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The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively. The centromere-specific histone H3 variant CENP-A is sufficient for centromere specification in many species. Cleveland and colleagues have used an elegant gene targeting strategy to define a two-step mechanism for how CENP-A acts in centromere targeting and kinetochore assembly and function.

Heterochromatin that depends on histone H3 lysine 9 methylation (H3K9me) renders embedded genes transcriptionally silent 1 – 3 . In the fission yeast Schizosaccharomyces pombe , H3K9me heterochromatin can be transmitted through cell division provided the counteracting demethylase Epe1 is absent 4 , 5 . Heterochromatin heritability might allow wild-type cells under certain conditions to acquire epimutations, which could influence phenotype through unstable gene silencing rather than DNA change 6 , 7 . Here we show that heterochromatin-dependent epimutants resistant to caffeine arise in fission yeast grown with threshold levels of caffeine. Isolates with unstable resistance have distinct heterochromatin islands with reduced expression of embedded genes, including some whose mutation confers caffeine resistance. Forced heterochromatin formation at implicated loci confirms that resistance results from heterochromatin-mediated silencing. Our analyses reveal that epigenetic processes promote phenotypic plasticity, letting wild-type cells adapt to unfavourable environments without genetic alteration. In some isolates, subsequent or coincident gene-amplification events augment resistance. Caffeine affects two anti-silencing factors: Epe1 is downregulated, reducing its chromatin association, and a shortened isoform of Mst2 histone acetyltransferase is expressed. Thus, heterochromatin-dependent epimutation provides a bet-hedging strategy allowing cells to adapt transiently to insults while remaining genetically wild type. Isolates with unstable caffeine resistance show cross-resistance to antifungal agents, suggesting that related heterochromatin-dependent processes may contribute to resistance of plant and human fungal pathogens to such agents. Fission yeast grown in sublethal levels of caffeine develop heterochromatin-dependent epimutations conferring unstable heritable gene silencing that conveys resistance to caffeine, while remaining genetically wild type.

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin–CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs) 1 , 2 . However, the principles on which cyclin–CDK complexes organize the temporal order of cell cycle events are contentious 3 . One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events 4 – 6 . A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity 7 , 8 . In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events 9 , 10 . Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs. The core cell cycle is largely driven by increasing total CDK activity together with minor differences in the substrate specificity of the CDKs initiating DNA replication and mitosis.

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for faithful chromosome segregation in mitosis. Cohesin also has vital roles in DNA repair and transcriptional regulation. The ring-shaped cohesin complex is thought to encircle sister DNA strands, but its molecular mechanism of action is poorly understood and the biochemical reconstitution of cohesin activity in vitro has remained an unattained goal. Here we reconstitute cohesin loading onto DNA using purified fission yeast cohesin and its loader complex, Mis4 Scc2 –Ssl3 Scc4 ( Schizosaccharomyces pombe gene names appear throughout with their more commonly known Saccharomyces cerevisiae counterparts added in superscript). Incubation of cohesin with DNA leads to spontaneous topological loading, but this remains inefficient. The loader contacts cohesin at multiple sites around the ring circumference, including the hitherto enigmatic Psc3 Scc3 subunit, and stimulates cohesin’s ATPase, resulting in efficient topological loading. The in vitro reconstitution of cohesin loading onto DNA provides mechanistic insight into the initial steps of the establishment of sister chromatid cohesion and other chromosomal processes mediated by cohesin. Many DNA processes require chromosomes to be held together by a ring-shaped complex called cohesin, but despite the importance of this protein, its interaction with DNA has not been reproduced in vitro ; here, using purified yeast proteins, cohesin loading is successfully recapitulated, offering mechanistic insight into how the loader complex mediates topological cohesin binding. How cohesin runs rings around DNA Many cellular DNA processes require the chromosomes to be held together by a ring-shaped protein complex, cohesin. Despite its importance, this reaction had not been fully reproduced in vitro . Yasuto Murayama and Frank Uhlmann have now successfully reconstituted cohesin loading with purified fission yeast proteins. The data offer insight into how the loader complex mediates topological binding of cohesin on DNA, and set the stage for further mechanistic studies of how sister chromatid cohesion is established.

Bub1 is a multi-task protein kinase required for proper chromosome segregation in eukaryotes. Impairment of Bub1 in humans may lead to chromosomal instability (CIN) or tumorigenesis. Yet, the primary cellular substrate of Bub1 has remained elusive. Here, we show that Bub1 phosphorylates the conserved serine 121 of histone H2A in fission yeast Schizosaccharomyces pombe. The h2a-SA mutant, in which all cellular H2A-S121 is replaced by alanine, phenocopies the bub1 kinase-dead mutant (bub1-KD) in losing the centromeric localization of shugoshin proteins. Artificial tethering of shugoshin to centromeres largely restores the h2a-SA or bub1-KD-related CIN defects, a function that is evolutionally conserved. Thus, Bub1 kinase creates a mark for shugoshin localization and the correct partitioning of chromosomes.

At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments 1 . They do this either by first dismantling and later reassembling the nuclear envelope in an ‘open mitosis’ or by reshaping an intact nucleus and then dividing it into two in a ‘closed mitosis’ 2 , 3 . Mitosis has been studied in a wide variety of eukaryotes for more than a century 4 , but how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment remains unknown 5 . Here, using the fission yeast Schizosaccharomyces pombe (a classical model for closed mitosis 5 ), genetics, live-cell imaging and electron tomography, we show that nuclear fission is achieved via local disassembly of nuclear pores within the narrow bridge that links segregating daughter nuclei. In doing so, we identify the protein Les1, which is localized to the inner nuclear envelope and restricts the process of local nuclear envelope breakdown to the bridge midzone to prevent the leakage of material from daughter nuclei. The mechanism of local nuclear envelope breakdown in a closed mitosis therefore closely mirrors nuclear envelope breakdown in open mitosis 3 , revealing an unexpectedly high conservation of nuclear remodelling mechanisms across diverse eukaryotes. In a study performed in Schizosaccharomyces pombe , ‘closed mitosis’ is shown to occur via local disassembly of the nuclear envelope within the narrow bridge connecting segregating daughter nuclei, and a key role is identified for Les1, which restricts nuclear envelope breakdown to the bridge.

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity 1 . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3′-end formation and termination 2 , but how this sequence is initiated remains unclear. In a chemical–genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)–cyclin T1 (CycT1) complex 3 . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe . Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 4 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat 5 . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing 6 ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling—a switch that is analogous to a Cdk1–PP1 circuit that controls mitotic progression 4 . The kinase Cdk9 and the phosphatase Dis2 regulate the termination of transcription in fission yeast in part by controlling the phosphorylation state of the elongation factor Spt5.

The crystal structure of the Atg101–Atg13 complex elucidates the function of Atg101 and sheds light on the molecular mechanisms of autophagy initiation in higher eukaryotes. Atg101 is an essential component of the autophagy-initiating ULK complex in higher eukaryotes, but it is absent from the functionally equivalent Atg1 complex in budding yeast. Here, we report the crystal structure of the fission yeast Atg101–Atg13 complex. Atg101 has a Hop1, Rev7 and Mad2 (HORMA) architecture similar to that of Atg13. Mad2 HORMA has two distinct conformations (O-Mad2 and C-Mad2), and, intriguingly, Atg101 resembles O-Mad2 rather than the C-Mad2–like Atg13. Atg13 HORMA from higher eukaryotes possesses an inherently unstable fold, which is stabilized by Atg101 via interactions analogous to those between O-Mad2 and C-Mad2. Mutational studies revealed that Atg101 is responsible for recruiting downstream factors to the autophagosome-formation site in mammals via a newly identified WF finger. These data define the molecular functions of Atg101, providing a basis for elucidating the molecular mechanisms of mammalian autophagy initiation by the ULK complex.

Polarized cell growth necessitates the dynamic remodeling of the plasma membrane, a process requiring BAR domain-containing proteins. While classical BAR proteins, with their crescent-shaped structure, are well characterized, the mechanisms underlying the localization and function of elongated F-BAR proteins remain unclear. Here, we demonstrate that the F-BAR domains of the fission yeast proteins Rga7 and Rga8 undergo liquid-liquid phase separation (LLPS). These domains form oligomers via hydrophobic interactions and assemble into condensates through electrostatic interactions mediated by the charged residues at their tips. Mutants deficient in phase separation fail to localize properly at cell poles, leading to defective polar distribution of key regulators, including the Rho GTPases, the exocyst complex, and glucan synthases, all crucial for maintaining cell integrity. We further show that Rga8 requires the actin transport system for tip localization, whereas Rga7 accumulates at cell tips via diffusion. The absence of both Rga7 and Rga8 causes cell lysis. Hence, our findings establish LLPS as a fundamental mechanism for the cortical accumulation and function of F-BAR proteins, providing new insights into their role in membrane dynamics.

Septins can function as scaffolds for protein recruitment, membrane-bound diffusion barriers, or membrane curvature sensors. Septins are important for cytokinesis, but their exact roles are still obscure. In fission yeast, four septins (Spn1–Spn4) accumulate at the rim of the division plane as rings. The octameric exocyst complex, which tethers exocytic vesicles to the plasma membrane, exhibits a similar localization and is essential for plasma membrane deposition during cytokinesis. Without septins, the exocyst spreads across the division plane but is absent from the rim during septum formation. These results suggest that septins and the exocyst physically interact for proper localization and function. Indeed, we predicted six pairs of interactions between septin and exocyst subunits by AlphaFold, most of them are confirmed by co-immunoprecipitation and yeast two-hybrid assays. Exocyst mislocalization results in mistargeting of secretory vesicles and their cargos, which leads to cell-separation delay in septin mutants. Our results indicate that septins guide the targeting of the exocyst complex on the plasma membrane for vesicle tethering during cytokinesis through physical interactions.