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An Amendment to this Article has been published and is linked from the HTML version of this paper.
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An Amendment to this Letter has been published and is linked from the HTML version of this paper.
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An Amendment to this Article has been published and is linked from the HTML version of this paper.
RESUMO
During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.
Assuntos
Meiose , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Fosfoproteínas/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Complexo Sinaptonêmico , CoesinasRESUMO
Cdb4 is a protein with unknown functions that binds to curved DNA in vitro in the fission yeast Schizosaccharomyces pombe. Homologues of Cdb4 were identified in a wide range of eukaryotes, including human Ebp1. Both S. pombe Cdb4 and human Ebp1 are nonpeptidase members of the methionine aminopeptidase family. It has been reported that Ebp1 homologues are involved in cell growth regulation and differentiation. However, opposing functions have also been considered and debated upon, and the precise biological functions of this conserved protein are largely unknown. S. pombe cdb4 is a nonessential gene, and no obvious phenotypes have been detected in cells with cdb4 gene deletion. In this study, we identified nup184, encoding a component of the nuclear pore complex, as a gene responsible for the synthetic lethal phenotype associated with cdb4. Furthermore, the synthetic lethal phenotype of Cdb4 was suppressed by over-expression of human Ebp1, suggesting that it has conserved crucial functions in S. pombe Cdb4 and human Ebp1. This synthetic lethal phenotype associated with Cdb4 and Nup184 provides a molecular genetics tool to study the functions of S. pombe Cdb4 and its conserved members of proteins, including human Ebp1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação a DNA/deficiência , Células HeLa , Humanos , Proteínas de Ligação a RNA/genética , Schizosaccharomyces/citologia , Mutações Sintéticas LetaisRESUMO
The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly in meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Sequência Conservada , Cinetocoros/metabolismo , Meiose , Animais , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Feminino , Humanos , Infertilidade/genética , Infertilidade/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Quinase 1 Polo-LikeRESUMO
In meiosis I, sister chromatids are captured by microtubules emanating from the same pole (mono-orientation), and centromeric cohesion is protected throughout anaphase. Shugoshin, which is localized to centromeres depending on the phosphorylation of histone H2A by Bub1 kinase, plays a central role in protecting meiotic cohesin Rec8 from separase cleavage. Another key meiotic kinetochore factor, meikin, may regulate cohesion protection, although the underlying molecular mechanisms remain elusive. Here, we show that fission yeast Moa1 (meikin), which associates stably with CENP-C during meiosis I, recruits Plo1 (polo-like kinase) to the kinetochores and phosphorylates Spc7 (KNL1) to accumulate Bub1. Consequently, in contrast to the transient kinetochore localization of mitotic Bub1, meiotic Bub1 persists at kinetochores until anaphase I. The meiotic Bub1 pool ensures robust Sgo1 (shugoshin) localization and cohesion protection at centromeres by cooperating with heterochromatin protein Swi6, which binds and stabilizes Sgo1. Furthermore, molecular genetic analyses show a hierarchical regulation of centromeric cohesion protection by meikin and shugoshin that is important for establishing meiosis-specific chromosome segregation. We provide evidence that the meiosis-specific Bub1 regulation is conserved in mouse.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Meiose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Animais , Adesão Celular , Células Cultivadas , Centrômero/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Cinetocoros , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Fosforilação , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento , Espermatócitos/citologia , Espermatócitos/metabolismo , Quinase 1 Polo-LikeRESUMO
Chromosome structure is dynamically regulated during cell division, and this regulation is dependent, in part, on condensin. The localization of condensin at chromosome arms is crucial for chromosome partitioning during anaphase. Condensin is also enriched at kinetochores but its precise role and loading machinery remain unclear. Here we show that fission yeast (Schizosaccharomyces pombe) kinetochore proteins Pcs1 and Mde4--homologues of budding yeast (Saccharomyces cerevisiae) monopolin subunits and known to prevent merotelic kinetochore orientation--act as a condensin 'recruiter' at kinetochores, and that condensin itself may act to clamp microtubule binding sites during metaphase. In addition to the regional recruitment factors, overall condensin association with chromatin is governed by the chromosomal passenger kinase Aurora B. Aurora-B-dependent phosphorylation of condensin promotes its association with histone H2A and H2A.Z, which we identify as conserved chromatin 'receptors' of condensin. Condensin phosphorylation and its deposition onto chromosome arms reach a peak during anaphase, when Aurora B kinase relocates from centromeres to the spindle midzone, where the separating chromosome arms are positioned. Our results elucidate the molecular basis for the spatiotemporal regulation of mitotic chromosome architecture, which is crucial for chromosome partitioning.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Mitose , Complexos Multiproteicos/metabolismo , Schizosaccharomyces/metabolismo , Aurora Quinase B , Aurora Quinases , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
During cell division microtubules capture chromosomes by binding to the kinetochore assembled in the centromeric region of chromosomes. In mitosis sister chromatids are captured by microtubules emanating from both spindle poles, a process called bipolar attachment, whereas in meiosis I sisters are attached to microtubules originating from one spindle pole, called monopolar attachment. For determining chromosome orientation, kinetochore geometry or structure might be an important target of regulation. However, the molecular basis of this regulation has remained elusive. Here we show the link between kinetochore orientation and cohesion within the centromere in fission yeast Schizosaccharomyces pombe by strategies developed to visualize the concealed cohesion within the centromere, and to introduce artificial tethers that can influence kinetochore geometry. Our data imply that cohesion at the core centromere induces the mono-orientation of kinetochores whereas cohesion at the peri-centromeric region promotes bi-orientation. Our study may reveal a general mechanism for the geometric regulation of kinetochores, which collaborates with previously defined tension-dependent reorientation machinery.
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Centrômero/metabolismo , Cinetocoros/metabolismo , Schizosaccharomyces/citologia , Centrômero/genética , Segregação de Cromossomos , Meiose , Microtúbulos/metabolismo , Mitose , Modelos BiológicosRESUMO
Nucleosomes containing the specific histone H3 variant CENP-A mark the centromere locus on each chromatin and initiate kinetochore assembly. For the common type of regional centromeres, little is known in molecular detail of centromeric chromatin organization, its propagation through cell division, and how distinct organization patterns may facilitate kinetochore assembly. Here, we show that in the fission yeast S. pombe, a relatively small number of CENP-A/Cnp1 nucleosomes are found within the centromeric core and that their positioning relative to underlying DNA varies among genetically homogenous cells. Consistent with the flexible positioning of Cnp1 nucleosomes, a large portion of the endogenous centromere is dispensable for its essential activity in mediating chromosome segregation. We present biochemical evidence that Cnp1 occupancy directly correlates with silencing of the underlying reporter genes. Furthermore, using a newly developed pedigree analysis assay, we demonstrated the epigenetic inheritance of Cnp1 positioning and quantified the rate of occasional repositioning of Cnp1 nucleosomes throughout cell generations. Together, our results reveal the plasticity and the epigenetically inheritable nature of centromeric chromatin organization.
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Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Nucleossomos/metabolismo , Schizosaccharomyces/genética , Autoantígenos/genética , Centrômero/ultraestrutura , Proteína Centromérica A , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Fúngicas/genética , Inativação Gênica , Genes Reporter , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Cinetocoros , Modelos Genéticos , Schizosaccharomyces/metabolismoRESUMO
The centromere of a chromosome is composed mainly of two domains, a kinetochore assembling core centromere and peri-centromeric heterochromatin regions. The crucial role of centromeric heterochromatin is still unknown, because even in simpler unicellular organisms such as the fission yeast Schizosaccharomyces pombe, the heterochromatin protein Swi6 (HP1 homologue) has several functions at centromeres, including silencing gene expression and recombination, enriching cohesin, promoting kinetochore assembly, and, ultimately, preventing erroneous microtubule attachment to the kinetochores. Here we show that the requirement of heterochromatin for mitotic chromosome segregation is largely replaced by forcibly enriching cohesin at centromeres in fission yeast. However, this enrichment of cohesin is not sufficient to replace the meiotic requirement for heterochromatin. We find that the heterochromatin protein Swi6 associates directly with meiosis-specific shugoshin Sgo1, a protector of cohesin at centromeres. A point mutation of Sgo1 (V242E), which abolishes the interaction with Swi6, impairs the centromeric localization and function of Sgo1. The forced centromeric localization of Sgo1 restores proper meiotic chromosome segregation in swi6 cells. We also show that the direct link between HP1 and shugoshin is conserved in human cells. Taken together, our findings suggest that the recruitment of shugoshin is the important primary role for centromeric heterochromatin in ensuring eukaryotic chromosome segregation.
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Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Homólogo 5 da Proteína Cromobox , Segregação de Cromossomos , Humanos , Meiose , Mitose , Ligação Proteica , Transporte Proteico , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , CoesinasRESUMO
In fission yeast, meiotic mono-orientation of sister kinetochores is established by cohesion at the core centromere, which is established by a meiotic cohesin complex and the kinetochore protein Moa1. The cohesin subunit Psm3 is acetylated by Eso1 and deacetylated by Clr6. We show that in meiosis, Eso1 is required for establishing core centromere cohesion during S phase, whereas Moa1 is required for maintaining this cohesion after S phase. The clr6-1 mutation suppresses the mono-orientation defect of moa1Δ cells, although the Clr6 target for this suppression is not Psm3. Thus, several acetylations are crucial for establishing and maintaining core centromere cohesion.
Assuntos
Meiose , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Acetilação , Replicação do DNA , Prófase Meiótica I , Modelos Biológicos , Mutação/genética , Subunidades Proteicas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Sister chromatid cohesion, mediated by a complex called cohesin, is crucial--particularly at centromeres--for proper chromosome segregation in mitosis and meiosis. In animal mitotic cells, phosphorylation of cohesin promotes its dissociation from chromosomes, but centromeric cohesin is protected by shugoshin until kinetochores are properly captured by the spindle microtubules. However, the mechanism of shugoshin-dependent protection of cohesin is unknown. Here we find a specific subtype of serine/threonine protein phosphatase 2A (PP2A) associating with human shugoshin. PP2A colocalizes with shugoshin at centromeres and is required for centromeric protection. Purified shugoshin complex has an ability to reverse the phosphorylation of cohesin in vitro, suggesting that dephosphorylation of cohesin is the mechanism of protection at centromeres. Meiotic shugoshin of fission yeast also associates with PP2A, with both proteins collaboratively protecting Rec8-containing cohesin at centromeres. Thus, we have revealed a conserved mechanism of centromeric protection of eukaryotic chromosomes in mitosis and meiosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Pareamento Cromossômico , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Meiose , Mitose , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Proteína Fosfatase 2 , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , CoesinasRESUMO
Meiosis is critically different from mitosis in that during meiosis, pairing and segregation of homologous chromosomes occur. During meiosis, the morphology of sister chromatids changes drastically, forming a prominent axial structure in the synaptonemal complex. The meiosis-specific cohesin complex plays a central role in the regulation of the processes required for recombination. In particular, the Rec8 subunit of the meiotic cohesin complex, which is conserved in a wide range of eukaryotes, has been analyzed for its function in modulating chromosomal architecture during the pairing and recombination of homologous chromosomes in meiosis. Here, we review the current understanding of Rec8 cohesin as a structural platform for meiotic chromosomes.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular/genética , Cromátides , Proteínas Cromossômicas não Histona/genética , Meiose/genética , CoesinasRESUMO
Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway.
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Domínios RING Finger , RNA Helicases/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Códon sem Sentido/metabolismo , Dados de Sequência Molecular , RNA Helicases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genéticaRESUMO
During meiosis, a single round of genome duplication is followed by two sequential rounds of chromosome segregation. Through this process, a diploid parent cell generates gametes with a haploid set of chromosomes. A characteristic of meiotic chromosome segregation is a stepwise loss of sister chromatid cohesion along chromosomal arms and at centromeres. Whereas arm cohesion plays an important role in ensuring homologue disjunction at meiosis I, persisting cohesion at pericentromeric regions throughout meiosis I is essential for the faithful equational segregation of sisters in the following meiosis II, similar to mitosis. A widely conserved pericentromeric protein called shugoshin, which associates with protein phosphatase 2A (PP2A), plays a critical role in this protection of cohesin. Another key aspect of meiosis I is the establishment of monopolar attachment of sister kinetochores to spindle microtubules. Cohesion or physical linkage at the core centromeres, where kinetochores assemble, may conjoin sister kinetochores, leading to monopolar attachment. A meiosis-specific kinetochore factor such as fission yeast Moa1 or budding yeast monopolin contributes to this regulation. We propose that cohesion at the core centromere and pericentromeric regions plays distinct roles, especially in defining the orientation of kinetochores.
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Centrômero/fisiologia , Segregação de Cromossomos/fisiologia , Cromossomos/fisiologia , Meiose/fisiologia , Animais , Proteínas de Ciclo Celular/fisiologia , Centrômero/ultraestrutura , Cromátides/fisiologia , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/fisiologia , Cromossomos/ultraestrutura , Cromossomos Fúngicos/fisiologia , Cromossomos Fúngicos/ultraestrutura , Endopeptidases/fisiologia , Humanos , Cinetocoros/fisiologia , Cinetocoros/ultraestrutura , Mitose/fisiologia , Modelos Genéticos , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Plantas/genética , Proteína Fosfatase 2/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiologia , Separase , Vertebrados , CoesinasRESUMO
The nuclear envelope (NE) consists of the inner and outer nuclear membranes (INM and ONM), and the nuclear pore complex (NPC), which penetrates the double membrane. ONM continues with the endoplasmic reticulum (ER). INM and NPC can interact with chromatin to regulate the genetic activities of the chromosome. Studies in the fission yeast Schizosaccharomyces pombe have contributed to understanding the molecular mechanisms underlying heterochromatin formation by the RNAi-mediated and histone deacetylase machineries. Recent studies have demonstrated that NE proteins modulate heterochromatin formation and functions through interactions with heterochromatic regions, including the pericentromeric and the sub-telomeric regions. In this review, we first introduce the molecular mechanisms underlying the heterochromatin formation and functions in fission yeast, and then summarize the NE proteins that play a role in anchoring heterochromatic regions and in modulating heterochromatin formation and functions, highlighting roles for a conserved INM protein, Lem2.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Heterocromatina/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Retículo Endoplasmático/metabolismo , Transporte Proteico , Telômero/metabolismoRESUMO
This protocol describes the live observation of chromosome segregation during fission yeast meiosis. To visualize one chromosome of interest, the lac operator (lacO array) is integrated at its centromere-proximal locus, and the lac repressor (lacI)-GFP fusion protein is expressed in a haploid strain. This haploid strain, in which mCherry-tagged tubulin is also expressed exogenously to monitor meiotic progression, is crossed with a nonlabeled haploid strain to induce meiosis. GFP and mCherry signals in resulting zygotes are observed by a fluorescent microscopy during the progression of meiosis.
Assuntos
Segregação de Cromossomos , Imageamento Tridimensional/métodos , Meiose , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Haploidia , Microscopia de Fluorescência , Zigoto/metabolismoRESUMO
In fission yeast Schizosaccharomyces pombe, initiation of meiosis is repressed by Pat1 kinase. This protocol describes how ectopic inactivation of the temperature-sensitive Pat1-114 kinase in G1-arrested h- /h- diploid cells carrying mat1-Pc induces a highly synchronized commitment to and execution of meiosis. Haploid or diploid pat1-114 mutants without mat1-Pc can also be used for convenience, although less synchrony may be attained compared with induction using true diploids. An essentially identical protocol can be used for induction via inhibition of genetically sensitized Pat1 kinase by ATP analogs.