Transcriptional Silencing at Saccharomyces Telomeres: Implications for Other Organisms

Transcriptional silencing at Saccharomyces telomeres: implications for other organisms

Wai-Hong Tham1 and Virginia A Zakian*,1

1Department of Molecular Biology, Princeton University, Princeton, New Jersey, NJ 08544, USA

Telomeres are the natural ends of eukaryotic chromo- including Sir2, Sir3, and Sir4p, as well as the two Rif somes. In most organisms, telomeres consist of simple, proteins, localize to telomeres (Bourns et al., 1998), repeated DNA with the strand running 5' to 3' towards presumably as a consequence of their ability to the end of the chromosome being rich in G residues. In interact, directly or indirectly, with Rap1p (Hardy et cases where the very end of the chromosome has been al., 1992; Moretti et al., 1994; Wotton and Shore, examined, the G-strand is extended to form a short, 1997). The Ku complex, which displays sequence non- single stranded tail. The chromatin structure of speci®c end binding activity and is comprised of two telomeric regions often has features that distinguish proteins Hdf1p and Hdf2p, also binds telomeres them from other parts of the genome. Because telomeres (Gravel et al., 1998; Martin et al., 1999). protect chromosome ends from degradation and end-to- Yeast has two classes of sub-telomeric repeats, Y' end fusions and prevent the loss of terminal DNA by and X (reviewed in Louis, 1995) (Figure 1a). The Y' serving as a substrate for telomerase, they are essential element, which comes in two sizes, 6.7 and 5.2 kb, is for the stable maintenance of eukaryotic chromosomes. found on about two-thirds of yeast telomeres. In addition to their essential functions, telomeres in Individual telomeres bear up to four tandem copies diverse organisms are specialized sites for gene of Y'; between tandem Y' elements, there are often expression. Transcription of genes located next to short C1±3A/TG1±3 tracts. X is quite heterogeneous telomeres is repressed, a phenomenon termed telomere but virtually all X elements have a core region of position eect (TPE). TPE is best characterized in the *500 bp that is found at almost every telomere (Louis yeast Saccharomyces cerevisiae. This article will focus et al., 1994). On telomeres having both Y' and X, Y' is on the silencing properties of Saccharomyces telomeres closer to the chromosome end. Y' and core X both and end with speculation on the role of TPE in yeasts contain a putative origin of DNA replication or ARS and other organisms. (autonomously replicating sequence) (Chan and Tye, Oncogene (2002) 21, 512 ± 521. DOI: 10.1038/sj/onc/ 1983a). 1205078 Unlike the telomeric C1±3A/TG1±3 tracts, sub- telomeric DNA is organized into nucleosomes (Wright Keywords: telomere; TPE; heterochromatin; telomer- et al., 1992). However, these nucleosomes have several ase; Ku complex; Sir proteins 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 DNA and chromatin structure of Saccharomyces methylase, suggesting that sub-telomeric DNA is less telomeres accessible and hence more compact than bulk chromatin (Gottschling, 1992). The amino terminal tails of histones Yeast telomeres consist of 350+75 bps of C1±3A/ H3 and H4 are hypoacetylated in sub-telomeric nucleo- TG1±3 DNA. The telomeric tract is encompassed in a somes compared to histones elsewhere (Braunstein et al., non-nucleosomal DNA-protein complex called the 1993; de Bruin et al., 2000; Monson et al., 1997). The telosome (Wright et al., 1992). The sequence speci®c Sir2, 3 and 4 proteins bind as a complex to sub-telomeric duplex DNA binding protein Rap1p is the major nucleosomes, brought there by their ability to interact protein in the telosome, with an estimated 10 ± 20 with the amino terminal tails of histones H3 and H4 molecules per chromosome end (Conrad et al., 1990; (Hecht et al., 1995; Strahl-Bolsinger et al., 1997). Loss of Gilson et al., 1993; Wright et al., 1992; Wright and any one of the Sir proteins eliminates binding of the Zakian, 1995). Cdc13p, a protein that binds to single other Sir proteins to sub-telomeric chromatin (Hecht et strand TG1±3 in vitro (Lin and Zakian, 1996; Nugent et al., 1996; Strahl-Bolsinger et al., 1997), whereas Sir4p al., 1996) is also telomere bound in vivo (Bourns et al., remains telomere bound, even in a sir3 strain (Bourns et 1998; Tsukamoto et al., 2001). Several other proteins, al., 1998). The Ku complex also associates with sub- telomeric nucleosomes (Martin et al., 1999). In contrast to its binding to chromosome ends, the association of Ku *Correspondence: VA Zakian; with sub-telomeric chromatin is Sir4p dependent (Martin E-mail: [email protected] et al., 1999). Telomere position effect W-H Tham and VA Zakian 513 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 Figure 1 Structure of natural (a) and truncated (b) telomeres. telomere position eect, TPE. TPE is not limited to the left telomere of 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 chromosome VII nor to the URA3 gene. TPE occurs arrowhead. (a) Most natural telomeres bear X and often bear Y'. at all tested truncated chromosomes, including the left Although X is heterogeneous, ranging from 0.3 to 3.75 kb in size telomere of chromosome VII, the right telomere of (Chan and Tye, 1983b), a *500 bp segment called core X is found at most telomeres (Louis et al., 1994). There are two major chromosomes V (Gottschling et al., 1990), and the left classes of Y',Y' long (6.7 kb) and Y' short (5.2 kb). Individual telomere of chromosome XV (Craven and Petes, 2000). telomeres can have zero or up to four tandem copies of Y'; the About half of natural telomeres exhibit TPE (Pryde telomere shown has a single Y' (indicated by bracket). STAR and Louis, 1999) (discussed in more detail below). Like (subtelomeric anti-silencing region) is a region within Y' that antagonizes silencing. The sub-telomeric repeats (STR) A, B, C, URA3, other RNA polymerase II transcribed genes, and D that are found between X and the telomere or between X such as TRP1, ADE2 and HIS3, as well as the RNA and Y', also have STAR activity. X and Y' both contain an ARS, polymerase III-transcribed tyrosyl tRNA gene, SUP4- which has a binding site for ORC, a multi-protein complex o, are subject to TPE (Gottschling et al., 1990; Huang needed for initiation of DNA replication. X contains a binding et al., 1997). 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 Although the transcriptional state at telomeres is chromosome VII. Many published studies on TPE were carried reversible, both the repressed and transcribed states are out on strains having URA3 adjacent to the left telomere of stable in wild-type strains. This stability is best chromosome VII. To make such strains, yeast is transformed with illustrated by the appearance of colonies generated by a fragment having a portion of ADH4, URA3, and an 81 bp tract cells with ADE2 next to a telomere (Gottschling et al., of C1±3A/TG1±3 DNA (denoted by black square). Recombina- tion within ADH4 results in URA3 integration and loss of the 1990). Cells expressing Ade2p generate white colonies, DNA distal of ADH4. A new telomere is formed on the 81 bp while lack of Ade2p results in red colonies. Most of the C1±3A/TG1±3 tract (Gottschling et al., 1990). The terminally colonies generated by a strain with ADE2 at the deleted version of chromosome VII is lost at the same rate as telomere fall into one of two types: largely white unmodi®ed chromosome VII (Sandell and Zakian, 1993). The X and Y' content of chromosome VII-L varies among dierent yeast colonies with red sectors and largely red colonies with strains 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. Assaying telomeric silencing Sectors of opposite expression states in largely red, and largely white colonies provide additional evidence that TPE was discovered during the course of constructing both states are reversible. strains to be used to determine the chromatin structure At truncated telomeres, there is a continuous domain of a single yeast telomere (Gottschling et al., 1990). of transcriptional silencing with the probability of Because yeast telomeres bear many kilo-basepairs of silencing inversely proportional to the distance from repetitive DNA, hybridization probes speci®c for a the telomere (Renauld et al., 1993). However, the single telomere are not readily available. To circumvent probability of silencing is not simply a function of this problem, we inserted the URA3 gene adjacent to distance, as the nature of the intervening DNA also the left telomere of chromosome VII, in such a way aects TPE. For example, the propagation of silencing that the sub-telomeric repeats are deleted, and a new from the telomere can be blocked by transcription of telomere is formed immediately distal of URA3. These an intervening gene. The extent of silencing is also terminally deleted or truncated chromosomes were sensitive to the length of the C1±3A/TG1±3 telomeric isolated by their Ura+ phenotype (Figure 1b). tract. In general, the longer the telomere, the greater URA3 was a fortuitous choice for this construction. the silencing of an adjacent gene (Buck and Shore, Cells that express Ura3p can not grow in the presence 1995; Kyrion et al., 1993). However, strains with long of the drug 5 ¯uoro orotic acid (FOA) (Boeke et al., telomeres can have reduced TPE, if lengthening is due

Oncogene Telomere position effect W-H Tham and VA Zakian 514 to loss of telomere bound silencing proteins (Kyrion et It is unclear why dierent telomeres have dierent al., 1992; Wiley and Zakian, 1995). silencing properties. However, neither X nor Y' is homogeneous, and major dierences in the silencing behavior of dierent telomeres may re¯ect seemingly Internal C1±3A/TG1±3 tracts are silencers minor dierences in sequence. There are four types of short, repeated elements termed sub-telomeric repeats An 81 bp tract of C1±3A/TG1±3 DNA inserted (STR A, B, C and D) within the distal portion of X *20 kb from the left telomere of chromosome VII (Figure 1a). Certain STR elements and the 0.14 kb does not repress transcription of an adjacent URA3 telomere proximal portion of Y' have anti-silencing or gene (Gottschling et al., 1990). However, a tel1 strain, STAR activity (subtelomeric anti-silencing region) which has telomeres of 4100 bps, has relatively (Fourel et al., 1999; Pryde and Louis, 1999) (Figure normal levels of TPE (Runge and Zakian, 1996). These 1a). When STAR elements are inserted between URA3 considerations led to the idea that telomeric silencing and the telomere, TPE decreases *20 ± 50-fold. When a cannot be conferred by C1±3A/TG1±3 repeats alone gene is bracketed by STAR elements, it is insulated but rather requires special features of telomeric DNA, from transcriptional silencing, indicating that STARs such as the single stranded TG1±3 tail and its have at least some of the properties of boundary associated end binding proteins (Gottschling et al., elements. However, TPE occurs at telomeres containing 1990). However, internal C1±3A/TG1±3 tracts of a STAR if they also contain a core X or the 0.76 kb *550 bp can repress nearby genes, a phenomenon centromere proximal region of Y'. Elements like core X termed C1±3A-based silencing, CBS (Stavenhagen and that block STAR action are termed sub-telomeric Zakian, 1994). Long internal C1±3A/TG1±3 tracts can silencing elements. Although sub-telomeric silencing silence even on a circular chromosome, demonstrating elements are not themselves sucient to silence a gene, that CBS does not require a telomere in cis. The level they are proposed to act as a relay system that helps of CBS is very sensitive to the length of the C1±3A/ propagate silencing initiated at the telomere to more TG1±3 tract, with *275 bp tracts showing little, if any internal regions of the chromosome. Taken together, silencing. Although there are naturally occurring tracts these data suggest that the sub-telomeric chromatin on of C1±3A/TG1±3 DNA at non-telomeric sites, all such natural chromosomes is a region of transcriptional tracts are relatively short: the eight longest range in repression punctuated by sub-domains that are permis- size from 52 to 159 bps (Mangahas et al., 2001). Thus, sive for transcription (Fourel et al., 1999; Pryde and there is no naturally occurring C1±3A/TG1±3 tract that Louis, 1999). This picture is very dierent from what is is long enough to act as a silencer on its own. seen at truncated telomeres where there is a continuous However, two *275 bp tracts of C1±3A/TG1±3 DNA zone of transcriptional repression that emanates from can act synergistically to silence a gene placed between the telomere and dissipates continuously towards the them, even though neither alone represses transcription centromere (Renauld et al., 1993). (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 Genes and conditions that aect silencing silencers. Since each of the eight longest, naturally occurring C1±3A/TG1±3 tracts is near a telomere A surprisingly large number of genes and conditions (Mangahas et al., 2001), these tracts might act to aect TPE. Deletion of certain non-essential genes, boost the level of silencing imposed by the telomere such as SIR2, SIR3, SIR4 (Aparicio et al., 1991), itself. 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 Silencing at natural telomeres genes, are also needed for transcriptional silencing at the silent mating type or HM loci as well as for CBS. Virtually all published studies on TPE use truncated A version of Rap1p that lacks the carboxyl terminal telomeres. TPE also occurs at many, but not all, *144 amino acids, the portion of Rap1p that interacts natural telomeres. When URA3 is inserted within the with the Sir complex, also abolishes TPE and CBS and sub-telomeric DNA of a natural telomere, its repres- reduces HM silencing yet still binds DNA and supplies sion varies dramatically, depending on its site of the essential functions of Rap1p (Kyrion et al., 1992, integration (Pryde and Louis, 1999). When URA3 is 1993; Stavenhagen and Zakian, 1994). Mutations in the inserted within Y', repression is low or undetectable. amino terminal tails of histones H3 and H4 that reduce Likewise, when URA3 is integrated near the ARS of their ability to interact with Sir proteins in vitro also the X element on telomeres III-R, IV-L, or X-R, TPE reduce TPE in vivo (Hecht et al., 1995). is very low. However, when URA3 is integrated at a Other telomere binding proteins have an inhibitory similar location at telomeres II-R, XI-L, or XIII-R or eect on TPE. For example, deletion of Rif1p and near this location on XIV-R, repression is much Rif2p increases TPE (Kyrion et al., 1993). Although higher, and in some cases as high as that seen when deleting Rif proteins results in telomere lengthening, URA3 is immediately adjacent to the truncated the enhancement of TPE seen in their absence seems to telomere of chromosome VII-L. be due primarily to their ability to compete with Sir3p

Oncogene Telomere position effect W-H Tham and VA Zakian 515 for binding to telomere-bound Rap1p (Mishra and distance from the telomere (Strahl-Bolsinger et al., Shore, 1999). Over-expression of structural components 1997). of telomeres can also impact TPE: for example, Sir3p over-expression increases (Renauld et al., 1993) and Sir4p over-expression decreases telomeric silencing Telomere folding: a structural requirement for TPE? (Cockell et al., 1995). The genetic requirements for TPE, determined using Rap1p is a sequence speci®c, duplex C1±3A/TG1±3 truncated telomeres, do not necessarily apply to binding protein that does not bind histones. None- native telomeres. Although TPE at natural telomeres theless, like the Sir and Hdf proteins, Rap1p is found requires the Sir proteins and Hdf1p, it is less in sub-telomeric chromatin (Strahl-Bolsinger et al., dependent on both than silencing at truncated 1997). This observation led to the proposal that the telomeres and in the case of Hdf1p, its dependence telomeric region has a folded structure. According to varies depending on the telomere being examined this model, telomere folding allows Rap1p-Sir interac- (Fourel et al., 1999; Pryde and Louis, 1999). Native tions within sub-telomeric chromatin (Strahl-Bolsinger telomeres are also less sensitive to the enhancement in et al., 1997). In addition to this biochemical evidence, TPE seen upon Sir3p over-expression. In addition, there is genetic support for telomere looping. In most deletion of SIR1 aects TPE at native ends (Fourel et locations, the yeast equivalent of an enhancer, the UAS al., 1999; Pryde and Louis, 1999) as well as at the (upstream activating sequence), activates transcription silent mating loci but not at the truncated chromo- only when it is positioned upstream of a gene. some VII-L telomere (Aparicio et al., 1991). Para- However, when a UAS is downstream of a telomere- doxically, certain mutations in subunits of ORC, a linked gene, expression of the gene is activated (de multi-protein complex that binds to ARS sequences Bruin et al., 2001). This ability is proposed to be due to and is required for initiation of DNA replication (Bell telomere folding, which places the UAS close to the and Stillman, 1992), result in decreased TPE at promoter of the telomere-linked gene. Conditions that truncated telomere VII-L, which does not contain an eliminate telomere looping disrupt TPE without ARS, but not at native telomeres, which do (Pryde aecting chromosome stability (de Bruin et al., 2000). and Louis, 1999). Telomere loops have also been detected in mammals In addition to genes whose functions are essential for (Grith et al., 1999), ciliates (Murti and Prescott, TPE, there are a surprisingly large number whose 1999), and trypanosomes (Munoz-Jordan et al., 2001). mutation causes more modest eects on TPE. For However, in these organisms, telomere loops form by example, deletion of RRM3, which encodes a DNA invasion of the 3' telomeric overhang into duplex helicase that is needed for normal fork progression telomeric DNA, while there is no evidence that looping through telomeric and sub-telomeric DNA, results in in S. cerevisiae involves base pairing of the telomere about a 10-fold drop in TPE (Ivessa et al., in with sub-telomeric DNA. 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 Eliminating TPE in cis transcribed state is cell cycle limited, occurring preferentially late in the cell cycle (Aparicio and Genes that aect TPE usually do so at all truncated Gottschling, 1994). Thus, genes that aect cell cycle telomeres in the cell. However, TPE can also be progression might also aect TPE, but these eects eliminated at a single telomere. The strategy for could be secondary to their impact on the cell cycle eliminating TPE in cis is based on the demonstration (Laman et al., 1995). that transcription from a strong promoter near an ARS The proteins whose loss has the most profound eect or centromere eliminates their functions (Hill and on TPE are all structural components of either the Bloom, 1987; Snyder et al., 1988). Using a parallel telosome, sub-telomeric chromatin, or both. When approach, the galactose inducible GAL UAS and these structural proteins are lost, the atypical chroma- TATA were inserted between URA3 and the telomere tin features that distinguish sub-telomeric chromatin on the truncated left arm of chromosome VII in such a from bulk chromatin, such as histone hypoacetylation way that transcription from the GAL UAS proceeds and reduced access to DNA modifying enzymes, are towards and through the telomeric tract (Sandell et al., also lost (de Bruin et al., 2000; Gottschling, 1992). 1994). When this strain is grown in galactose medium, These data led to a model in which Rap1p binds an abundant UG1±3 transcript about the length of the speci®cally to the telomeric repeats and initiates the telomeric C1±3A/TG1±3 tract is detected. The loss rate assembly of a multimeric protein complex that allows of chromosome VII is not increased by transcription the establishment and maintenance of TPE. The through the telomeric tract, demonstrating that the complex of Sir proteins is brought to the telomere by stability functions of the VII-L telomere are intact. its ability to interact with Rap1p. The Sir complex However, TPE is lost at the URA3 gene that is spreads to nearby nucleosomes by binding of Sir3p and adjacent to the transcribed telomere (Sandell et al., Sir4p to the amino terminal tails of histones H3 and 1994) while TPE at other telomeres in the same cell is H4 (Hecht et al., 1995). The concentration of Sir not aected (Tham et al., 2001). This system allows proteins on a native telomere decreases with increasing analysis of the behavior of a telomere that lacks TPE

Oncogene Telomere position effect W-H Tham and VA Zakian 516 in an otherwise silencing competent cell. Loss of TPE that a telomere adjacent gene is silenced when it is near at the transcribed VII-L telomere is accompanied by all the periphery and expressed when it is not. Although of the chromatin changes, such as histone acetylation, foci of silencing proteins are dispersed and silencing is that are associated with global loss of TPE. Again, the lost in a sir3 strain, the pattern of Y' localization is transcribed telomere is the only one in the cell whose largely unaltered (Gotta et al., 1996). In contrast, when chromatin structure is aected (de Bruin et al., 2000). one of the genes encoding a Ku subunit is deleted, foci Because transcription through the telomeric tract of silencing proteins are disrupted, the number of Y' results in rapid and severe loss of Rap1p from sub- foci increases, and only about half of the foci are at the telomeric chromatin, it eliminates looping at the periphery (Laroche et al., 1998). Interpretation of these aected telomere. In contrast, growth in medium experiments is complicated by the fact that the Y' lacking uracil or deletion of SIR3, results in, signal detects many, but not all telomeres, and is respectively, no or slower and less complete loss of heavily biased towards telomeres in clusters and those sub-telomeric Rap1p. These data suggest that telomere containing tandem copies of Y'. looping is necessary but not sucient for TPE. The chromosome visualization methods described in (Straight et al., 1996) have been used to follow the behavior of a single telomere under conditions where Telomeres are localized near the nuclear periphery: its TPE status is known (Tham et al., 2001). When an implications for TPE array of lac operator binding sites is inserted very near the end of a truncated chromosome VII-L in a strain Immuno-localization shows that several proteins that also expresses an inducible GFP (green ¯uorescent needed for telomeric silencing, including Rap1p, Sir2p, protein)-lac repressor fusion protein, the VII-L telo- Sir3p, Sir4p and Hdf2p are concentrated in six to eight mere is visualized as a compact green spot. The nuclear foci in diploid cells (Gotta et al., 1996, 1997; Laroche periphery is marked using an antibody to a nuclear et al., 1998; Palladino et al., 1993). At least for Rap1p, pore protein. Deconvolution microscopy is then used these foci are near the nuclear periphery (Gotta et al., to determine the position of the telomere relative to the 1996). Moreover, in situ hybridization reveals that periphery under a variety of conditions. This approach *70% of Y' DNA is similarly located (Gotta et al., shows that in most strains and conditions, the VII-L 1996; Laroche et al., 2000). Since the number of these telomere is localized near the nuclear periphery in the foci is considerably smaller than the 64 telomeres in a majority of cells. However, in silencing competent cells, diploid cell, each focus of silencing proteins is thought the level of telomere localization to the nuclear to contain multiple telomeres. periphery does not correlate with the level of telomeric There is increasing evidence that the nuclei of silencing. For example, the level of telomere VII-L eukaryotic cells are divided into permissive and localization is similar between a strain that has *80% restrictive compartments vis-aÁ-vis transcription. The repression at the VII-L telomere and a strain that has peripheral localization of silencing proteins and only 7% silent cells. In addition, deletion of SIR3 or chromosomal sites of transcriptional repression sug- HDF1 or growth in medium lacking uracil, each of gests that the yeast nuclear periphery is a region where which eliminates TPE at the VII-L telomere, does not transcription is inhibited. Perhaps the most compelling result in delocalization. Thus, telomeres are often support for this hypothesis is the demonstration that localized to the nuclear periphery, but this localization tethering a weak silencer to the nuclear periphery is separable from telomeric silencing. Nonetheless, no improves its ability to silence a nearby gene (Andrulis strain was identi®ed in which the VII-L telomere is et al., 1998). In addition, silencing at a non-telomeric both silenced and away from the nuclear periphery, site is increased when the site is brought closer to a suggesting that localization near the periphery is telomere (Maillet et al., 1996; Stavenhagen and Zakian, necessary, albeit not sucient, for silencing. 1994). Over-expression of Sir3p or Sir4p, which results Two conditions result in loss of localization of in dispersal of silencing proteins throughout the telomeres to the periphery (Tham et al., 2001). As cells nucleus, also improves silencing at non-telomeric sites. progress towards mitosis, the VII-L telomere moves Targeting Sir proteins to a site that is 20 kb from a away from the periphery, assuming a distribution truncated telomere, too far under normal conditions to indistinguishable from random. This is the same time be aected by TPE, allows silencing at that site in the cell cycle when a repressed telomeric gene has a (Marcand et al., 1996). These data suggest a model in higher probability of switching to a transcriptionally which silencing is limited by the abundance of silencing active state (Aparicio and Gottschling, 1994). In proteins. According to this model, transcriptional addition, transcription through the telomeric tract of repression can be increased by bringing a gene closer the VII-L telomere reduces its association with the to the periphery and hence closer to pools of silencing periphery at all stages of the cell cycle (Tham et al., proteins or by elevating and dispersing silencing 2001). As the transcribed telomere can switch tran- proteins throughout the nucleus, thereby increasing scriptional states during a G1 arrest (de Bruin et al., the probability of their binding a gene located in the 2000), release from the nuclear periphery may facilitate nuclear interior. switches in transcriptional state. It is not clear why Because silencing is reversible and not all Y' transcription through the telomeric tract results in sequences are at the periphery, an appealing model is telomere displacement from the nuclear periphery. One

Oncogene Telomere position effect W-H Tham and VA Zakian 517 possibility is that transcription of the telomere itself nucleus (Martin et al., 1999; McAinsh et al., 1999; dislodges a telomere binding protein that is essential Mills et al., 1999). This dispersal requires the DNA for tethering the VII-L telomere to the periphery. If damage sensing RAD9 pathway. Dispersal of silencing this model is correct, neither Sir3p nor a Ku subunit is proteins is accompanied by recruitment of the Ku the dislodged protein as localization of the VII-L complex to chromosome breaks, followed by move- telomere to the nuclear periphery is not altered in sir3 ment of the Sir complex to the same sites. Even a single or hdf1 strains (Tham et al., 2001). 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 Functions of TPE genetic backgrounds, Sir de®cient strains are hypersen- sitive to DNA damage (Martin et al., 1999; Mills et al., The genes near natural telomeric ends in S. cerevisiae 1999). These data suggest a model in which telomeres are often members of multigene families such as the provide a reservoir of repair proteins that can be SUC, MAL and MEL families (reviewed in Zakian, readily mobilized in response to DNA damage. 1996). This redundancy makes it dicult to determine Movement of silencing proteins away from telomeres if these genes are naturally regulated by TPE. When appears to be a genetically programmed step in the expression of four unique sub-telomeric genes is yeast aging process. As yeast cells age, the Sir complex analysed for dependency on SIR proteins, only redistributes from telomeres to the nucleolus, which YFR057w, an ORF of unknown function, is subject contains the genes that encode ribosomal RNA to natural telomeric silencing (Vega-Palas et al., 2000). (rRNA) (Kennedy et al., 1997). This re-localization is Although this ORF is repressed in wild-type strains, its thought to protect ribosomal DNA from breakage and expression is increased upon deletion of SIR2, 3 and 4. recombination (Gottlieb and Esposito, 1989; Kaeber- YFR057w is localized adjacent to telomere VI-R. lein et al., 1999). In addition, the NAD dependent There are several properties of this location that are deacetylase activity of Sir2p (Imai et al., 2000; Landry favorable for silencing. First, YFR057w is less than et al., 2000; Tanny et al., 1999) may repress rRNA 1 kb from the core X ARS on telomere VI-R. Second, transcription in aging cells, linking genomic silencing, Sir proteins spread as far as 3 kb from the end of the caloric intake and aging (Tissenbaum and Guarente, VI-R telomere, a region that encompasses YFR057w 2001). (Strahl-Bolsinger et al., 1997). Third, the VI-R telomere does not contain a TPE inhibiting STAR element. TPE in other organisms The retrotransposon Ty5-1, which resides 1.8 kb from the left telomere of chromosome III, is also TPE is not limited to S. cerevisiae. For example, TPE subject to natural telomeric silencing (Vega-Palas et al., occurs at truncated telomeres in ®ssion yeast, Schizo- 1997). Transcription of Ty5-1 is very low in wild-type saccharomyces pombe, an organism that is evolutiona- cells, is reduced even more in cells over-expressing rily distant from S. cerevisiae (Russell and Nurse, Sir3p, and is increased in a sir3 strain. Ty5 elements 1986). As in S. cerevisiae, URA3 shows a reversible, integrate preferentially near silent regions, like the meta-stable repression when placed next to a S. pombe silent mating type loci and telomeres (Zou et al., 1996). telomere (Nimmo et al., 1994). However, some features The variegated and semi-stable Ty5-1 transcription of the silencing process are dierent in the two yeasts. imposed by TPE probably reduces Ty5 transposition, For example, in S. pombe, some cells with a telomeric thereby limiting the genetic damage caused by its ade6+ gene produce uniformly pink colonies. This movement to new sites. colony class probably arises from a reduced, stable With the advent of genome wide analysis of level of ade6+ transcription in all cells in the colony, transcription, it is possible to ask if TPE has global rather than from a population in which ade6+ is eects on transcription of telomere-linked genes transcribed in some cells and repressed in others. This (Wyrick et al., 1999). There are 267 genes that are behavior diers from what is seen in the analogous within 20 kb of a telomere. In general, genes located situation in S. cerevisiae where colonies are either within 20 kb of chromosome ends are expressed at largely red or largely white. lower levels, an average of 0.5 mRNA molecules per Mitotic cells lacking the S. pombe duplex telomere cell, than genes located more internally, an average of DNA binding protein Taz1p have very long telomeres 2.4 mRNA molecules per cell. However, transcription and no TPE (Cooper et al., 1997, 1998; Nimmo et al., of only 20 of these genes is increased by deletion of a 1998). A similar massive telomere lengthening and loss SIR gene, and most of those aected are within 6 ± of TPE is seen in S. cerevisiae mutants that lack the 8 kb of a chromosome end. carboxyl terminal portion of Rap1p (Kyrion et al., Concentrating silencing proteins at telomeres may 1992, 1993). Unlike Rap1p, Taz1p is not essential. serve functions in addition to transcriptional silencing. However, S. pombe cells that lack Taz1p are seriously When chromosomes are damaged by treatments that compromised in meiosis: telomere clustering at spindle produce double strand breaks, Rap1p, Sir proteins and pole bodies is disrupted, chromosome movement is the Ku complex lose their punctate, peripheral defective, recombination is reduced, chromosomes mis- localization and become dispersed throughout the segregate, and spore viability is low (Cooper et al.,

Oncogene Telomere position effect W-H Tham and VA Zakian 518 1998; Nimmo et al., 1998). As in S. cerevisiae, in wild- for ectopic recombination (Freitas-Junior et al., 2000). type S. pombe, mitotic chromosomes are localized to In situ hybridization reveals that the 28 Plasmodium the nuclear periphery (Funabiki et al., 1993). Localiza- telomeres are clustered in 4 ± 7 foci at the nuclear tion of the S. cerevisiae VII-L telomere can be periphery with random localization of dierent telo- perturbed without compromising the mitotic stability meres among the dierent clusters. The high level of of chromosome VII (Tham et al., 2001). Perhaps loss recombination between heterologous chromosomes is of telomere localization in meiosis, as occurs in a taz17 thought to be a consequence of this clustering. S. pombe strain, has more dire eects on chromosome TPE was ®rst observed in Drosophila melanogaster behavior. (Levis et al., 1985). Drosophila telomeres are not Trypanosoma brucei, a protozoan parasite respon- comprised of telomerase generated simple repeats but sible for sleeping sickness, spreads among mammalian rather of at least two classes of retrotransposons, HeT- hosts through the tsetse ¯y (reviewed in Rudenko et A and TART (reviewed in Mason and Biessmann, al., 1998). The coats of individual parasites are 1995). However, the presence and identity of speci®c comprised of a single protein, encoded by a VSG sub-terminal middle repetitive sequences, rather than (variant-speci®c surface glycoprotein) gene. Switching the telomeric retrotransposons, may be the key the identity of the VSG coat enables the organism to regulators of TPE (Cryderman et al., 1999). The escape the mammalian immune system, a process that chromatin structure of these sub-telomeric repeats has occurs repeatedly during the course of an infection. several features reminiscent of telomeric chromatin in Although trypanosomes have close to 1000 VSG genes, S. cerevisiae (de Bruin et al., 2000), such as reduced there are only *50 sites from which these genes can be access to restriction enzymes (Cryderman et al., 1999). expressed, and each is adjacent to a telomere. In a The sensitivity of TPE to mutation of modifying genes given cell, only one of the many possible telomeric varies depending on the nature of the sub-telomeric expression sites is active, suggesting that the other sites repeats. For example, mutations in the chromosomal are subject to TPE. Indeed, promoters that are protein HP1 aect TPE at chromosome IV but not at normally active in the same cell type are stably chromosomes II and III, whose sub-telomeric repeats repressed when placed near a telomere (Horn and are related to each other but dierent from those on Cross, 1995). As with telomeric genes in S. cerevisiae, chromosome IV. Telomere placement is also regulated transcriptionally repressed VSG genes can switch to dierently at dierent telomeres. Chromosome IV transcriptionally active forms without DNA rearrange- telomeres are normally associated with the HP1 rich ments (and vice versa). In contrast to yeast where pericentric heterochromatin while chromosome II and individual telomeres act independently in terms of their III telomeres are localized to the nuclear periphery. transcriptional states (Tham et al., 2001), in trypano- When the telomere of the fourth chromosome is somes, all but one of the expression sites in a given cell translocated to chromosome two, its silencing activity is silent. It is not clear what enables one of the is diminished, although its residual silencing is still HP1 trypanosome expression sites to escape the transcrip- sensitive. This rearrangement also results in localiza- tional repression to which the rest are subject. tion of the translocated fourth telomere to the nuclear However, the telomeric DNA at inactive expression periphery (Cryderman et al., 1999). These studies sites, but not the active transcription site, contains high suggest that TPE in Drosophila is aected by the levels of a novel base, b-glucosyl-hydroxy-methyluracil, identity of the sub-telomeric repeats and their whose intriguing distribution suggests a possible role in associated proteins as well as by nuclear position. expression site control (Gommers-Ampt et al., 1993; Human somatic cells do not express telomerase, and van Leeuwen et al., 1998). Loss of TPE in S. cerevisiae hence their telomeres shorten by *50 ± 100 bps with by transcription through the telomeric tract provides a each cell division (Harley, 1995). If TPE occurs in paradigm for how TPE can be eliminated in cis at a human cells, it would provide a mechanism for age single telomere that might be relevant to VSG gene dependent changes in expression of telomere adjacent expression in trypanosomes (de Bruin et al., 2000; genes (Wright and Shay, 1992). Early attempts to Sandell et al., 1994; Tham et al., 2001). demonstrate TPE in mammalian cells were unsuccess- As in trypanosomes, sub-telomeric regions in ful (summarized in Baur et al., 2001). However, Plasmodium falciparum, the protozoan parasite that recently HeLa cells were found to express a telomere- causes malaria, contain multi-member gene families linked luciferase gene at a *10-fold lower level than an that encode virulence factors involved in antigenic internally located gene (Baur et al., 2001). This variation and cytoadhesion, such as the var genes. telomere-linked repression has two features that are After infection of erythrocytes, a Plasmodium cell reminiscent of TPE in S. cerevisiae. When telomeres expresses only one var gene at a time, suggesting that are lengthened by over-expression of telomerase, the other telomere-linked var genes are subject to TPE expression of the telomeric luciferase gene decreases (Scherf et al., 1998). Intriguingly, silencing of a plasmid 2 ± 10-fold. Incubation of cells with the histone containing a var promoter requires passage through S deacetylase inhibitor trichostatin A preferentially phase (Deitsch et al., 2001), a precondition for increases expression of telomere-linked luciferase genes, establishment of silent chromatin in many organisms. consistent with the possibility that hypoacetylation of Var genes on dierent chromosomes undergo recombi- telomeric chromatin is important for telomeric silen- nation at frequencies much higher than that expected cing in mammals as it is in yeast.

Oncogene Telomere position effect W-H Tham and VA Zakian 519 Future directions Hsu et al., 1999, 2000; Samper et al., 2000). In humans, the repair proteins RAD50 and MRE11 are telomere TPE has been reported in several single cell eukaryotes, associated (Zhu et al., 2000) while the yeast homo- ¯ies, and humans, indicating that it is a widespread logues of these proteins probably play roles in and perhaps even universal property of sub-telomeric recruiting telomerase to telomeres (Ritchie and Petes, regions. The S. cerevisiae analyses are a useful 2000; Tsukamoto et al., 2001). paradigm to guide future studies. There are questions Perhaps most intriguing for future studies is the that beg to be answered in other organisms. How far possibility that TPE provides a mechanism to modify from the telomere can a gene be located and still be gene expression in humans as a function of age. To test in¯uenced by TPE? Does the identity of sub-telomeric this idea, genes that show age related expression can be repeats, the size or presence of telomere loops, or the identi®ed by DNA micro-array analysis and their sub-nuclear localization of telomeres in¯uence silen- genomic positions determined. In yeast (Kyrion et al., cing? Probably the most critical next step is the 1993) and humans (Baur et al., 2001), long telomeres identi®cation of genes that regulate TPE. There are are more eective silencers than short telomeres. Thus, homologues of some but not all of the S. cerevisiae if TPE plays a role in age related gene expression, silencing proteins in higher eukaryotes (Brachmann et genes near telomeres are expected to be expressed at al., 1995; Li et al., 2000). Their analysis provides an higher levels in cells from older individuals. However, obvious starting point for genetic approaches. telomere adjacent genes might not be the only ones Identifying genes on which TPE depends is im- aected by age dependent changes in TPE. In yeast, portant because the phenotypes associated with their long internal tracts of telomeric DNA can repress loss will help determine the biological role of TPE. The transcription, and this repression depends on the same cellular function of TPE need not be identical in all silencing proteins that act at telomeres (Stavenhagen organisms. For example, although in S. cerevisiae the and Zakian, 1994). If internal silencing also occurs in transcription of many telomere-linked genes is aected mammals, as telomeres shorten with age, internal tracts by TPE (Wyrick et al., 1999), TPE is not needed for of telomeric DNA might compete more eectively with viability nor even for robust growth under laboratory telomeres for limiting silencing proteins. In this conditions. However, in parasitic protozoa, like scenario, genes near internal tracts of telomere DNA trypanosomes and Plasmodia, regulation of sub- are predicted to have reduced expression in aged cells. telomeric gene expression may be critical for pathogen- esis. 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 Acknowledgments between telomeric silencing and DNA repair. The We thank Mary Kate Alexander, Lara Goudsouzian, and mammalian Ku complex, like its yeast counterpart, is Michelle Mondoux for helpful comments on the manu- telomere associated and has roles in both DNA repair script and the National Institutes of Health for their and telomere function (d'Adda di Fagagna et al., 2001; support of the research in our laboratory.

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Oncogene