Condensin locates at transcriptional termination sites in mitosis, possibly releasing mitotic transcripts.

Condensin is an essential component of chromosome dynamics, including mitotic chromosome condensation and segregation, DNA repair, and development. Genome-wide localization of condensin is known to correlate with transcriptional activity. The functional relationship between condensin accumulation and transcription sites remains unclear, however. By constructing the auxin-inducible degron strain of condensin, herein we demonstrate that condensin does not affect transcription itself. Instead, RNA processing at transcriptional termination appears to define condensin accumulation sites during mitosis, in the fission yeast Schizosaccharomyces pombe. Combining the auxin-degron strain with the nda3 ß-tubulin cold-sensitive (cs) mutant enabled us to inactivate condensin in mitotically arrested cells, without releasing the cells into anaphase. Transcriptional activation and termination were not affected by condensin's degron-mediated depletion, at heat-shock inducible genes or mitotically activated genes. On the other hand, condensin accumulation sites shifted approximately 500 bp downstream in the auxin-degron of 5'-3' exoribonuclease Dhp1, in which transcripts became aberrantly elongated, suggesting that condensin accumulates at transcriptionally terminated DNA regions. Growth defects in mutant strains of 3'-processing ribonuclease and polyA cleavage factors were additive in condensin temperature-sensitive (ts) mutants. Considering condensin's in vitro activity to form double-stranded DNAs from unwound, single-stranded DNAs or DNA-RNA hybrids, condensin-mediated processing of mitotic transcripts at the 3'-end may be a prerequisite for faithful chromosome segregation.

Coordinated Roles of the Putative Ceramide-Conjugation Protein, Cwh43, and a Mn2+-Transporting, P-Type ATPase, Pmr1, in Fission Yeast.

Genetically controlled mechanisms of cell division and quiescence are vital for responding to changes in the nutritional environment and for cell survival. Previously, we have characterized temperature-sensitive (ts) mutants of the cwh43 gene in fission yeast, Schizosaccharomyces pombe, which is required for both cell proliferation and nitrogen starvation-induced G0 quiescence. Cwh43 encodes an evolutionarily conserved transmembrane protein that localizes in endoplasmic reticulum (ER). Defects in this protein fail to divide in low glucose and lose mitotic competence under nitrogen starvation, and also affect lipid metabolism. Here, we identified mutations of the pmr1 gene, which encodes an evolutionarily conserved Ca2+/Mn2+-transporting P-type ATPase, as potent extragenic suppressors of ts mutants of the cwh43 gene. Intriguingly, these pmr1 mutations specifically suppressed the ts phenotype of cwh43 mutants, among five P-type Ca2+- and/or Mn2+-ATPases reported in this organism. Cwh43 and Pmr1 co-localized in the ER. In cwh43 mutant cells, addition of excessive manganese to culture media enhanced the severe defect in cell morphology, and caused abnormal accumulation of a cell wall component, 1, 3-ß-glucan. In contrast, these abnormal phenotypes were abolished by deletion of the pmr1+ gene, as well as by removal of Mn2+ from the culture medium. Furthermore, nutrition-related phenotypes of cwh43 mutant cells were rescued in the absence of Pmr1. Our findings indicate that the cellular processes regulated by Cwh43 are appropriately balanced with Pmr1-mediated Mn2+ transport into the ER.

Negative Regulation of the Mis17-Mis6 Centromere Complex by mRNA Decay Pathway and EKC/KEOPS Complex in Schizosaccharomyces pombe.

The mitotic kinetochore forms at the centromere for proper chromosome segregation. Deposition of the centromere-specific histone H3 variant, spCENP-A/Cnp1, is vital for the formation of centromere-specific chromatin and the Mis17-Mis6 complex of the fission yeast Schizosaccharomyces pombe is required for this deposition. Here we identified extragenic suppressors for a Mis17-Mis6 complex temperature-sensitive (ts) mutant, mis17-S353P, using whole-genome sequencing. The large and small daughter nuclei phenotype observed in mis17-S353P was greatly rescued by these suppressors. Suppressor mutations in two ribonuclease genes involved in the mRNA decay pathway, exo2 and pan2, may affect Mis17 protein level, as mis17 mutant protein level was recovered in mis17-S353P exo2 double mutant cells. Suppressor mutations in EKC/KEOPS complex genes may not regulate Mis17 protein level, but restored centromeric localization of spCENP-A/Cnp1, Mis6 and Mis15 in mis17-S353P Therefore, the EKC/KEOPS complex may inhibit Mis17-Mis6 complex formation or centromeric localization. Mutational analysis in protein structure indicated that suppressor mutations in the EKC/KEOPS complex may interfere with its kinase activity or complex formation. Our results suggest that the mRNA decay pathway and the EKC/KEOPS complex negatively regulate Mis17-Mis6 complex-mediated centromere formation by distinct and unexpected mechanisms.

Casein kinase II-dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast.

DNA topoisomerase II (topo II) regulates the topological state of DNA and is necessary for DNA replication, transcription, and chromosome segregation. Topo II has essential functions in cell proliferation and therefore is a critical target of anticancer drugs. In this study, using Phos-tag SDS-PAGE analysis in fission yeast (Schizosaccharomyces pombe), we identified casein kinase II (Cka1/CKII)-dependent phosphorylation at the C-terminal residues Ser1363 and Ser1364 in topo II. We found that this phosphorylation decreases the inhibitory effect of an anticancer catalytic inhibitor of topo II, ICRF-193, on mitosis. Consistent with the constitutive activity of Cka1/CKII, Ser1363 and Ser1364 phosphorylation of topo II was stably maintained throughout the cell cycle. We demonstrate that ICRF-193-induced chromosomal mis-segregation is further exacerbated in two temperature-sensitive mutants, cka1-372 and cka1/orb5-19, of the catalytic subunit of CKII or in the topo II nonphosphorylatable alanine double mutant top2-S1363A,S1364A but not in cells of the phosphomimetic glutamate double mutant top2-S1363E,S1364E Our results suggest that Ser1363 and Ser1364 in topo II are targeted by Cka1/CKII kinase and that their phosphorylation facilitates topo II ATPase activity in the N-terminal region, which regulates protein turnover on chromosome DNA. Because CKII-mediated phosphorylation of the topo II C-terminal domain appears to be evolutionarily conserved, including in humans, we propose that attenuation of CKII-controlled topo II phosphorylation along with catalytic topo II inhibition may promote anticancer effects.

The putative ceramide-conjugation protein Cwh43 regulates G0 quiescence, nutrient metabolism and lipid homeostasis in fission yeast.

Cellular nutrient states control whether cells proliferate, or whether they enter or exit quiescence. Here, we report characterizations of fission yeast temperature-sensitive (ts) mutants of the evolutionarily conserved transmembrane protein Cwh43, and explore its relevance to utilization of glucose, nitrogen source and lipids. GFP-tagged Cwh43 localizes at ER associated with the nuclear envelope and the plasma membrane, as in budding yeast. We found that cwh43 mutants failed to divide in low glucose and lost viability during quiescence under nitrogen starvation. In cwh43 mutants, comprehensive metabolome analysis demonstrated dramatic changes in marker metabolites that altered under low glucose and/or nitrogen starvation, although cwh43 cells apparently consumed glucose in the culture medium. Furthermore, we found that cwh43 mutant cells had elevated levels of triacylglycerols (TGs) and coenzyme A, and that they accumulated lipid droplets. Notably, TG biosynthesis was required to maintain cell division in the cwh43 mutant. Thus, Cwh43 affects utilization of glucose and nitrogen sources, as well as storage lipid metabolism. These results may fit a notion developed in budding yeast stating that Cwh43 conjugates ceramide to glycosylphosphatidylinositol (GPI)-anchored proteins and maintains integrity of membrane organization.

ICRF-193, an anticancer topoisomerase II inhibitor, induces arched telophase spindles that snap, leading to a ploidy increase in fission yeast.

ICRF-193 [meso-4,4-(2,3-butanediyl)-bis(2,6-piperazinedione)] is a complex-stabilizing inhibitor of DNA topoisomerase II (topo II) that is used as an effective anticancer drug. ICRF-193 inhibits topo II catalytic activity in vitro and blocks nuclear division in vivo. Here, we examined the effects of ICRF-193 treatment on chromatin behavior and spindle dynamics using detailed live mitotic cell analysis in the fission yeast, Schizosaccharomyces pombe. Time-lapse movie analysis showed that ICRF-193 treatment leads to an elongation of presumed chromatin fibers connected to kinetochores during mid-mitosis. Anaphase spindles begin to arch, and eventually spindle poles come together abruptly, as if the spindle snapped at the point of spindle microtubule overlap in telophase. Segregating chromosomes appeared as elastic clumps and subsequently pulled back and merged. The snapped spindle phenotype was abolished by microtubule destabilization after thiabendazole treatment, accompanied by unequal chromosome segregation or severe defects in spindle extension. Thus, we conclude that ICRF-193-treated, unseparated sister chromatids pulling toward opposite spindle poles produce the arched and snapped telophase spindle. ICRF-193 treatment increased DNA content, suggesting that the failure of sister chromatids to separate properly in anaphase, causes the spindle to break in telophase, resulting in polyploidization.

RNA pol II transcript abundance controls condensin accumulation at mitotically up-regulated and heat-shock-inducible genes in fission yeast.

Condensin plays fundamental roles in chromosome dynamics. In this study, we determined the binding sites of condensin on fission yeast (Schizosaccharomyces pombe) chromosomes at the level of nucleotide sequences using chromatin immunoprecipitation (ChIP) and ChIP sequencing (ChIP-seq). We found that condensin binds to RNA polymerase I-, II- and III-transcribed genes during both mitosis and interphase, and we focused on pol II constitutive and inducible genes. Accumulation sites for condensin are distinct from those of cohesin and DNA topoisomerase II. Using cell cycle stage and heat-shock-inducible genes, we show that pol II-mediated transcripts cause condensin accumulation. First, condensin's enrichment on mitotically activated genes was abolished by deleting the sep1(+) gene that encodes an M-phase-specific forkhead transcription factor. Second, by raising the temperature, condensin accumulation was rapidly induced at heat-shock protein genes in interphase and even during mid-mitosis. In interphase, condensin accumulates preferentially during the postreplicative phase. Pol II-mediated transcription was neither repressed nor activated by condensin, as levels of transcripts per se did not change when mutant condensin failed to associate with chromosomal DNA. However, massive chromosome missegregation occurred, suggesting that abundant pol II transcription may require active condensin before proper chromosome segregation.