RESUMO
Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromossomos de Insetos/genética , Drosophila melanogaster/genética , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microscopia de Fluorescência/métodos , RNA/genética , Análise de Célula Única/métodos , Transcrição Gênica , Ativação Transcricional , Animais , Ciclo Celular/genética , Cromatina/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ Fluorescente , RNA/biossínteseRESUMO
Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development. VIDEO ABSTRACT.
Assuntos
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Inativação Gênica , Proteínas do Grupo Polycomb/metabolismo , Animais , Sistemas CRISPR-Cas , Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas do Grupo Polycomb/genéticaRESUMO
Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct "territories." Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.
Assuntos
Centrossomo/metabolismo , Eucromatina/genética , Heterocromatina/metabolismo , Animais , Estruturas Cromossômicas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eucromatina/metabolismo , Genoma/genética , Heterocromatina/genética , Heterocromatina/ultraestrutura , Histonas/genética , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR.
Assuntos
Cromossomos Fúngicos , Rearranjo Gênico , Recombinação Homóloga , Telômero , Mapeamento Cromossômico , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Instabilidade Genômica , Modelos Biológicos , Rad51 Recombinase/deficiência , Schizosaccharomyces/genéticaRESUMO
Ubiquinone is an essential factor for the electron transfer system and is also a known lipid antioxidant. The length of the ubiquinone isoprenoid side-chain differs amongst living organisms, with six isoprene units in the budding yeast Saccharomyces cerevisiae, eight units in Escherichia coli and 10 units in the fission yeast Schizosaccharomyces pombe and in humans. The length of the ubiquinone isoprenoid is determined by the product generated by polyprenyl diphosphate synthases (poly-PDSs), which are classified into homodimer (i.e. octa-PDS IspB in E. coli) and heterotetramer [i.e. deca-PDSs Dps1 and D-less polyprenyl diphosphate synthase (Dlp1) in Sc. pombe and in humans] types. In this study, we characterized the hexa-PDS (Coq1) of S. cerevisiae to identify whether this enzyme was a homodimer (as in bacteria) or a heteromer (as in fission yeast). When COQ1 was expressed in an E. coli ispB disruptant, only hexa-PDS activity and ubiquinone-6 were detected, indicating that the expression of Coq1 alone results in bacterial enzyme-like functionality. However, when expressed in fission yeast Deltadps1 and Deltadlp1 strains, COQ1 restored growth on minimal medium in the Deltadlp1 but not Deltadps1 strain. Intriguingly, ubiquinone-9 and ubiquinone-10, but not ubiquinone-6, were identified and deca-PDS activity was detected in the COQ1-expressing Deltadlp1 strain. No enzymatic activity or ubiquinone was detected in the COQ1-expressing Deltadps1 strain. These results indicate that Coq1 partners with Dps1, but not with Dlp1, to be functional in fission yeast. Binding of Coq1 and Dps1 was demonstrated by coimmunoprecipitation, and the formation of a tetramer consisting of Coq1 and Dps1 was detected in Sc. pombe. Thus, Coq1 is functional when expressed alone in E. coli and in budding yeast, but is only functional as a partner with Dps1 in fission yeast. This unusual observation indicates that different folding processes or protein modifications in budding yeast/E. coli versus those in fission yeast might affect the formation of an active enzyme. These results provide important insights into the process of how PDSs have evolved from homo- to hetero-types.
Assuntos
Dimetilaliltranstransferase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , DNA Complementar/isolamento & purificação , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Homologia de Sequência de Aminoácidos , Ubiquinona/biossíntese , Ubiquinona/químicaRESUMO
Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid-soluble antioxidant, is present in almost all organisms. Most cereal crops produce CoQ9, which has nine isoprene units. CoQ10, with 10 isoprene units, is a very popular food supplement. Here, we report the genetic engineering of rice to produce CoQ10 using the gene for decaprenyl diphosphate synthase (DdsA). The production of CoQ9 was almost completely replaced with that of CoQ10, despite the presence of endogenous CoQ9 synthesis. DdsA designed to express at the mitochondria increased accumulation of total CoQ amount in seeds.
Assuntos
Alquil e Aril Transferases/genética , Antioxidantes/fisiologia , Mitocôndrias/genética , Oryza/genética , Sementes/genética , Ubiquinona/análogos & derivados , Alquil e Aril Transferases/metabolismo , Sequência de Bases , Coenzimas , Produtos Agrícolas/enzimologia , Produtos Agrícolas/genética , Transporte de Elétrons/fisiologia , Engenharia Genética/métodos , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Oryza/enzimologia , Plantas Geneticamente Modificadas , Sementes/enzimologia , Terpenos/metabolismo , Ubiquinona/biossíntese , Ubiquinona/genéticaRESUMO
We previously constructed two Schizosaccahromyces pombe ubiquinone-10 (or Coenzyme Q10) less mutants, which are either defective for decaprenyl diphosphate synthase or p-hydroxybenzoate polyprenyl diphosphate transferase. To further confirm the roles of ubiquinone in S. pombe, we examined the phenotype of the abc1Sp (coq8Sp) mutant, which is highly speculated to be defective in ubiquinone biosynthesis. We show here that the abc1Sp defective strain did not produce UQ-10 and could not grow on minimal medium. The abc1Sp-deficient strain required supplementation with antioxidants such as cysteine or glutathione to grow on minimal medium. In support of the antioxidant function of ubiquinone, the abc1Sp-deficient strain is sensitive to H2O2 and Cu2+. In addition, expression of the stress inducible ctt1 gene was much induced in the ubiquinone less mutant than wild type. Interestingly, we also found that the abc1-deficient strain as well as other ubiquinone less mutants produced a significant amount of H2S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Thus, analysis of the phenotypes of S. pombe ubiquinone less mutants clearly demonstrate that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.
Assuntos
Mutação , Fenótipo , Schizosaccharomyces/genética , Ubiquinona/biossíntese , Ubiquinona/genética , Antioxidantes/administração & dosagem , Cobre/farmacologia , Cisteína/administração & dosagem , Glutationa/administração & dosagem , Peróxido de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/metabolismo , Estresse Oxidativo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismoRESUMO
Coenzyme Q (CoQ) is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3-9) that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana) to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants.
Assuntos
Arabidopsis/genética , Schizosaccharomyces/genética , Ubiquinona/genética , Arabidopsis/metabolismo , Vias Biossintéticas , Deleção de Genes , Genes Fúngicos , Genes de Plantas , Humanos , Schizosaccharomyces/metabolismo , Ubiquinona/metabolismoRESUMO
In most eukaryotes, centromeres are epigenetically defined by nucleosomes that contain the histone H3 variant centromere protein A (CENP-A). Specific targeting of the CENP-A-loading chaperone to the centromere is vital for stable centromere propagation; however, the existence of ectopic centromeres (neocentromeres) indicates that this chaperone can function in different chromatin environments. The mechanism responsible for accommodating the CENP-A chaperone at noncentromeric regions is poorly understood. Here, we report the identification of transient, immature neocentromeres in Schizosaccharomyces pombe that show reduced association with the CENP-A chaperone Scm3, owing to persistence of the histone H2A variant H2A.Z. After the acquisition of adjacent heterochromatin or relocation of the immature neocentromeres to subtelomeric regions, H2A.Z was depleted and Scm3 was replenished, thus leading to subsequent stabilization of the neocentromeres. These findings provide new insights into histone variant-mediated epigenetic control of neocentromere establishment.
Assuntos
Centrômero/fisiologia , Epigenômica , Histonas/metabolismo , Modelos Genéticos , Schizosaccharomyces/genética , Autoantígenos/química , Autoantígenos/metabolismo , Autoantígenos/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Centrômero/metabolismo , Centrômero/ultraestrutura , Proteína Centromérica A , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Histonas/fisiologia , Nucleossomos/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologiaRESUMO
The centromere functions as a unique chromosomal attachment site for microtubules. Appropriate microtubule attachment is fundamental for organized chromosome behavior during mitosis and meiosis. Hence, centromeres must function both smoothly and stably. However, centromeric DNA sequences are poorly conserved between species despite common functions and similar centromeric protein composition, which leads us to the question: how are centromeres established and maintained? In this review, we summarize the recent progress in deciphering the mechanisms of centromere function. Specifically, we focus our attention on mechanisms closely-related to centromeric DNA and chromatin. By gathering such information, we hope to reveal a new dimension to the true nature of centromeres.
Assuntos
Centrômero/genética , Cromatina/genética , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/genética , Animais , Autoantígenos/genética , Centrômero/classificação , Proteína Centromérica A , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Humanos , Meiose , Microtúbulos/metabolismo , Mitose , Análise de Sequência de DNARESUMO
The spindle assembly checkpoint monitors the state of spindle-kinetochore interaction to prevent premature onset of anaphase. Although checkpoint proteins, such as Mad2, are localized on kinetochores that do not interact properly with the spindle, it remains unknown how the checkpoint proteins recognize abnormalities in spindle-kinetochore interaction. Here, we report that Mad2 localization on kinetochores in fission yeast is regulated by two partially overlapping but distinct pathways: the Dam1/DASH and the Bub1 pathways. We show that Mad2 is localized on "unattached" as well as "tensionless" kinetochores. Our observations suggest that Bub1 is required for Mad2 to detect tensionless kinetochores, whereas Dam1/DASH is crucial for Mad2 to detect unattached kinetochores. In cells lacking both Bub1 and Dam1/DASH, Mad2 localization on kinetochores is diminished, and mitotic progression appears to be accelerated despite the frequent occurrence of abnormal chromosome segregation. Furthermore, we found that Dam1/DASH is required for promotion of spindle association with unattached kinetochores. In contrast, there is accumulating evidence that Bub1 is involved in resolution of erroneous spindle attachment on tensionless kinetochores. These pathways may act as molecular sensors determining the state of spindle association on each kinetochore, enabling proper regulation of the checkpoint activation as well as promotion/resolution of spindle attachment.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Fuso Acromático , Cromossomos/ultraestrutura , Deleção de Genes , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Mad2 , Microscopia de Fluorescência/métodos , Mitose , Plasmídeos/metabolismoRESUMO
The centromere is essential for the inheritance of genetic information on eukaryotic chromosomes. Epigenetic regulation of centromere identity has been implicated in genome stability, karyotype evolution, and speciation. However, little is known regarding the manner in which centromere dysfunction affects the chromosomal architectures. Here we show that in the fission yeast Schizosaccharomyces pombe, the conditional deletion of the centromere produces survivors that carry either a neocentromere-acquired chromosome at the subtelomeric region or an acentric chromosome rescued by intertelomere fusion with either of the remaining chromosomes. The ratio of neocentromere formation to telomere fusion is considerably decreased by the inactivation of genes involved in RNA interference-dependent heterochromatin formation. By affecting the modes of chromosomal reorganization, the genomic distribution of heterochromatin may influence the fate of karyotype evolution.
Assuntos
Centrômero/fisiologia , Cromossomos Fúngicos/fisiologia , Heterocromatina/metabolismo , Schizosaccharomyces/genética , Telômero/fisiologia , Imunoprecipitação da Cromatina , Segregação de Cromossomos , Replicação do DNA , Expressão Gênica , Genes Fúngicos , Histonas/metabolismo , Cariotipagem , Cinetocoros/metabolismo , Metilação , Mitose , Mutação , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
CENP-A is a centromere-specific histone H3 variant that is essential for kinetochore formation. Here, we report that the fission yeast Schizosaccharomyces pombe has at least two distinct CENP-A deposition phases across the cell cycle: S and G2. The S phase deposition requires Ams2 GATA factor, which promotes histone gene activation. In Delta ams2, CENP-A fails to retain during S, but it reaccumulates onto centromeres via the G2 deposition pathway, which is down-regulated by Hip1, a homologue of HIRA histone chaperon. Reducing the length of G2 in Delta ams2 results in failure of CENP-A accumulation, leading to chromosome missegregation. N-terminal green fluorescent protein-tagging reduces the centromeric association of CENP-A, causing cell death in Delta ams2 but not in wild-type cells, suggesting that the N-terminal tail of CENP-A may play a pivotal role in the formation of centromeric nucleosomes at G2. These observations imply that CENP-A is normally localized to centromeres in S phase in an Ams2-dependent manner and that the G2 pathway may salvage CENP-A assembly to promote genome stability. The flexibility of CENP-A incorporation during the cell cycle may account for the plasticity of kinetochore formation when the authentic centromere is damaged.
Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas Cromossômicas não Histona/química , Cromossomos Fúngicos/metabolismo , Fase G2 , Deleção de Genes , Proteínas de Fluorescência Verde/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Fase S , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/químicaRESUMO
Spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance system for chromosome missegregation. We isolated fission yeast Hos2, a component of the Dam1/DASH complex, as a multicopy suppressor of temperature-sensitive (ts) growth of nnf1-495 mutant that exhibits the minichromosome instability (mis) phenotype, producing lethal aneuploids without prominent mitotic delay. It remains elusive why SAC is satisfied in mis mutants despite the occurrence of missegregation. We found that Hos2 binds to the inner-kinetochore regions in both prometaphase and metaphase. Hos2 is essential for kinetochore localization of Dis1, a microtubule (MT) associated Dis1/XMAP215/TOG family protein that is required for proper MT dynamics. Cells lacking DASH exhibit cold-sensitive (cs) growth with the defective in sister-chromatid disjoining (dis) phenotype, which is characterized by hyper-condensed sister-chromatid pairs and elongated spindle MTs. Although DASH-deficient cells are viable at high temperatures, DASH-deletion transforms all the inner-kinetochore mis mutants so far tested into a constitutively active state of SAC, leading to the dis phenotype. We also discovered that Hos2 over-expression commonly suppresses growth retardation in a variety of inner-kinetochore mutants. These genetic interactions highlight the DASH-action(s) in satisfying SAC when aneuploids are formed during mitosis in the inner-kinetochore-defective mis mutants.