5). These results indicate that the locus-specific silencing phenotypes of null alleles of the SAS genes reflect the properties of the native HML and HMR silencers. Furthermore, these results suggest that the SAS genes do not normally contribute to silencing at HMR and that they are not redundant with SIR1 function at HMR as they are at HML.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

SAS genes define a new class of locus-specific regulators of silencing:
The analysis presented here established that SAS4 and SAS5, like SAS2, are positive regulators of silencing at HML and the telomeres and are negative regulators of silencing at HMR. Specifically, each is required for TPE, each contributes a function in silencing at HML that is redundant with SIR1 but is not redundant with the other SAS genes, and null alleles of each suppress silencing defects at HMR. These properties are unique among the genes known to regulate silencing, indicating that SAS2, SAS4, and SAS5 define a novel class of locus-specific regulators of silencing. By inference, the functions of Sas2p, Sas4p, and Sas5p are likely to be intimately related.

Role of locus-specific regulators of silencing:
As described above, the simplest interpretation of our data is that SAS2, SAS4, and SAS5 are locus-specific regulators that bring about silencing at HML and the telomeres, but not at HMR. Similarly, previous analysis of SIR1 suggests that it is also a locus-specific regulator of silencing that acts at HML and HMR but not at the telomeres. In this regard, the most informative clues to the role of the SAS genes may come from analysis of HML, where SIR1 and the SAS genes appear to play redundant roles in silencing. This redundancy raises the possibility that the SAS genes, like SIR1, contribute to the establishment of silencing. In particular, the SAS genes could contribute to the nucleation of silencing at the telomeres at HML, as SIR1 does at HML and HMR. By this model, the chromatin structures at HML, HMR, and in the telomeric regions would be predicted to be composed of identical components and differ only in the initial events that lead to their assembly. Furthermore, the differences in the efficiency of silencing in the different regions would be expected to result from differences in the efficiency of establishment.

What is the possible molecular role of the SAS genes in silencing at the telomeres and HML? Sas2p is a member of the MYST family of proteins (BORROW et al. 1996 ; REIFSNYDER et al. 1996 ; E. R. SMITH et al. 1998 ). The members of this family have similarity to protein acetylases, and two family members, Esa1p and Tip60, are histone acetylases (YAMAMOTO and HORIKOSHI 1997 ; E. R. SMITH et al. 1998 ). One model of SAS gene function is that Sas2p regulates silencing through the acetylation of a component of the silencing machinery. Given the phenotypic similarities among mutations in SAS2, SAS4, and SAS5, it is possible that Sas4p and Sas5p are components of a Sas2p-dependent acetylase complex. Alternatively, Sas4p and/or Sas5p could be the targets of a Sas2p-dependent acetylase.

Role of the SAS genes in regulation of silencing at HMR:
Three lines of evidence support a model in which null alleles of the SAS genes suppress silencing defects at HMR as an indirect consequence of disrupting TPE. First, SAS4 and SAS5 are required for TPE, as was previously shown for SAS2 (REIFSNYDER et al. 1996 ). Second, disruption of TPE can result in redistribution of the Sir proteins from the telomeres to other loci (COCKELL et al. 1995 ; GOTTA and GASSER 1996 ; GOTTA et al. 1996 , GOTTA et al. 1997 ; KENNEDY et al. 1997 ). Third, increased dosage of SIR1, SIR2, SIR3, or SIR4 was sufficient to suppress silencing defects at HMR. Collectively these observations support a model in which mutations in the SAS genes disrupt TPE, resulting in an increased concentration of the pool of free SIR proteins, which, in turn, can suppress silencing defects at HMR.

Differential regulation of HML and HMR:
Our observations that null alleles of the SAS genes cause silencing defects at HML and suppress defects at HMR provide strong evidence that there are important differences in the regulation of silencing at these two loci. One possible explanation for this observation is that silencing at HML and HMR may differ qualitatively. As described above, the data presented here are consistent with a model in which the SAS genes are locus-specific regulators of silencing that normally act at HML and the telomeres but not at HMR.

If the regulation of silencing at HML and HMR differs qualitatively, it is likely that additional previously unidentified molecules or mechanisms account for the greater efficiency of silencing at HMR relative to HML and the telomeres. One way that HMR is known to differ from HML is that the silencers at HMR are origins of replication, whereas the silencers at HML are not (DUBEY et al. 1991 ; RIVIER and RINE 1992 ; HURST and RIVIER 1999 ; RIVIER et al. 1999 ). It is possible that DNA replication, initiated at the HMR silencers, plays a role in the assembly or duplication of heterochromatin at HMR and that this function is lacking at HML. To date, a role for DNA replication in silencing at HMR has not been revealed; however, it is possible that the efficiency of silencing at HMR and the redundancy that is inherent in the HMR-E silencer has masked a possible contribution of replication to silencing at this locus.

	ACKNOWLEDGMENTS

We thank K. Replogle for help with strain construction, J. Ekena for help with mating analysis presented in Figure 5A. Belmont and C. Doe for discussions and comments on early versions of the manuscript, B. Cairns and S. Elgin for discussions, J. Rine for strains, and L. Pillus for discussion of unpublished work and for providing plasmids and strains. This work was supported by National Institutes of Health (NIH) grant GM-52103 (D.R.), by a Basil O'Connor Starter Scholar Research Award grant 5-FY96-0578 (D.R.), by the Charles M. Goodenberger Fund (D.R.), and by an NIH predoctoral training award 5T32-GM07283 (S.K.).

Manuscript received October 5, 1998; Accepted for publication May 4, 1999.

	LITERATURE CITED

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