RESUMO
Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily conserved histone H3 variant, which directs kinetochore assembly and hence centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity-selected solubilized fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. Chromatin association of Hap2 is Ies4-dependent. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naïve centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 facilitates transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilizes H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.
Assuntos
Cromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transcrição Gênica/fisiologia , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA Fúngico/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/metabolismoRESUMO
Heterochromatin that depends on histone H3 lysine 9 methylation (H3K9me) renders embedded genes transcriptionally silent1-3. In the fission yeast Schizosaccharomyces pombe, H3K9me heterochromatin can be transmitted through cell division provided the counteracting demethylase Epe1 is absent4,5. Heterochromatin heritability might allow wild-type cells under certain conditions to acquire epimutations, which could influence phenotype through unstable gene silencing rather than DNA change6,7. 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.
Assuntos
Farmacorresistência Fúngica/genética , Inativação Gênica , Heterocromatina/genética , Heterocromatina/metabolismo , Schizosaccharomyces/genética , Cafeína/farmacologia , Farmacorresistência Fúngica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Heterocromatina/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Fenótipo , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The relationship between DNA sequence, biochemical function, and molecular evolution is relatively well-described for protein-coding regions of genomes, but far less clear in noncoding regions, particularly, in eukaryote genomes. In part, this is because we lack a complete description of the essential noncoding elements in a eukaryote genome. To contribute to this challenge, we used saturating transposon mutagenesis to interrogate the Schizosaccharomyces pombe genome. We generated 31 million transposon insertions, a theoretical coverage of 2.4 insertions per genomic site. We applied a five-state hidden Markov model (HMM) to distinguish insertion-depleted regions from insertion biases. Both raw insertion-density and HMM-defined fitness estimates showed significant quantitative relationships to gene knockout fitness, genetic diversity, divergence, and expected functional regions based on transcription and gene annotations. Through several analyses, we conclude that transposon insertions produced fitness effects in 66-90% of the genome, including substantial portions of the noncoding regions. Based on the HMM, we estimate that 10% of the insertion depleted sites in the genome showed no signal of conservation between species and were weakly transcribed, demonstrating limitations of comparative genomics and transcriptomics to detect functional units. In this species, 3'- and 5'-untranslated regions were the most prominent insertion-depleted regions that were not represented in measures of constraint from comparative genomics. We conclude that the combination of transposon mutagenesis, evolutionary, and biochemical data can provide new insights into the relationship between genome function and molecular evolution.
Assuntos
Aptidão Genética , Genoma Fúngico , Schizosaccharomyces/genética , Modelos Genéticos , Mutagênese InsercionalAssuntos
Autoantígenos/metabolismo , Proteínas de Transporte/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteína Centromérica A , Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Centromere sequences are not conserved between species, and there is compelling evidence for epigenetic regulation of centromere identity, with location being dictated by the presence of chromatin containing the histone H3 variant CENP-A. Paradoxically, in most organisms CENP-A chromatin generally occurs on particular sequences. To investigate the contribution of primary DNA sequence to establishment of CENP-A chromatin in vivo, we utilised the fission yeast Schizosaccharomyces pombe. CENP-ACnp1 chromatin is normally assembled on â¼10 kb of central domain DNA within these regional centromeres. We demonstrate that overproduction of S. pombe CENP-ACnp1 bypasses the usual requirement for adjacent heterochromatin in establishing CENP-ACnp1 chromatin, and show that central domain DNA is a preferred substrate for de novo establishment of CENP-ACnp1 chromatin. When multimerised, a 2 kb sub-region can establish CENP-ACnp1 chromatin and form functional centromeres. Randomization of the 2 kb sequence to generate a sequence that maintains AT content and predicted nucleosome positioning is unable to establish CENP-ACnp1 chromatin. These analyses indicate that central domain DNA from fission yeast centromeres contains specific information that promotes CENP-ACnp1 incorporation into chromatin. Numerous transcriptional start sites were detected on the forward and reverse strands within the functional 2 kb sub-region and active promoters were identified. RNAPII is enriched on central domain DNA in wild-type cells, but only low levels of transcripts are detected, consistent with RNAPII stalling during transcription of centromeric DNA. Cells lacking factors involved in restarting transcription-TFIIS and Ubp3-assemble CENP-ACnp1 on central domain DNA when CENP-ACnp1 is at wild-type levels, suggesting that persistent stalling of RNAPII on centromere DNA triggers chromatin remodelling events that deposit CENP-ACnp1. Thus, sequence-encoded features of centromeric DNA create an environment of pervasive low quality RNAPII transcription that is an important determinant of CENP-ACnp1 assembly. These observations emphasise roles for both genetic and epigenetic processes in centromere establishment.
Assuntos
Autoantígenos/genética , Centrômero/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Epigênese Genética , Transcrição Gênica , Proteína Centromérica A , Montagem e Desmontagem da Cromatina/genética , DNA/genética , Heterocromatina/genética , Histonas/genética , Cinetocoros , SchizosaccharomycesRESUMO
Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feature of heterochromatin domains is the presence of hypoacetylated nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me). Here, we investigate the requirements for establishment, spreading and maintenance of heterochromatin using fission yeast centromeres as a paradigm. We show that establishment of heterochromatin on centromeric repeats is initiated at modular 'nucleation sites' by RNA interference (RNAi), ensuring the mitotic stability of centromere-bearing minichromosomes. We demonstrate that the histone deacetylases Sir2 and Clr3 and the chromodomain protein Swi6(HP1) are required for H3K9me spreading from nucleation sites, thus allowing formation of extended heterochromatin domains. We discovered that RNAi and Sir2 along with Swi6(HP1) operate in two independent pathways to maintain heterochromatin. Finally, we demonstrate that tethering of Sir2 is pivotal to the maintenance of heterochromatin at an ectopic locus in the absence of RNAi. These analyses reveal that Sir2, together with RNAi, are sufficient to ensure heterochromatin integrity and provide evidence for sequential establishment, spreading and maintenance steps in the assembly of centromeric heterochromatin.
Assuntos
Centrômero/genética , Montagem e Desmontagem da Cromatina/genética , Heterocromatina/metabolismo , Interferência de RNA/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Centrômero/metabolismo , Epigênese Genética/genética , Epigênese Genética/fisiologia , Heterocromatina/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/química , Histonas/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Biológicos , Organismos Geneticamente Modificados , Processamento de Proteína Pós-Traducional , Elementos Reguladores de Transcrição/genética , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.
Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Transporte/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/análise , Mutação , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/análise , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-A(Cnp1) deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites. FACT has little or no effect on CENP-A(Cnp1) assembly at endogenous centromeres where CENP-A(Cnp1) is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-A(Cnp1) at specific loci, including subtelomeric regions, where CENP-A(Cnp1) is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-A(Cnp1) chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-A(Cnp1) to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-A(Cnp1) chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification.
Assuntos
Aminopeptidases/genética , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Chaperonas Moleculares/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Transcrição Gênica , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , Epigênese Genética , Heterocromatina/genética , Histonas/genética , Cinetocoros , Nucleossomos/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Fission yeasts are an ancient group of fungal species that diverged from each other from tens to hundreds of million years ago. Among them is the preeminent model organism Schizosaccharomyces pombe, which has significantly contributed to our understandings of molecular mechanisms underlying fundamental cellular processes. The availability of the genomes of S. pombe and 3 other fission yeast species S. japonicus, S. octosporus, and S. cryophilus has enabled cross-species comparisons that provide insights into the evolution of genes, pathways, and genomes. Here, we performed genome sequencing on the type strain of the recently identified fission yeast species S. osmophilus and obtained a complete mitochondrial genome and a nuclear genome assembly with gaps only at rRNA gene arrays. A total of 5,098 protein-coding nuclear genes were annotated and orthologs for more than 95% of them were identified. Genome-based phylogenetic analysis showed that S. osmophilus is most closely related to S. octosporus and these 2 species diverged around 16 million years ago. To demonstrate the utility of this S. osmophilus reference genome, we conducted cross-species comparative analyses of centromeres, telomeres, transposons, the mating-type region, Cbp1 family proteins, and mitochondrial genomes. These analyses revealed conservation of repeat arrangements and sequence motifs in centromere cores, identified telomeric sequences composed of 2 types of repeats, delineated relationships among Tf1/sushi group retrotransposons, characterized the evolutionary origins and trajectories of Cbp1 family domesticated transposases, and discovered signs of interspecific transfer of 2 types of mitochondrial selfish elements.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Filogenia , Centrômero/genética , Centrômero/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-ACnp1 incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-ACnp1 overexpression bypasses heterochromatin dependency, suggesting that heterochromatin ensures exposure to conditions or locations permissive for CENP-ACnp1 assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-ACnp1 incorporation. We demonstrate that heterochromatin-independent de novo CENP-ACnp1 chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-ACnp1 assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation because target sequences are exposed to high levels of CENP-ACnp1 and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Epe1 histone demethylase restricts H3K9-methylation-dependent heterochromatin, preventing it from spreading over, and silencing, gene-containing regions in fission yeast. External stress induces an adaptive response allowing heterochromatin island formation that confers resistance on surviving wild-type lineages. Here we investigate the mechanism by which Epe1 is regulated in response to stress. Exposure to caffeine or antifungals results in Epe1 ubiquitylation and proteasome-dependent removal of the N-terminal 150 residues from Epe1, generating truncated Epe1 (tEpe1) which accumulates in the cytoplasm. Constitutive tEpe1 expression increases H3K9 methylation over several chromosomal regions, reducing expression of underlying genes and enhancing resistance. Reciprocally, constitutive non-cleavable Epe1 expression decreases resistance. tEpe1-mediated resistance requires a functional JmjC demethylase domain. Moreover, caffeine-induced Epe1-to-tEpe1 cleavage is dependent on an intact cell integrity MAP kinase stress signaling pathway, mutations in which alter resistance. Thus, environmental changes elicit a mechanism that curtails the function of this key epigenetic modifier, allowing heterochromatin to reprogram gene expression, thereby bestowing resistance to some cells within a population. H3K9me-heterochromatin components are conserved in human and crop-plant fungal pathogens for which a limited number of antifungals exist. Our findings reveal how transient heterochromatin-dependent antifungal resistant epimutations develop and thus inform on how they might be countered.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Antifúngicos/metabolismo , Cafeína/metabolismo , Citoplasma/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Centromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms, including the fission yeast, Schizosaccharomyces pombe, the RNAi pathway contributes to the formation of silent chromatin in pericentromeric regions. Little is known about how chromatin-remodeling factors contribute to heterochromatic integrity and centromere function. Here we show that the histone chaperone and remodeling complex FACT is required for centromeric-heterochromatin integrity and accurate chromosome segregation. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for pob3+ are viable and alleviate gene silencing at centromeric repeats and at the silent mating-type locus. Importantly, like heterochromatin and RNAi pathway mutants, Pob3 null strains exhibit lagging chromosomes on anaphase spindles. Whereas the processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, Swi6-association with the centromere is reduced. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in heterochromatin integrity and in centromere function.
Assuntos
Centrômero/metabolismo , Segregação de Cromossomos/fisiologia , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Fúngica da Expressão Gênica , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Interferência de RNA , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
BACKGROUND: Accurate chromosome segregation depends on the establishment of correct-amphitelic-kinetochore orientation. Merotelic kinetochore orientation is an error that occurs when a single kinetochore attaches to microtubules emanating from opposite spindle poles, a condition that hinders segregation of the kinetochore to a spindle pole in anaphase. To avoid chromosome missegregation resulting from merotelic kinetochore orientation, cells have developed mechanisms to prevent or correct merotelic attachment. A protein called Pcs1 has been implicated in preventing merotelic attachment in mitosis and meiosis II in the fission yeast S. pombe. RESULTS: We report that Pcs1 forms a complex with a protein called Mde4. Both Pcs1 and Mde4 localize to the central core of centromeres. Deletion of mde4(+), like that of pcs1(+), causes the appearance of lagging chromosomes during the anaphases of mitotic and meiosis II cells. We provide evidence that the kinetochores of lagging chromosomes in both pcs1 and mde4 mutant cells are merotelically attached. In addition, we find that lagging chromosomes in cells with defective centromeric heterochromatin also display features consistent with merotelic attachment. CONCLUSIONS: We suggest that the Pcs1/Mde4 complex is the fission yeast counterpart of the budding yeast monopolin subcomplex Csm1/Lrs4, which promotes the segregation of sister kinetochores to the same pole during meiosis I. We propose that the Pcs1/Mde4 complex acts in the central kinetochore domain to clamp microtubule binding sites together, the centromeric heterochromatin coating the flanking domains provides rigidity, and both systems contribute to the prevention of merotelic attachment.
Assuntos
Segregação de Cromossomos/fisiologia , Heterocromatina/metabolismo , Cinetocoros/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Proteínas de Ciclo Celular/metabolismo , Histona-Lisina N-Metiltransferase , Cinetocoros/metabolismo , Espectrometria de Massas , Meiose/fisiologia , Metiltransferases/metabolismo , Mitose/fisiologia , Complexos Multiproteicos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-A(Cnp1) chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-A(Cnp1) can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-A(Cnp1) chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-A(Cnp1) associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-A(Cnp1) for assembly into central domain chromatin, resulting in less CENP-A(Cnp1) and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-A(Cnp1) influence the extent of DNA at centromeres that is packaged in CENP-A(Cnp1) chromatin and the composition of this chromatin. Thus, CENP-A(Cnp1) chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-A(Cnp1) and other core histones.
Assuntos
Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Schizosaccharomyces/metabolismo , Centrômero/metabolismo , Proteína Centromérica A , Cromatina/metabolismo , DNA/química , Primers do DNA/química , Proteínas Fúngicas/química , Genoma Fúngico , Cinetocoros/metabolismo , Modelos Biológicos , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Fuso Acromático/metabolismoRESUMO
Post-translational modifications of core histones, particularly in the N terminus, seem to define different chromatin states in the genome. For instance, actively transcribed regions of euchromatin have a completely different set of 'marks' compared with silent heterochromatin. Recent analyses surprisingly demonstrate that, in addition to containing the histone H3 variant CENP-A, the centromeric chromatin that underlies the kinetochore bears a distinct combination of histone H3 modifications.
Assuntos
Centrômero/química , Cromatina/química , Cromatina/metabolismo , Sequência de Aminoácidos , Cromatina/genética , Histonas/química , Histonas/metabolismo , Dados de Sequência Molecular , Processamento de Proteína Pós-TraducionalRESUMO
During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here, we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine nine trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes.
Assuntos
Cromossomos , Heterocromatina , Mitose/genética , Animais , Cromossomos/química , Cromossomos/genética , Cromossomos/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Recombinante/química , DNA Recombinante/genética , DNA Recombinante/metabolismo , Células HeLa , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Schizosaccharomyces/genética , TransfecçãoRESUMO
The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1 promoter. To overcome these problems we have developed an improved system, SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for S. pombe expressed from the medium strength adh15 promoter. The SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3' end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the ade6 + or ura4 + genes by transformation of cells from asynchronous cultures. SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDIT system displayed reduced toxicity compared to currently available S. pombe editing systems. Thus, SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.
RESUMO
Fission yeast centromeres are composed of two domains: the central core and the outer repeats. Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins. Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin. Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure. The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region. Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain. Functional Mis6 is required for Sim4 localization at the kinetochore. Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore.
Assuntos
Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Segregação de Cromossomos/genética , Inativação Gênica/fisiologia , Cinetocoros/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Schizosaccharomyces/genética , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Testes Genéticos , Dados de Sequência Molecular , Mutação/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Elementos Silenciadores Transcricionais/genéticaRESUMO
The fission yeast multiprotein-component Sim4 complex plays a fundamental role in the assembly of a functional kinetochore. It affects centromere association of the histone H3 variant CENP-A as well as kinetochore association of the DASH complex. Here, multicopy suppressor analysis of a mutant version of the Sim4 complex component Mal2 identified the essential Fta2 kinetochore protein, which is required for bipolar chromosome attachment. Kinetochore localization of Mal2 and Fta2 depends on each other, and overexpression of one protein can rescue the phenotype of the mutant version of the other protein. fta2 mal2 double mutants were inviable, implying that the two proteins have an overlapping function. This close interaction with Fta2 is not shared by other Sim4 complex components, indicating the existence of functional subgroups within this complex. The Sim4 complex seems to be assembled in a hierarchical way, because Fta2 is localized correctly in a sim4 mutant. However, Fta2 kinetochore localization is reduced in a spc7 mutant. Spc7, a suppressor of the EB1 family member Mal3, is part of the conserved Ndc80-MIND-Spc7 kinetochore complex.