Cdc14p/FEAR Pathway Controls Segregation of Nucleolus in S. cerevisiae by Facilitating Condensin Targeting to rDNA Chromatin in Anaphase

The condensin complex is the chief molecular machine of mitotic chromosome condensation. Nucleolar concentration of condensin in mitosis was previously shown to correlate with proficiency of rDNA condensation and segregation. To uncover the mechanisms facilitating this targeting we conducted a screen for mutants that impair mitotic condensin congression to the nucleolus. Mutants in the cdc14, esp1 and cdc5 genes, which encode FEAR-network components, showed the most prominent defects in mitotic condensin localization. We established that Cdc14p activity released by the FEAR pathway was required for proper condensin-to-rDNA targeting in anaphase. The MEN pathway was dispensable for condensin-to-rDNA targeting, however MEN-mediated release of Cdc14p later in anaphase allowed for proper, albeit delayed, condensin targeting to rDNA and successful segregation of nucleolus in the slk19 FEAR mutant. Although condensin was physically dislodged from rDNA in the cdc14 mutant, it was properly assembled, phosphorylated and chromatin-bound, suggesting that condensin was mistargeted but active. This study identifies a novel pathway promoting condensin targeting to a specific chromosomal address, the rDNA locus.     

Cdc14 phosphatase induces rDNA condensation and resolves cohesin-independent cohesion during budding yeast anaphase

At anaphase onset, the protease separase triggers chromosome segregation by cleaving the chromosomal cohesin complex. Here, we show that cohesin destruction in metaphase is sufficient for segregation of much of the budding yeast genome, but not of the long arm of chromosome XII that contains the rDNA repeats. rDNA in metaphase, unlike most other sequences, remains in an undercondensed and topologically entangled state. Separase, concomitantly with cleaving cohesin, activates the phosphatase Cdc14. We find that Cdc14 exerts two effects on rDNA, both mediated by the condensin complex. Lengthwise condensation of rDNA shortens the chromosome XII arm sufficiently for segregation. This condensation depends on the aurora B kinase complex. Independently of condensation, Cdc14 induces condensin-dependent resolution of cohesin-independent rDNA linkage. Cdc14-dependent sister chromatid resolution at the rDNA could introduce a temporal order to chromosome segregation.     

Cdc14 protein phosphatase and topoisomerase II mediate rDNA dynamics and nucleophagic degradation of nucleolar proteins after TORC1 inactivation

Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase elicits nucleophagy degrading nucleolar proteins in budding yeast. After TORC1 inactivation, nucleolar proteins are relocated to sites proximal to the nucleus–vacuole junction (NVJ), where micronucleophagy occurs, whereas ribosomal DNA (rDNA encoding rRNA) escapes from the NVJ. Condensin-mediated rDNA condensation promotes the repositioning and nucleophagic degradation of nucleolar proteins. However, the molecular mechanism of TORC1 inactivation-induced chromosome condensation is still unknown. Here, we show that Cdc14 protein phosphatase and topoisomerase II (Topo II), which are engaged in rDNA condensation in mitosis, facilitate rDNA condensation after TORC1 inactivation. rDNA condensation after rapamycin treatment was compromised in cdc14-1 and top2-4 mutants. In addition, the repositioning of rDNA and nucleolar proteins and nucleophagic degradation of nucleolar proteins were impeded in these mutants. Furthermore, Cdc14 and Topo II were required for the survival of quiescent cells in prolonged nutrient-starved conditions. This study reveals that these factors are critical for starvation responses.     

Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA

Chromosome segregation is triggered by the cleavage of cohesins by separase. Here we show that in budding yeast separation of the ribosomal DNA (rDNA) and telomeres also requires Cdc14, a protein phosphatase known for its role in mitotic exit. Cdc14 shares this role with the FEAR network, which activates Cdc14 during early anaphase, but not the mitotic exit network, which promotes Cdc14 activity during late anaphase. We further show that CDC14 is necessary and sufficient to promote condensin enrichment at the rDNA locus and to trigger rDNA segregation in a condensin-dependent manner. We propose that Cdc14 released by the FEAR network mediates the partitioning of rDNA by facilitating the localization of condensin thereto. This dual role of the FEAR network in initiating mitotic exit and promoting chromosome segregation ensures that exit from mitosis is coupled to the completion of chromosome segregation.     

Putting the brake on FEAR: Tof2 promotes the biphasic release of Cdc14 phosphatase during mitotic exit

The completion of chromosome segregation during anaphase requires the hypercondensation of the ~1-Mb rDNA array, a reaction dependent on condensin and Cdc14 phosphatase. Using systematic genetic screens, we identified 29 novel genetic interactions with budding yeast condensin. Of these, FOB1, CSM1, LRS4, and TOF2 were required for the mitotic condensation of the tandem rDNA array localized on chromosome XII. Interestingly, whereas Fob1 and the monopolin subunits Csm1 and Lrs4 function in rDNA condensation throughout M phase, Tof2 was only required during anaphase. We show that Tof2, which shares homology with the Cdc14 inhibitor Net1/Cfi1, interacts with Cdc14 phosphatase and its deletion suppresses defects in mitotic exit network (MEN) components. Consistent with these genetic data, the onset of Cdc14 release from the nucleolus was similar in TOF2 and tof2Δ cells; however, the magnitude of the release was dramatically increased in the absence of Tof2, even when the MEN pathway was compromised. These data support a model whereby Tof2 coordinates the biphasic release of Cdc14 during anaphase by restraining a population of Cdc14 in the nucleolus after activation of the Cdc14 early anaphase release (FEAR) network, for subsequent release by the MEN.     

A Case of Selfish Nucleolar Segregation

Mitotic segregation of nucleolus in fission and budding yeast proceeds without disassembling its complex structure, creating challenging problems for transmission of nucleolus-organizing regions during nuclear division. The SMC complex called condensin, which plays a leading role in organizing mitotic structure of chromosomes in all eukaryotes, is essential for nucleolar segregation in budding yeast, where rDNA chromatin is the main target of mitotic condensin activity. Mitosis-specific condensin targeting to the nucleolus presents an attractive model to study mechanisms controlling condensin binding to specific chromatin domains. Recent reports suggest that the early-anaphase release of Cdc14 from the nucleolus (FEAR pathway) controls the proficiency of nucleolar segregation by promoting the mitotic condensin function in rDNA. This finding uncovers an essential function for the FEAR pathway and postulates the unique nucleolar self-regulatory mechanism, which evolved to recruit two essential enzymatic activities, Cdc14 phosphatase and condensin ATP-dependent supercoiling, for the specific task of segregating nucleoli without their disassembly.     

Nucleolus in the Spotlight

The recent contributions on chromatid segregation at the metaphase/anaphase transition demonstrate two distinct pathways in budding yeast. While segregation of most of the genome is a direct consequence of cohesin cleavage by separase, rDNA segregation requires a novel pathway involving Cdc14 phosphatase activation. This activation induces targeting of condensin to rDNA which in association with Aurora B kinase modulates rDNA compaction during anaphase. The resolution of rDNA sequences occurs after this step.     

Compacting DNA During the Interphase: Condensin Maintains rDNA Integrity

During mitosis, condensin is responsible for folding chromatin fibers into highly compact chromosomes, ensuring the faithful segregation of replicated chromosomes into daughter cells after each cell division. Our laboratory has unexpectedly found that condensin is capable of compacting DNA during the interphase: upon nutrient starvation, condensin is loaded to the rDNA array, leading to DNA condensation in this region. This subchromosomal DNA condensation appears to protect the integrity of the rDNA array. These observations provide the first microscopic evidence of DNA compaction by condensin outside mitosis. In addition, they show that condensin is also highly regulated during the interphase.     

The reverse, but coordinated, roles of Tor2 (TORC1) and Tor1 (TORC2) kinases for growth, cell cycle and separase-mediated mitosis in Schizosaccharomyces pombe

Target of rapamycin complexes (TORCs), which are vital for nutrient utilization, contain a catalytic subunit with the phosphatidyl inositol kinase-related kinase (PIKK) motif. TORC1 is required for cell growth, while the functions of TORC2 are less well understood. We show here that the fission yeast Schizosaccharomyces pombe TORC2 has a cell cycle role through determining the proper timing of Cdc2 Tyr15 dephosphorylation and the cell size under limited glucose, whereas TORC1 restrains mitosis and opposes securin-separase, which are essential for chromosome segregation. These results were obtained using the previously isolated TORC1 mutant tor2-L2048S in the phosphatidyl inositol kinase (PIK) domain and a new TORC2 mutant tor1-L2045D, which harbours a mutation in the same site. While mutated TORC1 and TORC2 displayed diminished kinase activity and FKBP12/Fkh1-dependent rapamycin sensitivity, their phenotypes were nearly opposite in mitosis. Premature mitosis and the G2-M delay occurred in TORC1 and TORC2 mutants, respectively. Surprisingly, separase/cut1-securin/cut2 mutants were rescued by TORC1/tor2-L2048S mutation or rapamycin addition or even Fkh1 deletion, whereas these mutants showed synthetic defect with TORC2/tor1-L2045D. TORC1 and TORC2 coordinate growth, mitosis and cell size control, such as Wee1 and Cdc25 do for the entry into mitosis.     

Lte1, Cdc14 and MEN‐controlled Cdk inactivation in yeast coordinate rDNA decompaction with late telophase progression

The mechanism of chromatin compaction in mitosis has been well studied, while little is known about what controls chromatin decompaction in early G1 phase. We have localized the Condensin subunit Brn1 to a compact spiral of rDNA in mitotic budding yeast cells. Brn1 release and the resulting rDNA decompaction in late telophase coincided with mitotic spindle dissociation, and occurred asymmetrically (daughter cells first). We immunoprecipitated the GTP‐exchange factor Lte1, which helps activate the mitotic exit network (MEN) in anaphase, with mitotic Brn1. In lteΔ cells Brn1 release was delayed, even at temperatures that do not impair mitotic exit. Mutations in MEN pathway components that act downstream of Lte1 similarly delayed rDNA decompaction. We found that Brn1 release in wild‐type cells coincided with the release of Cdc14 phosphatase from the nucleolus and with mitotic CDK inactivation, yet it could be selectively delayed by perturbation of the MEN pathway. This may argue that different levels of Cdk inactivation control spindle disassembly and chromatin decompaction. Mutation of lte1 also impaired rotation of the nucleus in early G1.     

Transcription of ribosomal genes can cause nondisjunction

Mitotic disjunction of the repetitive ribosomal DNA (rDNA) involves specialized segregation mechanisms dependent on the conserved phosphatase Cdc14. The reason behind this requirement is unknown. We show that rDNA segregation requires Cdc14 partly because of its physical length but most importantly because a fraction of ribosomal RNA (rRNA) genes are transcribed at very high rates. We show that cells cannot segregate rDNA without Cdc14 unless they undergo genetic rearrangements that reduce rDNA copy number. We then demonstrate that cells with normal length rDNA arrays can segregate rDNA in the absence of Cdc14 as long as rRNA genes are not transcribed. In addition, our study uncovers an unexpected role for the replication barrier protein Fob1 in rDNA segregation that is independent of Cdc14. These findings demonstrate that highly transcribed loci can cause chromosome nondisjunction.     

Dissection of the essential steps for condensin accumulation at kinetochores and rDNAs during fission yeast mitosis

The condensin complex has a fundamental role in chromosome dynamics. In this study, we report that accumulation of Schizosaccharomyces pombe condensin at mitotic kinetochores and ribosomal DNAs (rDNAs) occurs in multiple steps and is necessary for normal segregation of the sister kinetochores and rDNAs. Nuclear entry of condensin at the onset of mitosis requires Cut15/importin alpha and Cdc2 phosphorylation. Ark1/aurora and Cut17/Bir1/survivin are needed to dock the condensin at both the kinetochores and rDNAs. Furthermore, proteins that are necessary to form the chromatin architecture of the kinetochores (Mis6, Cnp1, and Mis13) and rDNAs (Nuc1 and Acr1) are required for condensin to accumulate specifically at these sites. Acr1 (accumulation of condensin at rDNA 1) is an rDNA upstream sequence binding protein that physically interacts with Rrn5, Rrn11, Rrn7, and Spp27 and is required for the proper accumulation of Nuc1 at rDNAs. The mechanism of condensin accumulation at the kinetochores may be conserved, as human condensin II fails to accumulate at kinetochores in hMis6 RNA interference-treated cells.     

Cdc14 Phosphatase Promotes TORC1-Regulated Autophagy in Yeast

Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.     

Condensin-dependent rDNA decatenation introduces a temporal pattern to chromosome segregation

The chromosomal condensin complex gives metaphase chromosomes structural stability. In addition, condensin is required for sister-chromatid resolution during their segregation in anaphase [1-7]. How condensin promotes chromosome resolution is poorly understood. Chromosome segregation during anaphase also fails after inactivation of topoisomerase II (topo II), the enzyme that removes catenation between sister chromatids left behind after completion of DNA replication [8, 9]. This has led to the proposal that condensin promotes DNA decatenation [3, 10, 11], but direct evidence for this is missing and alternative roles for condensin in chromosome resolution have been suggested [12-14]. Using the budding-yeast rDNA as a model, we now show that anaphase bridges in a condensin mutant are resolved by ectopic expression of a foreign (Chlorella virus) but not endogenous topo II. This suggests that catenation prevents sister-rDNA segregation but that yeast topo II is ineffective in decatenating the locus without condensin. Condensin and topo II colocalize along both rDNA and euchromatin, consistent with coordination of their activities. We investigate the physiological consequences of condensin-dependent rDNA decatenation and find that late decatenation determines the late segregation timing of this locus during anaphase. Regulation of decatenation therefore provides a means to fine tune the segregation timing of chromosomes in mitosis.     

Chromosome morphogenesis: condensin-dependent cohesin removal during meiosis

During meiosis, segregation of homologous chromosomes necessitates the coordination of sister chromatid cohesion, chromosome condensation, and recombination. Cohesion and condensation require the SMC complexes, cohesin and condensin, respectively. Here we use budding yeast Saccharomyces cerevisiae to show that condensin and Cdc5, a Polo-like kinase, facilitate the removal of cohesin from chromosomes prior to the onset of anaphase I when homologs segregate. This cohesin removal is critical for homolog segregation because it helps dissolve the recombination-dependent links between homologs that form during prophase I. Condensin enhances the association of Cdc5 with chromosomes and its phosphorylation of cohesin, which in turn likely stimulates cohesin removal. Condensin/Cdc5-dependent removal of cohesin underscores the potential importance of crosstalk between chromosome structural components in chromosome morphogenesis and provides a mechanism to couple chromosome morphogenesis with other meiotic events.     

Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle

Duplication and segregation of chromosomes involves dynamic reorganization of their internal structure by conserved architectural proteins, including the structural maintenance of chromosomes (SMC) complexes cohesin and condensin. Despite active investigation of the roles of these factors, a genome‐wide view of dynamic chromosome architecture at both small and large scale during cell division is still missing. Here, we report the first comprehensive 4D analysis of the higher‐order organization of the Saccharomyces cerevisiae genome throughout the cell cycle and investigate the roles of SMC complexes in controlling structural transitions. During replication, cohesion establishment promotes numerous long‐range intra‐chromosomal contacts and correlates with the individualization of chromosomes, which culminates at metaphase. In anaphase, mitotic chromosomes are abruptly reorganized depending on mechanical forces exerted by the mitotic spindle. Formation of a condensin‐dependent loop bridging the centromere cluster with the rDNA loci suggests that condensin‐mediated forces may also directly facilitate segregation. This work therefore comprehensively recapitulates cell cycle‐dependent chromosome dynamics in a unicellular eukaryote, but also unveils new features of chromosome structural reorganization during highly conserved stages of cell division.