Distinct Requirements for Pot1 in Limiting Telomere Length and Maintaining Chromosome Stability

Abstract

The fission yeast Pot1 (protection of telomeres) protein binds to the single-stranded extensions at the ends of telomeres, where its presence is critical for the maintenance of linear chromosomes. Homologs of Pot1 have been identified in a wide variety of eukaryotes, including plants, animals, and humans. We now show that Pot1 plays dual roles in telomere length regulation and chromosome end protection. Using a series of Pot1 truncation mutants, we have defined distinct areas of the protein required for chromosome stability and for limiting access to telomere ends by telomerase. We provide evidence that a large portion of Pot1, including the N-terminal DNA binding domain and amino acids close to the C terminus, is essential for its protective function. C-terminal Pot1 fragments were found to exert a dominant-negative effect by displacing endogenous Pot1 from telomeres. Reducing telomere-bound Pot1 in this manner resulted in dramatic lengthening of the telomere tract. Upon further reduction of Pot1 at telomeres, the opposite phenotype was observed: loss of telomeric DNA and chromosome end fusions. Our results demonstrate that cells must carefully regulate the amount of telomere-bound Pot1 to differentiate between allowing access to telomerase and catastrophic loss of telomeres.

Telomeres play a crucial role in ensuring genomic integrity by protecting the ends of chromosomes and by providing a mechanism for the complete replication of linear DNA (6, 16, 41). In most organisms, telomeres are comprised of arrays of GT-rich DNA bound by protein complexes. Telomeres shorten due to the inability of conventional DNA polymerases to fully replicate the ends of linear chromosomes (35). They are replenished by a ribonucleoprotein complex containing telomerase reverse transcriptase and an RNA subunit, which provides the template for telomere repeat synthesis (8). How cells measure telomere length and process this information to maintain telomere length homeostasis is a matter of great interest.

Recent work in budding yeast has demonstrated that telomeres cycle between open and closed states (56). The closed structure prevents access by telomerase for most of the cell cycle, while the open state allows telomere extension to occur in late S phase (39). Not every telomere switches to the open state during each S phase, with a higher probability for shorter telomeres to switch than for longer telomeres (56). Although the physical nature of the nonextendible state(s) is presently unknown, it is likely to be a structure in which access of telomerase to the single-stranded 3′ overhang is physically blocked. Experimental evidence for several such structures exists. Electron microscopic analysis of protozoan and vertebrate telomeres demonstrated that the 3′ overhang can fold back and invade internal telomeric duplex, giving rise to a t-loop (24, 44, 45, 48). Alternatively, single-stranded G-rich DNA can form G-quartets, which at least in vitro prevent extension by telomerase (64). Inhibition of telomerase can also be mediated by a family of specialized telomere end binding proteins that form a complex with the G-rich 3′ overhang and render it inaccessible (18, 20, 29, 58). Such factors include the telomere end binding proteins (TEBP) from hypotrichous ciliated protozoa, budding yeast Cdc13, and Pot1 proteins, which have been identified in diverse eukaryotes.

TEBP from Oxytricha nova was the first telomere binding protein to be isolated, cloned, and characterized (20, 23). Structural analysis revealed that TEBP efficiently protects telomeric DNA by burying the terminal 12 nucleotides (nt) between its α and β subunits (26). Two oligonucleotide/oligosaccharide binding (OB)-folds in the α subunit and one in the β subunit form the DNA binding site. A third OB-fold in the α subunit mediates protein-protein interactions with the β subunit or a second α subunit (53). Although purified TEBP has been shown to inhibit telomerase activity in vitro (18), its role in preventing access to telomeres in vivo has not been addressed.

In the budding yeast Saccharomyces cerevisiae, single-stranded telomeric overhangs are bound by the Cdc13 protein (19, 49). Inactivation of Cdc13 results in extensive degradation of the telomeric C-strand and is ultimately lethal. In addition to its role in chromosome end protection, Cdc13 functions in the recruitment of telomerase and the coordination of G- and C-strand synthesis (13, 34, 49, 50, 54). Cdc13 carries out its multiple roles by associating with several distinct protein complexes (21, 22, 50, 54). Although Cdc13 shares no apparent sequence similarity with ciliate TEBPs, structural analysis of the Cdc13 DNA-binding domain uncovered that the OB-fold constitutes a conserved motif for binding to single-stranded telomeric DNA (43).

Telomeres in the fission yeast Schizosaccharomyces pombe are protected by the Pot1 protein (3). Deletion of the pot1⁺ gene results in rapid loss of telomeric DNA, chromosome end fusions, and segregation defects. Although most pot1⁻ cells die, frequent survivors emerge in which all three chromosomes have circularized, eliminating the need for chromosome end maintenance. Chromosome circularization has also been observed in other fission yeast mutants that fail to maintain telomeres (46, 47) and is now a textbook example of bypass suppression (25).

Pot1 homologs have been identified in a wide variety of eukaryotes, including plants, microsporidia, other yeasts, and vertebrates (5, 51, 61). Consistent with a role in chromosome end protection, purified yeast, chicken, and human Pot1 bind to the G-rich strand of telomeric DNA with exceptionally high sequence specificity (3, 30, 32, 38, 61) and render the 3′ end inaccessible to nucleases and telomerase (29, 58). The mechanistic basis for this trait was uncovered by the crystal structure of an N-terminal Pot1 fragment in complex with DNA (31). While the protein domain adopts an OB-fold, the DNA forms a compact structure involving DNA self-recognition interactions that are essential for the stability of the protein-DNA complex. Single-nucleotide substitutions disrupt this folded conformation and prevent protein-DNA complex formation. A similar mode of DNA recognition by human Pot1 has recently also been revealed (32). Highly specific recognition of the telomeric overhang may be necessary to ensure efficient targeting of Pot1 to the chromosome ends and to preclude binding to single-stranded DNA that arises transiently at other sites in the genome.

Although N-terminal fragments of Pot1 are sufficient for highly specific DNA binding in vitro, expression of the DNA binding domain has no effect on telomere length in vivo (58). Here we show that amino acids close to the C terminus of Pot1 are equally important for its protective function as the DNA binding domain. Using a series of Pot1 truncations, we have identified distinct regions of the protein required for chromosome stability and for limiting telomerase access to telomere ends. Our results further demonstrate that cells must carefully regulate the amount of telomere-bound Pot1 to allow limited access to telomerase but prevent catastrophic loss of telomeres.

MATERIALS AND METHODS

Plasmids and strains.

Pot1 cDNA fragments were generated by PCR and inserted downstream of the thiamine-repressible nmt1 promoter (40) on S. pombe expression vector pPB32, a derivative of pNMT-TOPO (Invitrogen). The resulting plasmids were sequence verified and introduced into pot1⁺ haploid and pot1⁺/pot1::kan^r diploid strains by electroporation. Transformants were selected on Edinburgh minimal media (EMM; QBiogene) supplemented with histidine (150 mg/liter), uracil (150 mg/liter), adenine (7.5 mg/liter), and thiamine (3.4 mg/liter). Colonies were restreaked once prior to transfer into liquid EMM (haploids) or sporulation on 3% malt extract (diploids). Spores were treated with glusulase (Perkin-Elmer Life Sciences Inc.), washed with distilled water, and germinated on Pombe minimal glutamate (PMG) supplemented with histidine (150 mg/liter), uracil (150 mg/liter), and adenine (7.5 mg/liter). Thiamine (3.4 mg/liter) was added as indicated. Colonies were replica plated to yeast extract-adenine (YEA) plus or minus 100 μg/ml Geneticin disulfate (Sigma) to determine the ratio of pot1⁺ to pot1::kan^r haploids.

Immunostaining and fluorescence microscopy.

Cells expressing a Taz1-green fluorescent protein (GFP) fusion and V5-tagged Pot1 or Pot1 fragments were fixed in 3% formaldehyde-PEM [100 mM PIPES, NaOH, pH 6.9, 10 mM magnesium sulfate, and 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid] solution for 30 min, washed four times in PEM, and resuspended in 1 ml of PEM supplemented with 1.2 M sorbitol and 0.5 mg/ml Zymolyase-100T (US Biologicals). After 2 min at 37°C, cells were washed three times with PEM, followed by 3 min in PEM containing 1% Triton X-100. Blocking and incubations with antibodies were carried out in PEMBAL buffer (PEM with 1% globulin-free bovine serum albumin, 0.1% NaN₃, and 100 mM l-lysine-HCl). V5 antibody (Invitrogen) was used at a 1:200 dilution for an overnight incubation on a rotator at room temperature. After four 15-min washes with PEMBAL, goat anti-mouse-Alexa-Fluor 594 conjugate (Molecular Probes) was added at a 1:10,000 dilution for a minimum of 6 h at room temperature. Cells were washed twice with PEMBAL and twice with phosphate-buffered saline. During the last wash, 4′,6-diamidino-2-phenylindole (DAPI) was added to 1 μg/ml. Images were acquired on a Zeiss Axiovert microscope equipped with an Axiocam charge-coupled device camera and Axiovision software. Images were contrast adjusted and merged in Adobe Photoshop.

For live-cell analysis, S. pombe strains expressing Pot1 fragments and Taz1-GFP, Rap1-GFP, or Pot1-GFP were grown to ∼2 × 10⁶ cells/ml, washed with water, and resuspended in 0.5× yeast extract supplemented (YES; 0.5% yeast extract, 3% glucose, 0.1 g/liter histidine · HCl, uracil, leucine, adenine; pH 8.0) containing Hoechst 33342 at 10 μg/ml. Cells were mounted on glass slides, and images were acquired using a Zeiss LSM510 Meta confocal microscope and AIM software. To objectively and consistently distinguish between “strong” and “weak” Pot1-GFP foci, all images were acquired using identical settings and cells with visible GFP foci on the “raw” image were scored as “strong.” Cells with “weak foci” were identified on the basis of visible GFP foci following contrast adjustment. The remaining cells were scored as showing no detectable Pot1-GFP localization to telomeres.

Protein and DNA analysis.

S. pombe cells (1 × 10⁸) were lysed by vortexing in the presence of glass beads (0.5 mm in diameter) and 10% trichloroacetic acid at 4°C for 5 min. Beads were washed in 10% trichloroacetic acid, and precipitated proteins were collected by centrifugation at 16,000 × g for 10 min. Following an acetone wash, proteins were solubilized in 1× protein sample buffer (1× NuPAGE LDS sample buffer, 50 mM dithiothreitol, 2% sodium dodecyl sulfate). Samples were heated to 55°C for 5 min and centrifuged at 16,000 × g for 2 min. Aliquots of the soluble fractions were loaded onto NuPAGE 4 to 12% Bis-Tris gels with 1× MES (morpholineethanesulfonic acid) buffer, and electrophoresis was carried out under the manufacturer's recommended conditions. Proteins were transferred to Protran nitrocellulose membrane in a Bio-Rad Mini Trans-Blot cell at 100 V for 1 h. The membrane was blocked in 1× TTBS (20 mM Tris-HCl, pH 7.5, 0.1% Tween, 137 mM NaCl) plus 2% nonfat milk. Antibody Pot1N1-1 was generated in rabbits using the keyhole limpet hemocyanin-conjugated peptide MGEDVIDSLQLNELC-amide. Antibodies were used at the following dilutions: polyclonal anti-Pot1N1-1 at a 1:2,000 dilution, monoclonal anti-HA.11 (Covance Research Products) at 1:1,000, monoclonal anti-V5 (Invitrogen) and horseradish peroxidase-conjugated, goat anti-mouse and goat anti-rabbit (Pierce) at 1:5,000. Bands were visualized on Hyperfilm ECL (Amersham) by enhanced chemiluminescence.

S. pombe genomic DNA was prepared as follows. Frozen S. pombe cell pellets (approximately 5 × 10⁸ cells) were thawed, washed in 10 ml Z buffer (50 mM sodium citrate, 50 mM sodium monohydrogen phosphate, 40 mM EDTA) at pH 7.8, and resuspended in 2 ml Z buffer containing 0.5 mg/ml Zymolyase-100T and 2 mM dithiothreitol. The cell suspension was incubated at 37°C for 1 h, at which point sodium dodecyl sulfate was added to 4% (wt/vol), and incubation was continued at 65°C for 10 min. The volume was increased to 10 ml with 5× TE (50 mM Tris-HCl, pH 8.0, 5 mM EDTA) and proteinase K (Sigma) was added to 50 μg/ml. After 1 h of incubation at 50°C, 3 ml of potassium acetate solution (5 M) was added and samples were incubated on ice for 30 min. The precipitate was removed by centrifugation and the clarified supernatant was mixed with 1 volume of isopropanol to precipitate nucleic acids. After 20 min on ice, samples were subjected to centrifugation at 10,000 × g for 5 min. Nucleic acids were dried briefly and resuspended in 0.5 ml of 5× TE containing DNase-free RNase A (50 μg/ml). Following incubation at 37°C for 1 h, organic extraction, and ethanol precipitation, genomic DNA was resolubilized in 1× TE. Genomic DNA was processed and analyzed by Southern blotting as described previously (4).

Growth assays.

S. pombe strains expressing V5-tagged Pot1 protein or fragments thereof were grown overnight under vigorous shaking (250 rpm) at 32°C in 20 ml EMM supplemented with histidine (150 mg/liter), uracil (150 mg/liter), adenine (75 mg/liter), and thiamine (3.4 mg/liter). Cells were counted with a hemacytometer, and fresh cultures were inoculated at a density of 5 × 10⁵ cells/ml. After 24 h, the cell density was determined by counting, and cells were diluted into 20 ml of fresh supplemented EMM at 5 × 10⁵ cells/ml. The remaining cells were collected by centrifugation, washed once with water, and stored at −80°C for preparation of genomic DNA and protein extract. This procedure was repeated every 24 h, and cell counts were plotted over time. Once the growth rate had stabilized, thiamine was omitted from the media to induce expression of Pot1 fragments. Daily dilutions were continued for 8 days postinduction.

RESULTS

Domain requirements for telomere localization and end protection.

A series of Pot1 fragments were generated and introduced into fission yeast to dissect the Pot1 domain structure (Fig. 1A). Western blotting confirmed that Pot1 and its truncated derivatives were expressed (Fig. 1B). The use of an inducible promoter (nmt1, no message in thiamine) enabled us to monitor the effects of low- and high-level expression by adding or omitting thiamine from the media. Transcription from the nmt1 promoter is significantly reduced in the presence of thiamine, although some expression of heterologous genes placed under the control of this promoter is generally observed even in the repressed state (17).

FIG. 1.

(A) Bar diagram of Pot1 truncations. N and C denote amino- and carboxy-terminal fragments respectively. The number following N or C corresponds to the size of the fragment in kDa. Internal fragments are named by the positions of the first and last amino acids. The C-terminal V5-His₆ tags are represented by black squares. (B) Pot1 and truncation mutants were detected by Western blotting in crude extracts from cells grown in the absence of thiamine. Expression levels varied among fragments, and loading was adjusted to not exceed the linear range of enhanced chemiluminescence. Equal amounts of extract were loaded for N36, C42, C28, C14, and 314-508; 5-fold more was loaded for Pot1, and 50-fold more was loaded for 314-442 and the vector control.

To test whether any part of Pot1 is sufficient to mediate chromosome end protection on its own, the plasmids containing Pot1 fragments were introduced into a heterozygous diploid strain (pot1^+/−). Haploid cells were generated by sporulation, and those harboring a plasmid were selected on minimal media. We found that pot1⁻ cells containing the vector control were at a severe selective disadvantage under these conditions and only pot1⁺ cells formed colonies within 4 days (Table 1). In contrast, when the complete Pot1 open reading frame was present on the plasmid, cells containing or lacking the genomic copy of the pot1⁺ gene were recovered at a ratio of 1:1, indicating that the plasmid-encoded Pot1 protein complements the deficiency of the genomic copy. Complementation was also observed when cells were grown in the presence of thiamine, indicating that low-level expression from the repressed nmt1 promoter produced sufficient Pot1 protein for chromosome capping (Table 1). When Pot1 truncations were tested in this assay, none of them supported growth of pot1⁻ cells, suggesting that telomeres were not protected by any of the Pot1 fragments analyzed here (Table 1).

TABLE 1.

Large deletions compromise the essential function of Pot1^a

Plasmid	Presence of thiamine	No. of colonies		Complementation
		pot1+	pot1−
Vector	−	74	0	No
FIPot1	−	93	107	Yes
FIPot1	+	98	94	Yes
N36	−	200	0	No
N22	−	200	0	No
C42	−	96	0	No
C28	−	96	0	No
C14	−	96	0	No
314-442	−	96	0	No
314-508	−	96	0	No

^aSpores of a pot1^+/− strain harboring the indicated plasmids were plated to selective minimal media for germination. Under these conditions, functional Pot1 is essential for colony formation. The presence or absence of pot1⁺ at the genomic locus was verified by restreaking of colonies to the appropriate selective media. −, thiamine absent; +, thiamine present.

To investigate which, if any, Pot1 fragments localize to telomeres in vivo, we used a strain containing a fusion between the telomere binding protein Taz1 and green fluorescent protein (10). In most cells, Taz1-GFP localizes to one or two discrete nuclear foci, indicating that telomeres are clustered even in haploid cells. As expected from biochemical and genetic studies, full-length Pot1 colocalized with Taz1-GFP (Fig. 2A). Surprisingly, the C-terminal 42- and 28-kDa fragments of Pot1 (C42 and C28) also colocalized with Taz1 at telomeres, despite the absence of the N-terminal DNA binding domain in these fragments (Fig. 2B and C). In contrast, no discrete foci were observed for N-terminal or internal fragments of Pot1 (data not shown). It hence appears that Pot1 localizes to telomeres predominantly through protein-protein interactions mediated by its C-terminal domain rather than through binding to the G-rich overhang. Although telomeric foci were not detected for N-terminal and internal Pot1 fragments, it remains formally possible that these fragments localize to telomeres but are not detected by immunofluorescence microscopy due to obstruction of the epitope tags.

FIG. 2.

Pot1 and C-terminal Pot1 fragments colocalize with Taz1-GFP in the nuclear periphery. S. pombe cells were grown to a density of ∼5 × 10⁶ cells/ml and processed as described in Materials and Methods. Pot1 and Pot1 fragments were detected by immunofluorescence using a primary antibody against the V5 tag and an Alexa-Fluor 594-conjugated secondary antibody. Nuclei were visualized using the DNA intercalating dye 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride (Hoechst 33342) at 10 μg/ml. Two representative cells are shown for each construct. (A) Full-length Pot1. (B) The 42-kDa C-terminal Pot1 fragment. (C) The 28-kDa C-terminal fragment.

C-terminal fragment expression affects telomere length and Pot1 protein levels.

To examine the effects of Pot1 fragment expression on cell growth and telomere maintenance in the presence of endogenous Pot1, cells were grown by serial passaging in liquid culture for several days. Growth rates for strains harboring Pot1, Pot1 fragments, and empty vector were similar, with average division times ranging from 3 h 7 min to 3 h 16 min for all but the strain harboring the 314-508 internal fragment, which divided on average every 3 h 44 min.

Although growth rates did not change significantly, the presence of C-terminal Pot1 fragments had a dramatic effect on telomere length. Expression of C42 for approximately 60 generations caused a threefold lengthening of the telomeric repeat region to ∼600 nt (Fig. 3A, lanes 4 and 5). The effect was even more dramatic when C28 was expressed, with the average telomere length increasing to approximately 800 nt (lane 6). Further lengthening and increased heterogeneity of telomere length were observed when cultures were maintained under logarithmic growth conditions for several days (data not shown). Wild-type-length telomeres were maintained when either the internal or terminal half of C28 was expressed (lanes 7 and 9). Modest lengthening of telomeres to ∼280 nt was observed when full-length Pot1 was overexpressed or when an internal fragment of Pot1 (amino acids 314 to 508) was introduced (∼250 nt). Telomere length remained unchanged following the introduction of N36 (lane 3). In summary, profound telomere elongation was observed in the presence of C28 or C42, the same fragments that localized to telomeres.

FIG. 3.

(A) Telomere length analysis in strains expressing Pot1 fragments. Cells were grown in minimal media containing thiamine, resulting in low-level expression of Pot1 fragments prior to isolation of genomic DNA and digestion with EcoRI. The terminal restriction fragments consist of 790 nt of telomere-associated sequence in addition to a variable length of telomeric repeats. Samples were subjected to electrophoresis through a 1% agarose gel, equal loading was confirmed by ethidium bromide staining, and DNA was transferred to a nylon membrane for hybridization with a telomeric DNA fragment. (B) Expression of C42 and C28 results in reduced telomeric localization of Pot1-GFP. Cells were grown in minimal media containing thiamine, washed once, and resuspended in 0.5× YES, pH 7.5, containing Hoechst 33342 at 10 μg/ml for live-cell analysis. Scale bar, 10 μm. (C) Data from images such as those shown in panel B were quantified as described in Materials and Methods. A minimum of 100 cells were analyzed for each strain, and the percentage of cells in each group is plotted (red, no foci; green, weak foci; blue, strong foci). (D) Reduction of full-length Pot1 in cells expressing low levels of C42 or C28. Cells expressing HA-tagged Pot1 and the indicated fragments or a vector-only control were grown in minimal media containing thiamine. (Top panel) Probed with antibody against HA tag. The band marked by a star is due to cross-reactivity of the HA antibody with an S. pombe protein unrelated to Pot1 and serves as a loading control. (Lower panel) Probed with anti-V5 antibody to visualize Pot1 fragments.

Considerable elongation of telomeres has been observed in strains with deletion of genes coding for the telomere binding protein Taz1 or the Taz1-interacting protein Rap1 (10, 12, 28). We considered the possibility that the C-terminal fragments of Pot1 may interact directly or indirectly with Taz1 or Rap1 and reduce the fraction of Taz1 or Rap1 at telomeres. The predicted outcome would be telomere elongation. However, when C42 was introduced into strains expressing either GFP-tagged Taz1 or Rap1, the amount of telomere-bound Taz1 and Rap1 increased rather than decreased (data available upon request). Loss of Taz1 or Rap1 from telomeres is hence unlikely to be responsible for telomere elongation in the presence of C-terminal Pot1 fragments. On the contrary, our observations are consistent with the notion that C42-mediated telomere elongation generated additional binding sites for Taz1, resulting in brighter GFP foci for Taz1-GFP and Rap1-GFP.

We next examined whether the localization of a Pot1-GFP fusion was affected by the presence of Pot1 fragments. In the vector control, Pot1-GFP localized to discrete foci in the nuclear periphery (Fig. 3B, vector). These punctate Pot1-GFP signals were greatly reduced in the presence of C42 or C28, but not when C14 was introduced (compare panels in Fig. 3B). For quantification, images of cells containing the different fragments were acquired using identical settings and each cell was assigned to one of three categories depending on whether they showed strong, weak, or no GFP foci in the nuclear periphery. Strong GFP signals were observed in 97% of cells containing the vector control and 88% of cells containing C14 (Fig. 3C). In contrast, only 18 and 26% of cells harboring C42 and C28 were in this category, with the remainder showing weak or no detectable signal.

The reduced intensity of Pot1-GFP foci combined with the telomeric localization of C42 and C28 suggests that C-terminal fragments compete with the full-length protein for telomere binding. Due to cross-reactivity of the anti-GFP antibodies with another protein of similar electrophoretic mobility, we were not able to assess whether displaced Pot1-GFP is simply dispersed or degraded. Using a strain in which Pot1 is fused to three copies of the influenza virus hemagglutinin (HA) tag, a marked reduction in the total amount of Pot1-3HA was observed in strains expressing low levels of C28 or C42 (Fig. 3D). It hence appears that displacement of Pot1 from telomeres results in its degradation or that C-terminal fragment expression represses endogenous Pot1 synthesis.

C-terminal Pot1 fragments affect chromosome stability.

Use of the nmt1 promoter allowed us to increase expression of Pot1 fragments by omitting thiamine from the media. Twenty-four hours after induction, only small differences in growth rates were observed between cultures harboring different Pot1 fragments (Fig. 4A, day 1). However, on days 2 and 3, a severe growth defect was noted for strains harboring C28 and C42. The average generation time during the third day had increased to 23 h 20 min for C28 and 14 h 20 min for C42. During the same time period, generation times for strains harboring vector, full-length Pot1, and N36 had increased only slightly to 3 h 49 min, 3 h 53 min, and 3 h 54 min. An intermediate phenotype was observed with cells harboring internal Pot1 fragments (Fig. 4A).

FIG. 4.

(A) S. pombe cultures undergo crises following induction of high-level expression of C42 or C28. Growth curves were recorded as described in Materials and Methods. The cell density following each 24-h growth period is plotted. (B) Effects of high-level C42 expression on cell and nuclear morphology. Cells were harvested 24 h (top panels) and 48 h (lower panels) after induction and stained with DAPI to visualize nuclear DNA. Chromosomal abnormalities are marked by arrows (anaphase bridges), closed triangles (multinucleated cells), and open triangles (cut phenotype). Scale bar, 10 μm. (C) Pot1 expression increases following induction of C42 or C28. (Upper panel) Immunoblot for Pot1-HA detected in extract from day 0, 1, or 4 postinduction of the indicated fragments. (Lower panel) Same samples probed with V5 antibody to visualize Pot1 fragments. Tenfold-less extract has been loaded compared to the upper panel. (D) Live-cell analysis of Pot1-GFP localization 24 h after induction of C42. Cells were prepared as described in the legend to Fig. 3. Scale bar, 10 μm. (E) Same analysis as in panel D, but in cells expressing Taz1-GFP.

After 2 days, 77% of cells harboring C28 or C42 were elongated, indicative of a G₂ cell cycle arrest (Fig. 4B). DAPI staining revealed anaphase bridges, multinucleated cells, and cells undergoing cytokinesis prior to completion of chromosome segregation (cut phenotype) in 43% of cells expressing C42. These phenotypes were reminiscent of defects observed following a deletion of the pot1⁺ gene (3). As low-level expression of C28 and C42 resulted in a reduction in the amount of endogenous Pot1, we speculated that increasing C28 or C42 expression might cause a complete loss of endogenous Pot1 protein from the cell. Surprisingly, Western blotting revealed that full-length HA-tagged Pot1 levels increased rather than decreased upon induction of C42 or C28 (Fig. 4C, compare days 0 and 1).

Although Pot1 was now abundant in cells, the morphological phenotypes were consistent with a defect in chromosome end protection. Changes in full-length Pot1 localization following induction of C28 and C42 were analyzed in strains containing the Pot1-GFP fusion protein. Twenty-four hours after induction, three categories of cells were observed: those containing bright foci in the nuclear periphery (∼40%), those lacking a GFP signal altogether (∼40%), and those with GFP accumulation at discrete sites distant from the nucleus (∼20%). Notably, all severely elongated cells and cells with apparent chromosome segregation defects were in the second and third categories (Fig. 4D). We also examined the localization of Taz1-GFP under the same conditions and found Taz1 associated with nuclear DNA in the majority of cells (Fig. 4E). In cells with stretched nuclear DNA, Taz1-GFP was often found in the center of the DNA mass, suggesting that the chromosome segregation defect was indeed caused by an inability to separate telomeres (indicated by arrows in Fig. 4E). It thus appears that high levels of C28 and C42 displace endogenous Pot1 from telomeres, leading to telomere loss and chromosome end fusions.

C28 and C42 expression causes chromosome end fusions.

Analysis of different fission yeast mutants and growth conditions has uncovered at least three distinct mechanisms for how dysfunctional telomeres cause chromosome segregation defects. In the absence of functional telomerase, Pot1 or Rad3 and Tel1, telomeric DNA is lost and chromosome end fusions occur between internal DNA sequences, giving rise to circular or dicentric chromosomes (3, 46, 47). At least in strains lacking telomerase, fusions occur in the absence of the nonhomologous end-joining activities pku70⁺ and lig4⁺ (4). Chromosome fusions have also been observed in taz1⁻ cells starved for nitrogen (15). In this case, fusions occur between elongated telomeres and the events depend on the presence of pku70⁺ and lig4⁺. A third class of telomere-mediated chromosome segregation defects was observed in taz1⁻ cells grown at low temperature (42). However, in this case, telomeric fusions could not be detected by pulsed-field gel electrophoresis and the chromosome segregation defect is thought to be a consequence of topoisomerase-mediated telomere “entanglement.”

Like deletion of taz1⁺, expression of C28 or C42 resulted in telomere elongation, a phenotype that may facilitate telomere fusions and/or entanglement under certain conditions. On the other hand, chromosome segregation defects were only observed in cells lacking detectable amounts of Pot1 associated with chromosomes, a phenotype with obvious similarity to a pot1 deletion. To elucidate the mechanism underlying the chromosome segregation defects observed here, genomic DNA was digested with NotI and analyzed by pulsed-field gel electrophoresis (Fig. 5A). Using a probe that hybridizes to sequences located approximately 2.5 kb from the termini of intact chromosomes, the four terminal fragments C, I, L, and M were visualized in samples from all strains (Fig. 5B). Intriguingly, reduced hybridization was observed with DNA samples from strains harboring C42 and C28 (lanes 4 and 5), suggesting that telomeres and some subtelomeric DNA had been lost in a subset of cells. Reduced hybridization to a TAS2 probe has also been observed in trt1 and pot1 deletion strains, where it is concomitant with the occurrence of chromosome end fusions (3, 4). Indeed, bands of reduced electrophoretic mobility were detected when the same blot was treated with probes that hybridize to internal sites on the C, I, L, and M fragments (indicated by arrows in Fig. 5C). Based on their size and hybridization to combinations of I, L, and M probes, these additional bands were identified as the products of intra- and interchromosomal fusions. The fact that these products were not detected with the TAS2 probe indicates that fusions only occurred between chromosome ends that had lost at least 2.5 kb of subtelomeric DNA. The mechanism by which these fusions occur is currently unclear, but their stability in the absence of protein and divalent cations at 50°C for 48 h suggests that they either are the product of DNA end ligations or contain long stretches of heteroduplex DNA. These results suggest that high-level expression of C28 or C42 caused loss of endogenous Pot1 from telomeres, followed by telomere degradation and fusions of uncapped chromosome ends.

FIG. 5.

Pulsed-field gel electrophoresis and detection of terminal chromosome fragments. (A) Schematic showing the location of NotI restriction sites on S. pombe chromosomes (14). Only terminal fragments on chromosomes (Ch) I and II and are labeled; chromosome III lacks NotI sites. (B) Genomic DNA was prepared, NotI digested, and fractionated by pulsed-field gel electrophoresis as described previously (4). DNA was transferred to a nylon membrane and hybridized to the telomere-proximal TAS2 probe. The positions of C, I, L, and M fragments are indicated on the right. (C) The blot shown in panel B was reprobed with internal probes on the C, I, L, and M fragments. Bands corresponding to the products of chromosome end fusions are indicated by arrows.

Unlike the pot1 deletion, expression of C28 or C42 did not always result in survivors with circular chromosomes. Instead, we frequently observed that the high-level expression of Pot1 fragments diminished over the course of several days in liquid culture (e.g., Fig. 4C). Presumably, individual cells expressed different amounts of the plasmid-encoded protein, and selective pressure allowed weaker expressers to outgrow their competitors. After 5 days, the growth rate often returned to normal (Fig. 4A) and cell morphologies from cultures expressing different Pot1 fragments were indistinguishable (data not shown). However, low-level expression of Pot1 fragments persisted and telomeres remained elongated in cells containing C28 or C42 (data not shown). In other cultures, chromosomes circularized and moderate to high levels of C-terminal fragments were maintained during prolonged growth.

Balancing telomere accessibility and protection.

None of the Pot1 fragments described above complemented the failure of pot1⁻ spores to germinate and form colonies on minimal media, indicating that large truncations abolish the ability of Pot1 to mediate chromosome end protection (Table 1). To define the essential part of Pot1, a series of small truncations at the N and C termini of Pot1 were generated. We found that the N-terminal 23 amino acids are not essential for end protection as 45% of the recovered strains harboring Pot1ΔN23 lacked the genomic copy of Pot1 (Table 2) and maintained linear chromosomes. Interestingly, expression of Pot1ΔN23 in wild-type cells had a dominant effect on telomere length. In the presence of Pot1ΔN23, the telomere repeat tract was approximately twice as long as in wild-type cells (Fig. 6A, compare lanes 1 and 2 with lane 5). Even more dramatic lengthening was observed when Pot1ΔN23 was expressed in the absence of endogenous Pot1 (lanes 3 and 4).

TABLE 2.

Mapping the domain of Pot1 required for chromosome end protection^a

Plasmid	Presence of thiamine	No. of colonies		Complementation
		pot1+	pot1−
Pot1ΔN23	−	42	34	Yes
Pot1ΔN40	−	100	0	No
Pot1ΔC69	−	86	0	No
Pot1ΔC38	−	86	0	No
Pot1ΔC13	−	86	0	No
Pot1ΔC9	−	86	0	No
Pot1ΔC8	−	100	0	No
Pot1ΔC4	−	66	44	Yes
Pot1ΔC4	+	50	0	No
Pot1ΔC3	−	40	28	Yes
Pot1ΔC3	+	50	0	No
Pot1ΔC2	−	50	41	Yes
Pot1ΔC2	+	50	0	No
Pot1ΔC1	−	50	45	Yes
Pot1ΔC1	+	67	19	Yes

^aSpores of a pot1^+/− strain harboring the indicated plasmids were plated to selective minimal media lacking thiamine (−) for high-level expression or containing thiamine (+) for low-level expression. Complementation was assessed by scoring the number of pot1⁺ and pot1⁻ colonies.

FIG. 6.

Small terminal deletions in Pot1 affect telomere length and chromosome stability. (A) Telomere length analysis in strains expressing N-terminal deletions in the presence or absence of endogenous Pot1. (B) Telomere length in pot1⁺ and pot1⁻ strains expressing exogenous Pot1 lacking one or two C-terminal amino acids. (C) Expression of full-length Pot1 and C-terminal truncation mutants was analyzed in crude cell extract using antibody Pot1N1-1. Induced samples were grown in the absence of thiamine, and 50-fold less extract was loaded compared to uninduced samples grown in the presence of thiamine. The three marker bands correspond to 50, 60, and 80 kDa. (D) Effects of C-terminal deletions on cell morphology and chromosome segregation in pot1⁻ background. (Panel I) Cells expressing ΔC3 under induced conditions; panel II, ΔC1; panel III, ΔC2; panel IV, ΔC3, all under repressed conditions. Cells in panels II to IV were harvested 24 h after addition of thiamine to the media and were stained with DAPI to visualize nuclear DNA. Chromosomal abnormalities are marked by open triangles. Scale bar, 10 μm.

Deletion of an additional 17 amino acids (Pot1ΔN40) resulted in apparent loss of end protection and exclusive recovery of pot1⁺ strains among 100 isolates (Table 2). While Pot1ΔN40 failed to complement the essential function of Pot1 in chromosome end protection, it did cause telomere lengthening in pot1⁺ cells, presumably by displacing full-length Pot1 from telomeres (Fig. 6A, lanes 6 and 7). Taken together, our results show that N-terminal deletions as small as 23 or as large as 313 amino acids lead to profound lengthening of telomeres, when the truncated proteins are expressed in the presence of wild-type Pot1. The first 23 amino acids of Pot1 are not required for protection against telomere loss but are critical for maintaining wild-type telomere length.

We next generated a series of deletions from the C terminus of Pot1 ranging from a single amino acid deletion (Pot1ΔC1) to a version lacking the C-terminal 69 residues (Pot1ΔC69). When these were expressed from the induced nmt1 promoter, complementation of the telomere loss phenotype was only observed with truncations lacking four or fewer C-terminal amino acids (Table 2). We confirmed by Western blotting that all truncations were expressed at a similar level to full-length Pot1 (Fig. 6C) (data not shown). None of the C-terminal truncations had a significant effect on telomere length when expressed in pot1⁺ cells (Fig. 6B) (data not shown). However, we noted a slight increase in telomere length heterogeneity and a reduction in telomeric signal on Southern blots when ΔC2, ΔC3, or ΔC4 was expressed in a pot1⁻ background (e.g., Fig. 6B, lanes 5 and 6).

To investigate the possibility that these truncations only barely complemented the pot1⁻ telomere loss phenotype when highly expressed, we diluted cells into media containing thiamine. After 24 h, expression of plasmid-encoded Pot1 or Pot1 mutants had been reduced by approximately 500-fold (Fig. 6C). When the complementation assay was carried out under these conditions, full-length Pot1 and Pot1ΔC1 supported colony formation for pot1⁻ haploids (Tables 1 and 2) but no pot1⁻ colonies were observed when ΔC2, ΔC3 or ΔC4 was expressed at these lower levels (Table 2).

The observation that Pot1 lacking two to four C-terminal amino acids was able to complement when expressed from the induced but not from the repressed nmt1 promoter provided us with an opportunity to examine the events that occur when expression levels are lowered. Within 24 h, cells containing Pot1ΔC2 or Pot1ΔC3 displayed a number of phenotypes indicative of chromosomal instability. Morphological abnormalities, such as dramatic elongation or branching, were observed in 53% of cells containing Pot1ΔC2 and 69% of cells containing Pot1ΔC3. DAPI staining revealed the presence of multiple nuclei, unequal DNA content in daughter cells, and cut phenotypes in 52% of cells containing Pot1ΔC2 and 56% of cells containing Pot1ΔC3 (e.g., Fig. 6D, panels III and IV). In contrast, fewer than 5% of cells expressing high levels of Pot1ΔC3 (panel I) or low levels of Pot1ΔC1 (panel II) displayed morphological abnormalities.

To confirm that the morphological phenotypes indeed reflect the inability of Pot1ΔC2 and Pot1ΔC3 to provide chromosome end protection, genomic DNA from these strains was analyzed by pulsed-field gel electrophoresis and Southern blotting. Probing for the terminal NotI fragments of chromosomes I and II revealed abundant inter- and intrachromosomal fusions in the ΔC3 sample and, to a lesser extent, the ΔC2 sample (Fig. 7A). Chromosome fusions only occurred once telomeres and several kilobases of subtelomeric DNA had been lost from the chromosome termini and were hence not detected with a TAS1 probe (Fig. 7B). These results demonstrate that the most C-terminal amino acids of Pot1 are critical for chromosome end protection. Interestingly, a comparison of the C-terminal amino acids of Pot1 from different species revealed notable similarities. The most common sequence for the last six amino acids is comprised of two hydrophobic residues next to two polar (mostly charged) amino acids, followed by two additional hydrophobic residues (Fig. 7C). While the mechanistic basis for the critical function of this motif in chromosome end protection remains to be determined, its role may well be conserved in higher eukaryotes.

FIG. 7.

Detection of terminal chromosome fusions by pulsed-field gel electrophoresis. Cells were grown in the presence of thiamine for 24 h. (A) Hybridization to internal probes on the C, I, L, and M fragments. Bands corresponding to the products of chromosome end fusions are indicated by arrows on the right; molecular weight markers are shown on the left. (B) The blot shown in panel A hybridized to the telomere-proximal TAS1 probe. (C) Multiple sequence alignment of the carboxy-terminal amino acids of Pot1 proteins from S. pombe (Sp), Homo sapiens (Hs), Macaca fascicularis (Mf), Mus musculus (Mm), Rattus norvegicus (Rn), Gallus gallus (Gg), Xenopus laevis (Xl), and Neurospora crassa (Nc). Hydrophobic residues are shaded in light gray and conserved polar residues in dark gray.

DISCUSSION

Pot1 has an essential role in preventing the loss of telomeric DNA, as evidenced by the rapid degradation of telomeres following deletion of the pot1⁺ gene (3). In the absence of telomeric repeats, chromosome ends are no longer distinguished from DNA double-strand breaks and inappropriate “repair” leads to chromosome end fusions. Biochemical and cytological data are consistent with a model in which Pot1 binds the single-stranded G-rich extension at the ends of telomeres and prevents access to nucleases, telomerase, and other DNA-modifying enzymes. Here we demonstrate that the protective function of Pot1 requires the integrity of its C terminus, as well as the presence of the DNA binding domain. Using a series of Pot1 truncation mutants, we have uncovered a second role for Pot1 in limiting telomere elongation. Our results demonstrate that Pot1 is a positive and negative regulator of telomere length and illustrate the critical importance of regulating the amount of Pot1 present at telomeres.

Telomere length homeostasis.

In a screen for deletions causing telomere length changes, no fewer that 173 budding yeast genes were identified (2). As this screen did not examine over 1,000 essential genes and missed several genes that had previously been shown to affect telomere length, the actual number is likely to be in excess of 200. Although some cellular processes may affect telomere length only indirectly, it is clear that eukaryotic cells have evolved complex machinery to measure and adjust the length of the telomeric repeat tract. Such regulation is of particular importance during development of multicellular organisms, as telomere length is a determining factor for the number of divisions a cell can undergo in the absence of active telomerase (7).

Purified Pot1 protein limits nucleolytic degradation of single-stranded telomeric DNA and inhibits telomerase in vitro (58). Similarly, human Pot1 has been shown to act as an inhibitor of telomerase, most likely by sequestering the DNA substrate (29). We now provide evidence that low-level expression of a C-terminal Pot1 fragment causes telomere lengthening by reducing the local concentration of intact Pot1 at telomeres. As the C-terminal Pot1 fragments lack the DNA binding domain, their localization to telomeres is most likely mediated by interactions with other telomere-bound proteins. C-terminal fragments of human Pot1 that lack DNA binding activity in vitro (38) have also been found to localize to telomeres in vivo and cause telomere elongation (37). In this case, the interaction is mediated by Ptop/Pip1/Tint1, an adaptor protein that links hPot1 to a protein complex bound to the double-stranded part of the telomere containing Tin2, Trf1, and Trf2 (27, 36, 63). Although a search of the fission yeast genome did not reveal any sequence with apparent homology to Ptop/Pip1/Tint1, our results suggest that a functional homolog is likely to exist.

Other experiments that either reduce the amount of Pot1 at telomeres or limit its ability to interact with DNA further support a role for Pot1 as a negative regulator of telomere length. In cultured human cells, moderate reduction in hPot1 or Ptop/Pip1/Tint1 levels by RNA interference led to telomere lengthening, as did overexpression of a small peptide of Ptop/Pip1/Tint1 encompassing the Pot1 interaction domain or disruption of the Trf1 complex (36, 55, 59, 63). In fission yeast, a small deletion at the N terminus of Pot1 led to telomere elongation, most likely by affecting the integrity of the N-terminal DNA binding domain. Consistent with this hypothesis, expression of several point mutations designed to weaken the Pot1-DNA interactions based on the cocrystal structure also resulted in telomere elongation (P. Baumann., unpublished data).

Although the results described above strongly argue for Pot1 acting as a negative regulator of telomerase, the mechanism is unlikely to be as simple as Pot1 competing with telomerase for access to the 3′ termini. Otherwise, one would expect overexpression of Pot1 or expression of the Pot1 DNA binding domain to further restrict accessibility to telomerase and result in telomere shortening. However, overexpression of Pot1 caused telomere lengthening in fission yeast and human cells, while expression of the respective DNA binding domains caused lengthening or had no effect (1, 11, 36, 58). While further experiments are essential to elucidate the mechanism of Pot1-mediated telomere length regulation, the possibility that positive and negative effects can be mediated through the same protein is not without precedent and has been elegantly illustrated for budding yeast Cdc13 (9, 50). Recent biochemical experiments indeed suggest that hPot1 can have positive and negative effects on telomerase activity, depending on the 3′-terminal DNA sequence (33)

Telomere loss and chromosomal instability.

Telomere length regulation in vivo may involve a regulated reduction in the amount of telomere-bound Pot1 on short telomeres to increase accessibility to telomerase. Our results show, however, that some Pot1 must remain at telomeres to ensure chromosome stability. While low-level expression of C-terminal Pot1 fragments reduced the amount of endogenous Pot1 at telomeres, high-level expression of C28 or C42 resulted in apparent loss of Pot1 from chromosomes, followed by degradation and end-to-end fusions. In mammalian cells, only telomere lengthening was observed when expressing C-terminal hPot1 fragments (36, 37), suggesting that only part of the mechanism is conserved or that expression levels were too low to displace all endogenous Pot1 from telomeres. Consistent with the latter, chromosomal instability, anaphase bridges, and cell death were observed in a subset of cells following knockdown of Pot1 by RNA interference (60, 62). The remaining cells continued dividing but experienced telomere elongation, as predicted for cells that have reduced levels of Pot1 at telomeres (60, 63).

The observation that expression of C-terminal Pot1 fragments causes loss of endogenous Pot1 from telomeres suggests that recruitment of Pot1 to telomeres predominantly relies on protein-protein interactions and not on the ability of Pot1 to interact with single-stranded telomeric DNA. On the other hand, single-stranded DNA binding is nevertheless critical for Pot1 to maintain its role in chromosome end protection, as illustrated by partial or complete deletions of the DNA binding domain (Fig. 2 and 5). Equally, several point mutations designed to disrupt DNA binding abrogated the protective function of Pot1 (31).

Perhaps more surprising than the requirement for single-stranded DNA binding is the critical importance of the most C-terminal amino acids for maintaining telomeric sequences and thus chromosome stability. Structural and biochemical analyses of Pot1 suggest that the DNA binding domain is comprised of two OB-folds located in the N-terminal half of the protein (31, 58). At this point, we cannot exclude the possibility that the C-terminal domain of Pot1 modulates the interaction with DNA or that small deletions affect the overall folding or intracellular localization of Pot1. However, based on the similarities to hPot1 and TEBP, we favor a model in which the C terminus of Pot1 mediates essential protein-protein interactions.

The C-terminal half of hPot1 binds directly to Ptop/Pip1/Tint1, an interaction that appears to be essential for the recruitment of hPot1 to telomeres (36, 63). Recently, a direct interaction between hPot1 and Trf2 has also been reported (62). Expression of the dominant-negative Trf2^ΔBΔM mutant causes loss of Trf2 and hPot1 from telomeres and results in chromosome fusions. Based on the similarities between the most C-terminal amino acids of fission yeast and human Pot1, it will be interesting to determine whether these residues are required for the interaction with Ptop/Pip1/Tint1 and/or Trf2.

Similarity to ciliate TEBP provides further support for the C-terminal region of Pot1 representing a protein interaction domain. The α subunit of O. nova TEBP is comprised of three OB-folds, with the most C-terminal OB-fold mediating interactions with the β subunit of TEBP (26). While α can bind telomeric DNA on its own, the formation of α/β heterodimers dramatically increases the affinity for DNA and by inference the ability of the protein complex to protect telomeres (52). Although the available crystal structures of fission yeast and human Pot1 in complex with DNA only provide information about the DNA binding domain, sequence profile searches predicted the existence of a third OB-fold in the C-terminal regions of Pot1 family members (57; A. Mushegian and P. Baumann, unpublished data). It remains to be seen whether proteins that modulate the affinity of Pot1 for single-stranded DNA in a similar manner exist in yeast and mammalian cells.