Junko Kanoh

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.

Fission Yeast Pot1-Tpp1 Protects Telomeres and Regulates Telomere Length

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.

Telomere Binding Protein Taz1 Establishes Swi6 Heterochromatin Independently of RNAi at Telomeres

BACKGROUND The telomere is a specialized heterochromatin conserved among eukaryotes. However, it remains unknown how heterochromatin protein 1 (HP1) is recruited to telomeres and how telomere heterochromatin is formed. In fission yeast, the RNAi (RNA interference)-RITS (RNA-induced initiation of transcriptional silencing) pathway initiates heterochromatin formation at the centromeres and the silent mat locus by using common DNA sequences, the dg and dh repeats, as the templates for small interfering RNA (siRNA). RESULTS We found that telomeric repeats are sufficient for the establishment of Swi6 (a fission-yeast HP1 homolog) heterochromatin, and the establishment requires Taz1, a telomere binding protein of the TRF family. Additionally, Swi6 heterochromatin is established by a part of the subtelomere that contains sequences highly homologous to that of the dh repeat, and it is strikingly destabilized by the deletion of both Taz1 and RNAi-RITS. Transcripts from the telomeric dh-homologous region were specifically associated with RITS, and deletion of the telomeric dh-homologous region showed the phenotype similar to that of the rnai mutant in terms of the telomeric silencing, indicating that the RNAi-RITS pathway acts at the telomeric dh-homologous region to establish Swi6 heterochromatin. Furthermore, we found that Taz1 establishes Swi6 heterochromatin independently of the telomeric repeats and the RNAi-RITS pathway at the subtelomeres. CONCLUSION The telomere heterochromatin is regulated by at least two factors: One is Taz1, which is telomere specific, and the other is RNAi-RITS, which is commonly used at the constitutive heterochromatin regions.

Rapamycin sensitivity of the Schizosaccharomyces pombe tor2 mutant and organization of two highly phosphorylated TOR complexes by specific and common subunits

Nutrients are essential for cell growth and division. Screening of Schizosaccharomyces pombe temperature‐sensitive strains led to the isolation of a nutrient‐insensitive mutant, tor2‐287. This mutant produces a nitrogen starvation‐induced arrest phenotype in rich media, fails to recover from the arrest, and is hypersensitive to rapamycin. The L2048S substitution mutation in the catalytic domain in close proximity to the adenine base of ATP is unique as it is the sole known genetic cause of rapamycin hypersensitivity. Localization of Tor2 was speckled in the vegetative cytoplasm, and both speckled and membranous in the arrested cell cytoplasm. Using mass spectroscopic analysis, we identified six subunits (Tco89, Bit61, Toc1, Tel2, Tti1 and Cka1) that, in addition to the six previously identified subunits (Tor1, Tor2, Mip1/Raptor, Ste20/Rictor, Sin1/Avo1 and Wat1/Lst8), comprise the TOR complexes (TORCs). All of the subunits so far examined are multiply phosphorylated. Tel2 bound to Tti1 interacts with various phosphatidyl inositol kinase (PIK)‐related kinases including Tra1, Tra2 and Rad3, as well as Tor1 and Tor2. Schizosaccharomyces pombe TORCs should thus be functionally redundant and might be broadly regulated through different subunits that are either common or specific to the two TORCs, or even common to various PIK‐related kinases. Functional redundancy of the TORCs may explain the rapamycin hypersensitivity of tor2‐287.

Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast

Maintenance of genome integrity requires a checkpoint that restrains mitosis in response to DNA damage [1]. This checkpoint is enforced by Chk1, a protein kinase that targets Cdc25 [2--7]. Phosphorylated Cdc25 associates with 14-3-3 proteins, which appear to occlude a nuclear localization signal (NLS) and thereby inhibit Cdc25 nuclear import [6, 8--14]. Proficient checkpoint arrest is thought to require Cdc25 nuclear exclusion, although definitive evidence for this model is lacking. We have tested this hypothesis in fission yeast. We show that elimination of an NLS in Cdc25 causes Cdc25 nuclear exclusion and a mitotic delay, as predicted by the model. Attachment of an exogenous NLS forces nuclear inclusion of Cdc25 in damaged cells. However, forced nuclear localization of Cdc25 fails to override the damage checkpoint. Thus, nuclear exclusion of Cdc25 is unnecessary for checkpoint enforcement. We propose that direct inhibition of Cdc25 phosphatase activity by Chk1, as demonstrated in vitro with fission yeast and human Chk1 [15, 16], is sufficient for proficient checkpoint regulation of Cdc25 and may be the primary mechanism of checkpoint enforcement in fission yeast.

A conserved motif within RAP1 has diversified roles in telomere protection and regulation in different organisms

Repressor activator protein 1 (RAP1) is the most highly conserved telomere protein. It is involved in protecting chromosome ends in fission yeast and promoting gene silencing in Saccharomyces cerevisiae, whereas it represses homology-directed recombination at telomeres in mammals. To understand how RAP1 has such diverse functions at telomeres, we solved the crystal or solution structures of the RAP1 C-terminal (RCT) domains of RAP1 from multiple organisms in complex with their respective protein-binding partners. Our analysis establishes RAP1RCT as an evolutionarily conserved protein-protein interaction module. In mammalian and fission yeast cells, this module interacts with TRF2 and Taz1, respectively, targeting RAP1 to chromosome ends for telomere protection. In contrast, S. cerevisiae RAP1 uses its RCT domain to recruit Sir3 to telomeres to mediate gene silencing. Together, our results show that, depending on the organism, the evolutionarily conserved RAP1 RCT motif has diverse functional roles at telomeres.

Composition and conservation of the telomeric complex

The telomere is composed of telomeric DNA and telomere-associated proteins. Recently, many telomere-associated proteins have been identified, and various telomere functions have been uncovered. In budding yeast, scRap1 binds directly to telomeric DNA, and other telomere regulators (Sir proteins and Rif proteins) are recruited to the telomeres by interacting with scRap1. Cdc13 binds to the most distal end of the chromosome and recruits telomerase to the telomeres. In fission yeast and humans, TTAGGG repeat binding factor (TRF) family proteins bind directly to telomeric DNA, and Rap1 proteins and other telomere regulators are recruited to the telomeres by interacting with the TRF family proteins. Both organisms have Pot1 proteins at the most distal end of the telomere instead of a budding-yeast Cdc13-like protein. Therefore, fission yeast and humans have in part common telomeric compositions that differ from that of budding yeast, a result that suggests budding yeast has lost some telomere components during the course of evolution.

The fission yeast spSet1p is a histone H3-K4 methyltransferase that functions in telomere maintenance and DNA repair in an ATM kinase Rad3-dependent pathway.

We have characterized spSet1p, the Schizosaccharomyces pombe ortholog of the budding yeast histone H3 methyltransferase Set1p. SpSet1p catalyzes methylation of H3 at K4, in vivo and in vitro. Deleting spset1 partially affects telomeric and centromeric silencing. Strikingly, lack of spSet1p causes elongation of telomeres in wild-type cells and in most DNA damage checkpoint rad mutant cells, but not in cells lacking the ATM kinase Rad3 or its associated protein Rad26. Interestingly, spset1 deletion specifically causes a reduction in sensitivity to ultraviolet radiation of the PCNA-like checkpoint mutants hus1 and rad1, but not of cells devoid of Rad3. This partial suppression was not due to restoration of checkpoint function or to transcriptional induction of DNA repair genes. Moreover, spset1 allows recovery specifically of the crb2 checkpoint mutant upon treatment with the replication inhibitor hydroxyurea but not upon UV irradiation. Nevertheless, the pathway induced in spset1 cells cannot substitute for the Mus81/Rqh1 DNA damage tolerance pathway. Our results suggest that SpSet1p and the ATM kinase Rad3 function in a common genetic pathway linking chromatin to telomere length regulation and DNA repair.

Tel2 Is Required for Activation of the Mrc1-mediated Replication Checkpoint

Proteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2Δ strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1Δ cells especially at S phase. In contrast, the cds1Δswi1Δ mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity.

Telomeric DNA Ends Are Essential for the Localization of Ku at Telomeres in Fission Yeast

The Ku70-Ku80 heterodimer is a conserved protein complex essential for the non-homologous end-joining pathway. Ku proteins are also involved in telomere maintenance, although their precise roles remain to be elucidated. In fission yeast,pku70 +, the gene encoding the Ku70 homologue, has been reported. Here we report the identification and characterization of pku80 +, the gene encoding Ku80. Both pku70 + andpku80 + are essential for efficient non-homologous end-joining. We also found that the pku70and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. Thepku80 mutant shows telomere shortening and tandem amplification of a subtelomeric sequence but no defects in the telomere position effect, as was previously reported for the pku70mutant. By using the chromatin immunoprecipitation assay, we demonstrated that Pku70 and Pku80 physically interact with telomeric repeats and subtelomeric sequences. Interestingly, this telomere association of Pku proteins is independent of Taz1, a telomeric DNA-binding protein. We also showed that the Pku proteins do not associate with ectopically integrated telomeric repeats in the internal region of circular chromosomes. These results indicate that the physical end of DNA is necessary for the localization of Pku80 at telomeres.