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Molecular and Cellular Biology, April 2008, p. 2380-2390, Vol. 28, No. 7
0270-7306/08/$08.00+0     doi:10.1128/MCB.01648-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

Hong Ji, Christopher J. Adkins, Bethany R. Cartwright, and Katherine L. Friedman^*

Vanderbilt University, Department of Biological Sciences, VU Station B 351634, Nashville, Tennessee 37235

Received 5 September 2007/ Returned for modification 10 October 2007/ Accepted 10 January 2008

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In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

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Telomeres are nucleoprotein structures that protect chromosome ends from nucleolytic digestion and preclude the recognition of normal ends as double-strand breaks (6). In most eukaryotes, telomeres are maintained by telomerase, a ribonucleoprotein that uses a short region of its RNA subunit as a template for reverse transcription. In Saccharomyces cerevisiae, a reverse transcriptase (RT) (EST2) and RNA subunit (TLC1) form the catalytic core of telomerase (18, 21, 35). Est2p contains at least three functional domains: an N-terminal TEN domain that anchors Est2p on its DNA substrate (23, 24) and that may interact with other protein components (10), an RNA binding (RBD) region associates with that TLC1 RNA and contributes to dimerization (20), and an RT domain conserved among other telomerases and viral RTs (21).

While many organisms synthesize perfect TG-rich telomeric repeats, several protozoa, fungi, slime molds, and plants have heterogeneous telomere sequences (40). S. cerevisiae displays considerable degeneracy, with a consensus of 5'-[(TG)_0-6TGGGTGTG(G)]_n (9). Several models have been proposed to explain this heterogeneity. An analysis of wild-type (WT) telomeres and telomeres generated in the presence of template mutations suggests that the registration of the telomere terminus occurs preferentially at the 3' end of the template, with processive synthesis through a central core region and decreasing processivity at the 5' end of the template (9, 33). In contrast, telomere junction fragments generated during chromosome healing events are more consistent with nonprocessive synthesis and patterning driven by substrate/template alignment (32). In humanized yeast cells, in which the yeast RNA template is replaced with that of humans, Est2p generates perfect hexanucleotide repeats, suggesting that the template sequence can influence template usage (14).

In S. cerevisiae, the normal telomere length varies between 225 and 375 bp (43). The primary negative regulator of telomere length is Rap1p, a protein that associates directly with duplex telomeric DNA every 18 bp, on average (12). Artificially targeting Rap1p to an individual telomere proportionately shortens the TG_1-3 tract in cis (25, 26). This negative regulation requires Rif1p and Rif2p, proteins that bind to the C terminus of Rap1p and play overlapping, but not fully redundant, roles in telomere length homeostasis (13, 19, 41). The deletion of RIF1 or RIF2 increases the frequency of telomere elongation during a single cell cycle, suggesting that the Rap1p/Rif1p/Rif2p complex modulates telomerase access in a manner responsive to telomere length (39). Rap1p also mediates the silencing of genes near telomeres and has been implicated in the repression of nonhomologous end joining and telomere resection near a double-strand break (27-29).

While mutations in Est2p that abolish RT activity or disrupt complex assembly cause telomere shortening, three clusters of residues (see Fig. 1A) cause telomeres to overlengthen by up to 100 bp when mutated. A mutation in motif E (est2-motifE) of the RT domain increases nucleotide addition processivity in vitro (30). In contrast, the est2-up mutants in the finger subdomain (motifs 1 and 2) allow telomerase to escape inhibition by Pif1p helicase (7). We previously identified four est2-LT (for long telomere) mutations in the N-terminal TEN domain (16) that overelongate telomeres by an unknown mechanism. Here, we demonstrate that est2-LT strains do not increase telomeric Rap1p association or gene silencing, as has been observed upon telomere overelongation in other genetic backgrounds. The est2-LT phenotype is suppressed when Rap1p length regulation is compromised, indicating that the decreased binding of Rap1p per nucleotide within est2-LT telomeres causes telomere overelongation. Although telomere sequences are altered in est2-LT strains, the association of Rap1p with these sequences is not detectably changed in vitro. Indeed, the same sequence alterations occur in all est2 strains that abnormally increase telomere length. Taken together, these data provide evidence that a subset of residues in the catalytic subunit of S. cerevisiae telomerase modulates the association of Rap1p with telomeric DNA in vivo in a sequence-independent manner, thereby affecting the extent of telomere elongation.

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FIG. 1. Strains expressing different telomere-lengthening alleles of EST2 have distinguishable phenotypes. (A) Locations of long-telomere mutations within EST2. The domain structure of Est2p is shown (15). Mutations in three distinct regions cause longer-than-normal telomeres: est2-LT in the TEN domain, est2-up in motifs 1 and 2, and est2-MotifE in the RT domain (7, 16, 30). The est2-up34 allele utilized in this study (D460N) is indicated with an asterisk. (B) Telomere length in est2 strains. Genomic DNA from the indicated strains was cleaved with XhoI, separated in a 1.2% agarose gel, subjected to Southern blot analysis, and probed with a telomeric DNA fragment. The strains shown are identical to those used for the silencing (C) and ChIP assays (Fig. 2). M, DNA size marker. (C) Expression of a telomere-proximal ADE2 gene in est2 strains. Telomeric silencing was tested in an est2 ade2 strain containing an ectopic copy of ADE2 inserted near the right telomere of chromosome V (Chr. VR) (see the diagram at the top). This strain (YKF501) was transformed with a URA3 plasmid expressing the indicated EST2 allele. Shown are 10x serial dilutions of cells grown on medium lacking uracil or lacking uracil and adenine 75 generations after the introduction of the complementing plasmids. (D) Dependency of est2-mediated telomere overelongation on PIF1. Strains of genotype est2 (YKF501) and est2 pif1 (YKF503) were transformed with URA3 plasmids expressing WT EST2 (lanes 1 to 3 and 10) or the indicated est2 allele. Cells were grown for 75 generations to allow the telomere length to stabilize. Genomic DNA isolated from the indicated strains was digested with XhoI, subjected to Southern blot analysis, and probed with telomeric DNA. Telomeres containing a subtelomeric Y' element are shown. M, DNA size marker.

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Strains and plasmids. See Table 1 for detailed strain genotypes. est2-LT^E76K (glutamic acid 76 to lysine) was integrated into GA426 and FYBL1-23D by two-step gene replacement to create YKF500 and YKF510, respectively. EST2 was replaced with TRP1 in GA426 by transforming a SacI-SphI fragment from pKF404-HXTRPa (TRP1 from pRS314 cloned into the HindIII and XbaI sites of pKF404 [10]) to create YKF501. RIF2 was replaced in GA426 with TRP1 by using pFA6a-TRP1 (22) as a template to create strain YKF502.

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TABLE 1. Strains used in this study

PIF1 gene disruption was accomplished in YKF501 (containing pKF410 [proA-EST2 CEN URA3] [16]) by transforming a PCR fragment (the primers were 5'-GTAGTTTTGTGATGCTGTTCAG-3' and 5'-GTGAGTTAGTCTCCTTTGGC-3', and the template was genomic DNA from the pif1::Kan^r strain [Open Biosystems, Huntsville, AL]) to generate YKF503. TLC1 was replaced with tlc1-human as described previously (14) in strain YKF501 (containing pKF417 [EST2 CEN LEU2]) to create YKF504. Following transformation with pKF410 or its mutant derivatives, colonies were screened for the loss of pKF417. Southern blotting was performed as described previously (10).

Cloning and sequencing of telomeres. (i) Analysis of multiple cell cycles. YKF501 bearing plasmid pKF410 was restreaked on plates containing 5-fluoroorotic acid (5-FOA) and restreaked once to allow telomere shortening. After the reintroduction of pKF410 bearing WT or mutant EST2, transformants were restreaked three times (75 generations). Genomic DNA was extracted by glass bead lysis (34), and telomere PCR was performed (39). After gel purification (Qiagen), products were ligated into pGEM T-Easy (Promega) according to the manufacturer's instructions. Inserts were sequenced by FASTERIS (Plan les Quates, Switzerland) using the primer M13F.

(ii) Single-telomere extension (STEX). A fresh GA426 est2::TRP1 colony (recipient) was grown overnight in 5 ml synthetic dextrose medium lacking tryptophan (SD-Trp). A total of 1 x 10⁸ telomerase-deficient recipient cells were mixed with 1 x 10⁹ cells of strain FYBL1-23D or YKF510 (donor) as described previously (39) and were incubated at 30°C for 4 h on yeast extract-peptone-dextrose plates. The mating efficiency (monitored by comparing the growth on SD-Trp [recipients and diploids] to the growth on SD-Leu-Trp [diploids only]) was nearly 100% (data not shown). Telomere PCR was performed as described above.

ChIP and dot blot assays. Chromatin immunoprecipitation (ChIP) was performed as described previously, with some modifications (38). Yeast strains were grown to an optical density at 600 nm of 0.5 (50 ml in yeast extract-peptone-dextrose). Cross-linking was initiated with 1.4 ml 37% formaldehyde and terminated after 5 min by the addition of 2.5 ml 2.5 M glycine. Cells were pelleted and washed sequentially with HBS (50 mM HEPES, pH 7.5, 140 mM NaCl) and ChIP lysis buffer with phenylmethylsulfonyl fluoride (PMSF) (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, pH 8.0, 1% Igepal CA-630, 1% sodium deoxycholate, and 1 mM PMSF). Pellets were resuspended in 400 µl ChIP lysis buffer (plus one Roche Complete mini-protease inhibitor tablet per 10 ml) and disrupted by glass bead lysis. The lysate was obtained by centrifugation, and DNA was sonicated to fragments of 500 to 1,000 bp. Sonicated lysate was cleared twice by centrifugation at 7,000 x g for 2 min, and input samples (25 µl of 400 µl) were obtained. The remaining lysate was incubated on ice with 20 µl anti-Rap1p antibody (Santa Cruz Biotechnology). Samples were immunoprecipitated with 80 µl protein G Dynabeads (Invitrogen). Cross-linking was reversed by overnight incubation at 65°C, and DNA was detected by dot blot analysis. The probe was random-primed S. cerevisiae telomeric DNA or a random-primed PCR fragment from the ARO1 gene (the primer sequences were 5'-TGACTGGTACTACCGTAACGGTTC-3' and 5'-GAATACCATCTGGTAATTCTGTAGTTTTGAC-3'). The data analysis was described previously (36).

Analysis of telomere sequence data. The data analysis utilized newly synthesized sequences that were identified by the alignment of identical internal repeats. The frequency of GG-dinucleotide incorporation was calculated as the number of 5'-TGGGTGT sequences followed by GG divided by the total number of 5'-TGGGTGT sequences. When a core sequence overlapped with an adjacent repeat (5'-TGGGTGTGGGTGT), the 5' core (underlined) was considered to lack the GG sequence. Such overlapping core sequences generate a spacer with a value of –1. The frequency of overlapping core sequences is defined as the number of spacers in the –1 class divided by the total number of spacers. Predicted Rap1p binding sites were identified as described previously (33) with MatInspector (www.genomatix.de) using a 14-bp similarity matrix to assign confidence scores (17).

Rap1 protein expression, purification, and binding assays. Protein expression from pET28a(+)-Rap1p (XhoI-EcoRI) (11) was induced overnight in E. coli BL21 with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16°C. Recombinant His₆-tagged full-length Rap1p was enriched on nitrilotriacetic acid-agarose beads (Qiagen) by following the manufacturer's instructions and was purified by ion-exchange chromatography. Eluted protein was dialyzed in 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 20 mM KCl, 6% glycerol. The protein concentration was estimated by comparisons to bovine serum albumin standards.

For the gel shift assay, oligonucleotides (5'-ATATACACCCATACATTGA-3' and 5'-GTCAATGTATGGGTGTATA-3' [11]) were annealed to create a single Rap1p binding site (in boldface) and were labeled with Klenow polymerase (Roche) in the presence of [-³²P]dCTP. A total of 100 fmol of ³²P-labeled DNA was mixed with increasing amounts of unlabeled telomeric DNA (0 to 80 fmol). A total of 400 fmol of recombinant Rap1p was added to 20 µl binding buffer [20 mM Tris-HCl (pH 7.5), 70 mM KCl, 1 mM dithiothreitol, 6% (vol/vol) glycerol, 25 µg/ml bovine serum albumin, 2.5 µg/ml poly(dG-dC), 0.1 mM EDTA], and reaction mixtures were incubated for 10 min on ice and for 20 min at room temperature. Samples were separated on 6% polyacrylamide Tris-borate-EDTA gels and quantified by a phosphorimager.

For DNase I footprinting, ³²P end-labeled telomeric sequences (100 fmol) and a 20-fold excess of recombinant Rap1p were mixed in 20 µl binding buffer, incubated on ice for 10 min, and incubated for 1 h at room temperature. Samples were incubated with 0.5 U DNase I (Promega) for 1 min, followed by the addition of 45 µl stop solution (supplied by the manufacturer). DNA was phenol-chloroform extracted and ethanol precipitated. Samples were resuspended in 5 µl formaldehyde loading buffer, separated on 10% polyacrylamide gels, and analyzed by a phosphorimager.

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Rap1p association with est2-LT telomeres does not correlate with telomere length. The est2-up alleles were identified in a screen for mutations of EST2 that improve the transcriptional silencing of a telomere-proximal gene (7). Increased telomere length is thought to mediate this phenotype by increasing Rap1p association and the subsequent recruitment of the histone deacetylase Sir2p (27). Given that the entire EST2 open reading frame was subjected to mutagenesis, we were surprised that this screen failed to identify mutations in the EST2 TEN domain. We speculated that est2-LT strains, despite containing longer-than-normal telomeres, do not increase telomeric silencing. Two representative est2-LT alleles were chosen for analysis: est2-LT^E76K and est2-LT^N95A (asparagine 95 to alanine). est2-LT^E76K causes the telomere length to increase by 100 bp, while est2-LT^N95A has a slightly smaller, but reproducible, effect (Fig. 1B, lanes 2 and 3, respectively).

To assay telomeric gene silencing, plasmid-borne EST2 alleles were introduced into an est2 ade2 strain with ADE2 inserted near the right telomere of chromosome V (Fig. 1C). Cells expressing WT EST2 grow well on plates lacking adenine and are predominantly white, with small red sectors indicative of moderate ADE2 silencing (Fig. 1C). Consistently with a previous report, est2-up34 (the est2-up allele with the greatest effect on telomere length [D460N]) increases telomeric silencing, as indicated by the formation of red colonies with reduced growth in the absence of adenine. In contrast, telomeric silencing in cells expressing est2-LT^E76K, est2-LT^N95A, or est2-motifE is indistinguishable from that of the WT (Fig. 1C). The differences in silencing behavior of the mutant strains cannot be attributed to differences in telomere lengths, since all of them overlengthen telomeres to a similar extent (Fig. 1B). We conclude that telomere overlengthening in est2-LT and est2-motifE strains is not accompanied by increased telomeric silencing.

Given that the est2-up and est2-LT mutations differentially affect telomeric silencing and are located in distinct regions of the Est2 protein, we hypothesized that these alleles increase telomere length through distinct mechanisms. To test this hypothesis, we monitored a genetic interaction previously reported for the est2-up alleles. In a strain lacking the Pif1 helicase, a negative regulator of telomere length, est2-up34 does not cause any additional telomere lengthening (7). This result, combined with biochemical evidence, suggests that mutations in the finger subdomain (including est2-up34) render telomerase at least partially resistant to negative regulation by Pif1p (7). To test if other est2 alleles behave similarly, we created a pif1 est2 strain expressing plasmid-borne WT or mutant alleles of EST2. As shown in Fig. 1D, only est2-up34 fails to cause additional telomere lengthening in this background. We conclude that est2-LT and est2-motifE lengthen telomeres by a mechanism distinct from that of est2-up34.

Since Rap1p is required for telomeric silencing, our observation that increased telomere length is not always accompanied by increased gene silencing suggested that the longer telomeres of the est2-LT and est2-motifE strains do not undergo the expected increase in total Rap1p association. ChIP was used to address this possibility. To keep most telomeres intact, formaldehyde-cross-linked chromatin was sheared by sonication to 500 to 1,000 bp. Rap1p was immunoprecipitated, and the associated telomeric DNA was quantified by hybridization. To account for differences in telomere length, we expressed the amount of precipitated fragment as a percentage of the input telomeric DNA. Under these conditions, the efficiency of immunoprecipitation is proportional to the amount of Rap1p bound to each telomere. Values were normalized to those of a WT sample processed in parallel.

As predicted by the lack of increased telomeric silencing, the association of Rap1p with telomeres in est2-LT^E76K, est2-LT^N95A, and est2-motifE strains is indistinguishable from that of the WT (Fig. 2A). In contrast, the expression of est2-up34 significantly increases the efficiency of telomeric DNA immunoprecipitation by Rap1p (Fig. 2A). To rule out the possibility that est2-up34 abnormally increases Rap1p association, we monitored a control strain (rif2) in which the extent of telomere overelongation is similar to that observed in est2-LT^E76K and est2-up34 strains (see below). As expected from Rif2p's role downstream of Rap1p (41), the elongated telomeres of a rif2 strain bind significantly more Rap1p than WT (Fig. 2A).

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FIG. 2. Association of Rap1p with WT and mutant telomeres correlates with telomeric silencing but not with telomere length. (A) Association of Rap1p with WT and mutant telomeres. ChIP was used to measure the association of Rap1p with telomeric DNA. Values represent the fraction of total telomeric DNA immunoprecipitated with anti-Rap1p antibody normalized to the value for the WT in each independent experiment. Error bars represent standard deviations from at least three experiments. A paired t test was used to determine significance. P values are indicated (brackets) only for those samples that had values that differed significantly from those of the WT (P < 0.05). The left and center panels show an analysis of strains in the YKF501 (GA426 est2::TRP1) background. The indicated proA-EST2 alleles (WT, est2-LTE76K, est2-motifE, est2-up34, and est2-LTN95A) were expressed from plasmid pKF410. The strains are identical to those utilized in Fig. 1B and C. The graph on the right shows an analysis of strains in the GA426 background: GA426 (WT), YKF500 (GA426 with est2-LTE76K integrated at the endogenous locus), and YKF502 (GA426 rif2::TRP1). (B) Measurement of relative telomeric DNA abundance. Telomeric DNA abundance was quantified in input extracts from the samples shown in panel A by measuring the ratio of signal generated with a telomeric DNA probe to that of a single-copy sequence (a 372-bp fragment of ARO1). Values were normalized to those of the WT in each independent experiment. Error bars represent standard deviations from at least three experiments. A paired t test was used to determine significance. P values are indicated (brackets) for those samples that had values that differed significantly from those of the WT (P < 0.05).

To confirm that differences in Rap1p association cannot be attributed to differences in the extent of telomere overelongation, we measured the ratio of hybridization of a telomere-specific probe to that of a single-copy control sequence (ARO1) in input samples from each strain. While the relative abundance of telomeric DNA was significantly increased over that of the WT, the extent of telomere overelongation in the different mutant strains was statistically indistinguishable (Fig. 2B). The observation that the longer telomeres of est2-LT^E76K, est2-LT^N95A, and est2-motifE strains bind no more Rap1p than do WT-length telomeres is consistent with an overall reduction (per nucleotide) in the association of Rap1p within telomeres in these mutant strains. This result led us to address the role of Rap1p in the generation of the long-telomere phenotype.

Generation of the long-telomere phenotype in est2-LT strains requires the association of Rap1p with telomeric DNA. Since telomere elongation is inhibited in a manner proportional to the amount of bound Rap1p, a reduction in the frequency of Rap1p association with telomeres should cause telomere overelongation (as previously observed for certain mutations in the telomerase template [31]). An equilibrium length is reached when telomeres bind, on average, an amount of Rap1p equivalent to that normally associated with WT telomeres. The observation that transcriptional silencing and total Rap1p association at the elongated telomeres of est2-LT and est2-motifE strains are equivalent to WT levels fits this prediction and suggests that telomere overlengthening is a response to reduced binding by Rap1p. If true, telomere overelongation in est2-LT strains should be Rap1p dependent.

We initially set out to eliminate Rap1p function by deleting its binding partners, Rif1p and Rif2p. Unfortunately, the extreme length and heterogeneity of telomeres in this background made it impossible to determine if the est2 alleles cause additional elongation (data not shown). To circumvent this difficulty, we took advantage of a strain in which the yeast RNA template is replaced with that of the human telomerase RNA, dictating the synthesis of the hexanucleotide sequence (5'-TTAGGG) normally found at human telomeres (14). This humanized sequence does not bind Rap1p, but telomeres are short and stable due to an alternate, Tbf1p-dependent mechanism (1, 2, 4). An est2 strain expressing the humanized TLC1 variant (tlc1-human) from its endogenous locus was grown for approximately 50 generations in the absence of telomerase to allow telomere shortening. This strain subsequently was transformed with plasmids expressing WT or mutant est2, and telomerase was allowed to synthesize telomeres in the presence of the humanized RNA. Although the internal telomeric sequences in these strains retain WT sequence and are capable of binding Rap1p, any additional DNA synthesized by the mutant enzymes is derived from the humanized template. As one would predict for mutations dependent on Rap1p, neither est2-LT^N95A nor est2-LT^E76K increases the telomere length compared to the length of WT telomeres when the humanized RNA is used (Fig. 3A and B). In contrast, est2-up34 retains the ability to cause telomere overlengthening in this background, which is consistent with a Rap1p-independent mode of action (Fig. 3C). Surprisingly, est2-motifE also overelongates telomeres in this background (Fig. 3A, lanes 12 to 15). Telomeres cloned from the est2-LT^E76K and est2-motifE strains contain perfect 6-nucleotide repeats at the chromosome terminus (data not shown), confirming that Rap1p binding is eliminated within the newly synthesized sequences. The failure of est2-LT^E76K and est2-LT^N95A to lengthen telomeres in the humanized background suggests that telomere lengthening occurs in response to the reduced frequency of telomeric Rap1p association in vivo, although we cannot eliminate the possibility that altering the template sequence has other unanticipated effects on the est2-LT phenotype. We also conclude that est2-motifE affects telomere length in a manner that is at least partially Rap1p independent.

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FIG. 3. Telomere overlengthening by the est2-LT alleles is suppressed in a background of humanized TLC1. (A) est2 strains expressing WT TLC1 (lanes 1 to 3) or a tlc1-human variant (lanes 4 to 15) were transformed with plasmids bearing EST2, est2-LTE76K, or est2-motifE as indicated. DNA was cleaved with PstI, separated in a 1.5% agarose gel, subjected to Southern blot analysis, and probed with a mixture of two DNA fragments (S. cerevisiae telomere DNA and the humanized [TTAGGG]n sequence). M, DNA size marker. (B) est2 strains expressing WT TLC1 (lanes 1 and 2) or a tlc1-human variant (lanes 3 to 10) were transformed with plasmids bearing EST2 or est2-LTN95A as indicated. Samples were treated as described in the legend to panel A. (C) est2 strains expressing a tlc1-human variant were transformed with plasmids bearing EST2 or est2-up34 as indicated. Samples were treated as described in the legend to panel A.

Telomere overelongation is associated with altered telomere sequences in vivo. We sought to understand the mechanism through which the frequency of Rap1p association is decreased at est2-LT telomeres. Given that Rap1p associates directly with telomeric DNA, altering the sequence of the telomeric repeat also could have this effect. To examine this possibility, telomere sequences were obtained in two ways that allowed the unambiguous identification of repeats synthesized by the mutant enzyme. In an analysis of telomere addition over multiple cell cycles, telomeres were allowed to shorten for 50 generations in the absence of EST2. WT or mutant EST2 was introduced, the telomere length was recovered for 75 generations, and multiple clonal isolates of a single telomere were sequenced (9). Because yeast telomerase generates irregular sequences, repeats synthesized after the introduction of the complementing plasmid were identified by their divergence from the identical, internal repeats. In a second approach (STEX), telomere repeats generated during a single S phase were analyzed (39). est2 recipient cells with shortened telomeres were mated with donor cells expressing EST2 or est2-LT^E76K at the endogenous locus. After zygotes proceeded through the first postzygotic S phase, marked telomeres originating in the recipient strain were cloned and sequenced. Again, newly synthesized repeats were identified as those sequences that did not align between different telomeres. The frequency and extent of telomere elongation by est2-LT^E76K telomerase during a single S phase were not statistically different from those of the WT, with a marked preference for the elongation of the shortest telomeres, as previously reported (Fig. 4) (39).

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FIG. 4. Analysis of telomere addition in a single cell cycle. An est2 recipient strain was mated with strains expressing either WT EST2 (center) or est2-LTE76K (right). Telomeres were cloned and sequenced after the completion of the first postzygotic S phase. The lengths of individual telomeres are plotted. For each telomere, identical TG1-3 sequences resulting from semiconservative DNA replication (gray) and divergent sequences representing new telomere additions (black) are shown. Based on the low frequency of telomere additions in the absence of mating (left) and on the observed significant differences in the telomere sequence following mating to an EST2 or est2-LTE76K donor (Table 1), we infer that most divergent sequences result from telomere extension by telomerase. The average amounts of new telomere addition are 82 (EST2) and 58 (est2-LTE76K) nucleotides (nt). These differences are not statistically significant (P = 0.084; nonparametric Kruskal-Wallis test). Likewise, the frequency of telomere addition is statistically indistinguishable between the two strains (P = 0.09; Fisher's exact test). Telomeres utilized for experiments shown in Fig. 6 and 7 are indicated with asterisks.

A detailed inspection of est2-LT^E76K telomeres did indeed reveal a change in the telomere sequence. As previously described, approximately 90% of repeats in WT telomeres contain a 7-nucleotide core sequence (5'-TGGGTGT) (Fig. 5A), half of which are followed by two consecutive G nucleotides (as templated by TLC1) (9). est2-LT^E76K telomeres contain this GG dinucleotide at an increased frequency (67 to 77%) (Table 2). A combined probability test that examined the magnitude of the effect in telomeres generated by both experimental regimens indicated statistical significance (P = 0.025) (37).

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FIG. 5. Pattern of telomere repeat addition is altered in est2-LTE76K strains. (A) Model of yeast telomere repeat heterogeneity (9). Processive reverse transcription of a central portion of the telomerase template RNA (gray) incorporates the 5'-TGGGTGT core sequence in 90% of repeats. Heterogeneity is proposed to arise from a combination of poor processivity (gray nucleotides), multiple registers of alignment, and abortive reverse transcription at the 3' end of the template. The GG dinucleotide is produced by the reverse transcription of 470CC471 (box). (B) The number of nucleotides between core sequences is significantly altered in est2-LTE76K telomeres. The number of nucleotides between core sequences (5'-TGGGTGT) was graphed from WT and est2-LTE76K telomeres synthesized during a single cell cycle; –1 spacing occurs when the 3' T of one core sequence is identical to the 5' T of the next. Data are derived from 65 WT repeats and 55 mutant repeats. The distributions of absolute values are statistically different (P < 0.02) by the chi-square contingency test. (C) The majority of predicted Rap1p binding sites overlap. Rap1p sites predicted within a representative WT sequence (see Materials and Methods) are underlined. Overlap is indicated by negative values (parentheses). (D) Predicted Rap1p binding sites are more dispersed in est2-LTE76K telomeres than in WT telomeres. The amount of overlap between 31 predicted Rap1p binding sites in WT telomeres and 31 sites in est2-LTE76K telomeres was determined by using the method illustrated in panel C, and the frequency of each class was plotted. Distances of greater than two nucleotides were grouped. Distributions of absolute values are statistically different (P = 0.042) by the chi-square contingency test. nt, nucleotide.

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TABLE 2. Comparison of GG dinucleotide incorporation by WT and mutant telomerase

While the majority of core sequences in WT telomeres are separated by two or more nucleotides, nearly 30% overlap to generate the sequence 5'-TGGGTGTGGGTGT (a spacing of –1). Telomeres synthesized in a single cell cycle by Est2-LT^E76Kp show a greater than 50% reduction in the –1 spacer class (Table 3) and an overall shift in the distribution of spacer lengths (P = 0.03) (Fig. 5B). This is the first reported example of a mutation within the yeast catalytic subunit that affects the patterning of telomere repeats in vivo.

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TABLE 3. Comparison of telomere sequences added by WT and mutant telomerase

To address the potential impact on Rap1p binding, a computer algorithm (MatInspector) was used to identify putative Rap1p sites in WT and mutant sequences (5). As previously described, nearly all predicted Rap1p binding sites fall into three classes corresponding to 13/13, 12/13, and 11/13 matches to the consensus sequence (33). est2-LT^E76K telomeres shift the distribution among these classes, with a relative paucity of the 5'-TGGGTGTGGGTGTG sequence (11/13 matches) reflecting the reduction in overlapping core sequences (Table 4). In addition, the extent of overlap between adjacent predicted Rap1p sites is reduced in est2-LT^E76K telomeres (P = 0.042) (Fig. 5C and D). This effect is small and does not decrease the total number of predicted sites across the length of a telomere. However, we speculated that changing the relative spacing of adjacent sites could disproportionately affect Rap1p occupancy.

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TABLE 4. Predicted Rap1p binding sites in WT and est2-LTE76K telomeres

We reasoned that if the sequence alterations observed in est2-LT^E76K telomeres directly account for the reduced frequency of Rap1p binding (Fig. 2A), similar changes should be observed in other strains that exhibit this phenotype (est2-LT^N95A and est2-motifE). Indeed, the sequencing of telomeres generated in both strains during multiple generations reveals sequence changes reminiscent of those observed in est2-LT^E76K (Tables 2 and 3), with similar changes in the distribution of predicted Rap1p binding sites (data not shown). As a control, we examined telomere sequences from the est2-up34 strain. Since Rap1p binding is increased at est2-up34 telomeres in vivo (Fig. 2A), we predicted no change in the telomere sequence. Contrary to our expectation, est2-up34 telomeres also display an increased GG-dinucleotide frequency and a reduced amount of core sequence overlap compared to those of the WT. Indeed, the telomere sequence changes among the four mutant strains are statistically indistinguishable (Tables 2 and 3) (P = 0.98 and 0.87 between GG incorporation and overlapping core sequences, respectively, in a two-by-four chi-square contingency test). Given that changes in the telomere sequence do not correlate with the extent of Rap1p binding in vivo, we conclude that altered telomere patterning is a consequence, rather than a cause, of telomere overelongation in these mechanistically distinct est2 strains.

Rap1p association at WT and est2-LT^E76K telomeres is indistinguishable in vitro. Although telomere sequence alterations do not appear to be directly responsible for reduced Rap1p binding in vivo, we wanted to determine the consequence of the predicted changes in Rap1p binding sites observed at est2-LT^E76K telomeres. To address whether these altered sequences reduce Rap1p association, we assayed the binding of recombinant Rap1p to cloned WT and mutant telomeres in vitro. Matched WT and mutant clones containing approximately the same amounts of telomeric DNA (230 or 180 bp) were chosen from the STEX experiment (Fig. 4). In the mutant telomeres, 91% (E184) or 48% (E229) of the total TG_1-3 sequence was synthesized by est2-LT^E76K telomerase. In general, sequences on these telomeres are representative of the overall trend of increased GG-dinucleotide incorporation and decreased core sequence overlap observed among all est2-LT^E76K telomeres (Fig. 6A). The variability in the GG-dinucleotide rate observed in the WT telomere sequences reflects the stochastic variation given the small number of repeats in each clone. Consistently with the relatively small change in overlap between predicted Rap1p binding sites that we observed among all mutant telomeres (Fig. 5C and D), the total number of predicted Rap1p binding sites is similar between size-matched WT and est2-LT^E76K telomere clones (Fig. 6A).

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FIG. 6. est2-LTE76K telomeres bind Rap1p at predicted sites in vitro. (A) The characteristics of four cloned telomere fragments utilized in panel B and Fig. 7 are shown. Two WT telomeres (W239 and W178) and two est2-LTE76K telomeres (E229 and E184) were generated by telomere addition in a single cell cycle. Numbers indicate the lengths of the TG1-3 repeats. In est2-LTE76K telomeres, only a portion of this sequence is generated by the mutant enzyme (91% for E184 and 48% for E229). The percentage of core sequences followed by GG, the percentage of overlapping core sequences, and the number of predicted Rap1p binding sites (see Materials and Methods) were determined for the entire TG1-3 sequence. Each DNA fragment also contains 100 bp of identical subtelomeric and vector-derived sequences. (B) DNase I footprinting analysis of Rap1p bound to telomeric substrates. Rap1p (20-fold molar excess) was bound to 100 fmol of 32P end-labeled telomeric DNA (W239, W178, E229, or E184 as indicated) prior to DNase I treatment. Control reaction mixtures (0) were incubated in the absence of Rap1p. A black bar indicates a 13/13 or 12/13 match to the Rap1p consensus sequence, and a gray bar indicates an 11/13 match (see Materials and Methods and Table 4). Not all Rap1p sites are shown due to the poor resolution near the top of the gel. For E229 and E184, the transition between sequences synthesized by est2-LTE76K telomerase (at the labeled end of the DNA molecule) and the WT telomerase is indicated (arrow).

To address the occupancy of predicted Rap1p sites, we monitored the binding of full-length, recombinant Rap1p to WT and est2-LT^E76K-derived sequences by DNase I footprinting. In all four telomeres, regions of protection correspond well with our predictions (Fig. 6B). The strong protection observed on est2-LT^E76K telomeres suggests that the majority of substrates are bound, arguing against a substantial decrease in binding efficiency.

To quantify Rap1p association more carefully, we measured the ability of the size-matched WT and est2-LT^E76K telomeres to compete for Rap1p binding to a short double-stranded substrate by gel shifting. Unlabeled telomeric competitor DNA quantitatively reduces the association of Rap1p with the radiolabeled substrate (Fig. 7A). Because each unlabeled DNA contains 14 to 17 potential Rap1p binding sites (Fig. 6A), effective competition occurs at low molar ratios. This assay is quite sensitive, as we could distinguish between two WT telomere sequences differing by less than 30% in length (239 bp versus 178 bp) (Fig. 7B). The ability of the longer sequence to compete for nearly half of the binding to the short oligonucleotide at a 1/20 molar ratio suggests that most or all of the 16 predicted Rap1p sites on this fragment are bound by Rap1p. est2-LT^E76K telomeres compete at least as well for Rap1p binding as their size-matched controls. For example, at the 1/20 molar ratio, est2-LT^E76K and WT telomeres of 180 bp reduce the shifted product by 25.5% ± 1.5% and 18.9% ± 10.2%, respectively. The longer est2-LT^E76K telomere (E229) competes even better than its WT counterpart, perhaps reflecting the additional Rap1p site predicted within this sequence (Fig. 6A). We conclude that changes in the telomere sequence cannot account for the reduced frequency with which Rap1p associates with est-2LT telomeres in vivo.

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FIG. 7. Association of Rap1p with est2-LTE76K telomeres is not reduced in vitro. (A) Increasing amounts (0, 5, 10, 20, 30, 50, 60, and 80 fmol) of WT (W239) unlabeled telomeric DNA were mixed with 100 fmol of an 18-bp 32P-labeled double-stranded DNA and 400 fmol of recombinant Rap1p. Binding reactions were separated by polyacrylamide gel electrophoresis and visualized by a phosphorimager. (B) Quantification of multiple experiments performed as described for panel A using WT (W239 and W178) or mutant (E229 and E184) competitor DNAs. The percent competition was calculated by normalizing the fraction of oligonucleotide bound to Rap1p in each sample to the value observed in the absence of competitor. Error bars represent standard deviations from three independent experiments.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Telomerase negatively influences telomere length through three distinct mechanisms corresponding to the three classes of est2 mutations that overelongate telomeres (7, 16, 30, and this work). Unexpectedly, some of these mutants (est2-LT and est2-motifE) decrease the frequency of Rap1p association with telomeric chromatin in vivo. While overelongation is accompanied by changes in the telomere sequence, such alterations are not restricted to strains in which Rap1p binding is reduced and do not decrease the binding of Rap1p to telomeric DNA in vitro. These data provide evidence that the catalytic subunit of telomerase directly or indirectly influences the association of Rap1p with telomeric DNA in a manner that is independent of the telomere sequence, suggesting that telomerase plays roles upstream and downstream of Rap1p in the regulation of telomere length.

Mechanisms of telomere overelongation by est2 alleles. Previous studies identified telomere-overelongating mutations in three regions of EST2. The est2-up mutations in the finger subdomain alter the interaction of Est2p with Pif1p (7), a helicase that disrupts the association of telomerase with its DNA substrate (3). In contrast to the est2-up mutations, both est2-LT alleles tested here cause additional telomere lengthening in the absence of PIF1 (Fig. 1D), indicating that these mutant proteins preserve normal interactions with Pif1p. Differential effects of est2-up34 and the est2-LT mutations on Rap1p association with telomeric DNA in vivo underscore this distinction (Fig. 2).

The mechanism of telomere overelongation by the est2- motifE allele is more complex. This mutation mimics the viral consensus sequence within motif E of the RT domain and increases the nucleotide addition processivity in vitro (30), a phenotype not shared by est2-LT^E76K or est2-LT^N95A (16). Despite this difference, est2-motifE decreases Rap1p association with telomeres in vivo in a manner indistinguishable from that of est2-LT^E76K (Fig. 2). However, the persistence of the est2-motifE long-telomere phenotype in the absence of Rap1p association (Fig. 3A) suggests that altered enzymatic activity contributes to telomere overelongation.

Generation of abnormal sequences in long-telomere strains. The properties of yeast telomerase that influence the telomere sequence in vivo are poorly understood. A previous study revealed normal sequences in strains with short but stable telomeres (tel1 and ku70) (33). For the first time, we report a consistent change in the telomere sequence in est2 strains with long telomeres. The ubiquity of this effect suggests that the telomerase activity is altered under conditions of overelongation. Alternatively, each est2 mutation may similarly affect telomerase catalysis in vivo, despite increasing the telomere length through genetically distinct pathways. The est2-LT^E76K mutation increases repeat addition processivity of telomerase on certain primers in vitro (24). However, our observation that the number of nucleotides added per telomere-lengthening event is not increased in this mutant (an average of 82 nucleotides in the WT versus 58 nucleotides in the est2-LT^E76K strain; P = 0.084) (Fig. 4) suggests that this effect does not contribute to telomere overelongation in vivo.

The most obvious explanation for increased GG-dinucleotide incorporation is an increased propensity of telomerase to reach the end of the template before dissociating or translocating. We addressed this possibility by using an RNA template mutant containing a U at position 469 (Fig. 5A) (9). WT and est2-LT^E76K strains incorporate the complementary A at indistinguishable rates (10.6 and 9.9% of total repeats, respectively). Therefore, any change in nucleotide addition processivity is restricted to template residue C⁴⁷⁰. This interpretation assumes that excess GG dinucleotides are templated by residues ⁴⁷¹CC⁴⁷⁰, as previously demonstrated for the WT (9). Indeed, a mutation that eliminates the templating CC dinucleotide (⁴⁷²CAC⁴⁷¹ to ACA) abolishes GG dinucleotides in est2-LT^E76K telomeres (data not shown). Taken together, these observations are inconsistent with large changes in nucleotide addition processivity. We propose that the spectrum of permitted primer/template alignments is altered under conditions in which the normal negative regulation of the telomere length is compromised.

Association of Rap1p with telomeres in vivo. We were surprised to discover that est2-LT and est2-motifE strains do not increase Rap1p association with telomeres in vivo, despite increasing the telomere length by 30%. The failure to detect this increase is not an experimental limitation, since est2-up34 and rif2 strains similarly overlengthen telomeres but significantly increase the immunoprecipitation of telomeric DNA with Rap1p (Fig. 2). We hypothesize that telomere overelongation in an est2-LT^E76K strain is a response to reduced Rap1p association, since lengthening is not observed when Rap1p's regulatory function is bypassed in the humanized strain (Fig. 3). We cannot exclude the possibility that the increased processivity of Est2-LT^E76Kp contributes to telomere overelongation (and is suppressed by the humanized TLC1 template). However, a model in which reduced Rap1p association causes telomere lengthening is consistent with our previous observation that the expression of the est2-LT^E76K phenotype requires Tel1p, a downstream effector of the Rap1p pathway (16). Furthermore, this model also explains the lack of significant changes in the frequency or extent of telomere addition by est2-LT^E76K telomerase in a single cell cycle (Fig. 4), since the telomeric substrates undergoing elongation have not previously encountered the mutant enzyme and are expected to bind Rap1p normally.

What accounts for the reduced association of Rap1p with est2-LT and est2-motifE telomeres in vivo? Our original hypothesis, that Rap1p binding is reduced by changes in the telomere sequence, is inconsistent with the observation that est2-up34 telomerase generates similarly altered telomere sequences but increases overall Rap1p association in vivo. Furthermore, sequences generated by est2-LT^E76K telomerase bind recombinant Rap1p at least as well as the WT in vitro (Fig. 7). Conflicts between our observations can be reconciled if a mechanism independent of the DNA sequence influences Rap1p association with telomeres in vivo. One possibility is that telomerase, either directly or indirectly, promotes the loading of Rap1p onto newly synthesized terminal sequences. Upon the reduction of this activity in est2-LT strains, telomeres continue to elongate until Rap1p binds at a level equivalent to that found at normal-length telomeres. In this light, it is interesting that several subunits of the INO80 chromatin-remodeling complex recently have been shown to negatively regulate telomere length, perhaps through a physical interaction with the telomerase complex (42). Therefore, telomerase may participate in the normal formation of telomeric chromatin. The identification of other long-telomere strains that fail to increase Rap1p association will address these intriguing possibilities.

 
We thank V. Zakian, J. Lingner, T. Weil, P. Liu, and K. Runge for generous gifts of protocols and reagents; D. McCauley for help with statistical analysis; D. Kaplan, T. Graham, E. Fanning, and X. Jiang for helpful suggestions and reagents; and J. Talley for help in protein purification. We appreciate critical comments on the manuscript from R. Bairley and M. Platts. We are grateful to the anonymous reviewers for helpful comments and suggestions.

This work was supported by American Cancer Society Research Scholar grant RSG-04-048-GMC and National Science Foundation grant MCB-0721595 to K.L.F., grant no. 52003905 of the Howard Hughes Medical Institute Professors Program, and a Dissertation Enhancement Grant from Vanderbilt University to H.J.

 
* Corresponding author. Mailing address: Vanderbilt University, Department of Biological Sciences, 1161 21st Ave. S, Nashville, TN 37235. Phone: (615) 322-5143. Fax: (615) 343-6707. E-mail: katherine.friedman{at}vanderbilt.edu

Published ahead of print on 22 January 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Molecular and Cellular Biology, April 2008, p. 2380-2390, Vol. 28, No. 7
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