RESUMEN
Crossovers (COs) between homologous chromosomes ensure their faithful segregation during meiosis. We identify C. elegans COSA-1, a cyclin-related protein conserved in metazoa, as a key component required to convert meiotic double-strand breaks (DSBs) into COs. During late meiotic prophase, COSA-1 localizes to foci that correspond to the single CO site on each homolog pair and indicate sites of eventual concentration of other conserved CO proteins. Chromosomes gain and lose competence to load CO proteins during meiotic progression, with competence to load COSA-1 requiring prior licensing. Our data further suggest a self-reinforcing mechanism maintaining CO designation. Modeling of a nonlinear dose-response relationship between IR-induced DSBs and COSA-1 foci reveals efficient conversion of DSBs into COs when DSBs are limiting and a robust capacity to limit cytologically differentiated CO sites when DSBs are in excess. COSA-1 foci serve as a unique live cell readout for investigating CO formation and CO interference.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Intercambio Genético , Ciclinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Meiosis , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cromosomas/metabolismo , Ciclinas/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Modelos Moleculares , MutaciónRESUMEN
Faithful inheritance of genetic information through sexual reproduction relies on the formation of crossovers between homologous chromosomes during meiosis, which, in turn, relies on the formation and repair of numerous double-strand breaks (DSBs). As DSBs pose a potential threat to the genome, mechanisms that ensure timely and error-free DSB repair are crucial for successful meiosis. Here, we identify NBS-1, the Caenorhabditis elegans ortholog of the NBS1 (mutated in Nijmegen Breakage Syndrome) subunit of the conserved MRE11-RAD50-NBS1/Xrs2 (MRN) complex, as a key mediator of DSB repair via homologous recombination (HR) during meiosis. Loss of nbs-1 leads to severely reduced loading of recombinase RAD-51, ssDNA binding protein RPA, and pro-crossover factor COSA-1 during meiotic prophase progression; aggregated and fragmented chromosomes at the end of meiotic prophase; and 100% progeny lethality. These phenotypes reflect a role for NBS-1 in processing of meiotic DSBs for HR that is shared with its interacting partners MRE-11-RAD-50 and COM-1 (ortholog of Com1/Sae2/CtIP). Unexpectedly, in contrast to MRE-11 and RAD-50, NBS-1 is not required for meiotic DSB formation. Meiotic defects of the nbs-1 mutant are partially suppressed by abrogation of the nonhomologous end-joining (NHEJ) pathway, indicating a role for NBS-1 in antagonizing NHEJ during meiosis. Our data further reveal that NBS-1 and COM-1 play distinct roles in promoting HR and antagonizing NHEJ. We propose a model in which different components of the MRN-C complex work together to couple meiotic DSB formation with efficient and timely engagement of HR, thereby ensuring crossover formation and restoration of genome integrity before the meiotic divisions.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Embrión no Mamífero/metabolismo , Recombinación Homóloga , Meiosis , Animales , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/metabolismo , Embrión no Mamífero/citologíaRESUMEN
For most organisms, chromosome segregation during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs) and repair of a subset of these DSBs as inter-homolog crossovers (COs). However, timing and levels of DSB formation must be tightly controlled to avoid jeopardizing genome integrity. Here we identify the DSB-2 protein, which is required for efficient DSB formation during C. elegans meiosis but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of DSB formation, and its disappearance coincides with a decline in RAD-51 foci marking early recombination intermediates and precedes appearance of COSA-1 foci marking CO-designated sites. These and other data suggest that DSB-2 and its paralog DSB-1 promote competence for DSB formation. Further, immunofluorescence analyses of wild-type gonads and various meiotic mutants reveal that association of DSB-2 with chromatin is coordinated with multiple distinct aspects of the meiotic program, including the phosphorylation state of nuclear envelope protein SUN-1 and dependence on RAD-50 to load the RAD-51 recombinase at DSB sites. Moreover, association of DSB-2 with chromatin is prolonged in mutants impaired for either DSB formation or formation of downstream CO intermediates. These and other data suggest that association of DSB-2 with chromatin is an indicator of competence for DSB formation, and that cells respond to a deficit of CO-competent recombination intermediates by prolonging the DSB-competent state. In the context of this model, we propose that formation of sufficient CO-competent intermediates engages a negative feedback response that leads to cessation of DSB formation as part of a major coordinated transition in meiotic prophase progression. The proposed negative feedback regulation of DSB formation simultaneously (1) ensures that sufficient DSBs are made to guarantee CO formation and (2) prevents excessive DSB levels that could have deleterious effects.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Intercambio Genético , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Meiosis/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Redes Reguladoras de Genes , Recombinación Homóloga/genética , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Morquio A syndrome (mucopolysaccharidosis IVA) is an autosomal recessive disorder that results from deficient activity of the enzyme N-acetylgalactosamine-6-sulfatase (GALNS) due to alterations in the GALNS gene, which causes major skeletal and connective tissue abnormalities and effects on multiple organ systems. The GALNS alterations associated with Morquio A are numerous and heterogeneous, and new alterations are continuously identified. To aid detection and interpretation of GALNS alterations, from previously published research, we provide a comprehensive and up-to-date listing of 277 unique GALNS alterations associated with Morquio A identified from 1,091 published GALNS alleles. In agreement with previous findings, most reported GALNS alterations are missense changes and even the most frequent alterations are relatively uncommon. We found that 48% of patients are assessed as homozygous for a GALNS alteration, 39% are assessed as heterozygous for two identified GALNS alterations, and in 13% of patients only one GALNS alteration is detected. We report here the creation of a locus-specific database for the GALNS gene (http://galns.mutdb.org/) that catalogs all reported alterations in GALNS to date. We highlight the challenges both in alteration detection and genotype-phenotype interpretation caused in part by the heterogeneity of GALNS alterations and provide recommendations for molecular testing of GALNS.
Asunto(s)
Condroitinsulfatasas/genética , Bases de Datos de Ácidos Nucleicos , Mucopolisacaridosis IV/genética , Mutación , Alelos , Animales , Biomarcadores , Modelos Animales de Enfermedad , Frecuencia de los Genes , Estudios de Asociación Genética , Genotipo , Geografía , Humanos , Recién Nacido , Mucopolisacaridosis IV/diagnóstico , Mucopolisacaridosis IV/terapia , Tamizaje Neonatal , Fenotipo , Sitios de Carácter CuantitativoRESUMEN
Meiotic recombination is initiated by the programmed induction of double-strand DNA breaks (DSBs), lesions that pose a potential threat to the genome. A subset of the DSBs induced during meiotic prophase become designated to be repaired by a pathway that specifically yields interhomolog crossovers (COs), which mature into chiasmata that temporarily connect the homologs to ensure their proper segregation at meiosis I. The remaining DSBs must be repaired by other mechanisms to restore genomic integrity prior to the meiotic divisions. Here we show that HIM-6, the Caenorhabditis elegans ortholog of the RecQ family DNA helicase BLM, functions in both of these processes. We show that him-6 mutants are competent to load the MutSγ complex at multiple potential CO sites, to generate intermediates that fulfill the requirements of monitoring mechanisms that enable meiotic progression, and to accomplish and robustly regulate CO designation. However, recombination events at a subset of CO-designated sites fail to mature into COs and chiasmata, indicating a pro-CO role for HIM-6/BLM that manifests itself late in the CO pathway. Moreover, we find that in addition to promoting COs, HIM-6 plays a role in eliminating and/or preventing the formation of persistent MutSγ-independent associations between homologous chromosomes. We propose that HIM-6/BLM enforces biased outcomes of recombination events to ensure that both (a) CO-designated recombination intermediates are reliably resolved as COs and (b) other recombination intermediates reliably mature into noncrossovers in a timely manner.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Intercambio Genético , Animales , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Meiosis/genéticaRESUMEN
Most promoters in yeast contain a nucleosome-depleted region (NDR), but the mechanisms by which NDRs are established and maintained in vivo are currently unclear. We have examined how genome-wide nucleosome placement is altered in the absence of two distinct types of nucleosome remodeling activity. In mutants of both SNF2, which encodes the ATPase component of the Swi/Snf remodeling complex, and ASF1, which encodes a histone chaperone, distinct sets of gene promoters carry excess nucleosomes in their NDRs relative to wild-type. In snf2 mutants, excess promoter nucleosomes correlate with reduced gene expression. In both mutants, the excess nucleosomes occupy DNA sequences that are energetically less favorable for nucleosome formation, indicating that intrinsic histone-DNA interactions are not sufficient for nucleosome positioning in vivo, and that Snf2 and Asf1 promote thermodynamic equilibration of nucleosomal arrays. Cells lacking SNF2 or ASF1 still accomplish the changes in promoter nucleosome structure associated with large-scale transcriptional reprogramming. However, chromatin reorganization in the mutants is reduced in extent compared to wild-type cells, even though transcriptional changes proceed normally. In summary, active remodeling is required for distributing nucleosomes to energetically favorable positions in vivo and for reorganizing chromatin in response to changes in transcriptional activity.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Regulación Fúngica de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Chaperonas Moleculares/genética , Mutación , ARN Mensajero/análisis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Transcripción Genética , Activación TranscripcionalRESUMEN
Several well-studied promoters in yeast lose nucleosomes upon transcriptional activation and gain them upon repression, an observation that has prompted the model that transcriptional activation and repression requires nucleosome remodeling of regulated promoters. We have examined global nucleosome positioning before and after glucose-induced transcriptional reprogramming, a condition under which more than half of all yeast genes significantly change expression. The majority of induced and repressed genes exhibit no change in promoter nucleosome arrangement, although promoters that do undergo nucleosome remodeling tend to contain a TATA box. Rather, we found multiple examples where the pre-existing accessibility of putative transcription factor binding sites before glucose addition determined whether the corresponding gene would change expression in response to glucose addition. These results suggest that selection of appropriate transcription factor binding sites may be dictated to a large extent by nucleosome prepositioning but that regulation of expression through these sites is dictated not by nucleosome repositioning but by changes in transcription factor activity.
Asunto(s)
Cromatina/genética , Perfilación de la Expresión Génica , Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Teorema de Bayes , Sitios de Unión , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genoma Fúngico/genética , Glucosa/farmacología , Cadenas de Markov , Nucleosomas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , TATA Box/genética , Factores de Transcripción/metabolismoRESUMEN
Analysis of transcriptional silencing in Saccharomyces has provided valuable insights into heterochromatin formation and function. However, most of these studies revealed only the average behaviors of populations of cells. Here, we examined transcriptional silencing by monitoring individual yeast cells carrying distinguishable fluorescent reporter genes inserted at two different silent loci. These studies showed that two silent loci in a single cell behave independently, demonstrating that heterochromatin formation is locus autonomous. Furthermore, some silencing mutants with an intermediate phenotype, such as sir1, consist of two distinct populations, one repressed and one derepressed, while other mutants, such as those inactivating the SAS-I histone H4 K16 acetylase, consist of cells all with an intermediate level of expression. Finally, both establishment and decay of silencing can be influenced by specific gene activators, with establishment occurring stochastically over several generations. Thus, quantifying silencing in individual cells reveals aspects of silencing not evident from population-wide measurements.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Saccharomyces/genética , Transcripción Genética , Acetiltransferasas/metabolismo , Colorantes Fluorescentes , Genes Reporteros , Histona Acetiltransferasas , Histonas/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Activación TranscripcionalRESUMEN
In addition to its previously established roles in cAMP relay and cAMP chemotaxis, loss of signal transduction through the RasC protein was found to impact a number of vegetative cell functions. Vegetative rasC- cells exhibited reduced random motility, were less polarized and had altered F-actin distribution. Cells lacking RasC also contained more protein and were larger in size than wild type cells. These increases were associated with increased liquid phase endocytosis. Despite the increase in cell size, cytokinesis was relatively normal and there was no change in the rate of cell division. rasC- cells also chemotaxed poorly to folate and exhibited reduced F-actin accumulation, reduced ERK2 phosphorylation and reduced Akt/PKB phosphorylation in response to folate, indicating that RasC was also involved in transducing chemotactic signals in vegetative cells.