Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae.

Rap1p binds to sites embedded within the Saccharomyces cerevisiae telomeric TG1-3 tract. Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere. To test this, we tethered Sir3p adjacent to the telomere via LexA binding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 165 amino acids thought to contain sites for Sir3p association. Tethering of LexA-Sir3p adjacent to the telomere is sufficient to restore telomeric silencing, indicating that Sir3p can nucleate silencing at the telomere. Tethering of LexA-Sir3p or the LexA-Sir3p(N2O5) gain-of-function protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild-type cells. Hence, Sir3p recruitment to the telomere is limiting in telomeric silencing. In addition, LexA-Sir3p(N2O5) hyperrepresses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract. This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can interact with Rap1p-associated factors at the telomere. We also demonstrate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cis with a short tract of telomeric TG1-3 sequences is sufficient to confer silencing at an internal chromosomal position. Internal silencing is enhanced in rap1-17 strains. We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing.     

Mutational Analysis Defines a C-Terminal Tail Domain of Rap1 Essential for Telomeric Silencing in Saccharomyces Cerevisiae

Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene. The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids. Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region. These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing. Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain. Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control. The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length. Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles. In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction. These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing. These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing.     

Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction

The previously identified LRS (Loss of rDNA Silencing) domain of the nucleosome is critically important for silencing at both ribosomal DNA and telomeres. To understand the function of the LRS surface in silencing, we performed an EMS mutagenesis screen to identify suppressors of the H3 A75V LRS allele. We identified dominant and recessive mutations in histones H3, H4, and dominant mutations in the BAH (Bromo Adjacent Homology) domain of SIR3. We further characterized a surface of Sir3p critical for silencing via the LRS surface. We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles. Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4. Mutations in H3 and H4 genes that lose silencing tend to make the LRS surface more electronegative, whereas mutations that increase silencing make it less electronegative. Conversely, suppressors of LRS alleles in either SIR3, histone H3, or H4 also tend to make their respective surfaces less electronegative. Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain. Based on these findings, we propose an electrostatic model for how an extensive surface on the Sir3p BAH domain may regulate docking onto the LRS surface.     

Sir3p domains involved in the initiation of telomeric silencing in Saccharomyces cerevisiae.

Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3(N205) mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD "active" site is under both positive and negative control mediated by multiple Sir3p domains.     

The yeast silent information regulator Sir4p anchors and partitions plasmids.

Circular plasmids containing telomeric TG1-3 arrays or the HMR E silencer segregate efficiently between dividing cells of the yeast Saccharomyces cerevisiae. Subtelomeric X repeats augment the TG1-3 partitioning activity by a process that requires the SIR2, SIR3, and SIR4 genes, which are also required for silencer-based partitioning. Here we show that targeting Sir4p to DNA directly via fusion to the bacterial repressor LexA confers efficient mitotic segregation to otherwise unstable plasmids. The Sir4p partitioning activity resides within a 300-amino-acid region (residues 950 to 1262) which precedes the coiled-coil dimerization motif at the extreme carboxy end of the protein. Using a topology-based assay, we demonstrate that the partitioning domain also retards the axial rotation of LexA operators in vivo. The anchoring and partitioning properties of LexA-Sir4p chimeras persist despite the loss of the endogenous SIR genes, indicating that these functions are intrinsic to Sir4p and not to a complex of Sir factors. In contrast, inactivation of the Sir4p-interacting protein Rap1p reduces partitioning by a LexA-Sir4p fusion. The data are consistent with a model in which the partitioning and anchoring domain of Sir4p (PAD4 domain) attaches to a nuclear component that divides symmetrically between cells at mitosis; DNA linked to Sir4p by LexA serves as a reporter of protein movement in these experiments. We infer that the segregation behavior of telomere- and silencer-based plasmids is, in part, a consequence of these Sir4p-mediated interactions. The assays presented herein illustrate two novel approaches to monitor the intracellular dynamics of nuclear proteins.     

The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae

We have developed a novel technique for combined immunofluorescence/in situ hybridization on fixed budding yeast cells that maintains the three-dimensional structure of the nucleus as monitored by focal sections of cells labeled with fluorescent probes and by staining with a nuclear pore antibody. Within the resolution of these immunodetection techniques, we show that proteins encoded by the SIR3, SIR4, and RAP1 genes colocalize in a statistically significant manner with Y' telomere- associated DNA sequences. In wild-type cells the Y' in situ hybridization signals can be resolved by light microscopy into fewer than ten foci per diploid nucleus. This suggests that telomeres are clustered in vegetatively growing cells, and that proteins essential for telomeric silencing are concentrated at their sites of action, i.e., at telomeres and/or subtelomeric regions. As observed for Rap1, the Sir4p staining is diffuse in a sir3- strain, and similarly, Sir3p staining is no longer punctate in a sir4- strain, although the derivatized Y' probe continues to label discrete sites in these strains. Nonetheless, the Y' FISH is altered in a qualitative manner in sir3 and sir4 mutant strains, consistent with the previously reported phenotypes of shortened telomeric repeats and loss of telomeric silencing.     

Localization of Sir2p: the nucleolus as a compartment for silent information regulators.

In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.     

A cytosolic NAD-dependent deacetylase, Hst2p, can modulate nucleolar and telomeric silencing in yeast

In budding yeast, the silent information regulator Sir2p is a nuclear NAD-dependent deacetylase that is essential for both telomeric and rDNA silencing. All eukaryotic species examined to date have multiple homologues of Sir two (HSTs), which share a highly conserved globular core domain. Here we report that yeast Hst2p and a mammalian Hst2p homologue, hSirT2p, are cytoplasmic in yeast and human cells, in contrast to yHst1p and ySir2p which are exclusively nuclear. Although yHst2p cannot restore silencing in a sir2 deletion, overexpression of yHst2p influences nuclear silencing events in a SIR2 strain, derepressing subtelomeric silencing while increasing repression in the rDNA. In contrast, a form of ySir2p carrying a point mutation in the conserved core domain disrupts both telomeric position effect (TPE) and rDNA repression at low expression levels. This argues that non-nuclear yHst2p can compete for a substrate or ligand specifically required for telomeric, and not rDNA repression.     

C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.     

The nuclear GTPase Gsp1p can affect proper telomeric function through the Sir4 protein in Saccharomyces cerevisiae

The small Ras-like GTPase Ran/Gsp1p is a highly conserved nuclear protein required for the nucleocytoplasmic trafficking of macromolecules. Recent findings suggest that the Ran/Gsp1p pathway may have additional roles in several aspects of nuclear structure and function, including spindle assembly, nuclear envelope formation, nuclear pore complex assembly and RNA processing. Here, we provide evidence that Gsp1p can regulate telomeric function in Saccharomyces cerevisiae. We show that overexpression of PRP20, encoding the Gsp1p GDP/GTP nuclear exchange factor, specifically weakens telomeric silencing without detectably affecting nucleocytoplasmic transport. In addition to this silencing defect, we show that Rap1p and Sir3p delocalize from their normal telomeric foci. Interestingly, Gsp1p was found to interact genetically and physically with the telomeric component Sir4p. Taken together, these results suggest that the GSP1 pathway could regulate proper telomeric function in yeast through Sir4p.     

Yeast telomeric sequences function as chromosomal anchorage points in vivo.

Site-specific recombination in Saccharomyces cerevisiae was used to generate non-replicative DNA rings containing yeast telomeric sequences. In topoisomerase mutants expressing Escherichia coli topoisomerase I, the rings adopted a novel DNA topology consistent with the ability of yeast telomeric DNA to block or retard the axial rotation of DNA. DNA fragments bearing portions of the terminal repeat sequence C1-3 A/TG1-3 were both necessary and sufficient to create a barrier to DNA rotation. Synthetic oligonucleotide sequences containing Rap1p binding sites, a well represented motif in naturally occurring C1-3A arrays, also conferred immobilization; mutant Rap1p binding sites and telomeric sequences from other organisms were not sufficient. DNA anchoring was diminished by addition of competing telomeric sequences, implicating a role for an as yet unidentified limiting trans-acting factor. Though Rap1p is a likely protein constituent of the DNA anchor, deletion of the non-essential C-terminal domain did not affect the topology of telomeric DNA rings. Similarly, disruption of SIR2, SIR3 and SIR4, genes which influence a variety of telomere functions in yeast, also had no effect. We propose that telomeric DNA supports the formation of a SIR-independent macromolecular protein-DNA assembly that hinders the motion of DNA because of its linkage to an insoluble nuclear structure. Potential roles for DNA anchoring in telomere biology are discussed.     

The Rap1p-telomere complex does not determine the replicative capacity of telomerase-deficient yeast

Telomeres are nucleoprotein structures that cap the ends of chromosomes and thereby protect their stability and integrity. In the presence of telomerase, the enzyme that synthesizes telomeric repeats, telomere length is controlled primarily by Rap1p, the budding yeast telomeric DNA binding protein which, through its C-terminal domain, nucleates a protein complex that limits telomere lengthening. In the absence of telomerase, telomeres shorten with every cell division, and eventually, cells enter replicative senescence. We have set out to identify the telomeric property that determines the replicative capacity of telomerase-deficient budding yeast. We show that in cells deficient for both telomerase and homologous recombination, replicative capacity is dependent on telomere length but not on the binding of Rap1p to the telomeric repeats. Strikingly, inhibition of Rap1p binding or truncation of the C-terminal tail of Rap1p in Kluyveromyces lactis and deletion of the Rap1p-recruited complex in Saccharomyces cerevisiae lead to a dramatic increase in replicative capacity. The study of the role of telomere binding proteins and telomere length on replicative capacity in yeast may have significant implications for our understanding of cellular senescence in higher organisms.