Fission Yeast Telomeres Research Articles

Taz1 Binding to a Fission Yeast Model Telomere: FORMATION OF TELOMERIC LOOPS AND HIGHER ORDER STRUCTURES

Similar to its human homologues TRF1 and TRF2, fission yeast Taz1 protein is a component of telomeric chromatin regulating proper telomere maintenance. As mammalian TRF1 and TRF2 proteins have been shown to directly bind telomeric DNA to form protein arrays and looped structures, termed t-loops, the ability of Taz1p to act on fission yeast telomeric DNA in similar ways was examined using purified protein and model DNA templates. When incubated with Taz1p, model telomeres containing 3' single-stranded telomeric overhangs formed t-loops at a frequency approaching 13%. Termini with blunt ends and non-telomeric overhangs were deficient in t-loop formation. In addition, we observed arrays of multiple Taz1p molecules bound to the telomeric regions, resembling the pattern of TRF1 binding. The presence of t-loops larger than the telomeric tract, a high frequency of end-bound DNAs and a donut shape of the Taz1p complex suggest that Taz1p binds the 3' overhang then extrudes a loop that grows in size as the donut slides along the duplex DNA. Based on these in vitro results we discuss possible general implications for fission yeast telomere dynamics.

The Telomere Protein Taz1 Is Required to Prevent and Repair Genomic DNA Breaks

One fundamental function of telomeres is to prevent the ends of chromosomes from being sensed and treated as DNA damage. Here we present evidence for additional roles of telomeres in promoting proper chromosome segregation and DNA repair. We find that the fission yeast telomere protein Taz1p is required for cell cycle progression at 20°C, a temperature at which taz1Δ cells exhibit a G2/M DNA damage checkpoint delay, chromosome missegregation, and DNA double-strand breaks (DSBs). Spindle assembly checkpoint components and a checkpoint-independent function of Rad3p are required for taz1Δ cells to survive at 20°C. Disruption of topoisomerase II activity suppresses the cold sensitivity of taz1Δ cells, suggesting a scenario in which telomeric entanglement is the primary defect. Furthermore, hypersensitivity to treatments that induce DSBs suggests that Taz1p is involved in DSB repair. Our observations imply roles for Taz1p-containing telomeres in preventing and repairing DNA breaks throughout the genome.

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(+) and pku80(+) are essential for efficient non-homologous end-joining. We also found that the pku70 and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. The pku80 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 pku70 mutant. 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.

Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres.

Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.

Histone deacetylase homologs regulate epigenetic inheritance of transcriptional silencing and chromosome segregation in fission yeast.

Position-effect control at the silent mat2-mat3 interval and at centromeres and telomeres in fission yeast is suggested to be mediated through the assembly of heterochromatin-like structures. Therefore, trans-acting genes that affect silencing may encode either chromatin proteins, factors that modify them, or factors that affect chromatin assembly. Here, we report the identification of an essential gene, clr6 (cryptic loci regulator), which encodes a putative histone deacetylase that when mutated affects epigenetically maintained repression at the mat2-mat3 region and at centromeres and reduces the fidelity of chromosome segregation. Furthermore, we show that the Clr3 protein, when mutated, alleviates recombination block at mat region as well as silencing at donor loci and at centromeres and telomeres, also shares strong homology to known histone deacetylases. Genetic analyses indicate that silencing might be regulated by at least two overlapping histone deacetylase activities. We also found that transient inhibition of histone deacetylase activity by trichostatin A results in the increased missegregation of chromosomes in subsequent generations and, remarkably, alters the imprint at the mat locus, causing the heritable conversion of the repressed epigenetic state to the expressed state. This work supports the model that the level of histone deacetylation has a role in the assembly of repressive heterochromatin and provides insight into the mechanism of epigenetic inheritance.

Fission yeast Taz1 protein is required for meiotic telomere clustering and recombination.

The alignment of homologous chromosomes during meiosis is essential for their recombination and segregation. Telomeres form and protect the ends of eukaryotic linear chromosomes, and are composed of tandem repeats of a simple DNA sequence and the proteins that bind to these repeats. A role for telomeres in meiosis was suspected from observations of telomere clustering in meiotic cells, and has now been supported experimentally by the dramatic rearrangement of telomere locations during premeiotic stages in fission yeast. Here we show that the fission yeast telomere protein, Taz1, is required for stable association between telomeres and spindle pole bodies during meiotic prophase. In the absence of Taz1, telomere clustering at the spindle pole bodies is disrupted, meiotic recombination is reduced, and both spore viability and the ability of zygotes to re-enter mitosis are impaired to a level that would be expected if chromosome segregation were occurring randomly. Such telomeric association mediated by telomere-specific proteins may also be important for proper chromosome alignment and recombination during meiosis in humans.

Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast.

Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 and cut7 mutants defective in tubulin of a kinesin-related motor, cells are blocked in early stages of mitosis due to the absence of the spindle, and centromeres dissociate but remain close to the SPB, whereas in a metaphase-arrested nuc2 mutant, they reside at the middle of the spindle. FISH is therefore a powerful tool for analyzing mitotic chromosome movement and disjunction using various mutants. Surprisingly, in top2 defective in DNA topoisomerase II, while most chromatid DNAs remain undivided, sister centromeres are separated. Significance of this finding is discussed. In contrast, most chromatid DNAs are separated but telomeric DNAs are not in cut1 mutant. In cut1, the dependence of SPB duplication on the completion of mitosis is abolished. In crm1 mutant cells defective in higher-order chromosome organization, the interphase arrangements of centromeres and telomeres are disrupted.