Fumie Masuda

Heterochromatin integrity affects chromosome reorganization after centromere dysfunction.

The centromere is essential for the inheritance of genetic information on eukaryotic chromosomes. Epigenetic regulation of centromere identity has been implicated in genome stability, karyotype evolution, and speciation. However, little is known regarding the manner in which centromere dysfunction affects the chromosomal architectures. Here we show that in the fission yeast Schizosaccharomyces pombe, the conditional deletion of the centromere produces survivors that carry either a neocentromere-acquired chromosome at the subtelomeric region or an acentric chromosome rescued by intertelomere fusion with either of the remaining chromosomes. The ratio of neocentromere formation to telomere fusion is considerably decreased by the inactivation of genes involved in RNA interference–dependent heterochromatin formation. By affecting the modes of chromosomal reorganization, the genomic distribution of heterochromatin may influence the fate of karyotype evolution.

Two distinct pathways responsible for the loading of CENP-A to centromeres in the fission yeast cell cycle

CENP-A is a centromere-specific histone H3 variant that is- essential for faithful chromosome segregation in all eukaryotes thus far investigated. We genetically identified two factors, Ams2 and Mis6, each of which is required for the correct centromere localization of SpCENP-A (Cnp1), the fission yeast homologue of CENP-A. Ams2 is a cell-cycle-regulated GATA factor that localizes on the nuclear chromatin, including on centromeres, during the S phase. Ams2 may be responsible for the replication-coupled loading of SpCENP-A by facilitating nucleosomal formation during the S phase. Consistently, overproduction of histone H4, but not that of H3, suppressed the defect of SpCENP-A localization in Ams2-deficient cells. We demonstrated the existence of at least two distinct phases for SpCENP-A loading during the cell cycle: the S phase and the late-G2 phase. Ectopically induced SpCENP-A was efficiently loaded onto the centromeres in G2-arrested cells, indicating that SpCENP-A probably undergoes replication-uncoupled loading after the completion of S phase. This G2 loading pathway of SpCENP-A may require Mis6, a constitutive centromere-binding protein that is also implicated in the Mad2-dependent spindle attachment checkpoint response. Here, we discuss the functional relationship between the flexible loading mechanism of CENP-A and the plasticity of centromere chromatin formation in fission yeast.

Epigenetic Inactivation and Subsequent Heterochromatinization of a Centromere Stabilize Dicentric Chromosomes

BACKGROUND The kinetochore is a multiprotein complex that forms on a chromosomal locus designated as the centromere, which links the chromosome to the spindle during mitosis and meiosis. Most eukaryotes, with the exception of holocentric species, have a single distinct centromere per chromosome, and the presence of multiple centromeres on a single chromosome is predicted to cause breakage and/or loss of that chromosome. However, some stably maintained non-Robertsonian translocated chromosomes have been reported, suggesting that the excessive centromeres are inactivated by an as yet undetermined mechanism. RESULTS We have developed systems to generate dicentric chromosomes containing two centromeres by fusing two chromosomes in fission yeast. Although the majority of cells harboring the artificial dicentric chromosome are arrested with elongated cell morphology in a manner dependent on the DNA structure checkpoint genes, a portion of the cells survive by converting the dicentric chromosome into a stable functional monocentric chromosome; either centromere was inactivated epigenetically or by DNA rearrangement. Mutations compromising kinetochore formation increased the frequency of epigenetic centromere inactivation. The inactivated centromere is occupied by heterochromatin and frequently reactivated in heterochromatin- or histone deacetylase-deficient mutants. CONCLUSIONS Chromosomes with multiple centromeres are stabilized by epigenetic centromere inactivation, which is initiated by kinetochore disassembly. Consequent heterochromatinization and histone deacetylation expanding from pericentric repeats to the central domain prevent reactivation of the inactivated centromere.

Hsk1- and SCFPof3-Dependent Proteolysis of S. pombe Ams2 Ensures Histone Homeostasis and Centromere Function

Summary Schizosaccharomyces pombe GATA factor Ams2 is responsible for cell cycle-dependent transcriptional activation of all the core histone genes peaking at G1/S phase. Intriguingly, its own protein level also fluctuates concurrently. Here, we show that Ams2 is ubiquitylated and degraded through the SCF (Skp1-Cdc53/Cullin-1-F-box) ubiquitin ligase, in which F box protein Pof3 binds this protein. Ams2 is phosphorylated at multiple sites, which is required for SCFPof3-dependent proteolysis. Hsk1/Cdc7 kinase physically associates with and phosphorylates Ams2. Even mild overexpression of Ams2 induces constitutive histone expression and chromosome instability, and its toxicity is exaggerated when Hsk1 function is compromised. This is partly attributable to abnormal incorporation of canonical H3 into the central CENP-A/Cnp1-rich centromere, thereby reversing specific chromatin structures to apparently normal nucleosomes. We propose that Hsk1 plays a vital role during post S phase in genome stability via SCFPof3-mediated degradation of Ams2, thereby maintaining centromere integrity.

Mechanisms of expression and translocation of major fission yeast glucose transporters regulated by CaMKK/phosphatases, nuclear shuttling, and TOR

Glucose transporters play a pivotal role in glucose homeostasis. The fission yeast high-affinity glucose transporter Ght5 is regulated with regard to transcription and localization via CaMKK and TOR pathways. These results clarify the evolutionarily conserved mechanisms underlying glucose homeostasis that prevent hyperglycemia in humans.

Glucose restriction induces transient G2 cell cycle arrest extending cellular chronological lifespan

While glucose is the fundamental source of energy in most eukaryotes, it is not always abundantly available in natural environments, including within the human body. Eukaryotic cells are therefore thought to possess adaptive mechanisms to survive glucose-limited conditions, which remain unclear. Here, we report a novel mechanism regulating cell cycle progression in response to abrupt changes in extracellular glucose concentration. Upon reduction of glucose in the medium, wild-type fission yeast cells undergo transient arrest specifically at G2 phase. This cell cycle arrest is dependent on the Wee1 tyrosine kinase inhibiting the key cell cycle regulator, CDK1/Cdc2. Mutant cells lacking Wee1 are not arrested at G2 upon glucose limitation and lose viability faster than the wild-type cells under glucose-depleted quiescent conditions, suggesting that this cell cycle arrest is required for extension of chronological lifespan. Our findings indicate the presence of a novel cell cycle checkpoint monitoring glucose availability, which may be a good molecular target for cancer therapy.

Characterisation of functional domains in fission yeast Ams2 that are required for core histone gene transcription

Histone gene expression is regulated in a cell cycle-dependent manner, with a peak at S phase, which is crucial for cell division and genome integrity. However, the detailed mechanisms by which expression of histone genes are tightly regulated remain largely unknown. Fission yeast Ams2, a GATA-type zinc finger motif-containing factor, is required for activation of S phase-specific core histone gene transcription. Here we report the molecular characterisation of Ams2. We show that the zinc finger motif in Ams2 is necessary to bind the histone gene promoter region and to activate histone gene transcription. An N-terminal region of Ams2 acts as a self-interaction domain. Intriguingly, N-terminally truncated Ams2 binds to the histone gene promoters, but does not fully activate histone gene transcription. These observations imply that Ams2 self-interactions are required for efficient core histone gene transcription. Moreover, we show that Ams2 interacts with Teb1, which itself binds to the core histone gene promoters. We discuss the relationships between Ams2 domains and efficient transcription of the core histone genes in fission yeast.