RESUMO
Programmed DNA double-strand break (DSB) formation is essential for achieving accurate chromosome segregation during meiosis. DSB repair timing and template choice are tightly regulated. However, little is known about how DSB distribution and the choice of repair pathway are regulated along the length of chromosomes, which has direct effects on the recombination landscape and chromosome remodeling at late prophase I. Here, we use the spatiotemporal resolution of meiosis in the Caenorhabditis elegans germline along with genetic approaches to study distribution of DSB processing and its regulation. High-resolution imaging of computationally straightened chromosomes immunostained for the RAD-51 recombinase marking DSB repair sites reveals that the pattern of RAD-51 foci throughout pachytene resembles crossover distribution in wild type. Specifically, RAD-51 foci occur primarily along the gene-poor distal thirds of the chromosomes in both early and late pachytene, and on both the X and the autosomes. However, this biased off-center distribution can be abrogated by the formation of excess DSBs. Reduced condensin function, but not an increase in total physical axial length, results in a homogeneous distribution of RAD-51 foci, whereas regulation of H3K9 methylation is required for the enrichment of RAD-51 at off-center positions. Finally, the DSB recognition heterodimer cKU-70/80, but not the non-homologous end-joining canonical ligase LIG-4, contributes to the enriched off-center distribution of RAD-51 foci. Taken together, our data supports a model by which regulation of the chromatin landscape, DSB levels, and DSB detection by cKU-70/80 collaborate to promote DSB processing by homologous recombination at off-center regions of the chromosomes in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Quebras de DNA de Cadeia Dupla , Cromatina/genética , Cromatina/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Troca Genética , Reparo do DNA , Cromossomos/genética , Cromossomos/metabolismo , Meiose/genéticaRESUMO
Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals. gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 coordinates the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Humanos , Camundongos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Pareamento Cromossômico , Segregação de Cromossomos , Mamíferos/genética , Meiose , Prófase Meiótica I , Complexo Sinaptonêmico/metabolismoRESUMO
Epigenetic modifiers are emerging as important regulators of the genome. However, how they regulate specific processes during meiosis is not well understood. Methylation of H3K79 by the histone methyltransferase Dot1 has been shown to be involved in the maintenance of genomic stability in various organisms. In S. cerevisiae, Dot1 modulates the meiotic checkpoint response triggered by synapsis and/or recombination defects by promoting Hop1-dependent Mek1 activation and Hop1 distribution along unsynapsed meiotic chromosomes, at least in part, by regulating Pch2 localization. However, how this protein regulates meiosis in metazoans is unknown. Here, we describe the effects of H3K79me depletion via analysis of dot-1.1 or zfp-1 mutants during meiosis in Caenorhabditis elegans. We observed decreased fertility and increased embryonic lethality in dot-1.1 mutants suggesting meiotic dysfunction. We show that DOT-1.1 plays a role in the regulation of pairing, synapsis and recombination in the worm. Furthermore, we demonstrate that DOT-1.1 is an important regulator of mechanisms surveilling chromosome synapsis during meiosis. In sum, our results reveal that regulation of H3K79me plays an important role in coordinating events during meiosis in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Histona-Lisina N-Metiltransferase/genética , Meiose/genética , Fatores de Transcrição/genética , Animais , Pareamento Cromossômico/genética , Cromossomos , Proteínas de Ligação a DNA/genética , Mutação/genética , Proteínas Nucleares/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Exposure to diethylhexyl phthalate (DEHP), the most abundant plasticizer used in the production of polyvinyl-containing plastics, has been associated to adverse reproductive health outcomes in both males and females. While the effects of DEHP on reproductive health have been widely investigated, the molecular mechanisms by which exposure to environmentally-relevant levels of DEHP and its metabolites impact the female germline in the context of a multicellular organism have remained elusive. Using the Caenorhabditis elegans germline as a model for studying reprotoxicity, we show that exposure to environmentally-relevant levels of DEHP and its metabolites results in increased meiotic double-strand breaks (DSBs), altered DSB repair progression, activation of p53/CEP-1-dependent germ cell apoptosis, defects in chromosome remodeling at late prophase I, aberrant chromosome morphology in diakinesis oocytes, increased chromosome non-disjunction and defects during early embryogenesis. Exposure to DEHP results in a subset of nuclei held in a DSB permissive state in mid to late pachytene that exhibit defects in crossover (CO) designation/formation. In addition, these nuclei show reduced Polo-like kinase-1/2 (PLK-1/2)-dependent phosphorylation of SYP-4, a synaptonemal complex (SC) protein. Moreover, DEHP exposure leads to germline-specific change in the expression of prmt-5, which encodes for an arginine methyltransferase, and both increased SC length and altered CO designation levels on the X chromosome. Taken together, our data suggest a model by which impairment of a PLK-1/2-dependent negative feedback loop set in place to shut down meiotic DSBs, together with alterations in chromosome structure, contribute to the formation of an excess number of DSBs and altered CO designation levels, leading to genomic instability.
Assuntos
Troca Genética , Quebras de DNA de Cadeia Dupla , Dietilexilftalato/toxicidade , Oogênese , Oogônios/efeitos dos fármacos , Plastificantes/toxicidade , Animais , Apoptose , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Instabilidade Genômica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oogônios/citologia , Oogônios/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The impact of exposure to microbial pathogens on animal reproductive capacity and germline physiology is not well understood. The nematode Caenorhabditis elegans is a bacterivore that encounters pathogenic microbes in its natural environment. How pathogenic bacteria affect host reproductive capacity of C. elegans is not well understood. Here, we show that exposure of C. elegans hermaphrodites to the Gram-negative pathogen Pseudomonas aeruginosa causes a marked reduction in brood size with concomitant reduction in the number of nuclei in the germline and gonad size. We define 2 processes that are induced that contribute to the decrease in the number of germ cell nuclei. First, we observe that infection with P. aeruginosa leads to the induction of germ cell apoptosis. Second, we observe that this exposure induces mitotic quiescence in the proliferative zone of the C. elegans gonad. Importantly, these processes appear to be reversible; when animals are removed from the presence of P. aeruginosa, germ cell apoptosis is abated, germ cell nuclei numbers increase, and brood sizes recover. The reversible germline dynamics during exposure to P. aeruginosa may represent an adaptive response to improve survival of progeny and may serve to facilitate resource allocation that promotes survival during pathogen infection.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Pseudomonas aeruginosa/metabolismo , Divisão Celular , Proteínas de Caenorhabditis elegans/genética , Células Germinativas/metabolismo , ApoptoseRESUMO
N,N-diethyl-meta-toluamide (DEET) is a commonly used synthetic insect repellent. Although the neurological effects of DEET have been widely investigated, its effects on the germline are less understood. Here, we show that exposure of the nematode Caenorhabditis elegans, which is highly predictive of mammalian reprotoxicity, resulting in internal DEET levels within the range detected in human biological samples, causes activation of p53/CEP-1-dependent germ cell apoptosis, altered meiotic recombination, chromosome abnormalities, and missegregation. RNA-sequencing analysis links DEET-induced alterations in the expression of genes related to redox processes and chromatin structure to reduced mitochondrial function, impaired DNA double-strand break repair progression, and defects during early embryogenesis. We propose that Caenorhabditis elegans exposure to DEET interferes with gene expression, leading to increased oxidative stress and altered chromatin structure, resulting in germline effects that pose a risk to reproductive health.
RESUMO
[This corrects the article DOI: 10.1016/j.isci.2023.108699.].
RESUMO
Loss of function during aging is accompanied by transcriptional drift, altering gene expression and contributing to a variety of age-related diseases. CREB-regulated transcriptional coactivators (CRTCs) have emerged as key regulators of gene expression that might be targeted to promote longevity. Here we define the role of the Caenorhabditis elegans CRTC-1 in the epigenetic regulation of longevity. Endogenous CRTC-1 binds chromatin factors, including components of the COMPASS complex, which trimethylates lysine 4 on histone H3 (H3K4me3). CRISPR editing of endogenous CRTC-1 reveals that the CREB-binding domain in neurons is specifically required for H3K4me3-dependent longevity. However, this effect is independent of CREB but instead acts via the transcription factor AP-1. Strikingly, CRTC-1 also mediates global histone acetylation levels, and this acetylation is essential for H3K4me3-dependent longevity. Indeed, overexpression of an acetyltransferase enzyme is sufficient to promote longevity in wild-type worms. CRTCs, therefore, link energetics to longevity by critically fine-tuning histone acetylation and methylation to promote healthy aging.
Assuntos
Caenorhabditis elegans , Longevidade , Animais , Caenorhabditis elegans/genética , Epigênese Genética , Histonas/química , Longevidade/genética , Fatores de Transcrição/genéticaRESUMO
The Vasa family of proteins comprises several conserved DEAD box RNA helicases important for mRNA regulation whose exact function in the germline is still unknown. In Caenorhabditis elegans, there are six known members of the Vasa family, and all of them are associated with P granules. One of these proteins, VBH-1, is important for oogenesis, spermatogenesis, embryo development, and the oocyte/sperm switch in this nematode. We decided to extend our previous work in C. elegans to sibling species Caenorhabditis remanei to understand what is the function of the VBH-1 homolog in this gonochoristic species. We found that Cre-VBH-1 is present in the cytoplasm of germ cells and it remains associated with P granules throughout the life cycle of C. remanei. Several aspects between VBH-1 and Cre-VBH-1 function are conserved like their role during oogenesis, spermatogenesis, and embryonic development. However, Cre-vbh-1 silencing in C. remanei had a stronger effect on spermatogenesis and spermatid activation than in C. elegans. An unexpected finding was that silencing of vbh-1 in the C. elegans caused a decrease in germ cell apoptosis in the hermaphrodite gonad, while silencing of Cre-vbh-1 in C. remanei elicited germ cell apoptosis in the male gonad. These data suggest that VBH-1 might play a role in germ cell survival in both species albeit it appears to have an opposite role in each one.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/citologia , Sobrevivência Celular , RNA Helicases DEAD-box/genética , Oócitos/fisiologia , Espermatozoides/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Sequência Conservada , Grânulos Citoplasmáticos/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/fisiologia , Desenvolvimento Embrionário , Feminino , Fertilidade , Inativação Gênica , Gônadas/citologia , Gônadas/metabolismo , Masculino , Dados de Sequência Molecular , Oócitos/metabolismo , Oogênese , Especificidade de Órgãos , Transporte Proteico , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Espermatogênese , Espermatozoides/metabolismoRESUMO
To generate gametes, sexually reproducing organisms need to achieve a reduction in ploidy, via meiosis. Several mechanisms are set in place to ensure proper reductional chromosome segregation at the first meiotic division (MI), including chromosome remodeling during late prophase I. Chromosome remodeling after crossover formation involves changes in chromosome condensation and restructuring, resulting in a compact bivalent, with sister kinetochores oriented to opposite poles, whose structure is crucial for localized loss of cohesion and accurate chromosome segregation. Here, we review the general processes involved in late prophase I chromosome remodeling, their regulation, and the strategies devised by different organisms to produce bivalents with configurations that promote accurate segregation.
Assuntos
Segregação de Cromossomos , Prófase Meiótica I , Segregação de Cromossomos/genética , Cinetocoros , Meiose/genética , Prófase Meiótica I/genéticaRESUMO
DNA double-strand breaks (DSBs) are deleterious lesions, which must be repaired precisely to maintain genomic stability. During meiosis, programmed DSBs are repaired via homologous recombination (HR) while repair using the nonhomologous end joining (NHEJ) pathway is inhibited, thereby ensuring crossover formation and accurate chromosome segregation.1,2 How DSB repair pathway choice is implemented during meiosis is unknown. In C. elegans, meiotic DSB repair takes place in the context of the fully formed, highly dynamic zipper-like structure present between homologous chromosomes called the synaptonemal complex (SC).3,4,5,6,7,8,9 The SC consists of a pair of lateral elements bridged by a central region composed of the SYP proteins in C. elegans. How the structural components of the SC are regulated to maintain the architectural integrity of the assembled SC around DSB repair sites remained unclear. Here, we show that SYP-4, a central region component of the SC, is phosphorylated at Serine 447 in a manner dependent on DSBs and the ATM/ATR DNA damage response kinases. We show that this SYP-4 phosphorylation is critical for preserving the SC structure following exogenous (γ-IR-induced) DSB formation and for promoting normal DSB repair progression and crossover patterning following SPO-11-dependent and exogenous DSBs. We propose a model in which ATM/ATR-dependent phosphorylation of SYP-4 at the S447 site plays important roles both in maintaining the architectural integrity of the SC following DSB formation and in warding off repair via the NHEJ repair pathway, thereby preventing aneuploidy.
Assuntos
Proteínas de Caenorhabditis elegans , Quebras de DNA de Cadeia Dupla , Animais , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Reparo do DNA , Meiose , DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismoRESUMO
Interhomolog crossovers (COs) are a prerequisite for achieving accurate chromosome segregation during meiosis [1, 2]. COs are not randomly positioned, occurring at distinct genomic intervals during meiosis in all species examined [3-10]. The role of CO position as a major determinant of accurate chromosome segregation has not been previously directly analyzed in a metazoan. Here, we use spo-11 mutants, which lack endogenous DNA double-strand breaks (DSBs), to induce a single DSB by Mos1 transposon excision at defined chromosomal locations in the C. elegans germline and show that the position of the resulting CO directly affects the formation of distinct chromosome subdomains during meiotic chromosome remodeling. CO formation in the typically CO-deprived center region of autosomes leads to premature loss of sister chromatid cohesion and chromosome missegregation, whereas COs at an off-centered position, as in wild type, can result in normal remodeling and accurate segregation. Ionizing radiation (IR)-induced DSBs lead to the same outcomes, and modeling of IR dose-response reveals that the CO-unfavorable center region encompasses up to 6% of the total chromosome length. DSBs proximal to telomeres rarely form COs, likely because of formation of unstable recombination intermediates that cannot be sustained as chiasmata until late prophase. Our work supports a model in which regulation of CO position early in meiotic prophase is required for proper designation of chromosome subdomains and normal chromosome remodeling in late meiotic prophase I, resulting in accurate chromosome segregation and providing a mechanism to prevent aneuploid gamete formation.