spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast.

Telomeres are essential for genome integrity. scRap1 (S. cerevisiae Rap1) directly binds to telomeric DNA and regulates telomere length and telomere position effect (TPE) by recruiting two different groups of proteins to its RCT (Rap1 C-terminal) domain. The first group, Rif1 and Rif2, regulates telomere length. The second group, Sir3 and Sir4, is involved in heterochromatin formation. On the other hand, human TRF1 and TRF2, as well as their fission yeast homolog, Taz1, directly bind to telomeric DNA and negatively regulate telomere length. Taz1 also plays important roles in TPE and meiosis. Human Rap1, the ortholog of scRap1, negatively regulates telomere length and appears to be recruited to telomeres by interacting with TRF2. Here, we describe two novel fission yeast proteins, spRap1 (S. pombe Rap1) and spRif1 (S. pombe Rif1), which are orthologous to scRap1 and scRif1, respectively. spRap1 and spRif1 are independently recruited to telomeres by interacting with Taz1. The rap1 mutant is severely defective in telomere length control, TPE, and telomere clustering toward the spindle pole body (SPB) at the premeiotic horsetail stage, indicating that spRap1 has critical roles in these telomere functions. The rif1 mutant also shows some defects in telomere length control and meiosis. Our results indicate that Taz1 provides binding sites for telomere regulators, spRap1 and spRif1, which perform the essential telomere functions. This study establishes the similarity of telomere organization in fission yeast and humans.     

Shelterin promotes tethering of late replication origins to telomeres for replication‐timing control

DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere‐binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere‐like sequence near the origins. Here, we showed using a lacO/LacI‐GFP system that Taz1‐dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication‐timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication‐timing control and telomeric association of Taz1‐dependent late origins, and this requirement was bypassed by a minishelterin Tpz1‐Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin‐mediated telomeric association of the origins at the onset of S phase.     

Meiotic proteins bqt1 and bqt2 tether telomeres to form the bouquet arrangement of chromosomes

In many organisms, meiotic chromosomes are bundled at their telomeres to form a "bouquet" arrangement. The bouquet formation plays an important role in homologous chromosome pairing and therefore progression of meiosis. As meiotic telomere clustering occurs in response to mating pheromone signaling in fission yeast, we looked for factors essential for bouquet formation among genes induced under mating pheromone signaling. This genome-wide search identified two proteins, Bqt1 and Bqt2, that connect telomeres to the spindle-pole body (SPB; the centrosome equivalent in fungi). Neither Bqt1 nor Bqt2 alone functions as a connector, but together the two proteins form a bridge between Rap1 (a telomere protein) and Sad1 (an SPB protein). Significantly, when both Bqt1 and Bqt2 are ectopically expressed in mitotic cells, they also form a bridge between Rap1 and Sad1. Thus, a complex including Bqt1 and Bqt2 is essential for connecting telomeres to the SPB.     

Telomere binding of the Rap1 protein is required for meiosis in fission yeast.

Telomeres are essential for chromosome integrity, protecting the ends of eukaryotic linear chromosomes during cell proliferation. Telomeres also function in meiosis; a characteristic clustering of telomeres beneath the nuclear membrane is observed during meiotic prophase in many organisms from yeasts to plants and humans, and the role of the telomeres in meiotic pairing and the recombination of homologous chromosomes has been demonstrated in the fission yeast Schizosaccharomyces pombe and in the budding yeast Saccharomyces cerevisiae. Here we report that S. pombe Rap1 is a telomeric protein essential for meiosis. While Rap1 is conserved in budding yeast and humans, schemes for telomere binding vary among species: human RAP1 binds to the telomere through interaction with the telomere binding protein TRF2; S. cerevisiae Rap1, however, binds telomeric DNA directly, and no orthologs of TRF proteins have been identified in this organism. In S. pombe, unlike in S. cerevisiae, an ortholog of human TRF has been identified. This ortholog, Taz1, binds directly to telomere repeats [18] and is necessary for telomere clustering in meiotic prophase. Our results demonstrate that S. pombe Rap1 binds to telomeres through interaction with Taz1, similar to human Rap1-TRF2, and that Taz1-mediated telomere localization of Rap1 is necessary for telomere clustering and for the successful completion of meiosis. Moreover, in taz1-disrupted cells, molecular fusion of Rap1 with the Taz1 DNA binding domain recovers telomere clustering and largely complements defects in meiosis, indicating that telomere localization of Rap1 is a key requirement for meiosis.     

Fission Yeast Shelterin Regulates DNA Polymerases and Rad3ATR Kinase to Limit Telomere Extension

Studies in fission yeast have previously identified evolutionarily conserved shelterin and Stn1-Ten1 complexes, and established Rad3^ATR/Tel1^ATM-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 as the critical post-translational modification for telomerase recruitment to telomeres. Furthermore, shelterin subunits Poz1, Rap1 and Taz1 have been identified as negative regulators of Thr93 phosphorylation and telomerase recruitment. However, it remained unclear how telomere maintenance is dynamically regulated during the cell cycle. Thus, we investigated how loss of Poz1, Rap1 and Taz1 affects cell cycle regulation of Ccq1 Thr93 phosphorylation and telomere association of telomerase (Trt1^TERT), DNA polymerases, Replication Protein A (RPA) complex, Rad3^ATR-Rad26^ATRIP checkpoint kinase complex, Tel1^ATM kinase, shelterin subunits (Tpz1, Ccq1 and Poz1) and Stn1. We further investigated how telomere shortening, caused by trt1Δ or catalytically dead Trt1-D743A, affects cell cycle-regulated telomere association of telomerase and DNA polymerases. These analyses established that fission yeast shelterin maintains telomere length homeostasis by coordinating the differential arrival of leading (Polε) and lagging (Polα) strand DNA polymerases at telomeres to modulate Rad3^ATR association, Ccq1 Thr93 phosphorylation and telomerase recruitment.     

Taz1, Rap1 and Rif1 act both interdependently and independently to maintain telomeres

Telomere protection and maintenance are accomplished through the coordinated actions of telomere-specific DNA binding proteins and their interacting partners. The fission yeast ortholog of human TRF1/2, Taz1, binds telomeric DNA and regulates numerous aspects of telomere function. Here, we ask which aspects of Taz1 function are mediated through its interacting proteins, Rap1 and Rif1. We demonstrate that rap1+ deletion phenocopies some, but not all, aspects of taz1Delta telomere dysfunction, while Rif1 exhibits a very different functional spectrum. Rap1 acts in a Taz1-dependent pathway to prevent chromosome end fusions and regulate telomeric 3' overhang formation, while Rif1 is dispensable for these functions. Telomerase inhibition by Taz1 is mediated by two separate pathways, one involving Rap1 and the other involving Rif1. In contrast, Taz1 is uniquely required to prevent chromosomal entanglements and missegregation at cold temperatures. Strikingly, while rap1+ deletion exacerbates the cold sensitivity of taz1Delta cells, rif1+ deletion restores full viability. Thus, Rap1 and Rif1 are each required for a subset of the functions of Taz1, but each acquires Taz1-independent functions in its absence. Furthermore, Taz1 can function independently of its known binding partners.     

Recombination-based telomere maintenance is dependent on Tel1-MRN and Rap1 and inhibited by telomerase, Taz1, and Ku in fission yeast

Fission yeast cells survive loss of the telomerase catalytic subunit Trt1 (TERT) through recombination-based telomere maintenance or through chromosome circularization. Although trt1Δ survivors with linear chromosomes can be obtained, they often spontaneously circularize their chromosomes. Therefore, it was difficult to establish genetic requirements for telomerase-independent telomere maintenance. In contrast, when the telomere-binding protein Taz1 is also deleted, taz1Δ trt1Δ cells are able to stably maintain telomeres. Thus, taz1Δ trt1Δ cells can serve as a valuable tool in understanding the regulation of telomerase-independent telomere maintenance. In this study, we show that the checkpoint kinase Tel1 (ATM) and the DNA repair complex Rad32-Rad50-Nbs1 (MRN) are required for telomere maintenance in taz1Δ trt1Δ cells. Surprisingly, Rap1 is also essential for telomere maintenance in taz1Δ trt1Δ cells, even though recruitment of Rap1 to telomeres depends on Taz1. Expression of catalytically inactive Trt1 can efficiently inhibit recombination-based telomere maintenance, but the inhibition requires both Est1 and Ku70. While Est1 is essential for recruitment of Trt1 to telomeres, Ku70 is dispensable. Thus, we conclude that Taz1, TERT-Est1, and Ku70-Ku80 prevent telomere recombination, whereas MRN-Tel1 and Rap1 promote recombination-based telomere maintenance. Evolutionarily conserved proteins in higher eukaryotic cells might similarly contribute to telomere recombination.     

Rap1-independent telomere attachment and bouquet formation in mammalian meiosis

Attachment of telomeres to the nuclear envelope (NE) and their clustering in a chromosomal bouquet during meiotic prophase I is an evolutionary conserved event that promotes chromosome pairing and recombination. In fission yeast, bouquet formation fails when the telomeric protein Rap1 is absent or when the telomeric protein Taz1 fails to recruit Rap1 to telomeres. The mammalian Rap1 orthologue is a component of the shelterin complex and localises to telomeres through an interaction with a Taz1-like telomeric DNA binding factor, TRF2. Here, we investigated the role of mammalian Rap1 in meiotic telomere attachment and clustering by analysing spermatogenesis in Rap1-deficient mice. The results establish that the meiotic three-dimensional nuclear architecture and recombination are not affected by the absence of Rap1. Furthermore, Rap1-deficient meiotic telomeres assemble the SUN1 nuclear membrane protein, attach to the NE, and undergo bouquet formation indistinguishable from the wild-type setting. Thus, the role of Rap1 in meiosis is not conserved between fission yeast and mammals, suggesting that mammals have alternative modes for connecting telomeres to SUN proteins on the meiotic nuclear envelope.     

Tpz1-Ccq1 and Tpz1-Poz1 Interactions within Fission Yeast Shelterin Modulate Ccq1 Thr93 Phosphorylation and Telomerase Recruitment

In both fission yeast and humans, the shelterin complex plays central roles in regulation of telomerase recruitment, protection of telomeres against DNA damage response factors, and formation of heterochromatin at telomeres. While shelterin is essential for limiting activation of the DNA damage checkpoint kinases ATR and ATM at telomeres, these kinases are required for stable maintenance of telomeres. In fission yeast, Rad3^ATR and Tel1^ATM kinases are redundantly required for telomerase recruitment, since Rad3^ATR/Tel1^ATM-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 promotes interaction between Ccq1 and the telomerase subunit Est1. However, it remained unclear how protein-protein interactions within the shelterin complex (consisting of Taz1, Rap1, Poz1, Tpz1, Pot1 and Ccq1) contribute to the regulation of Ccq1 Thr93 phosphorylation and telomerase recruitment. In this study, we identify domains and amino acid residues that are critical for mediating Tpz1-Ccq1 and Tpz1-Poz1 interaction within the fission yeast shelterin complex. Using separation of function Tpz1 mutants that maintain Tpz1-Pot1 interaction but specifically disrupt either Tpz1-Ccq1 or Tpz1-Poz1 interaction, we then establish that Tpz1-Ccq1 interaction promotes Ccq1 Thr93 phosphorylation, telomerase recruitment, checkpoint inhibition and telomeric heterochromatin formation. Furthermore, we demonstrate that Tpz1-Poz1 interaction promotes telomere association of Poz1, and loss of Poz1 from telomeres leads to increases in Ccq1 Thr93 phosphorylation and telomerase recruitment, and telomeric heterochromatin formation defect. In addition, our studies establish that Tpz1-Poz1 and Tpz1-Ccq1 interactions redundantly fulfill the essential telomere protection function of the shelterin complex, since simultaneous loss of both interactions caused immediate loss of cell viability for the majority of cells and generation of survivors with circular chromosomes. Based on these findings, we suggest that the negative regulatory function of Tpz1-Poz1 interaction works upstream of Rad3^ATR kinase, while Tpz1-Ccq1 interaction works downstream of Rad3^ATR kinase to facilitate Ccq1 Thr93 phosphorylation and telomerase recruitment.     

Membrane proteins Bqt3 and -4 anchor telomeres to the nuclear envelope to ensure chromosomal bouquet formation

In many organisms, telomeres cluster to form a bouquet arrangement of chromosomes during meiotic prophase. Previously, we reported that two meiotic proteins, Bqt1 and -2, are required for tethering telomeres to the spindle pole body (SPB) during meiotic prophase in fission yeast. This study has further identified two novel, ubiquitously expressed inner nuclear membrane (INM) proteins, Bqt3 and -4, which are required for bouquet formation. We found that in the absence of Bqt4, telomeres failed to associate with the nuclear membranes in vegetative cells and consequently failed to cluster to the SPB in meiotic prophase. In the absence of Bqt3, Bqt4 protein was degraded during meiosis, leading to a phenotype similar to that of the bqt4-null mutant. Collectively, these results show that Bqt4 anchors telomeres to the INM and that Bqt3 protects Bqt4 from protein degradation. Interestingly, the functional integrity of telomeres is maintained even when they are separated from the nuclear envelope in vegetative cells.     

Bqt2p is essential for initiating telomere clustering upon pheromone sensing in fission yeast

The telomere bouquet, i.e., telomere clustering on the nuclear envelope (NE) during meiotic prophase, is thought to promote homologous chromosome pairing. Using a visual screen, we identified bqt2/im295, a mutant that disrupts telomere clustering in fission yeast. Bqt2p is required for linking telomeres to the meiotic spindle pole body (SPB) but not for attachment of telomeres or the SPB to the NE. Bqt2p is expressed upon pheromone sensing and colocalizes thereafter to Sad1p, an SPB protein. This localization only depends on Bqt1p, not on other identified proteins required for telomere clustering. Upon pheromone sensing, generation of Sad1p foci next to telomeres depends on Bqt2p. However, depletion of Bqt2p from the SPB is dispensable for dissolving the telomere bouquet at the end of meiotic prophase. Therefore, telomere bouquet formation requires Bqt2p as a linking component and is finely regulated during meiotic progression.     

Fission yeast Rap1 homolog is a telomere-specific silencing factor and interacts with Taz1p

Taz1p is the fission yeast orthologue of human TRF2, a telomeric repeat-binding protein. Delta(taz1) mutants are defective in telomeric silencing, telomere length control, and meiotic recombination events. A recent report demonstrated that the human Rap1p homolog (hRap1) is recruited to telomere by interaction with TRF2, arguing that the telomere control mechanism of higher eukaryotes is distinct from that of the budding yeast. Taz1p showed a significant similarity to human TRF2, but not with the budding yeast Rap1p (scRap1p). This suggests that Taz1p and TRF2 share common features in telomere regulation. To assess the roles of Taz1p in telomere-related functions in detail, we attempted to identify a protein(s) that interacts with Taz1p by using two-hybrid screening. Interestingly, the sequence analysis of a positive clone revealed a perfect match with a Rap1 homolog in S. pombe (spRap1), which showed a significant homology with scRap1p and hRap1p. Here we show that the spRap1 deficiency in haploid cells is viable, which results in increased telomere length regulation, disruption of telomere silencing, and aberrant meiosis (like the delta(taz1) mutant). This suggests that spRap1p might be recruited to the telomere by Taz1p and play crucial roles in telomere function. Interestingly, the delta(rap1) mutants in fission yeast are defective only for telomere silencing. Therefore, the role of spRap1p may be distinct from that of scRap1p, which is involved in the silencing at both the telomere and mating type locus. Our data, therefore, suggest that the regulation mechanisms of telomere in fission yeast resemble that of higher eukaryotic cells rather than the budding yeast.     

Rap1 affects the length and heterogeneity of human telomeres

Telomere length is controlled in part by cis-acting negative regulators that limit telomere extension by telomerase. In budding yeast, the major telomere length regulator scRap1 binds to telomeric DNA and acts to inhibit telomere elongation in cis. Because the human Rap1 ortholog hRap1 does not bind to telomeric DNA directly but is recruited to telomeres by TRF2, we examined its role in telomere length control. The data are consistent with hRap1 being a negative regulator of telomere length, indicating functional conservation. Deletion mapping confirmed that hRap1 is tethered to telomeres through interaction of its C terminus with TRF2. The telomere length phenotypes of hRap1 deletion mutants implicated both the BRCT and Myb domain as protein interaction domains involved in telomere length regulation. By contrast, scRap1 binds to telomeres with its Myb domains and uses its C terminus to recruit the telomere length regulators Rif1 and Rif2. Together, our data show that although the role of Rap1 at telomeres has been largely conserved, the domains of Rap1 have undergone extensive functional changes during eukaryotic evolution. Surprisingly, hRap1 alleles lacking the BRCT domain diminished the heterogeneity of human telomeres, indicating that hRap1 also plays a role in the regulation of telomere length distribution.     

Rap1p telomere association is not required for mitotic stability of a C(3)TA(2) telomere in yeast

Telomeric DNA usually consists of a repetitive sequence: C(1-3)A/TG(1-3) in yeast, and C(3)TA(2)/T(2)AG(3) in vertebrates. In yeast, the sequence-specific DNA- binding protein Rap1p is thought to be essential for telomere function. In a tlc1h mutant, the templating region of the telomerase RNA gene is altered so that telomerase adds the vertebrate telomere sequence instead of the yeast sequence to the chromosome end. A tlc1h strain has short but stable telomeres and no growth defect. We show here that Rap1p and the Rap1p-associated Rif2p did not bind to a telomere that contains purely vertebrate repeats, while the TG(1-3) single-stranded DNA binding protein Cdc13p and the normally non-telomeric protein Tbf1p did bind this telomere. A chromosome with one entirely vertebrate-sequence telomere had a wild-type loss rate, and the telomere was maintained at a short but stable length. However, this telomere was unable to silence a telomere-adjacent URA3 gene, and the strain carrying this telomere had a severe defect in meiosis. We conclude that Rap1p localization to a C(3)TA(2) telomere is not required for its essential mitotic functions.