RESUMEN
Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We have discovered the machinery and mechanism underlying the dynamic enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during C. elegans dosage compensation and demonstrated H4K20me1's pivotal role in regulating higher-order chromosome structure and X-chromosome-wide gene expression. The structure and the activity of the dosage compensation complex (DCC) subunit DPY-21 define a Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminates H4K20me1 enrichment in somatic cells, elevates X-linked gene expression, reduces X chromosome compaction, and disrupts X chromosome conformation by diminishing the formation of topologically associating domains (TADs). Unexpectedly, DPY-21 also associates with autosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Our findings demonstrate the direct link between chromatin modification and higher-order chromosome structure in long-range regulation of gene expression.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Cromosoma X/química , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/genética , Compensación de Dosificación (Genética) , Embrión no Mamífero/metabolismo , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/metabolismo , Modelos Moleculares , Mutación , Piperidinas/metabolismo , Alineación de Secuencia , Tiofenos/metabolismoRESUMEN
The biological roles of nucleic acid methylation, other than at the C5-position of cytosines in CpG dinucleotides, are still not well understood. Here, we report genetic evidence for a critical role for the putative DNA demethylase NMAD-1 in regulating meiosis in C. elegans. nmad-1 mutants have reduced fertility. They show defects in prophase I of meiosis, which leads to reduced embryo production and an increased incidence of males due to defective chromosomal segregation. In nmad-1 mutant worms, nuclear staging beginning at the leptotene and zygotene stages is disorganized, the cohesin complex is mislocalized at the diplotene and diakinesis stages, and chromosomes are improperly condensed, fused, or lost by the end of diakinesis. RNA sequencing of the nmad-1 germline revealed reduced induction of DNA replication and DNA damage response genes during meiosis, which was coupled with delayed DNA replication, impaired DNA repair and increased apoptosis of maturing oocytes. To begin to understand how NMAD-1 regulates DNA replication and repair, we used immunoprecipitation and mass spectrometry to identify NMAD-1 binding proteins. NMAD-1 binds to multiple proteins that regulate DNA repair and replication, including topoisomerase TOP-2 and co-localizes with TOP-2 on chromatin. Moreover, the majority of TOP-2 binding to chromatin depends on NMAD-1. These results suggest that NMAD-1 functions at DNA replication sites to regulate DNA replication and repair during meiosis.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Reparación del ADN , Replicación del ADN , Dioxigenasas/genética , Oxidorreductasas N-Desmetilantes/genética , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Segregación Cromosómica , Dioxigenasas/metabolismo , Masculino , Meiosis , Mutación , Oxidorreductasas N-Desmetilantes/metabolismo , Análisis de Secuencia de ARNRESUMEN
The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (â¼1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/metabolismo , Compensación de Dosificación (Genética)/fisiología , Complejos Multiproteicos/metabolismo , Cromosoma X/metabolismo , Animales , Proteínas de Caenorhabditis elegans/genética , Compensación de Dosificación (Genética)/genética , Femenino , Regulación de la Expresión Génica , Hibridación Fluorescente in Situ , Masculino , Unión Proteica , Análisis de Secuencia de ARN , Cromosoma X/genéticaRESUMEN
Crossover recombination events between homologous chromosomes are required to form chiasmata, temporary connections between homologues that ensure their proper segregation at meiosis I. Despite this requirement for crossovers and an excess of the double-strand DNA breaks that are the initiating events for meiotic recombination, most organisms make very few crossovers per chromosome pair. Moreover, crossovers tend to inhibit the formation of other crossovers nearby on the same chromosome pair, a poorly understood phenomenon known as crossover interference. Here we show that the synaptonemal complex, a meiosis-specific structure that assembles between aligned homologous chromosomes, both constrains and is altered by crossover recombination events. Using a cytological marker of crossover sites in Caenorhabditis elegans, we show that partial depletion of the synaptonemal complex central region proteins attenuates crossover interference, increasing crossovers and reducing the effective distance over which interference operates, indicating that synaptonemal complex proteins limit crossovers. Moreover, we show that crossovers are associated with a local 0.4-0.5-micrometre increase in chromosome axis length. We propose that meiotic crossover regulation operates as a self-limiting system in which meiotic chromosome structures establish an environment that promotes crossover formation, which in turn alters chromosome structure to inhibit other crossovers at additional sites.
Asunto(s)
Caenorhabditis elegans/genética , Cromosomas/genética , Cromosomas/metabolismo , Intercambio Genético , Meiosis , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/metabolismo , Emparejamiento Cromosómico , Segregación Cromosómica , Cromosomas/química , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga , Meiosis/genética , Proteínas Nucleares/deficiencia , Proteínas Nucleares/metabolismo , Complejo Sinaptonémico/metabolismoRESUMEN
Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.
Asunto(s)
Microscopía Fluorescente/métodos , Algoritmos , Animales , Supervivencia Celular , Drosophila melanogaster/citología , Saccharomyces cerevisiae/citología , Programas InformáticosRESUMEN
Centrosome aberrations caused by misregulated centrosome maturation result in defective spindle and genomic instability. Here we report that the fission yeast homolog of the human transcription factor EAP30, Dot2, negatively regulates meiotic spindle pole body (SPB, the yeast equivalent of centrosome) maturation. dot2 mutants show excess electron-dense material accumulating near SPBs, which we refer to as aberrant microtubule organization centers (AMtOCs). These AMtOCs assemble multipolar spindles, leading to chromosome missegregation. SPB aberrations were associated with elevated levels of Pcp1, the fission yeast ortholog of pericentrin/kentrin, and reducing pcp1(+) expression significantly suppressed AMtOCs in dot2-439 cells. Our findings, therefore, uncover meiosis-specific regulation of SPB maturation and provide evidence that a member of the conserved EAP30 family is required for maintenance of genome stability through regulation of SPB maturation. EAP30 is part of a transcription factor complex associated with acute myeloid leukemia, so these results may have relevance to human cancer.
Asunto(s)
Meiosis , Centro Organizador de los Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/ultraestructura , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Centrosoma/metabolismo , Inestabilidad Cromosómica , Cromosomas Fúngicos , Humanos , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fase S , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which enter the specimen through each objective lens. The resulting illumination intensity pattern contains high spatial frequency components both axially and laterally. In addition, the emission is collected by both objective lenses coherently, and combined interferometrically on a single camera, resulting in a detection transfer function with axially extended support. These two effects combine to produce near-isotropic resolution. Experimental images of test samples and biological specimens confirm the theoretical predictions.
Asunto(s)
Aumento de la Imagen/instrumentación , Imagenología Tridimensional/instrumentación , Lentes , Microscopía/instrumentación , Nanotecnología/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Microscopía/métodos , Nanotecnología/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: The fission yeast Schizosaccharomyces pombe has a cylindrical cell shape, for which growth is strictly limited to both ends, and serves as an excellent model system for genetic analysis of cell-polarity determination. Previous studies identified a cell-end marker protein, Tea1, that is transported by cytoplasmic microtubules to cell tips and recruits other cell-end factors, including the Dyrk-family Pom1 kinase. The deltatea1 mutant cells cannot grow in a bipolar fashion and show T-shaped morphology after heat shock. RESULTS: We identified Wsh3/Tea4 as a novel protein that interacts with Win1 MAP kinase kinase kinase (MAPKKK) of the stress-activated MAP kinase cascade. Wsh3 forms a complex with Tea1 and is transported to cell tips by growing microtubules. The deltawsh3 mutant shows monopolar growth with abnormal Tea1 aggregate at the non-growing cell end; this abnormal aggregate fails to recruit Pom1 kinase. Consistent with the observed interaction between Win1 and Wsh3, cells lacking Wsh3 or Tea1 show more severe cell-polarity defects under osmolarity and heat-stress stimuli that are known to activate the stress MAPK cascade. Furthermore, mutants of the stress MAPK also exhibit cell-polarity defects when exposed to the same stress. CONCLUSIONS: Wsh3/Tea4 is an essential component of the Tea1 cell-end complex. In addition to its role in bipolar growth during the normal cell cycle, the Wsh3-Tea1 complex, together with the stress-signaling MAPK cascade, contributes to cell-polarity maintenance under stress conditions.
Asunto(s)
Polaridad Celular/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Glutatión Transferasa , Proteínas Asociadas a Microtúbulos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Transporte de Proteínas/fisiología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The regulation and timing of spindle pole body (SPB) duplication and maturation in fission yeast was examined by transmission electron microscopy. When cells are arrested at G1 by nitrogen starvation, the SPB is unduplicated. On release from G1, the SPBs were duplicated after 1-2 h. In cells arrested at S by hydroxyurea, SPBs are duplicated but not mature. In G1 arrest/release experiments with cdc2.33 cells at the restrictive temperature, SPBs remained single, whereas in cells at the permissive temperature, SPBs were duplicated. In cdc10 mutant cells, the SPBs seem not only to be duplicated but also to undergo partial maturation, including invagination of the nuclear envelope underneath the SPB. There may be an S-phase-specific inhibitor of SPB maturation whose expression is under control of cdc10(+). This model was examined by induction of overreplication of the genome by overexpression of rum1p or cdc18p. In cdc18p-overexpressing cells, the SPBs are duplicated but not mature, suggesting that cdc18p is one component of this feedback mechanism. In contrast, cells overexpressing rum1p have large, deformed SPBs accompanied by other features of maturation and duplication. We propose a feedback mechanism for maturation of the SPB that is coupled with exit from S to trigger morphological changes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fase G1 , Fase S , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Huso Acromático/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Fase G1/efectos de los fármacos , Regulación Fúngica de la Expresión Génica , Hidroxiurea/farmacología , Microscopía Electrónica , Nitrógeno/deficiencia , Nitrógeno/metabolismo , Fase S/efectos de los fármacos , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/ultraestructura , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/ultraestructura , Factores de Tiempo , Factores de TranscripciónRESUMEN
In meiotic prophase of many eukaryotic organisms, telomeres attach to the nuclear envelope and form a polarized configuration called the bouquet. Bouquet formation is hypothesized to facilitate homologous chromosome pairing. In fission yeast, bouquet formation and telomere clustering occurs in karyogamy and persists throughout the horsetail stage. Here we report the isolation and characterization of six mutants from our screen for meiotic mutants. These mutants show defective telomere clustering as demonstrated by mislocalization of Swi6::GFP, a heterochromatin-binding protein, and Taz1p::GFP, a telomere-specific protein. These mutants define four complementation groups and are named dot1 to dot4-defective organization of telomeres. dot3 and dot4 are allelic to mat1-Mm and mei4, respectively. Immunolocalization of Sad1, a protein associated with the spindle pole body (SPB), in dot mutants showed an elevated frequency of multiple Sad1-nuclei signals relative to wild type. Many of these Sad1 foci were colocalized with Taz1::GFP. Impaired SPB structure and function were further demonstrated by failure of spore wall formation in dot1, by multiple Pcp1::GFP signals (an SPB component) in dot2, and by abnormal microtubule organizations during meiosis in dot mutants. The coincidence of impaired SPB functions with defective telomere clustering suggests a link between the SPB and the telomere cluster.
Asunto(s)
Meiosis/fisiología , Schizosaccharomyces/genética , Huso Acromático/metabolismo , Proteínas de Unión a Telómeros , Telómero/metabolismo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Microtúbulos/metabolismo , Mutación , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe , Factores de Transcripción , Ubiquitina TiolesterasaRESUMEN
Schizosaccharomyces pombe is an excellent organism for studying microtubule dynamics owing to the presence of well-defined microtubule arrays that undergo dramatic rearrangements during various stages of the cell cycle. Using sensitive time-lapse video microscopy and kymographic analysis, we have determined the polymerization/depolymerization kinetics of individual microtubules within these arrays throughout the fission yeast cell cycle. Interphase bundles are composed of 4-7 microtubules that act autonomously, demonstrating that individual microtubules are responsible for mediating the functions ascribed to these arrays. The nucleation and growth of cytoplasmic microtubules is inhibited upon cellular transition into mitosis, leading to their gradual disappearance. At the onset of mitosis, microtubules form on the nuclear face of the spindle pole body and exhibit dramatically increased dynamics. The presence of these intra-nuclear astral microtubules (INA) is reminiscent of spindle assembly and the search and chromosome capture mechanism observed in metazoan cells. Consistent with other in vivo studies, we do not observe microtubule flux in the anaphase B spindle. Finally, the depolymerization of individual microtubules alternates between each half-spindle, resulting in spindle collapse during telophase. On the basis of these observations, we conclude that microtubules in these diverse cytoskeletal arrays have autonomous behaviors that are an essential component of any model describing cell-cycle-dependent changes in the behavior and function of microtubule arrays.
Asunto(s)
Núcleo Celular/fisiología , Microtúbulos/fisiología , Schizosaccharomyces/fisiología , Huso Acromático/fisiología , Anafase/fisiología , Interfase/fisiología , Microscopía por Video , Schizosaccharomyces/ultraestructuraRESUMEN
Human damaged DNA-binding protein (DDB) is a heterodimer of p48/DDB2 and p127/DDB1 subunits. Mutations in DDB2 are responsible for Xeroderma Pigmentosum group E, but no mutants of mammalian DDB1 have been described. To study DDB1, the Schizosaccharomyces pombe DDB1 sequence homologue (ddb1(+)) was cloned, and a ddb1 deletion strain was constructed. The gene is not essential; however, mutant cells showed a 37% impairment in colony-forming ability, an elongated phenotype, and abnormal nuclei. The ddb1Delta strain was sensitive to UV irradiation, X-rays, methylmethane sulfonate, and thiabendazole, and these sensitivities were compared with those of the well characterized rad13Delta, rhp51Delta, and cds1Delta mutant strains. Ddb1p showed nuclear and nucleolar localization, and the aberrant nuclear structures observed in the ddb1Delta strain suggest a role for Ddb1p in chromosome segregation.