Population genomics of the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has been widely used as a model eukaryote to study a diverse range of biological processes. However, population genetic studies of this species have been limited to date, and we know very little about the evolutionary processes and selective pressures that are shaping its genome. Here, we sequenced the genomes of 32 worldwide S. pombe strains and examined the pattern of polymorphisms across their genomes. In addition to introns and untranslated regions (UTRs), intergenic regions also exhibited lower levels of nucleotide diversity than synonymous sites, suggesting that a considerable amount of noncoding DNA is under selective constraint and thus likely to be functional. A number of genomic regions showed a reduction of nucleotide diversity probably caused by selective sweeps. We also identified a region close to the end of chromosome 3 where an extremely high level of divergence was observed between 5 of the 32 strains and the remain 27, possibly due to introgression, strong positive selection, or that region being responsible for reproductive isolation. Our study should serve as an important starting point in using a population genomics approach to further elucidate the biology of this important model organism

Noncompetitive Counteractions of DNA Polymerase ɛ and ISW2/yCHRAC for Epigenetic Inheritance of Telomere Position Effect in Saccharomyces cerevisiae

Relocation of euchromatic genes near the heterochromatin region often results in mosaic gene silencing. In Saccharomyces cerevisiae, cells with the genes inserted at telomeric heterochromatin-like regions show a phenotypic variegation known as the telomere-position effect, and the epigenetic states are stably passed on to following generations. Here we show that the epigenetic states of the telomere gene are not stably inherited in cells either bearing a mutation in a catalytic subunit (Pol2) of replicative DNA polymerase ɛ (Pol ɛ) or lacking one of the nonessential and histone fold motif-containing subunits of Pol ɛ, Dpb3 and Dpb4. We also report a novel and putative chromatin-remodeling complex, ISW2/yCHRAC, that contains Isw2, Itc1, Dpb3-like subunit (Dls1), and Dpb4. Using the single-cell method developed in this study, we demonstrate that without Pol ɛ and ISW2/yCHRAC, the epigenetic states of the telomere are frequently switched. Furthermore, we reveal that Pol ɛ and ISW2/yCHRAC function independently: Pol ɛ operates for the stable inheritance of a silent state, while ISW2/yCHRAC works for that of an expressed state. We therefore propose that inheritance of specific epigenetic states of a telomere requires at least two counteracting regulators

Coordinating upper limbs for octave playing on the piano via neuro-musculoskeletal modeling.

Understanding the coordination of multiple biomechanical degrees of freedom in biological organisms is crucial for unraveling the neurophysiological control of sophisticated motor tasks. This study focuses on the cooperative behavior of upper-limb motor movements in the context of octave playing on the piano. While the vertebrate locomotor system has been extensively investigated, the coherence and precision timing of rhythmic movements in the upper-limb system remain incompletely understood. Inspired by the spinal cord neuronal circuits (central pattern generator, CPG), a computational neuro-musculoskeletal model is proposed to explore the coordination of upper-limb motor movements during octave playing across varying tempos and volumes. The proposed model incorporates a CPG-based nervous system, a physiologically-informed mechanical body, and a piano environment to mimic human joint coordination and expressiveness. The model integrates neural rhythm generation, spinal reflex circuits, and biomechanical muscle dynamics while considering piano playing quality and energy expenditure. Based on real-world human subject experiments, the model has been refined to study tempo transitions and volume control during piano playing. This computational approach offers insights into the neurophysiological basis of upper-limb motor coordination in piano playing and its relation to expressive features

The roles of DNA polymerace ε and yCHRAC of budding yeast in epigenetic inheritance of telomere position effect

Relocation of euchromatic genes near a heterochromatin region often results in mosaic gene silencing in eukaryotes. In Saccharotmyces cerevisiae, when a wild-type gene is located near a telomere, it is subjected to telomere position-effect variegation (TPE), which includes transcriptional silencing and which provides heritable silent and expressed states as well as reversible switching between the epigenetic states. The silent state of a telomeric gene is attributable to heterochromatin-like structure, which is composed of several proteins such as Sir-proteins and hypoacetylated histones and which spreads from the telomeric end. While many modifiers of telomeric heterochromatin have been identified, the molecular nature for the switching and heritable propagation of epigenetic states is not well understood. To explain the stochastic nature of phenotypic variegation and stable inheritance of the epigenetic states, it has been proposed that these phenomena are attributed to competition between the assembly of heterochromatin and the establishment of active-chromatin. Because the silent state of a telomeric gene is altered to the expressed state by a trans-activator in G2/M-phase-arrested cells but not in G1- or early S-phase-arrested cells, it has been suggested that progression through the S-phase is required in TPE for switching from the silent to the expressed state. Furthermore, mutant forms of the replication protein PCNA are defective in silencing and interaction with CAF-1, a replication-coupled chromatin assembly factor, suggesting that DNA replication machinery is linked to silencing at heterochromatin. One of replicative polymerases, DNA polymerase ε (Pol ε), is composed of the catalytic-subunit Pol2, Dpb2, Dpb3 and Dpb4 in S. cerevisiae, and the feature of the subunit composition of Pol ε is evolutionally conserved in eukaryotes. Although Pol2 and Dpb2 are essential for DNA replication, Dpb3 and Dpb4, which contain the histone-fold motif related to chromatin metabolisms, are dispensable, and their function is not clear. To gain insight into possible roles of Pol ε in chromatin configuration, I examined TPE in mutant cells defective in Pol ε. In the assay of silencing with telomeric URA3, dpb3Δ, dpb4Δ and pol2-11 (C-terminal mutant of POL2) cells displayed a partial defect of silencing, and this defect was most evident in dpb3Δ cells. The silencing defect of pol2-11 cells was completely suppressed by simultaneous introduction of high copy DPB3 and DPB4. Moreover, the silencing level in the dpb3Δ dpb4Δ double mutant was similar to that in dpb4Δ, indicating that the dpb4Δ mutation is epistatic to the dpb3Δ mutation. In parallel, I also observed the expression of telomeric ADE2, which gave rise to red and white sector colonies in the wild-type strain because of mosaic gene silencing by TPE. I found that dpb4Δ mutant cells form non-sectoring light pink colonies and dpb3Δ mutant cells form white colonies. These results suggest that the mutations in Pol ε increase the switching frequency between alternative epigenetic states in TPE. To monitor the switching between a silent and an expressed state in each cell division, I developed a single-cell telomeric silencing assay, with which I could distinguish between a silent state (off) and an expressed state (on) of telomeric α2 gene in a single cell on an α-factor-containing medium. With this assay, I measured switching rates from "on" to "off\u27 and from "off\u27 to "on", and found that both switching rates increased in dpb4Δ cells, whereas in dpb3Δ cells the switching rate horn "off\u27 to "on" specifically increased. These results suggest that Dpb4 plays a role in the stable inheritance of both silent and expressed states, while Dpb3 is involved only in the stable inheritance of a silent state. The epistasis of dpb4Δ to dpb3Δ, together with different switching patterns in these mutants, suggests that Dpb4 is shared by Pol ε and an unknown complex that plays a counteracting role in TPE. I thus purified protein complexes containing Dpb4 using anti-Flag antibody and the 5Flag-epitope tagged Dpb4 protein, and found that two distinct protein complexes, Pol ε and yCHRAC, share Dpb4. yCHRAC is a putative homologue of the chromatin accessibility complex CHRAC in higher eukaryotes, and composed of a WAC-motif protein Itc1, an ISWI-chromatin remodeling factor homologue Isw2, a novel histone-fold protein Dpb31, and Dpb4. Since Pol ε and CHRAC in human cells also share a histone-fold protein that is a counterpart of Dpb4, it is suggested that the relationship between Pol ε and the CHRAC-like complex is evolutionally conserved. I next addressed whether yCHRAC counteracts Pol ε for TPE. In the assays with telomeric URA3 and ADE2, the itc1Δ and dpb31Δ mutations enhanced telomere silencing and restored it in dpb3Δ cells to the level of dpb4Δ cells, whereas they did not affect it in dpb4Δ cells. Moreover, in contrast to the dpb3Δ mutation, the dpb31Δ mutation increased the switching rate from "on" to "off\u27, but did not affect that from "off\u27 to "on". Therefore, these results suggest that yCHRAC regulates the stable inheritance of the expressed state of TPE independent of Pol ε, while yCHRAC and Pol ε counteract each other for TPE. In conclusion, position effect variegation at yeast telomeres is caused not only by simple competition between the assembly of heterochromatin and the establishment of active-chromatin but also by specific factors such as Pol ε and yCHRAC, which serve for the stable inheritance of the epigenetic states

Soft robotic tactile perception of softer objects based on learning of spatiotemporal pressure patterns

The softness perception of objects with lower stiffness than that of robotic skin is challenging, as the proportion of the deformation of skin to that of an object's surface is unknown. This makes it difficult to derive the indentation depth typically used for stiffness estimation. To overcome this challenge, we implemented human-inspired softness sensing in a soft anthropomorphic finger based on tactile information alone without using the information about indentation depth or displacement. In the experiments where LSTM networks were trained to discriminate viscoelastic soft objects, we demonstrated that the sensorized robotic finger using tactile information from barometric sensors embedded in its soft skin could successfully learn to discriminate soft objects. By dissociating the relative contribution of the dynamic pattern of pressure distribution and that of local pressure, we further investigated how differences in available tactile information could impact the ability to distinguish the softness of viscoelastic objects. The results demonstrated that the pressure distribution and its change on the soft contact area of the robotic finger provided information to discriminate the softness of viscoelastic objects and that the tactile information about softness was spatiotemporal in nature. The results further implied that nonlinear local dynamics such as hysteresis in local pressure changes can provide additional information about the viscoelasticity of touched objects.EPSRC EP/T00519X/1 SHERO project Horizon2020 (FET) 828818 Horizon 2020 101034337 JSPS KAKENHI (JP21H05823, JP21KK0182, JP22H00988

Cellular responses to compound stress induced by atmospheric-pressure plasma in fission yeast

ORCID　0000-0002-2401-4808The stress response is one of the most fundamental cellular processes. Although the molecular mechanisms underlying responses to a single stressor have been extensively studied, cellular responses to multiple stresses remain largely unknown. Here, we characterized fission yeast cellular responses to a novel stress inducer, non-thermal atmospheric-pressure plasma. Plasma irradiation generates ultraviolet radiation, electromagnetic fields and a variety of chemically reactive species simultaneously, and thus can impose multiple stresses on cells. We applied direct plasma irradiation to fission yeast and showed that strong plasma irradiation inhibited fission yeast growth. We demonstrated that mutants lacking sep1 and ace2, both of which encode transcription factors required for proper cell separation, were resistant to plasma irradiation. Sep1-target transcripts were downregulated by mild plasma irradiation. We also demonstrated that plasma irradiation inhibited the target of rapamycin kinase complex 1 (TORC1). These observations indicate that two pathways, namely the Sep1-Ace2 cell separation pathway and TORC1 pathway, operate when fission yeast cope with multiple stresses induced by plasma irradiation

Two different Argonaute complexes are required for siRNA generation and heterochromatin assembly in fission yeast

The RNA-induced transcriptional silencing (RITS) complex, containing Ago1, Chp1, Tas3 and centromeric small interfering RNAs (siRNAs), is required for heterochromatic gene silencing at centromeres. Here, we identify a second fission yeast Argonaute complex (Argonaute siRNA chaperone, ARC), which contains, in addition to Ago1, two previously uncharacterized proteins, Arb1 and Arb2, both of which are required for histone H3 Lys9 (H3-K9) methylation, heterochromatin assembly and siRNA generation. Furthermore, whereas siRNAs in the RITS complex are mostly single-stranded, siRNAs associated with ARC are mostly double-stranded, indicating that Arb1 and Arb2 inhibit the release of the siRNA passenger strand from Ago1. Consistent with this observation, purified Arb1 inhibits the slicer activity of Ago1 in vitro, and purified catalytically inactive Ago1 contains only double-stranded siRNA. Finally, we show that slicer activity is required for the siRNA-dependent association of Ago1 with chromatin and for the spreading of histone H3-K9 methylation

MOESM1 of RNAi-dependent heterochromatin assembly in fission yeast Schizosaccharomyces pombe requires heat-shock molecular chaperones Hsp90 and Mas5

Additional file 1: Figure S1. Fission yeast Hsp70 proteins and their homologs. (A) Phylogenic tree of Hsp70 proteins. Scale-bar unit indicates the number of amino acid substitutions per site. Names of proteins are associated with two-letter abbreviations and color-coded to indicate the species: “sp” for Schizosaccharomyces pombe (red), “sc” for Saccharomyces cerevisiae (black), and “dm” for Drosophila melanogaster (blue). (B) Domain structure of Hsp70 proteins. Protein names are depicted as in (A) and their amino acid lengths are shown. Protein domains are drawn as boxes according to the CATH-Gene3D classification. The N-terminal ATPase domains (ATPase) are composed of three internal domains that belong to the CATH superfamilies 3.30.420.40, 3.30.30.30, and 3.90.640.10. Substrate-binding domains (SB) belong to the 2.60.34.10 superfamily. The C-terminal lid domains (Lid) belong to the 1.20.1270.10 superfamily. Predicted localization signals for mitochondria (TargetP-M) and for the endoplasmic reticulum or beyond (TargetP-S) are shown as shaded boxes. Locations of the proteins in the cell are indicated. For clarity, D. melanogaster Hsp70 proteins other than Hsc70-4 are omitted. Figure S2. Hsp40 family proteins in fission yeast. Domain structure of all 26 fission yeast Hsp40 proteins are shown. Protein names and amino acid lengths are indicated. Protein domains are drawn as boxes according to the CATH-Gene3D, Pfam, and Prosite classifications. Predicted localization signals are shown as in Additional file 1: Figure S1. Predicted trans-membrane helix regions (TMhelix) are indicated as gray boxes. The DnaJ domains belong to the CATH superfamily 1.10.287.110. Type-I Hsp40 proteins are characterized by C-terminal (purple) and central (pink) domains that belong to the CATH superfamilies 2.60.260.20 and 2.10.230.10, respectively. The type-II Hsp40 protein Psi1 also contains the C-terminal domain but lacks the central domain. The other proteins are classified as the type-III Hsp40 proteins. Figure S3. Fission yeast type-I and -II Hsp40 proteins and their homologs. (A) Phylogenic tree of Hsp40 proteins. Scale-bar unit indicates the number of amino acid substitutions per site. Names of proteins are depicted as in Additional file 1: Figure S1. (B) Domain structure of Hsp40 proteins. Protein names are depicted as in (A) and amino acid lengths are shown. Protein domains and predicted localization signals are drawn as in Additional file 1: Figure S2. Locations of proteins in the cell are indicated. For clarity, D. melanogaster Hsp40 proteins other than Droj2 are omitted. Figure S4. Schematic representation of marker integration sites. The ade6+ and ura4+ marker genes were located in the SphI site of otr1R (otr1R(SphI)::ade6+) and in the NcoI site of imr1L (imr1L(NcoI)::ura4+), respectively. Figure S5. Hsp90 and Mas5 are dispensable for red pigment formation. Cells were spotted on normal YES plates (YES) and YES containing limited amount of adenine (Low adenine), and incubated at 30°C for six days. Figure S6. Derepression of marker genes inserted in the pericentromere. (A) Cells were serially diluted, spotted on normal EMMS plates and EMMS lacking adenine (EMMS - adenine) or uracil (EMMS - uracil), and incubated at 30°C for 6 days. (B) Strand-specific RT-qPCR for the ura4 marker gene. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 3). (C) Strand-specific RT-qPCR for the ade6 and ura4 genes located in the endogenous loci (ade6+ and ura4+) and in the pericentromere (otr1R::ade6+ and imr1L::ura4+). Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 3). **P < 0.01 (Student’s t-test). Figure S7. Hsp90 and Mas5 are dispensable for the silencing at the mating-type locus. Strand-specific RT-qPCR for the cenH transcript from the mating-type locus. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 6). **P < 0.01 (Student’s t-test). Figure S8. Reduction of Ago1 protein level in hsp90-A4 and mas5∆ cells. Two-fold serially diluted whole-cell extracts were separated by SDS-PAGE followed by western blotting and Coomassie brilliant blue staining. The membrane for detecting FLAG-Ago1 with an antibody against FLAG epitope was reprobed with an antibody against α-tubulin. Figure S9. mRNA expression levels of arb1 and tas3 genes. Strand-specific RT-qPCR for the arb1 and tas3 genes. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 6). *P < 0.05, **P < 0.01 (Student’s t-test). Figure S10. Protein expression level of Tas3. Detection of proteins in whole-cell extracts by western blotting. The membrane for detecting Tas3-Myc with an antibody against Myc epitope was reprobed with an antibody against α-tubulin to confirm that equal amounts of samples were loaded. For the mas5 deletion, four independently constructed clones were tested. Figure S11. Colony colors of otr1R::ade6+ strains are dependent on the ade6 alleles in the endogenous ade6 locus. Cells were streaked on normal YES plates (YES) and YES containing limited amount of adenine (Low adenine), and incubated at 30°C for four days. Strains with otr1R::ade6+ in the ade6-DN/N genetic background formed darker colonies. Note that the colony colors of canonical heterochromatin mutants (i.e. clr4∆) in the ade6-m210 background were faint pink, and are not as white as those of strains with the native ade6+ allele in the endogenous ade6 locus. Figure S12. Reintroduction of the R33C mutation phenocopied the original A4 isolate. (A) Schematic diagram showing the introduction of the mutation (R33C) found in the original A4 isolate. (B) Cells were streaked on normal YES plates (YES) and YES containing limited amount of adenine (Low adenine), and incubated at 30°C for four days. In HKM-1565 and HKM-1618, the G418-resistant kanMX cassette was located upstream of the hsp90 promoter. Both strains were generated by introducing kanMX-containing DNA fragments with or without the R33C mutation into the genome of the wild-type strain HKM-1100. Figure S13. Silver staining of immunoprecipitated proteins that were analyzed by mass spectrometry. Immunoprecipitates from indicated cells were separated by SDS-PAGE and silver-stained (see Methods). Each lane in the gel was sliced as indicated by red lines. Proteins in each gel piece were analyzed by nano-liquid chromatography tandem mass spectrometry. Black arrows indicate visible protein bands that are absent or barely seen in the untagged control (no-tag)