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Fission Yeast Pot1-Tpp1 Protects Telomeres and Regulates Telomere Length

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Science  06 Jun 2008:
Vol. 320, Issue 5881, pp. 1341-1344
DOI: 10.1126/science.1154819

Abstract

Telomeres are specialized chromatin structures that protect chromosomal ends. Protection of telomeres 1 (Pot1) binds to the telomeric G-rich overhang, thereby protecting telomeres and regulating telomerase. Mammalian POT1 and TPP1 interact and constitute part of the six-protein shelterin complex. Here we report that Tpz1, the TPP1 homolog in fission yeast, forms a complex with Pot1. Tpz1 binds to Ccq1 and the previously undiscovered protein Poz1 (Pot1-associated in Schizosaccharomyces pombe), which protect telomeres redundantly and regulate telomerase in positive and negative manners, respectively. Thus, the Pot1-Tpz1 complex accomplishes its functions by recruiting effector molecules Ccq1 and Poz1. Moreover, Poz1 bridges Pot1-Tpz1 and Taz1-Rap1, thereby connecting the single-stranded and double-stranded telomeric DNA regions. Such molecular architectures are similar to those of mammalian shelterin, indicating that the overall DNA-protein architecture is conserved across evolution.

Telomeres consist of long arrays of double-stranded (ds) G-rich telomeric repeats terminated by G-rich single-stranded (ss) overhangs at the 3′-terminus (G-tail). The Oxytricha nova telomere end–binding protein (TEBP)–α and –β heterodimers are prototypes of G-tail–binding proteins (1, 2). TEBP-α and -β contain oligonucleotide/oligosaccharide-binding (OB) fold domains involved in DNA binding and protein-protein interaction (3). Protection of telomeres 1 (Pot1), the homolog of TEBP-α, binds to the G-tail and is essential for telomere protection (4, 5). Mammalian POT1 and the TEBP-β homolog TPP1 form a complex that protects telomeres and regulates telomerase (69). POT1 and TPP1 constitute the shelterin complex together with TIN2, RAP1, and ds telomeric DNA–binding proteins TRF1 and TRF2 (10). It has been proposed that mammalian POT1 transduces signals from TRF1 to negatively control telomerase reaction (11).

To elucidate how fission yeast Pot1 functions, we purified proteins associated with Pot1. We identified Ccq1 and two uncharacterized proteins, encoded SPAC19G12.13 and SPAC6F6.16, by mass spectrometry of Pot1 immunoprecipitates (fig. S1 and table S1). Ccq1 is a telomere protein that recruits a Snf2/histone deacetylase (HDAC)–containing repressor complex (SHREC) (12, 13). SPAC19G12.13 produces the previously undiscovered Pot1 complex component Poz1 (Pot1-associated in Schizosaccharomyces pombe) (GenBank accession number AB433171). We found that the presence of introns in SPAC6F6.16 resulted in encoding a protein larger than that predicted in the database (fig. S2). Secondary structure–based fold-recognition programs predicted that the N terminus of the SPAC6F6.16 product (residues 1 to 158) contains an OB fold domain that is most closely related to those of TEBP-β and human TPP1 (fig. S2 and table S3). Moreover, the product and Pot1 form a complex that protects telomeres and regulates telomerase. We therefore conclude that SPAC6F6.16 encodes the fission yeast Tpp1 homolog. Because the gene name tpp1+ is already being used by another gene in fission yeast, we will call this gene tpz1+ (TPP1 homolog in Schizosaccharomyces pombe) (GenBank accession number AB433170).

The physical association between Pot1 and Ccq1, Tpz1, or Poz1 was confirmed by immunoprecipitation (Fig. 1A). Immunofluorescence experiments showed that Ccq1, Tpz1, and Poz1 colocalize with Pot1 (fig. S3). A chromatin immunoprecipitation (ChIP) assay also demonstrated that Ccq1, Tpz1, and Poz1 are specifically bound to telomeres (Fig. 1B). Thus, Ccq1, Tpz1, and Poz1 are closely associated with Pot1 at telomeres. Ccq1 directly associates with Clr3, an HDAC in SHREC (12) (fig. S4). However, Clr3 was not detected in the Pot1-precipitated fraction and is not involved in controlling telomere length, indicating that the Pot1 complex identified in this study is distinct from SHREC.

Fig. 1.

S. pombe Pot1, Ccq1, Tpz1, and Poz1 form a complex at telomeres. (A) Pot1 interacts with Ccq1, Tpz1, and Poz1. Immunoprecipitates (IP) were obtained from whole-cell extracts (WCEs) with the use of indicated antibodies and analyzed by immunoblotting. Arrows indicate positions of tagged proteins. HA, hemagglutinin. (B) Tpz1, Poz1, and Ccq1 are bound to telomeres. The ChIP assay was performed with subtelomeric and histidine1+ (his1+) gene primer sets. Relative enrichment at telomeres as compared with the his1+ locus in the precipitated fractions was determined by the quantitative polymerase chain reaction system. Error bars represent SD (n = 3 individual experiments). (C) Physical association between Tpz1 and Ccq1, Pot1, or Poz1. Protein interaction was detected by the yeast two-hybrid assay. The GAL4 DNA-binding domain (GAL4 DNA-BD) and GAL4 activation domain (GAL4 AD) were fused to indicated peptides. OB1 and OB2 refer to the OB fold domains that were experimentally established and predicted, respectively (5, 20). (D) Tpz1 is required for Pot1 complex formation. WCEs were prepared from strains expressing the indicated tagged proteins in the presence or absence of tpz1+ in trt1Δ cells harboring self-circularized chromosomes.

We used a yeast two-hybrid assay to examine interactions between Tpz1 and Pot1, Ccq1, or Poz1. Tpz1 associated with Pot1, Ccq1, and Poz1 (Fig. 1C). However, we did not observe any substantial interaction between Poz1 and Pot1 or Ccq1 (fig. S5). Deletion analyses revealed that the N terminus of Tpz1 (amino acids 2 to 223) is sufficient for the interaction with Pot1 but not with Ccq1 or Poz1 [Pot1-binding domain (PBD)]. The N-terminal OB fold (OB1) of Pot1 (amino acids 1 to 138) is dispensable for the interaction. In contrast, the C terminus of Tpz1 (amino acids 379- to 508) interacts with Ccq1 and Poz1 but not with Pot1[Ccq1/Poz1-binding domain (CPBD)]. Taken together, Tpz1 is the core molecule interacting with Pot1, Ccq1, and Poz1. The relative positions of the regions responsible for Pot1-Tpz1 interaction in Pot1 and Tpz1 are similar to those in TEBP-α/β and human TPP1 and POT1 (fig. S12). In fission yeast, telomere-defective cells can survive by self-circularizing the three chromosomes. Pot1-Flag was coimmunoprecipitated with Ccq1-Myc in telomerase-defective cells [telomerase reverse transcriptase-1Δ (trt1Δ); trt1+ encodes the catalytic subunit of telomerase] that maintain circular chromosomes lacking telomeric repeat sequences (14, 15), suggesting that the Pot1 complex is formed independently of the presence of either ds or ss telomeric DNAs or physical chromosomal ends (Fig. 1D). When tpz1+ was deleted, the Pot1-Ccq1 association was abolished in circular chromosome–containing cells, indicating that Tpz1 plays a crucial role in complex assembly.

tpz1Δ cells did not grow or formed very small colonies (fig. S2). In the surviving haploid tpz1Δ cells, telomeric signals were completely lost (Fig. 2A), chromosome I–derived telomere-containing Not I fragments (I and L) disappeared in pulsed-field gel electrophoresis (PFGE), and a band corresponding to I+L–fused fragments was detected (Fig. 2B and fig. S6D). Such findings are diagnostic of self-circularized chromosomes, a hallmark of telomere deprotection (15), and are observed in pot1Δ cells (4), indicating that tpz1+ is also essential for telomere protection. To test whether Tpz1 protects telomeres independently of Pot1, a glutathione S-transferase (GST)–fused N-terminal fragment of Tpz1 (amino acids 2 to 223) containing PBD was overproduced. GST-Tpz1 (amino acids 2 to 223) interacted with Pot1, and the interaction between endogenous Tpz1 and Pot1 was substantially reduced (fig. S6). In this condition, telomeric DNA was completely lost and self-circularized chromosomes appeared, indicating that the physical interaction between Pot1 and Tpz1 is required for end protection, as was reported for mammalian POT1 and TPP1 (9, 16). In contrast to tpz1Δ, poz1Δ and ccq1Δ single mutants grew normally. Poz1 and Ccq1 are recruited to telomeres independently of each other (fig. S7). However, when ccq1+ and poz1+ were simultaneously deleted, cells rapidly lost telomeres and survived by forming self-circularized chromosomes (Fig. 2, A and B). Therefore, the Pot1-Tpz1 complex is essential to protect telomeres from fusions, and Ccq1 and Poz1 contribute redundantly to this pathway.

Fig. 2.

The Pot1 complex protects telomeres and associates with telomerase activity. (A) Telomere length in tpz1Δ, ccq1Δ, poz1Δ, and ccq1Δpoz1Δ. Digested genomic DNAs from indicated strains were subjected to Southern hybridization with indicated probes (21). (B) Chromosome circularization in tpz1Δ and ccq1Δpoz1Δ. Not I–digested DNAs were separated by PFGE. The ethidium bromide–stained gel (left) and Southern hybridization with probes specific for I and L fragments (right) are shown. (C) Tpz1 coimmunoprecipitates telomerase activity in a Ccq1-dependent manner. Telomerase activity was measured in the precipitates prepared from strains expressing indicated proteins in the presence or absence of ribonuclease as described in (22) (top). 32P-labeled 19-, 20-, and 31-nucleotide oligomers served as size markers. Amounts of precipitated proteins were determined by immunoblotting (bottom).

poz1Δ and ccq1Δ cells possessed elongated (up to 2 kilo–base pair long) and shortened (by ∼200 base pairs) telomeres, respectively (Fig. 2A) (13). Therefore, Poz1 and Ccq1 regulate telomere length negatively and positively, respectively. Tpz1 immunoprecipitates exhibited telomerase activity that gave rise to products similar to those of Trt1 immunoprecipitates (Fig. 2C). The Tpz1-associated telomerase activity was abrogated in ccq1Δ but not in poz1Δ cells. Moreover, ccq1Δtrt1Δ cells showed similar telomere length to trt1Δ cells (fig. S8). The ccq1+ deletion did not affect the activity of Trt1 immunoprecipitates (Fig. 2C). These results suggest that Ccq1 promotes telomere elongation by facilitating telomerase recruitment or stabilizing the Tpz1-telomerase complex but is not required for the catalytic activity of telomerase per se.

Telomeric DNA is maintained primarily by telomerase. Telomere elongation in poz1Δ cells was abolished by the deletion of trt1+ (Fig. 3A). These results indicate that telomeres in poz1Δ cells are elongated by telomerase and suggest that Poz1 negatively regulates telomerase. Taz1, the homolog of vertebrate ds telomeric DNA-binding proteins TRF1 and TRF2, associates with Rap1. Telomeres are elongated in taz1Δ or rap1Δ cells (1719), and the elongation in taz1Δ cells depends on telomerase (14). taz1Δ or rap1Δ is epistatic to poz1Δ in telomere-length control (Fig. 3B). The yeast two-hybrid assay showed that Poz1 specifically interacts with Rap1 but not Taz1 (Fig. 3C). Coimmunoprecipitation experiments demonstrated that Poz1 interacts with Rap1 in a Taz1-independent manner in vivo (Fig. 3D). These results suggest that Poz1 acts as a transducer downstream of the Taz1-Rap1 pathway so as to negatively control telomerase.

Fig. 3.

Poz1 negatively controls telomere length through association with Taz1-Rap1. (A) poz1+ deletion leads to telomerase-dependent telomere elongation. Genomic DNAs were prepared from indicated haploid cells derived from heterozygous poz1Δ/poz1+ trt1Δ/trt1+ diploid cells (results from two independent tetrads a and b are shown) and subjected to Southern hybridization. (B) taz1Δ or rap1Δ is epistatic to poz1Δ in telomere-length control. (C) Poz1 and Rap1 protein interaction was detected by the yeast two-hybrid assay. (D) Poz1 interacts with Rap1 in a Taz1-independent manner in vivo. Immunoprecipitates were obtained from WCEs using antibodies to Flag and analyzed by immunoblotting. (E) Poz1 and Pot1 are localized at internal ds telomeric repeats in a Taz1- or Rap1-dependent manner. A cassette containing TAS-ura4+-telomeric repeats was inserted into the ade6 locus of circular chromosome III. Fold enrichments of ura4+ and lys1+ in indicated immunoprecipitates in a ChIP assay are shown.

To examine how the Pot1 complex is recruited to telomeres, telomeric repeats were inserted into self-circularized chromosomes lacking original telomere repeats in the trt1Δ background (Fig. 3E). ChIP assays demonstrated that Poz1 and Pot1 associate with the internal telomeric repeats in a Taz1- and Rap1-dependent manner, indicating that the Pot1 complex can be recruited by ds telomeric DNA in the absence of ss telomeric DNA or physical DNA ends. These results also suggest that Taz1-Rap1 is responsible for bridging ds telomeric DNA and the Pot1 complex. Conversely, we observed that Poz1 and Pot1 are bound to native telomeric ends in taz1Δ or rap1Δ cells (fig. S9), indicating that the Pot1 complex is recruited to telomeres in a Taz1-Rap1–independent manner. We did not observe complete loss of telomeric DNA in ccq1Δtaz1Δ or ccq1Δrap1Δ cells, in contrast to ccq1Δpoz1Δ cells (fig. S10), suggesting that Poz1 is recruited to and protects telomeres independently of Taz1 or Rap1. Taken together, we suggest that Pot1 is recruited to telomeres by two distinct modes: in ds telomeric DNA–dependent and G-tail–dependent manners.

We found that ccq1Δpoz1Δ cells lost telomeres much more rapidly than trt1Δpoz1Δ cells did (Figs. 2A and 3A), raising the possibility that Ccq1 maintains telomeres in a telomerase-independent manner in addition to its role in regulating elongation by telomerase. Telomeres are recognized as double-strand breaks in ccq1Δ cells to activate the DNA damage checkpoint (fig. S11). trt1Δ cells cultured in liquid medium showed progressive telomere shortening and poor growth. Then, the cells resumed growth by amplifying terminal DNAs containing telomeric repeats and subtelomeric sequences [telomere-associated sequence (TAS)] via homologous recombination (HR) (14). ccq1Δtrt1Δ cells started to show amplified telomere+TAS signals earlier than trt1Δ cells, suggesting that ccq1Δ accelerates HR-mediated telomere lengthening in the trt1Δ background (fig. S11). Although telomere signals persisted in rhp55Δ, rhp51Δ, and ccq1Δ single mutants, the concomitant deletion of ccq1+ and rhp55+ or rhp51+ led to the complete loss of telomeric DNA and chromosome circularization (Fig. 4, A and B), indicating that terminal DNAs in ccq1Δ cells are maintained via HR. Thus, Ccq1 protects telomeres from HR. In ccq1Δ cells, the deregulated HR activity at telomeres contributes to the survival of cells without circular chromosome formation.

Fig. 4.

Ccq1 inhibits HR at telomeres. (A) Telomeres are lost in ccq1Δrhp51Δ and ccq1Δrhp55Δ. (B) HR prevents chromosome circularization in ccq1Δ. Not I–digested chromosomal DNAs were fractionated by PFGE, stained with ethidium bromide (left), and subjected to Southern hybridization (right). (C) Model of Pot1-complex–mediated switching between closed and semi-open telomere configurations. The Pot1 complex exists at telomeres in two distinguishable modes (closed and semi-open configurations) that regulate telomerase in opposite manners. In both cases, Ccq1 and Poz1 protect telomeres from degradation, HR, and fusion.

We propose that the Pot1 complex exists at telomeres in two distinguishable modes. When telomeres are relatively long, the Pot1 complex associates with ds telomeric DNA-Taz1-Rap1 via Poz1 because of a high concentration of local Taz1-Rap1 proteins, leading to inhibition of telomerase action (a closed configuration). When telomeres shorten, the Pot1 complex is dissociated from Taz1-Rap1, facilitating telomerase action aided by Ccq1 (a semi-open configuration). The structure and function of fission yeast telomeres revealed in this study highlight the conservation of major features of the shelterin complex across a wide evolutionary distance (fig. S12). We also predict that shelterin functions by recruiting unidentified effector molecules in higher eukaryotes, similar to the recruitment of Ccq1 in fission yeast.

Supporting Online Material

www.sciencemag.org/cgi/content/full/320/5881/1341/DC1

Materials and Methods

Figs. S1 to S12

Tables S1 to S3

References

References and Notes

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