Transcriptional silencing at Saccharomyces telomeres - Nature

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Oncogene (2002) 21, 512 ± 521 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

Transcriptional silencing at Saccharomyces telomeres: implications for other organisms Wai-Hong Tham1 and Virginia A Zakian*,1 1

Department of Molecular Biology, Princeton University, Princeton, New Jersey, NJ 08544, USA

Telomeres are the natural ends of eukaryotic chromosomes. In most organisms, telomeres consist of simple, repeated DNA with the strand running 5' to 3' towards the end of the chromosome being rich in G residues. In cases where the very end of the chromosome has been examined, the G-strand is extended to form a short, single stranded tail. The chromatin structure of telomeric regions often has features that distinguish them from other parts of the genome. Because telomeres protect chromosome ends from degradation and end-toend fusions and prevent the loss of terminal DNA by serving as a substrate for telomerase, they are essential for the stable maintenance of eukaryotic chromosomes. In addition to their essential functions, telomeres in diverse organisms are specialized sites for gene expression. Transcription of genes located next to telomeres is repressed, a phenomenon termed telomere position eect (TPE). TPE is best characterized in the yeast Saccharomyces cerevisiae. This article will focus on the silencing properties of Saccharomyces telomeres and end with speculation on the role of TPE in yeasts and other organisms. Oncogene (2002) 21, 512 ± 521. DOI: 10.1038/sj/onc/ 1205078 Keywords: telomere; TPE; heterochromatin; telomerase; Ku complex; Sir proteins DNA and chromatin structure of Saccharomyces telomeres Yeast telomeres consist of 350+75 bps of C1 ± 3A/ TG1 ± 3 DNA. The telomeric tract is encompassed in a non-nucleosomal DNA-protein complex called the telosome (Wright et al., 1992). The sequence speci®c duplex DNA binding protein Rap1p is the major protein in the telosome, with an estimated 10 ± 20 molecules per chromosome end (Conrad et al., 1990; Gilson et al., 1993; Wright et al., 1992; Wright and Zakian, 1995). Cdc13p, a protein that binds to single strand TG1 ± 3 in vitro (Lin and Zakian, 1996; Nugent et al., 1996) is also telomere bound in vivo (Bourns et al., 1998; Tsukamoto et al., 2001). Several other proteins, *Correspondence: VA Zakian; E-mail: [email protected]

including Sir2, Sir3, and Sir4p, as well as the two Rif proteins, localize to telomeres (Bourns et al., 1998), presumably as a consequence of their ability to interact, directly or indirectly, with Rap1p (Hardy et al., 1992; Moretti et al., 1994; Wotton and Shore, 1997). The Ku complex, which displays sequence nonspeci®c end binding activity and is comprised of two proteins Hdf1p and Hdf2p, also binds telomeres (Gravel et al., 1998; Martin et al., 1999). Yeast has two classes of sub-telomeric repeats, Y' and X (reviewed in Louis, 1995) (Figure 1a). The Y' element, which comes in two sizes, 6.7 and 5.2 kb, is found on about two-thirds of yeast telomeres. Individual telomeres bear up to four tandem copies of Y'; between tandem Y' elements, there are often short C1 ± 3A/TG1 ± 3 tracts. X is quite heterogeneous but virtually all X elements have a core region of *500 bp that is found at almost every telomere (Louis et al., 1994). On telomeres having both Y' and X, Y' is closer to the chromosome end. Y' and core X both contain a putative origin of DNA replication or ARS (autonomously replicating sequence) (Chan and Tye, 1983a). Unlike the telomeric C1 ± 3A/TG1 ± 3 tracts, subtelomeric DNA is organized into nucleosomes (Wright et al., 1992). However, these nucleosomes have several features that distinguish them from nucleosomes in most other parts of the genome. Sub-telomeric nucleosomes are refractory in vivo to modi®cation by the dam methylase, suggesting that sub-telomeric DNA is less accessible and hence more compact than bulk chromatin (Gottschling, 1992). The amino terminal tails of histones H3 and H4 are hypoacetylated in sub-telomeric nucleosomes compared to histones elsewhere (Braunstein et al., 1993; de Bruin et al., 2000; Monson et al., 1997). The Sir2, 3 and 4 proteins bind as a complex to sub-telomeric nucleosomes, brought there by their ability to interact with the amino terminal tails of histones H3 and H4 (Hecht et al., 1995; Strahl-Bolsinger et al., 1997). Loss of any one of the Sir proteins eliminates binding of the other Sir proteins to sub-telomeric chromatin (Hecht et al., 1996; Strahl-Bolsinger et al., 1997), whereas Sir4p remains telomere bound, even in a sir3 strain (Bourns et al., 1998). The Ku complex also associates with subtelomeric nucleosomes (Martin et al., 1999). In contrast to its binding to chromosome ends, the association of Ku with sub-telomeric chromatin is Sir4p dependent (Martin et al., 1999).

Telomere position effect W-H Tham and VA Zakian

Figure 1 Structure of natural (a) and truncated (b) telomeres. This ®gure is not drawn to scale. The *350 bp tract of C1 ± 3A/ TG1 ± 3 at the end of the chromosome is depicted as a black arrowhead. (a) Most natural telomeres bear X and often bear Y'. Although X is heterogeneous, ranging from 0.3 to 3.75 kb in size (Chan and Tye, 1983b), a *500 bp segment called core X is found at most telomeres (Louis et al., 1994). There are two major classes of Y', Y' long (6.7 kb) and Y' short (5.2 kb). Individual telomeres can have zero or up to four tandem copies of Y'; the telomere shown has a single Y' (indicated by bracket). STAR (subtelomeric anti-silencing region) is a region within Y' that antagonizes silencing. The sub-telomeric repeats (STR) A, B, C, and D that are found between X and the telomere or between X and Y', also have STAR activity. X and Y' both contain an ARS, which has a binding site for ORC, a multi-protein complex needed for initiation of DNA replication. X contains a binding site for Abf1p, a protein involved in both transcription and ARS function. (b) The ADH4 gene is about 15 kb from the left end of chromosome VII. Many published studies on TPE were carried out on strains having URA3 adjacent to the left telomere of chromosome VII. To make such strains, yeast is transformed with a fragment having a portion of ADH4, URA3, and an 81 bp tract of C1 ± 3A/TG1 ± 3 DNA (denoted by black square). Recombination within ADH4 results in URA3 integration and loss of the DNA distal of ADH4. A new telomere is formed on the 81 bp C1 ± 3A/TG1 ± 3 tract (Gottschling et al., 1990). The terminally deleted version of chromosome VII is lost at the same rate as unmodi®ed chromosome VII (Sandell and Zakian, 1993). The X and Y' content of chromosome VII-L varies among dierent yeast strains

Assaying telomeric silencing TPE was discovered during the course of constructing strains to be used to determine the chromatin structure of a single yeast telomere (Gottschling et al., 1990). Because yeast telomeres bear many kilo-basepairs of repetitive DNA, hybridization probes speci®c for a single telomere are not readily available. To circumvent this problem, we inserted the URA3 gene adjacent to the left telomere of chromosome VII, in such a way that the sub-telomeric repeats are deleted, and a new telomere is formed immediately distal of URA3. These terminally deleted or truncated chromosomes were isolated by their Ura+ phenotype (Figure 1b). URA3 was a fortuitous choice for this construction. Cells that express Ura3p can not grow in the presence of the drug 5 ¯uoro orotic acid (FOA) (Boeke et al.,

1984). Hence, growth on FOA medium identi®es cells that have lost or mutated the URA3 gene. Surprisingly, despite their Ura+ phenotype, 20 ± 60% of cells with the telomeric URA3 gene are also FOA-resistant (FOAR) (Gottschling et al., 1990). When the FOAR colonies are replica plated to media lacking uracil, most cells grow. Because the FOAR phenotype is reversible, it is not due to loss or mutation of URA3. Northern analysis shows that the amount of mRNA from the telomeric URA3 gene is reduced compared to expression of a non-telomeric URA3 gene in cells growing in medium containing uracil, a condition that supports basal expression of URA3. In contrast, mRNA levels are essentially normal in medium lacking uracil, where URA3 expression is induced (de Bruin et al., 2000; Gottschling et al., 1990). This reversible repression of URA3 transcription is referred to as telomere position eect, TPE. TPE is not limited to the left telomere of chromosome VII nor to the URA3 gene. TPE occurs at all tested truncated chromosomes, including the left telomere of chromosome VII, the right telomere of chromosomes V (Gottschling et al., 1990), and the left telomere of chromosome XV (Craven and Petes, 2000). About half of natural telomeres exhibit TPE (Pryde and Louis, 1999) (discussed in more detail below). Like URA3, other RNA polymerase II transcribed genes, such as TRP1, ADE2 and HIS3, as well as the RNA polymerase III-transcribed tyrosyl tRNA gene, SUP4o, are subject to TPE (Gottschling et al., 1990; Huang et al., 1997). Although the transcriptional state at telomeres is reversible, both the repressed and transcribed states are stable in wild-type strains. This stability is best illustrated by the appearance of colonies generated by cells with ADE2 next to a telomere (Gottschling et al., 1990). Cells expressing Ade2p generate white colonies, while lack of Ade2p results in red colonies. Most of the colonies generated by a strain with ADE2 at the telomere fall into one of two types: largely white colonies with red sectors and largely red colonies with white sectors. Since it takes *25 cell divisions to generate a yeast colony, both the transcribed and repressed states are stable for many cell divisions. Sectors of opposite expression states in largely red, and largely white colonies provide additional evidence that both states are reversible. At truncated telomeres, there is a continuous domain of transcriptional silencing with the probability of silencing inversely proportional to the distance from the telomere (Renauld et al., 1993). However, the probability of silencing is not simply a function of distance, as the nature of the intervening DNA also aects TPE. For example, the propagation of silencing from the telomere can be blocked by transcription of an intervening gene. The extent of silencing is also sensitive to the length of the C1 ± 3A/TG1 ± 3 telomeric tract. In general, the longer the telomere, the greater the silencing of an adjacent gene (Buck and Shore, 1995; Kyrion et al., 1993). However, strains with long telomeres can have reduced TPE, if lengthening is due

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to loss of telomere bound silencing proteins (Kyrion et al., 1992; Wiley and Zakian, 1995). Internal C1 ± 3A/TG1 ± 3 tracts are silencers An 81 bp tract of C1 ± 3A/TG1 ± 3 DNA inserted *20 kb from the left telomere of chromosome VII does not repress transcription of an adjacent URA3 gene (Gottschling et al., 1990). However, a tel1 strain, which has telomeres of 4100 bps, has relatively normal levels of TPE (Runge and Zakian, 1996). These considerations led to the idea that telomeric silencing cannot be conferred by C1 ± 3A/TG1 ± 3 repeats alone but rather requires special features of telomeric DNA, such as the single stranded TG1 ± 3 tail and its associated end binding proteins (Gottschling et al., 1990). However, internal C1 ± 3A/TG1 ± 3 tracts of *550 bp can repress nearby genes, a phenomenon termed C1 ± 3A-based silencing, CBS (Stavenhagen and Zakian, 1994). Long internal C1 ± 3A/TG1 ± 3 tracts can silence even on a circular chromosome, demonstrating that CBS does not require a telomere in cis. The level of CBS is very sensitive to the length of the C1 ± 3A/ TG1 ± 3 tract, with *275 bp tracts showing little, if any silencing. Although there are naturally occurring tracts of C1 ± 3A/TG1 ± 3 DNA at non-telomeric sites, all such tracts are relatively short: the eight longest range in size from 52 to 159 bps (Mangahas et al., 2001). Thus, there is no naturally occurring C1 ± 3A/TG1 ± 3 tract that is long enough to act as a silencer on its own. However, two *275 bp tracts of C1 ± 3A/TG1 ± 3 DNA can act synergistically to silence a gene placed between them, even though neither alone represses transcription (Stavenhagen and Zakian, 1994). In addition, when relatively short C1 ± 3A/TG1 ± 3 tracts are moved closer and closer to a telomere, they become better and better silencers. Since each of the eight longest, naturally occurring C1 ± 3A/TG1 ± 3 tracts is near a telomere (Mangahas et al., 2001), these tracts might act to boost the level of silencing imposed by the telomere itself. Silencing at natural telomeres Virtually all published studies on TPE use truncated telomeres. TPE also occurs at many, but not all, natural telomeres. When URA3 is inserted within the sub-telomeric DNA of a natural telomere, its repression varies dramatically, depending on its site of integration (Pryde and Louis, 1999). When URA3 is inserted within Y', repression is low or undetectable. Likewise, when URA3 is integrated near the ARS of the X element on telomeres III-R, IV-L, or X-R, TPE is very low. However, when URA3 is integrated at a similar location at telomeres II-R, XI-L, or XIII-R or near this location on XIV-R, repression is much higher, and in some cases as high as that seen when URA3 is immediately adjacent to the truncated telomere of chromosome VII-L.

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It is unclear why dierent telomeres have dierent silencing properties. However, neither X nor Y' is homogeneous, and major dierences in the silencing behavior of dierent telomeres may re¯ect seemingly minor dierences in sequence. There are four types of short, repeated elements termed sub-telomeric repeats (STR A, B, C and D) within the distal portion of X (Figure 1a). Certain STR elements and the 0.14 kb telomere proximal portion of Y' have anti-silencing or STAR activity (subtelomeric anti-silencing region) (Fourel et al., 1999; Pryde and Louis, 1999) (Figure 1a). When STAR elements are inserted between URA3 and the telomere, TPE decreases *20 ± 50-fold. When a gene is bracketed by STAR elements, it is insulated from transcriptional silencing, indicating that STARs have at least some of the properties of boundary elements. However, TPE occurs at telomeres containing a STAR if they also contain a core X or the 0.76 kb centromere proximal region of Y'. Elements like core X that block STAR action are termed sub-telomeric silencing elements. Although sub-telomeric silencing elements are not themselves sucient to silence a gene, they are proposed to act as a relay system that helps propagate silencing initiated at the telomere to more internal regions of the chromosome. Taken together, these data suggest that the sub-telomeric chromatin on natural chromosomes is a region of transcriptional repression punctuated by sub-domains that are permissive for transcription (Fourel et al., 1999; Pryde and Louis, 1999). This picture is very dierent from what is seen at truncated telomeres where there is a continuous zone of transcriptional repression that emanates from the telomere and dissipates continuously towards the centromere (Renauld et al., 1993). Genes and conditions that aect silencing A surprisingly large number of genes and conditions aect TPE. Deletion of certain non-essential genes, such as SIR2, SIR3, SIR4 (Aparicio et al., 1991), HDF1 and HDF2 (Boulton and Jackson, 1998; Laroche et al., 1998), abolishes or nearly abolishes TPE. The SIR genes, but not the Ku encoding HDF genes, are also needed for transcriptional silencing at the silent mating type or HM loci as well as for CBS. A version of Rap1p that lacks the carboxyl terminal *144 amino acids, the portion of Rap1p that interacts with the Sir complex, also abolishes TPE and CBS and reduces HM silencing yet still binds DNA and supplies the essential functions of Rap1p (Kyrion et al., 1992, 1993; Stavenhagen and Zakian, 1994). Mutations in the amino terminal tails of histones H3 and H4 that reduce their ability to interact with Sir proteins in vitro also reduce TPE in vivo (Hecht et al., 1995). Other telomere binding proteins have an inhibitory eect on TPE. For example, deletion of Rif1p and Rif2p increases TPE (Kyrion et al., 1993). Although deleting Rif proteins results in telomere lengthening, the enhancement of TPE seen in their absence seems to be due primarily to their ability to compete with Sir3p


for binding to telomere-bound Rap1p (Mishra and Shore, 1999). Over-expression of structural components of telomeres can also impact TPE: for example, Sir3p over-expression increases (Renauld et al., 1993) and Sir4p over-expression decreases telomeric silencing (Cockell et al., 1995). The genetic requirements for TPE, determined using truncated telomeres, do not necessarily apply to native telomeres. Although TPE at natural telomeres requires the Sir proteins and Hdf1p, it is less dependent on both than silencing at truncated telomeres and in the case of Hdf1p, its dependence varies depending on the telomere being examined (Fourel et al., 1999; Pryde and Louis, 1999). Native telomeres are also less sensitive to the enhancement in TPE seen upon Sir3p over-expression. In addition, deletion of SIR1 aects TPE at native ends (Fourel et al., 1999; Pryde and Louis, 1999) as well as at the silent mating loci but not at the truncated chromosome VII-L telomere (Aparicio et al., 1991). Paradoxically, certain mutations in subunits of ORC, a multi-protein complex that binds to ARS sequences and is required for initiation of DNA replication (Bell and Stillman, 1992), result in decreased TPE at truncated telomere VII-L, which does not contain an ARS, but not at native telomeres, which do (Pryde and Louis, 1999). In addition to genes whose functions are essential for TPE, there are a surprisingly large number whose mutation causes more modest eects on TPE. For example, deletion of RRM3, which encodes a DNA helicase that is needed for normal fork progression through telomeric and sub-telomeric DNA, results in about a 10-fold drop in TPE (Ivessa et al., in preparation). Many genes that have mild eects probably act indirectly. For example, the ability of a telomeric gene to switch from a repressed to a transcribed state is cell cycle limited, occurring preferentially late in the cell cycle (Aparicio and Gottschling, 1994). Thus, genes that aect cell cycle progression might also aect TPE, but these eects could be secondary to their impact on the cell cycle (Laman et al., 1995). The proteins whose loss has the most profound eect on TPE are all structural components of either the telosome, sub-telomeric chromatin, or both. When these structural proteins are lost, the atypical chromatin features that distinguish sub-telomeric chromatin from bulk chromatin, such as histone hypoacetylation and reduced access to DNA modifying enzymes, are also lost (de Bruin et al., 2000; Gottschling, 1992). These data led to a model in which Rap1p binds speci®cally to the telomeric repeats and initiates the assembly of a multimeric protein complex that allows the establishment and maintenance of TPE. The complex of Sir proteins is brought to the telomere by its ability to interact with Rap1p. The Sir complex spreads to nearby nucleosomes by binding of Sir3p and Sir4p to the amino terminal tails of histones H3 and H4 (Hecht et al., 1995). The concentration of Sir proteins on a native telomere decreases with increasing

distance from the telomere (Strahl-Bolsinger et al., 1997).

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Telomere folding: a structural requirement for TPE? Rap1p is a sequence speci®c, duplex C1 ± 3A/TG1 ± 3 binding protein that does not bind histones. Nonetheless, like the Sir and Hdf proteins, Rap1p is found in sub-telomeric chromatin (Strahl-Bolsinger et al., 1997). This observation led to the proposal that the telomeric region has a folded structure. According to this model, telomere folding allows Rap1p-Sir interactions within sub-telomeric chromatin (Strahl-Bolsinger et al., 1997). In addition to this biochemical evidence, there is genetic support for telomere looping. In most locations, the yeast equivalent of an enhancer, the UAS (upstream activating sequence), activates transcription only when it is positioned upstream of a gene. However, when a UAS is downstream of a telomerelinked gene, expression of the gene is activated (de Bruin et al., 2001). This ability is proposed to be due to telomere folding, which places the UAS close to the promoter of the telomere-linked gene. Conditions that eliminate telomere looping disrupt TPE without aecting chromosome stability (de Bruin et al., 2000). Telomere loops have also been detected in mammals (Grith et al., 1999), ciliates (Murti and Prescott, 1999), and trypanosomes (Munoz-Jordan et al., 2001). However, in these organisms, telomere loops form by invasion of the 3' telomeric overhang into duplex telomeric DNA, while there is no evidence that looping in S. cerevisiae involves base pairing of the telomere with sub-telomeric DNA. Eliminating TPE in cis Genes that aect TPE usually do so at all truncated telomeres in the cell. However, TPE can also be eliminated at a single telomere. The strategy for eliminating TPE in cis is based on the demonstration that transcription from a strong promoter near an ARS or centromere eliminates their functions (Hill and Bloom, 1987; Snyder et al., 1988). Using a parallel approach, the galactose inducible GAL UAS and TATA were inserted between URA3 and the telomere on the truncated left arm of chromosome VII in such a way that transcription from the GAL UAS proceeds towards and through the telomeric tract (Sandell et al., 1994). When this strain is grown in galactose medium, an abundant UG1 ± 3 transcript about the length of the telomeric C1 ± 3A/TG1 ± 3 tract is detected. The loss rate of chromosome VII is not increased by transcription through the telomeric tract, demonstrating that the stability functions of the VII-L telomere are intact. However, TPE is lost at the URA3 gene that is adjacent to the transcribed telomere (Sandell et al., 1994) while TPE at other telomeres in the same cell is not aected (Tham et al., 2001). This system allows analysis of the behavior of a telomere that lacks TPE Oncogene


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in an otherwise silencing competent cell. Loss of TPE at the transcribed VII-L telomere is accompanied by all of the chromatin changes, such as histone acetylation, that are associated with global loss of TPE. Again, the transcribed telomere is the only one in the cell whose chromatin structure is aected (de Bruin et al., 2000). Because transcription through the telomeric tract results in rapid and severe loss of Rap1p from subtelomeric chromatin, it eliminates looping at the aected telomere. In contrast, growth in medium lacking uracil or deletion of SIR3, results in, respectively, no or slower and less complete loss of sub-telomeric Rap1p. These data suggest that telomere looping is necessary but not sucient for TPE. Telomeres are localized near the nuclear periphery: implications for TPE Immuno-localization shows that several proteins needed for telomeric silencing, including Rap1p, Sir2p, Sir3p, Sir4p and Hdf2p are concentrated in six to eight foci in diploid cells (Gotta et al., 1996, 1997; Laroche et al., 1998; Palladino et al., 1993). At least for Rap1p, these foci are near the nuclear periphery (Gotta et al., 1996). Moreover, in situ hybridization reveals that *70% of Y' DNA is similarly located (Gotta et al., 1996; Laroche et al., 2000). Since the number of these foci is considerably smaller than the 64 telomeres in a diploid cell, each focus of silencing proteins is thought to contain multiple telomeres. There is increasing evidence that the nuclei of eukaryotic cells are divided into permissive and restrictive compartments vis-aÁ-vis transcription. The peripheral localization of silencing proteins and chromosomal sites of transcriptional repression suggests that the yeast nuclear periphery is a region where transcription is inhibited. Perhaps the most compelling support for this hypothesis is the demonstration that tethering a weak silencer to the nuclear periphery improves its ability to silence a nearby gene (Andrulis et al., 1998). In addition, silencing at a non-telomeric site is increased when the site is brought closer to a telomere (Maillet et al., 1996; Stavenhagen and Zakian, 1994). Over-expression of Sir3p or Sir4p, which results in dispersal of silencing proteins throughout the nucleus, also improves silencing at non-telomeric sites. Targeting Sir proteins to a site that is 20 kb from a truncated telomere, too far under normal conditions to be aected by TPE, allows silencing at that site (Marcand et al., 1996). These data suggest a model in which silencing is limited by the abundance of silencing proteins. According to this model, transcriptional repression can be increased by bringing a gene closer to the periphery and hence closer to pools of silencing proteins or by elevating and dispersing silencing proteins throughout the nucleus, thereby increasing the probability of their binding a gene located in the nuclear interior. Because silencing is reversible and not all Y' sequences are at the periphery, an appealing model is

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that a telomere adjacent gene is silenced when it is near the periphery and expressed when it is not. Although foci of silencing proteins are dispersed and silencing is lost in a sir3 strain, the pattern of Y' localization is largely unaltered (Gotta et al., 1996). In contrast, when one of the genes encoding a Ku subunit is deleted, foci of silencing proteins are disrupted, the number of Y' foci increases, and only about half of the foci are at the periphery (Laroche et al., 1998). Interpretation of these experiments is complicated by the fact that the Y' signal detects many, but not all telomeres, and is heavily biased towards telomeres in clusters and those containing tandem copies of Y'. The chromosome visualization methods described in (Straight et al., 1996) have been used to follow the behavior of a single telomere under conditions where its TPE status is known (Tham et al., 2001). When an array of lac operator binding sites is inserted very near the end of a truncated chromosome VII-L in a strain that also expresses an inducible GFP (green ¯uorescent protein)-lac repressor fusion protein, the VII-L telomere is visualized as a compact green spot. The nuclear periphery is marked using an antibody to a nuclear pore protein. Deconvolution microscopy is then used to determine the position of the telomere relative to the periphery under a variety of conditions. This approach shows that in most strains and conditions, the VII-L telomere is localized near the nuclear periphery in the majority of cells. However, in silencing competent cells, the level of telomere localization to the nuclear periphery does not correlate with the level of telomeric silencing. For example, the level of telomere VII-L localization is similar between a strain that has *80% repression at the VII-L telomere and a strain that has only 7% silent cells. In addition, deletion of SIR3 or HDF1 or growth in medium lacking uracil, each of which eliminates TPE at the VII-L telomere, does not result in delocalization. Thus, telomeres are often localized to the nuclear periphery, but this localization is separable from telomeric silencing. Nonetheless, no strain was identi®ed in which the VII-L telomere is both silenced and away from the nuclear periphery, suggesting that localization near the periphery is necessary, albeit not sucient, for silencing. Two conditions result in loss of localization of telomeres to the periphery (Tham et al., 2001). As cells progress towards mitosis, the VII-L telomere moves away from the periphery, assuming a distribution indistinguishable from random. This is the same time in the cell cycle when a repressed telomeric gene has a higher probability of switching to a transcriptionally active state (Aparicio and Gottschling, 1994). In addition, transcription through the telomeric tract of the VII-L telomere reduces its association with the periphery at all stages of the cell cycle (Tham et al., 2001). As the transcribed telomere can switch transcriptional states during a G1 arrest (de Bruin et al., 2000), release from the nuclear periphery may facilitate switches in transcriptional state. It is not clear why transcription through the telomeric tract results in telomere displacement from the nuclear periphery. One


possibility is that transcription of the telomere itself dislodges a telomere binding protein that is essential for tethering the VII-L telomere to the periphery. If this model is correct, neither Sir3p nor a Ku subunit is the dislodged protein as localization of the VII-L telomere to the nuclear periphery is not altered in sir3 or hdf1 strains (Tham et al., 2001). Functions of TPE The genes near natural telomeric ends in S. cerevisiae are often members of multigene families such as the SUC, MAL and MEL families (reviewed in Zakian, 1996). This redundancy makes it dicult to determine if these genes are naturally regulated by TPE. When expression of four unique sub-telomeric genes is analysed for dependency on SIR proteins, only YFR057w, an ORF of unknown function, is subject to natural telomeric silencing (Vega-Palas et al., 2000). Although this ORF is repressed in wild-type strains, its expression is increased upon deletion of SIR2, 3 and 4. YFR057w is localized adjacent to telomere VI-R. There are several properties of this location that are favorable for silencing. First, YFR057w is less than 1 kb from the core X ARS on telomere VI-R. Second, Sir proteins spread as far as 3 kb from the end of the VI-R telomere, a region that encompasses YFR057w (Strahl-Bolsinger et al., 1997). Third, the VI-R telomere does not contain a TPE inhibiting STAR element. The retrotransposon Ty5-1, which resides 1.8 kb from the left telomere of chromosome III, is also subject to natural telomeric silencing (Vega-Palas et al., 1997). Transcription of Ty5-1 is very low in wild-type cells, is reduced even more in cells over-expressing Sir3p, and is increased in a sir3 strain. Ty5 elements integrate preferentially near silent regions, like the silent mating type loci and telomeres (Zou et al., 1996). The variegated and semi-stable Ty5-1 transcription imposed by TPE probably reduces Ty5 transposition, thereby limiting the genetic damage caused by its movement to new sites. With the advent of genome wide analysis of transcription, it is possible to ask if TPE has global eects on transcription of telomere-linked genes (Wyrick et al., 1999). There are 267 genes that are within 20 kb of a telomere. In general, genes located within 20 kb of chromosome ends are expressed at lower levels, an average of 0.5 mRNA molecules per cell, than genes located more internally, an average of 2.4 mRNA molecules per cell. However, transcription of only 20 of these genes is increased by deletion of a SIR gene, and most of those aected are within 6 ± 8 kb of a chromosome end. Concentrating silencing proteins at telomeres may serve functions in addition to transcriptional silencing. When chromosomes are damaged by treatments that produce double strand breaks, Rap1p, Sir proteins and the Ku complex lose their punctate, peripheral localization and become dispersed throughout the

nucleus (Martin et al., 1999; McAinsh et al., 1999; Mills et al., 1999). This dispersal requires the DNA damage sensing RAD9 pathway. Dispersal of silencing proteins is accompanied by recruitment of the Ku complex to chromosome breaks, followed by movement of the Sir complex to the same sites. Even a single double strand break induces these events. The role of the Ku complex in DNA repair is well-documented (reviewed in Haber, 1999). Moreover, in at least some genetic backgrounds, Sir de®cient strains are hypersensitive to DNA damage (Martin et al., 1999; Mills et al., 1999). These data suggest a model in which telomeres provide a reservoir of repair proteins that can be readily mobilized in response to DNA damage. Movement of silencing proteins away from telomeres appears to be a genetically programmed step in the yeast aging process. As yeast cells age, the Sir complex redistributes from telomeres to the nucleolus, which contains the genes that encode ribosomal RNA (rRNA) (Kennedy et al., 1997). This re-localization is thought to protect ribosomal DNA from breakage and recombination (Gottlieb and Esposito, 1989; Kaeberlein et al., 1999). In addition, the NAD dependent deacetylase activity of Sir2p (Imai et al., 2000; Landry et al., 2000; Tanny et al., 1999) may repress rRNA transcription in aging cells, linking genomic silencing, caloric intake and aging (Tissenbaum and Guarente, 2001).

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TPE in other organisms TPE is not limited to S. cerevisiae. For example, TPE occurs at truncated telomeres in ®ssion yeast, Schizosaccharomyces pombe, an organism that is evolutionarily distant from S. cerevisiae (Russell and Nurse, 1986). As in S. cerevisiae, URA3 shows a reversible, meta-stable repression when placed next to a S. pombe telomere (Nimmo et al., 1994). However, some features of the silencing process are dierent in the two yeasts. For example, in S. pombe, some cells with a telomeric ade6+ gene produce uniformly pink colonies. This colony class probably arises from a reduced, stable level of ade6+ transcription in all cells in the colony, rather than from a population in which ade6+ is transcribed in some cells and repressed in others. This behavior diers from what is seen in the analogous situation in S. cerevisiae where colonies are either largely red or largely white. Mitotic cells lacking the S. pombe duplex telomere DNA binding protein Taz1p have very long telomeres and no TPE (Cooper et al., 1997, 1998; Nimmo et al., 1998). A similar massive telomere lengthening and loss of TPE is seen in S. cerevisiae mutants that lack the carboxyl terminal portion of Rap1p (Kyrion et al., 1992, 1993). Unlike Rap1p, Taz1p is not essential. However, S. pombe cells that lack Taz1p are seriously compromised in meiosis: telomere clustering at spindle pole bodies is disrupted, chromosome movement is defective, recombination is reduced, chromosomes missegregate, and spore viability is low (Cooper et al., Oncogene


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1998; Nimmo et al., 1998). As in S. cerevisiae, in wildtype S. pombe, mitotic chromosomes are localized to the nuclear periphery (Funabiki et al., 1993). Localization of the S. cerevisiae VII-L telomere can be perturbed without compromising the mitotic stability of chromosome VII (Tham et al., 2001). Perhaps loss of telomere localization in meiosis, as occurs in a taz17 S. pombe strain, has more dire eects on chromosome behavior. Trypanosoma brucei, a protozoan parasite responsible for sleeping sickness, spreads among mammalian hosts through the tsetse ¯y (reviewed in Rudenko et al., 1998). The coats of individual parasites are comprised of a single protein, encoded by a VSG (variant-speci®c surface glycoprotein) gene. Switching the identity of the VSG coat enables the organism to escape the mammalian immune system, a process that occurs repeatedly during the course of an infection. Although trypanosomes have close to 1000 VSG genes, there are only *50 sites from which these genes can be expressed, and each is adjacent to a telomere. In a given cell, only one of the many possible telomeric expression sites is active, suggesting that the other sites are subject to TPE. Indeed, promoters that are normally active in the same cell type are stably repressed when placed near a telomere (Horn and Cross, 1995). As with telomeric genes in S. cerevisiae, transcriptionally repressed VSG genes can switch to transcriptionally active forms without DNA rearrangements (and vice versa). In contrast to yeast where individual telomeres act independently in terms of their transcriptional states (Tham et al., 2001), in trypanosomes, all but one of the expression sites in a given cell is silent. It is not clear what enables one of the trypanosome expression sites to escape the transcriptional repression to which the rest are subject. However, the telomeric DNA at inactive expression sites, but not the active transcription site, contains high levels of a novel base, b-glucosyl-hydroxy-methyluracil, whose intriguing distribution suggests a possible role in expression site control (Gommers-Ampt et al., 1993; van Leeuwen et al., 1998). Loss of TPE in S. cerevisiae by transcription through the telomeric tract provides a paradigm for how TPE can be eliminated in cis at a single telomere that might be relevant to VSG gene expression in trypanosomes (de Bruin et al., 2000; Sandell et al., 1994; Tham et al., 2001). As in trypanosomes, sub-telomeric regions in Plasmodium falciparum, the protozoan parasite that causes malaria, contain multi-member gene families that encode virulence factors involved in antigenic variation and cytoadhesion, such as the var genes. After infection of erythrocytes, a Plasmodium cell expresses only one var gene at a time, suggesting that the other telomere-linked var genes are subject to TPE (Scherf et al., 1998). Intriguingly, silencing of a plasmid containing a var promoter requires passage through S phase (Deitsch et al., 2001), a precondition for establishment of silent chromatin in many organisms. Var genes on dierent chromosomes undergo recombination at frequencies much higher than that expected

for ectopic recombination (Freitas-Junior et al., 2000). In situ hybridization reveals that the 28 Plasmodium telomeres are clustered in 4 ± 7 foci at the nuclear periphery with random localization of dierent telomeres among the dierent clusters. The high level of recombination between heterologous chromosomes is thought to be a consequence of this clustering. TPE was ®rst observed in Drosophila melanogaster (Levis et al., 1985). Drosophila telomeres are not comprised of telomerase generated simple repeats but rather of at least two classes of retrotransposons, HeTA and TART (reviewed in Mason and Biessmann, 1995). However, the presence and identity of speci®c sub-terminal middle repetitive sequences, rather than the telomeric retrotransposons, may be the key regulators of TPE (Cryderman et al., 1999). The chromatin structure of these sub-telomeric repeats has several features reminiscent of telomeric chromatin in S. cerevisiae (de Bruin et al., 2000), such as reduced access to restriction enzymes (Cryderman et al., 1999). The sensitivity of TPE to mutation of modifying genes varies depending on the nature of the sub-telomeric repeats. For example, mutations in the chromosomal protein HP1 aect TPE at chromosome IV but not at chromosomes II and III, whose sub-telomeric repeats are related to each other but dierent from those on chromosome IV. Telomere placement is also regulated dierently at dierent telomeres. Chromosome IV telomeres are normally associated with the HP1 rich pericentric heterochromatin while chromosome II and III telomeres are localized to the nuclear periphery. When the telomere of the fourth chromosome is translocated to chromosome two, its silencing activity is diminished, although its residual silencing is still HP1 sensitive. This rearrangement also results in localization of the translocated fourth telomere to the nuclear periphery (Cryderman et al., 1999). These studies suggest that TPE in Drosophila is aected by the identity of the sub-telomeric repeats and their associated proteins as well as by nuclear position. Human somatic cells do not express telomerase, and hence their telomeres shorten by *50 ± 100 bps with each cell division (Harley, 1995). If TPE occurs in human cells, it would provide a mechanism for age dependent changes in expression of telomere adjacent genes (Wright and Shay, 1992). Early attempts to demonstrate TPE in mammalian cells were unsuccessful (summarized in Baur et al., 2001). However, recently HeLa cells were found to express a telomerelinked luciferase gene at a *10-fold lower level than an internally located gene (Baur et al., 2001). This telomere-linked repression has two features that are reminiscent of TPE in S. cerevisiae. When telomeres are lengthened by over-expression of telomerase, expression of the telomeric luciferase gene decreases 2 ± 10-fold. Incubation of cells with the histone deacetylase inhibitor trichostatin A preferentially increases expression of telomere-linked luciferase genes, consistent with the possibility that hypoacetylation of telomeric chromatin is important for telomeric silencing in mammals as it is in yeast.


Future directions TPE has been reported in several single cell eukaryotes, ¯ies, and humans, indicating that it is a widespread and perhaps even universal property of sub-telomeric regions. The S. cerevisiae analyses are a useful paradigm to guide future studies. There are questions that beg to be answered in other organisms. How far from the telomere can a gene be located and still be in¯uenced by TPE? Does the identity of sub-telomeric repeats, the size or presence of telomere loops, or the sub-nuclear localization of telomeres in¯uence silencing? Probably the most critical next step is the identi®cation of genes that regulate TPE. There are homologues of some but not all of the S. cerevisiae silencing proteins in higher eukaryotes (Brachmann et al., 1995; Li et al., 2000). Their analysis provides an obvious starting point for genetic approaches. Identifying genes on which TPE depends is important because the phenotypes associated with their loss will help determine the biological role of TPE. The cellular function of TPE need not be identical in all organisms. For example, although in S. cerevisiae the transcription of many telomere-linked genes is aected by TPE (Wyrick et al., 1999), TPE is not needed for viability nor even for robust growth under laboratory conditions. However, in parasitic protozoa, like trypanosomes and Plasmodia, regulation of subtelomeric gene expression may be critical for pathogenesis. If this turns out to be the case, treatments that interfere with TPE could have practical value. There are hints in mammals, as in yeast, for links between telomeric silencing and DNA repair. The mammalian Ku complex, like its yeast counterpart, is telomere associated and has roles in both DNA repair and telomere function (d'Adda di Fagagna et al., 2001;

Hsu et al., 1999, 2000; Samper et al., 2000). In humans, the repair proteins RAD50 and MRE11 are telomere associated (Zhu et al., 2000) while the yeast homologues of these proteins probably play roles in recruiting telomerase to telomeres (Ritchie and Petes, 2000; Tsukamoto et al., 2001). Perhaps most intriguing for future studies is the possibility that TPE provides a mechanism to modify gene expression in humans as a function of age. To test this idea, genes that show age related expression can be identi®ed by DNA micro-array analysis and their genomic positions determined. In yeast (Kyrion et al., 1993) and humans (Baur et al., 2001), long telomeres are more eective silencers than short telomeres. Thus, if TPE plays a role in age related gene expression, genes near telomeres are expected to be expressed at higher levels in cells from older individuals. However, telomere adjacent genes might not be the only ones aected by age dependent changes in TPE. In yeast, long internal tracts of telomeric DNA can repress transcription, and this repression depends on the same silencing proteins that act at telomeres (Stavenhagen and Zakian, 1994). If internal silencing also occurs in mammals, as telomeres shorten with age, internal tracts of telomeric DNA might compete more eectively with telomeres for limiting silencing proteins. In this scenario, genes near internal tracts of telomere DNA are predicted to have reduced expression in aged cells.

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Acknowledgments We thank Mary Kate Alexander, Lara Goudsouzian, and Michelle Mondoux for helpful comments on the manuscript and the National Institutes of Health for their support of the research in our laboratory.

References Andrulis ED, Neiman AM, Zappulla DC and Sternglanz R. (1998). Nature, 394, 592 ± 595. Aparicio OM, Billington BL and Gottschling DE. (1991). Cell, 66, 1279 ± 1287. Aparicio OM and Gottschling DE. (1994). Genes Dev., 8, 1133 ± 1146. Baur JA, Zou Y, Shay JW and Wright WE. (2001). Science, 292, 2075 ± 2077. Bell SP and Stillman B. (1992). Nature, 357, 128 ± 134. Boeke JD, LaCroute F and Fink GR. (1984). Mol. Gen. Genet., 197, 345 ± 346. Boulton SJ and Jackson SP. (1998). EMBO J., 17, 1819 ± 1828. Bourns BD, Alexander MK, Smith AM and Zakian VA. (1998). Mol. Cell. Biol., 18, 5600 ± 5608. Brachmann CB, Sherman JM, Devine SE, Cameron EE, Pillus L and Boeke JD. (1995). Genes Dev., 9, 2888 ± 2902. Braunstein M, Rose AB, Holmes SG, Allis CD and Broach JR. (1993). Genes Dev., 7, 592 ± 604. Buck SW and Shore D. (1995). Genes Dev., 9, 370 ± 384. Chan CS and Tye BK. (1983a). J. Mol. Biol., 168, 505 ± 523. Chan CSM and Tye B-K. (1983b). Cell, 33, 563 ± 573.

Cockell M, Palladino F, Laroche T, Kyrion G, Liu C, Lustig AJ and Gasser SM. (1995). J. Cell Biol., 129, 909 ± 924. Conrad MN, Wright JH, Wolf AJ and Zakian VA. (1990). Cell, 63, 739 ± 750. Cooper JP, Nimmo ER, Allshire RC and Cech TR. (1997). Nature, 385, 744 ± 747. Cooper JP, Watanabe Y and Nurse P. (1998). Nature, 392, 828 ± 831. Craven RJ and Petes TD. (2000). Mol. Cell. Biol., 20, 2378 ± 2384. Cryderman DE, Morris EJ, Biessmann H, Elgin SC and Wallrath LL. (1999). EMBO J., 18, 3724 ± 3735. d'Adda di Fagagna F, Hande MP, Tong W, Roth D, Lansdorp PM, Wang Z and Jackson SP. (2001). Curr. Biol., 11, 1192 ± 1196. de Bruin D, Kantrow SM, Liberatore RA and Zakian VA. (2000). Mol. Cell. Biol., 20, 7991 ± 8000. de Bruin D, Zaman Z, Liberatore RA and Ptashne M. (2001). Nature, 409, 109 ± 113. Deitsch KW, Calderwood MS and Wellems TE. (2001). Nature, 412, 875 ± 876.

Oncogene


520

Oncogene

Fourel G, Revardel E, Koering CE and Gilson E. (1999). EMBO J., 18, 2522 ± 2537. Freitas-Junior LH, Bottius E, Pirrit LA, Deitsch KW, Scheidig C, Guinet F, Nehrbass U, Wellems TE and Scherf A. (2000). Nature, 407, 1018 ± 1022. Funabiki H, Hagan I, Uzawa S and Yanagida M. (1993). J. Cell Biol., 121, 961 ± 976. Gilson E, Roberge M, Giraldo R, Rhodes D and Gasser SM. (1993). J. Mol. Biol., 231, 293 ± 310. Gommers-Ampt JH, Van Leeuwen F, de Beer AL, Vliegenthart JF, Dizdaroglu M, Kowalak JA, Crain PF and Borst P. (1993). Cell, 75, 1129 ± 1136. Gotta M, Laroche T, Formenton A, Maillet L, Scherthan H and Gasser SM. (1996). J. Cell Biol., 134, 1349 ± 1363. Gotta M, Strahl-Bolsinger S, Renauld H, Laroche T, Kennedy BK, Grunstein M and Gasser SM. (1997). EMBO J., 16, 3243 ± 3255. Gottlieb S and Esposito RE. (1989). Cell, 56, 771 ± 776. Gottschling DE. (1992). Proc. Natl. Acad. Sci. USA, 89, 4062 ± 4065. Gottschling DE, Aparicio OM, Billington BL and Zakian VA. (1990). Cell, 63, 751 ± 762. Gravel S, Larrivee M, Labrecque P and Wellinger RJ. (1998). Science, 280, 741 ± 744. Grith JD, Comeau L, Rosen®eld S, Stansel RM, Bianchi A, Moss H and de Lange T. (1999). Cell, 97, 503 ± 514. Haber JE. (1999). Cell, 97, 829 ± 832. Hardy CF, Sussel L and Shore D. (1992). Genes Dev., 6, 801 ± 814. Harley CB. (1995). Telomeres. Blackburn, EH and Greider, CW (eds). Cold Spring Harbor Laboratory Press: Plainview, NY, pp 247 ± 263. Hecht A, Laroche T, Strahl-Bolsinger S, Gasser SM and Grunstein M. (1995). Cell, 80, 583 ± 592. Hecht A, Strahl-Bolsinger S and Grunstein M. (1996). Nature, 383, 92 ± 96. Hill A and Bloom K. (1987). Mol. Cell. Biol., 7, 2397 ± 2405. Horn D and Cross GA. (1995). Cell, 83, 555 ± 561. Hsu HL, Gilley D, Blackburn EH and Chen DJ. (1999). Proc. Natl. Acad. Sci. USA, 96, 12454 ± 12458. Hsu HL, Gilley D, Galande SA, Hande MP, Allen B, Kim SH, Li GC, Campisi J, Kohwi-Shigematsu T and Chen DJ. (2000). Genes Dev., 14, 2807 ± 2812. Huang H, Kahana A, Gottschling DE, Prakash L and Liebman SW. (1997). Mol. Cell. Biol., 17, 6693 ± 6699. Imai S, Armstrong CM, Kaeberlein M and Guarente L. (2000). Nature, 403, 795 ± 800. Kaeberlein M, McVey M and Guarente L. (1999). Genes Dev., 13, 2570 ± 2580. Kennedy BK, Gotta M, Sinclair DA, Mills K, McNabb DS, Murthy M, Pak SM, Laroche T, Gasser SM and Guarente L. (1997). Cell, 89, 381 ± 391. Kyrion G, Boakye KA and Lustig AJ. (1992). Mol. Cell. Biol., 12, 5159 ± 5173. Kyrion G, Liu K, Liu C and Lustig AJ. (1993). Genes Dev., 7, 1146 ± 1159. Laman H, Balderes D and Shore D. (1995). Mol. Cell Biol, 15, 3608 ± 3617. Landry J, Slama JT and Sternglanz R. (2000). Biochem. Biophys. Res. Commun., 278, 685 ± 690. Laroche T, Martin SG, Gotta M, Gorham HC, Pryde FE, Louis EJ and Gasser SM. (1998). Curr. Biol., 8, 653 ± 656. Laroche T, Martin SG, Tsai-P¯ugfelder M and Gasser SM. (2000). J. Struct. Biol., 129, 159 ± 174. Levis R, Hazelrigg T and Rubin GM. (1985). Science, 229, 558 ± 561. Li B, Oestreich S and de Lange T. (2000). Cell, 101, 471 ± 483.

Lin JJ and Zakian VA. (1996). Proc. Natl. Acad. Sci. USA, 93, 13760 ± 13765. Louis EJ. (1995). Yeast, 11, 1553 ± 1574. Louis EJ, Naumova ES, Lee A, Naumov G and Haber JE. (1994). Genetics, 136, 789 ± 802. Maillet L, Boscheron C, Gotta M, Marcand S, Gilson E and Gasser SM. (1996). Genes Dev., 10, 1796 ± 1811. Mangahas JL, Alexander MK, Sandell LL and Zakian VA. (2001). Mol. Biol. Cell., 12, 4078 ± 4089. Marcand S, Buck SW, Moretti P, Gilson E and Shore D. (1996). Genes Dev., 10, 1297 ± 1309. Martin SG, Laroche T, Suka N, Grunstein M and Gasser SM. (1999). Cell, 97, 621 ± 633. Mason JM and Biessmann H. (1995). Trends Genet., 11, 58 ± 62. McAinsh AD, Scott-Drew S, Murray JA and Jackson SP. (1999). Curr. Biol., 9, 963 ± 966. Mills KD, Sinclair DA and Guarente L. (1999). Cell, 97, 609 ± 620. Mishra K and Shore D. (1999). Curr. Biol., 9, 1123 ± 1126. Monson EK, de Bruin D and Zakian VA. (1997). Proc. Natl. Acad. Sci. USA, 94, 13081 ± 13086. Moretti P, Freeman K, Coodly L and Shore D. (1994). Genes Dev., 8, 2257 ± 2269. Munoz-Jordan JL, Cross GA, de Lange T and Grith JD. (2001). EMBO J., 20, 579 ± 588. Murti KG and Prescott DM. (1999). Proc. Natl. Acad. Sci. USA, 96, 14436 ± 14439. Nimmo ER, Cranston G and Allshire RC. (1994). EMBO J., 13, 3801 ± 3811. Nimmo ER, Pidoux AL, Perry PE and Allshire RC. (1998). Nature, 392, 825 ± 828. Nugent CI, Hughes TR, Lue NF and Lundblad V. (1996). Science, 274, 249 ± 252. Palladino F, Laroche T, Gilson E, Axelrod A, Pillus L and Gasser SM. (1993). Cell, 75, 543 ± 555. Pryde FE and Louis EJ. (1999). EMBO J., 18, 2538 ± 2550. Renauld H, Aparicio OM, Zierath PD, Billington BL, Chhablani SK and Gottschling DE. (1993). Genes Dev., 7, 1133 ± 1145. Ritchie KB and Petes TD. (2000). Genetics, 155, 475 ± 479. Rudenko G, Cross M and Borst P. (1998). Trends Microbiol., 6, 113 ± 116. Runge KW and Zakian VA. (1996). Mol. Cell. Biol., 16, 3094 ± 3105. Russell P and Nurse P. (1986). Cell, 45, 781 ± 782. Samper E, Goytisolo FA, Slijepcevic P, van Buul PP and Blasco MA. (2000). EMBO Rep., 1, 244 ± 252. Sandell LL, Gottschling DE and Zakian VA. (1994). Proc. Natl. Acad. Sci. USA, 91, 12061 ± 12065. Sandell LL and Zakian VA. (1993). Cell, 75, 729 ± 739. Scherf A, Hernandez-Rivas R, Buet P, Bottius E, Benatar C, Pouvelle B, Gysin J and Lanzer M. (1998). EMBO J., 17, 5418 ± 5426. Snyder M, Spolsky RJ and Davis RW. (1988). Mol. Cell. Biol., 8, 2184 ± 2194. Stavenhagen JB and Zakian VA. (1994). Genes Dev., 8, 1411 ± 1422. Strahl-Bolsinger S, Hecht A, Luo K and Grunstein M. (1997). Genes Dev., 11, 83 ± 93. Straight AF, Belmont AS, Robinett CC and Murray AW. (1996). Curr. Biol., 6, 1599 ± 1608. Tanny JC, Dowd GJ, Huang J, Hilz H and Moazed D. (1999). Cell, 99, 735 ± 745. Tham WH, Wyithe JS, Ferrigno PK, Silver PA and Zakian VA. (2001). Mol. Cell, 8, 189 ± 199.


Tissenbaum HA and Guarente L. (2001). Nature, 410, 227 ± 230. Tsukamoto Y, Taggart AKP and Zakian VA. (2001). Curr. Biol., 11, 1328 ± 1335. van Leeuwen F, Taylor MC, Mondragon A, Moreau H, Gibson W, Kieft R and Borst P. (1998). Proc. Natl. Acad. Sci. USA, 95, 2366 ± 2371. Vega-Palas MA, Martin-Figueroa E and Florencio FJ. (2000). Mol. Gen. Genet., 263, 287 ± 291. Vega-Palas MA, Venditti S and Di Mauro E. (1997). Nat. Genet., 15, 232 ± 233. Wiley E and Zakian VA. (1995). Genetics, 139, 67 ± 79. Wotton D and Shore D. (1997). Genes Dev . 11, 748 ± 760. Wright JH, Gottschling DE and Zakian VA. (1992). Genes Dev., 6, 197 ± 210.

Wright JH and Zakian VA. (1995). Nuc. Acids Res., 23, 1454 ± 1460. Wright WE and Shay JW. (1992). Trends. Genet., 8, 193 ± 197. Wyrick JJ, Holstege FC, Jennings EG, Causton HC, Shore D, Grunstein M, Lander ES and Young RA. (1999). Nature, 402, 418 ± 421. Zakian VA. (1996). Annu. Rev. Genet., 30, 141 ± 172. Zhu XD, Kuster B, Mann M, Petrini JH and de Lange T. (2000). Nat. Genet., 25, 347 ± 352. Zou S, Ke N, Kim JM and Voytas DF. (1996). Genes Dev., 10, 634 ± 645.

521

Oncogene