Bqt4 affects relative movement between SPB and nucleolus in fission yeast.

Movement dynamics in the nucleus involve various biological processes, including DNA repair, which is crucial for cancer prevention. Changes in the movement of the components of the nucleus indicate the changes in movement dynamics in the nucleus. In Schizosaccharomyces pombe, the inner nuclear membrane protein Bqt4 plays an essential role in attaching telomeres to the nuclear envelope. We observed that the deletion of bqt4+ caused a significant decrease in the mean square displacement (MSD) calculated from the distance between the nucleolar center and spindle pole body (SPB), hereafter referred to as MSD(SPB-Nucleolus). The MSD(SPB-Nucleolus) decrease in bqt4Δ was microtubule-dependent. The Rap1-binding ability loss mutant, bqt4F46A, and nonspecific DNA-binding ability mutants, bqt43E-A, did not exhibit an MSD(SPB-Nucleolus) decrease compared to the WT. Moreover, the bqt43E-Arap1Δ double mutant and 1-262 amino acids truncated mutant bqt4ΔN (263-432), which does not have either Rap1-binding or nonspecific DNA-binding abilities, did not exhibit the MSD(SPB-Nucleolus) decrease to the same extent as bqt4Δ. These results suggest that the unknown function of Bqt4 in the C-terminal domain is essential for the maintenance of the pattern of relative movement between SPB and the nucleolus.

Subtelomeres: hotspots of genome variation.

Eukaryotic cells contain multiple types of duplicated sequences. Typical examples are tandem repeat sequences including telomeres, centromeres, rDNA genes and transposable elements. Most of these sequences are unstable; thus, their copy numbers or sequences change rapidly in the course of evolution. In this review, I will describe roles of subtelomere regions, which are located adjacent to telomeres at chromosome ends, and recent discoveries about their sequence variation.

Roles of Specialized Chromatin and DNA Structures at Subtelomeres in Schizosaccharomyces pombe.

Eukaryotes have linear chromosomes with domains called telomeres at both ends. The telomere DNA consists of a simple tandem repeat sequence, and multiple telomere-binding proteins including the shelterin complex maintain chromosome-end structures and regulate various biological reactions, such as protection of chromosome ends and control of telomere DNA length. On the other hand, subtelomeres, which are located adjacent to telomeres, contain a complex mosaic of multiple common segmental sequences and a variety of gene sequences. This review focused on roles of the subtelomeric chromatin and DNA structures in the fission yeast Schizosaccharomyces pombe. The fission yeast subtelomeres form three distinct chromatin structures; one is the shelterin complex, which is localized not only at the telomeres but also at the telomere-proximal regions of subtelomeres to form transcriptionally repressive chromatin structures. The others are heterochromatin and knob, which have repressive effects in gene expression, but the subtelomeres are equipped with a mechanism that prevents these condensed chromatin structures from invading adjacent euchromatin regions. On the other hand, recombination reactions within or near subtelomeric sequences allow chromosomes to be circularized, enabling cells to survive in telomere shortening. Furthermore, DNA structures of the subtelomeres are more variable than other chromosomal regions, which may have contributed to biological diversity and evolution while changing gene expression and chromatin structures.

Fission yeast Stn1 maintains stability of repetitive DNA at subtelomere and ribosomal DNA regions.

Telomere binding protein Stn1 forms the CST (Cdc13/CTC1-STN1-TEN1) complex in budding yeast and mammals. Likewise, fission yeast Stn1 and Ten1 form a complex indispensable for telomere protection. We have previously reported that stn1-1, a high-temperature sensitive mutant, rapidly loses telomere DNA at the restrictive temperature due to frequent failure of replication fork progression at telomeres and subtelomeres, both containing repetitive sequences. It is unclear, however, whether Stn1 is required for maintaining other repetitive DNAs such as ribosomal DNA. In this study, we have demonstrated that stn1-1 cells, even when grown at the permissive temperature, exhibited dynamic rearrangements in the telomere-proximal regions of subtelomere and ribosomal DNA repeats. Furthermore, Rad52 and γH2A accumulation was observed at ribosomal DNA repeats in the stn1-1 mutant. The phenotypes exhibited by the stn1-1 allele were largely suppressed in the absence of Reb1, a replication fork barrier-forming protein, suggesting that Stn1 is involved in the maintenance of the arrested replication forks. Collectively, we propose that Stn1 maintains the stability of repetitive DNAs at subtelomeres and rDNA regions.

Complete sequences of Schizosaccharomyces pombe subtelomeres reveal multiple patterns of genome variation.

Genome sequences have been determined for many model organisms; however, repetitive regions such as centromeres, telomeres, and subtelomeres have not yet been sequenced completely. Here, we report the complete sequences of subtelomeric homologous (SH) regions of the fission yeast Schizosaccharomyces pombe. We overcame technical difficulties to obtain subtelomeric repetitive sequences by constructing strains that possess single SH regions of a standard laboratory strain. In addition, some natural isolates of S. pombe were analyzed using previous sequencing data. Whole sequences of SH regions revealed that each SH region consists of two distinct parts with mosaics of multiple common segments or blocks showing high variation among subtelomeres and strains. Subtelomere regions show relatively high frequency of nucleotide variations among strains compared with the other chromosomal regions. Furthermore, we identified subtelomeric RecQ-type helicase genes, tlh3 and tlh4, which add to the already known tlh1 and tlh2, and found that the tlh1-4 genes show high sequence variation with missense mutations, insertions, and deletions but no severe effects on their RNA expression. Our results indicate that SH sequences are highly polymorphic and hot spots for genome variation. These features of subtelomeres may have contributed to genome diversity and, conversely, various diseases.

Telomere DNA length-dependent regulation of DNA replication timing at internal late replication origins.

DNA replication is initiated at replication origins on chromosomes at their scheduled time during S phase of the cell cycle. Replication timing control is highly conserved among eukaryotes but the underlying mechanisms are not fully understood. Recent studies have revealed that some telomere-binding proteins regulate replication timing at late-replicating origins throughout the genome. To investigate the molecular basis of this process, we analyzed the effects of excessive elongation of telomere DNA on replication timing by deleting telomere-associated shelterin proteins in Schizosaccharomyces pombe. We found that rap1∆ and poz1∆ cells showed abnormally accelerated replication at internal late origins but not at subtelomere regions. These defects were suppressed by removal of telomere DNA and by deletion of the telomere-binding protein Taz1. Furthermore, Sds21-a counter protein phosphatase against Dbf4-dependent kinase (DDK)-accumulated at elongated telomeres in a Taz1-dependent manner but was depleted at internal late origins, indicating that highly elongated telomeres sequester Sds21 at telomeres and perturb replication timing at internal regions. These results demonstrate that telomere DNA length is an important determinant of replication timing at internal regions of chromosomes in eukaryotes.

Casein kinase 2 regulates telomere protein complex formation through Rap1 phosphorylation.

Telomeres located at the ends of linear chromosomes play important roles in the maintenance of life. Rap1, a component of the shelterin telomere protein complex, interacts with multiple proteins to perform various functions; further, formation of shelterin requires Rap1 binding to other components such as Taz1 and Poz1, and telomere tethering to the nuclear envelope (NE) involves interactions between Rap1 and Bqt4, a nuclear membrane protein. Although Rap1 is a hub for telomere protein complexes, the regulatory mechanisms of its interactions with partner proteins are not fully understood. Here, we show that Rap1 is phosphorylated by casein kinase 2 (CK2) at multiple sites, which promotes interactions with Bqt4 and Poz1. Among the multiple CK2-mediated phosphorylation sites of Rap1, phosphorylation at Ser496 was found to be crucial for both Rap1-Bqt4 and Rap1-Poz1 interactions. These mechanisms mediate proper telomere tethering to the NE and the formation of the silenced chromatin structure at chromosome ends.

Alpha satellite DNA-repeat OwlAlp1 forms centromeres in Azara's owl monkey.

Centromeres play crucial roles in faithful chromosome segregation and genome integrity. In simian primates, centromeres possess tandem array of alpha satellite DNA (also referred to as alphoid DNA). Average sizes of alpha satellite repeat units vary between species, for example, 171 bp in human and 343-344 bp in many platyrrhini species (New World monkeys). Interestingly, Azara's owl monkey (Aotus azarae), a platyrrhini species, possesses alpha satellite DNA of two distinct unit sizes, OwlAlp1 (185 bp) and OwlAlp2 (344 bp), both of which present as megasatellite DNAs in the genome. It is, however, unknown which repeat sequence is responsible for functional centromere formation. To investigate the localization of centromeres in vivo, we carried out chromatin immunoprecipitation (ChIP) assay using Azara's owl monkey cells. We found that CENP-A, a histone H3 variant essential for centromere formation, was enriched at OwlAlp1, but not at OwlAlp2. Moreover, CENP-A was detected only at constricted regions of chromosomes by immunofluorescent microscopy. In contrast, trimethylation of histone H3-K9 (H3K9me3), a marker of heterochromatin, was enriched at both OwlAlp1 and OwlAlp2. Our results show that the shorter alpha satellite repeat, OwlAlp1, is selectively used for centromere formation in this monkey.

The Inner Nuclear Membrane Protein Bqt4 in Fission Yeast Contains a DNA-Binding Domain Essential for Telomere Association with the Nuclear Envelope.

Telomeres, the protective caps at the end of the chromosomes, are often associated with the nuclear envelope (NE). Telomere positioning to the NE is dynamically regulated during mitosis and meiosis. One inner nuclear membrane protein, Bqt4, in Schizosaccharomyces pombe plays essential roles in connecting telomeres to the NE. However, the structural basis of Bqt4 in mediating telomere-NE association is not clear. Here, we report the crystal structure of the N-terminal domain of Bqt4. The N-terminal domain of Bqt4 structurally resembles the APSES-family DNA-binding domain and has a moderate double-stranded DNA-binding activity. Disruption of Bqt4-DNA interaction results in telomere detachment from the NE. These data suggest that the DNA-binding activity of Bqt4 may function to prime the chromosome onto the NE and promote telomere-NE association.

Structural insights into chromosome attachment to the nuclear envelope by an inner nuclear membrane protein Bqt4 in fission yeast.

The dynamic association of chromosomes with the nuclear envelope (NE) is essential for chromosome maintenance. Schizosaccharomyces pombe inner nuclear membrane protein Bqt4 plays a critical role in connecting telomeres to the NE, mainly through a direct interaction with the telomeric protein Rap1. Bqt4 also interacts with Lem2 for pericentric heterochromatin maintenance. How Bqt4 coordinates the interactions with different proteins to exert their functions is unclear. Here, we report the crystal structures of the N-terminal domain of Bqt4 in complexes with Bqt4-binding motifs from Rap1, Lem2, and Sad1. The structural, biochemical and cellular analyses reveal that the N-terminal domain of Bqt4 is a protein-interaction module that recognizes a consensus motif and plays essential roles in telomere-NE association and meiosis progression. Phosphorylation of Bqt4-interacting proteins may act as a switch to regulate these interactions during cell cycles. Our studies provide structural insights into the identification and regulation of Bqt4-mediated interactions.

Unexpected roles of a shugoshin protein at subtelomeres.

A chromosome is composed of structurally and functionally distinct domains. Telomeres, which are located at the ends of linear chromosomes, play crucial roles in genome stability. Although substantial knowledge of telomeres has been accumulated, the regulation and function of subtelomeres, which are the domains adjacent to telomeres, remain largely unknown. In this review, I describe recent discoveries about the multiple roles of a shugoshin family protein, Sgo2, which is localized at centromeres in mitosis and contributes to precise chromosome segregation, in defining chromatin structure and functions of the subtelomeres in fission yeast. Sgo2 becomes enriched at the subtelomeres, particularly during G2 phase, and is essential for the formation of a highly condensed subtelomeric chromatin body called the knob. Furthermore, Sgo2 maintains the expression levels of subtelomeric genes and the timing of DNA replication at subtelomeric late origins.

Structure of the fission yeast S. pombe telomeric Tpz1-Poz1-Rap1 complex.

Telomeric shelterin complex caps chromosome ends and plays a crucial role in telomere maintenance and protection. In the fission yeast Schizosaccharomyces pombe, shelterin is composed of telomeric single- and double-stranded DNA-binding protein subcomplexes Pot1-Tpz1 and Taz1-Rap1, which are bridged by their interacting protein Poz1. However, the structure of Poz1 and how Poz1 functions as an interaction hub in the shelterin complex remain unclear. Here we report the crystal structure of Poz1 in complex with Poz1-binding motifs of Tpz1 and Rap1. The crystal structure shows that Poz1 employs two different binding surfaces to interact with Tpz1 and Rap1. Unexpectedly, the structure also reveals that Poz1 adopts a dimeric conformation. Mutational analyses suggest that proper interactions between Tpz1, Poz1, and Rap1 in the shelterin core complex are required for telomere length homeostasis and heterochromatin structure maintenance at telomeres. Structural resemblance between Poz1 and the TRFH domains of other shelterin proteins in fission yeast and humans suggests a model for the evolution of shelterin proteins.

Subtelomeres constitute a safeguard for gene expression and chromosome homeostasis.

The subtelomere, a telomere-adjacent chromosomal domain, contains species-specific homologous DNA sequences, in addition to various genes. However, the functions of subtelomeres, particularly subtelomeric homologous (SH) sequences, remain elusive. Here, we report the first comprehensive analyses of the cellular functions of SH sequences in the fission yeast, Schizosaccharomyces pombe. Complete removal of SH sequences from the genome revealed that they are dispensable for mitosis, meiosis and telomere length control. However, when telomeres are lost, SH sequences prevent deleterious inter-chromosomal end fusion by facilitating intra-chromosomal circularization. Surprisingly, SH-deleted cells sometimes survive telomere loss through inter-chromosomal end fusions via homologous loci such as LTRs, accompanied by centromere inactivation of either chromosome. Moreover, SH sequences function as a buffer region against the spreading of subtelomeric heterochromatin into the neighboring gene-rich regions. Furthermore, we found a nucleosome-free region at the subtelomeric border, which may be a second barrier that blocks heterochromatin spreading into the subtelomere-adjacent euchromatin. Thus, our results demonstrate multiple defense functions of subtelomeres in chromosome homeostasis and gene expression.

CK2 phospho-independent assembly of the Tel2-associated stress-signaling complexes in Schizosaccharomyces pombe.

An evolutionarily conserved protein Tel2 regulates a variety of stress signals. In mammals, TEL2 associates with TTI1 and TTI2 to form the Triple T (TTT: TEL2-TTI1-TTI2) complex as well as with all the phosphatidylinositol 3-kinase-like kinases (PIKKs) and the R2TP (Ruvbl1-Ruvbl2-Tah1-Pih1 in budding yeast)/prefoldin-like complex that associates with HSP90. The phosphorylation of TEL2 by casein kinase 2 (CK2) enables direct binding of PIHD1 (mammalian Pih1) to TEL2 and is important for the stability and the functions of PIKKs. However, the regulatory mechanisms of Tel2 in fission yeast Schizosaccharomyces pombe remain largely unknown. Here, we report that S. pombe Tel2 is phosphorylated by CK2 at Ser490 and Thr493. Tel2 forms the TTT complex with Tti1 and Tti2 and also associates with PIKKs, Rvb2, and Hsp90 in vivo; however, the phosphorylation of Tel2 affects neither the stability of the Tel2-associated proteins nor their association with Tel2. Thus, Tel2 stably associates with its binding partners irrespective of its phosphorylation. Furthermore, the Tel2 phosphorylation by CK2 is not required for the various stress responses to which PIKKs are pivotal. Our results suggest that the Tel2-containing protein complexes are conserved among eukaryotes, but the molecular regulation of their formation has been altered during evolution.

Shugoshin forms a specialized chromatin domain at subtelomeres that regulates transcription and replication timing.

A chromosome is composed of structurally and functionally distinct domains. However, the molecular mechanisms underlying the formation of chromatin structure and the function of subtelomeres, the telomere-adjacent regions, remain obscure. Here we report the roles of the conserved centromeric protein Shugoshin 2 (Sgo2) in defining chromatin structure and functions of the subtelomeres in the fission yeast Schizosaccharomyces pombe. We show that Sgo2 localizes at the subtelomeres preferentially during G2 phase and is essential for the formation of a highly condensed subtelomeric chromatin body 'knob'. Furthermore, the absence of Sgo2 leads to the derepression of the subtelomeric genes and premature DNA replication at the subtelomeric late origins. Thus, the subtelomeric specialized chromatin domain organized by Sgo2 represses both transcription and replication to ensure proper gene expression and replication timing.

Receptor for activated C-kinase (RACK1) homolog Cpc2 facilitates the general amino acid control response through Gcn2 kinase in fission yeast.

General amino acid control (GAAC) is crucial for sensing and adaptation to nutrient availability. Amino acid starvation activates protein kinase Gcn2, which plays a central role in the GAAC response by phosphorylating the α-subunit of eukaryotic initiation factor 2 (eIF2α), leading to the translational switch to stimulate selective expression of stress-responsive genes. We report here that in fission yeast Schizosaccharomyces pombe, Cpc2, a homolog of mammalian receptor for activated C-kinase (RACK1), is important for the GAAC response. Deletion of S. pombe cpc2 impairs the amino acid starvation-induced phosphorylation of eIF2α and the expression of amino acid biosynthesis genes, thereby rendering cells severely sensitive to amino acid limitation. Unlike the Saccharomyces cerevisiae Cpc2 ortholog, which normally suppresses the GAAC response, our findings suggest that S. pombe Cpc2 promotes the GAAC response. We also found that S. pombe Cpc2 is required for starvation-induced Gcn2 autophosphorylation, which is essential for Gcn2 function. These results indicate that S. pombe Cpc2 facilitates the GAAC response through the regulation of Gcn2 activation and provide a novel insight for the regulatory function of RACK1 on Gcn2-mediated GAAC response.