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
The mechanisms that extend lifespan in humans are poorly understood. Here we show that extended longevity in humans is associated with a distinct transcriptome signature in the cerebral cortex that is characterized by downregulation of genes related to neural excitation and synaptic function. In Caenorhabditis elegans, neural excitation increases with age and inhibition of excitation globally, or in glutamatergic or cholinergic neurons, increases longevity. Furthermore, longevity is dynamically regulated by the excitatory-inhibitory balance of neural circuits. The transcription factor REST is upregulated in humans with extended longevity and represses excitation-related genes. Notably, REST-deficient mice exhibit increased cortical activity and neuronal excitability during ageing. Similarly, loss-of-function mutations in the C. elegans REST orthologue genes spr-3 and spr-4 elevate neural excitation and reduce the lifespan of long-lived daf-2 mutants. In wild-type worms, overexpression of spr-4 suppresses excitation and extends lifespan. REST, SPR-3, SPR-4 and reduced excitation activate the longevity-associated transcription factors FOXO1 and DAF-16 in mammals and worms, respectively. These findings reveal a conserved mechanism of ageing that is mediated by neural circuit activity and regulated by REST.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Longevidade , Neurônios/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Envelhecimento , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Caenorhabditis elegans , Fatores de Transcrição Forkhead/metabolismo , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Interferência de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
The position of recombination events established along chromosomes in early prophase I and the chromosome remodeling that takes place in late prophase I are intrinsically linked steps of meiosis that need to be tightly regulated to ensure accurate chromosome segregation and haploid gamete formation. Here, we show that RAD-51 foci, which form at the sites of programmed meiotic DNA double-strand breaks (DSBs), exhibit a biased distribution toward off-centered positions along the chromosomes in wild-type Caenorhabditis elegans, and we identify two meiotic roles for chromatin-associated protein HIM-17 that ensure normal chromosome remodeling in late prophase I. During early prophase I, HIM-17 regulates the distribution of DSB-dependent RAD-51 foci and crossovers on chromosomes, which is critical for the formation of distinct chromosome subdomains (short and long arms of the bivalents) later during chromosome remodeling. During late prophase I, HIM-17 promotes the normal expression and localization of protein phosphatases GSP-1/2 to the surface of the bivalent chromosomes and may promote GSP-1 phosphorylation, thereby antagonizing Aurora B kinase AIR-2 loading on the long arms and preventing premature loss of sister chromatid cohesion. We propose that HIM-17 plays distinct roles at different stages during meiotic progression that converge to promote normal chromosome remodeling and accurate chromosome segregation.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Meiose/fisiologia , Recombinação Genética/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Segregação de Cromossomos/genética , Cromossomos/metabolismo , Troca Genética/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Rad51 Recombinase/metabolismo , Recombinação Genética/genéticaRESUMO
Histone methylation is dynamically regulated to shape the epigenome and adjust central nuclear processes including transcription, cell cycle control and DNA repair. Lysine-specific histone demethylase 2 (LSD2) has been implicated in multiple types of human cancers. However, its functions remain poorly understood. This study investigated the histone demethylase LSD2 homolog AMX-1 in C. elegans and uncovered a potential link between H3K4me2 modulation and DNA interstrand crosslink (ICL) repair. AMX-1 is a histone demethylase and mainly localizes to embryonic cells, the mitotic gut and sheath cells. Lack of AMX-1 expression resulted in embryonic lethality, a decreased brood size and disorganized premeiotic tip germline nuclei. Expression of AMX-1 and of the histone H3K4 demethylase SPR-5 is reciprocally up-regulated upon lack of each other and the mutants show increased H3K4me2 levels in the germline, indicating that AMX-1 and SPR-5 regulate H3K4me2 demethylation. Loss of AMX-1 function activates the CHK-1 kinase acting downstream of ATR and leads to the accumulation of RAD-51 foci and increased DNA damage-dependent apoptosis in the germline. AMX-1 is required for the proper expression of mismatch repair component MutL/MLH-1 and sensitivity against ICLs. Interestingly, formation of ICLs lead to ubiquitination-dependent subcellular relocalization of AMX-1. Taken together, our data suggest that AMX-1 functions in ICL repair in the germline.
Assuntos
Reparo do DNA/genética , Histona Desmetilases/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans , Núcleo Celular/metabolismo , Dano ao DNA/genética , Reparo do DNA/fisiologia , Células Germinativas/metabolismo , Histona Desmetilases/fisiologia , Histonas/genética , Metilação , Processamento de Proteína Pós-Traducional/genética , UbiquitinaçãoRESUMO
N-terminal acetylation of the first two amino acids on proteins is a prevalent cotranslational modification. Despite its abundance, the biological processes associated with this modification are not well understood. Here, we mapped the pattern of protein N-terminal acetylation in Caenorhabditis elegans, uncovering a conserved set of rules for this protein modification and identifying substrates for the N-terminal acetyltransferase B (NatB) complex. We observed an enrichment for global protein N-terminal acetylation and also specifically for NatB substrates in the nucleus, supporting the importance of this modification for regulating biological functions within this cellular compartment. Peptide profiling analysis provides evidence of cross-talk between N-terminal acetylation and internal modifications in a NAT substrate-specific manner. In vivo studies indicate that N-terminal acetylation is critical for meiosis, as it regulates the assembly of the synaptonemal complex (SC), a proteinaceous structure ubiquitously present during meiosis from yeast to humans. Specifically, N-terminal acetylation of NatB substrate SYP-1, an SC structural component, is critical for SC assembly. These findings provide novel insights into the biological functions of N-terminal acetylation and its essential role during meiosis.
Assuntos
Caenorhabditis elegans/metabolismo , Acetiltransferase N-Terminal B/metabolismo , Complexo Sinaptonêmico/metabolismo , Acetilação , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Meiose/genética , Mutação , Acetiltransferase N-Terminal B/genética , Proteínas Nucleares/metabolismo , Proteoma , Complexo Sinaptonêmico/química , Complexo Sinaptonêmico/genéticaRESUMO
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
Chemicals that are highly prevalent in our environment, such as phthalates and pesticides, have been linked to problems associated with reproductive health. However, rapid assessment of their impact on reproductive health and understanding how they cause such deleterious effects, remain challenging due to their fast-growing numbers and the limitations of various current toxicity assessment model systems. Here, we performed a high-throughput screen in C. elegans to identify chemicals inducing aneuploidy as a result of impaired germline function. We screened 46 chemicals that are widely present in our environment, but for which effects in the germline remain poorly understood. These included pesticides, phthalates, and chemicals used in hydraulic fracturing and crude oil processing. Of the 46 chemicals tested, 41% exhibited levels of aneuploidy higher than those detected for bisphenol A (BPA), an endocrine disruptor shown to affect meiosis, at concentrations correlating well with mammalian reproductive endpoints. We further examined three candidates eliciting aneuploidy: dibutyl phthalate (DBP), a likely endocrine disruptor and frequently used plasticizer, and the pesticides 2-(thiocyanomethylthio) benzothiazole (TCMTB) and permethrin. Exposure to these chemicals resulted in increased embryonic lethality, elevated DNA double-strand break (DSB) formation, activation of p53/CEP-1-dependent germ cell apoptosis, chromosomal abnormalities in oocytes at diakinesis, impaired chromosome segregation during early embryogenesis, and germline-specific alterations in gene expression. This study indicates that this high-throughput screening system is highly reliable for the identification of environmental chemicals inducing aneuploidy, and provides new insights into the impact of exposure to three widely used chemicals on meiosis and germline function.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Poluentes Ambientais/toxicidade , Células Germinativas/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Aneugênicos/toxicidade , Aneuploidia , Animais , Animais Geneticamente Modificados , Benzotiazóis/toxicidade , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Quebras de DNA de Cadeia Dupla , Dibutilftalato/toxicidade , Exposição Ambiental , Inseticidas/toxicidade , Meiose/efeitos dos fármacos , Permetrina/toxicidade , Plastificantes/toxicidade , Tiocianatos/toxicidadeRESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Reparo do DNA , Replicação do DNA , Dioxigenases/genética , Oxirredutases N-Desmetilantes/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Dioxigenases/metabolismo , Masculino , Meiose , Mutação , Oxirredutases N-Desmetilantes/metabolismo , Análise de Sequência de RNARESUMO
The proteinaceous zipper-like structure known as the synaptonemal complex (SC), which forms between pairs of homologous chromosomes during meiosis from yeast to humans, plays important roles in promoting interhomolog crossover formation, regulating cessation of DNA double-strand break (DSB) formation following crossover designation, and ensuring accurate meiotic chromosome segregation. Recent studies are starting to reveal critical roles for different protein modifications in regulating SC dynamics. Protein SUMOylation, N-terminal acetylation, and phosphorylation have been shown to be essential for the regulated assembly and disassembly of the SC. Moreover, phosphorylation of specific SC components has been found to link changes in SC dynamics with meiotic recombination. This review highlights the latest findings on how protein modifications regulate SC dynamics and functions.
Assuntos
Segregação de Cromossomos/genética , Troca Genética/genética , Quebras de DNA de Cadeia Dupla , Processamento de Proteína Pós-Traducional , Complexo Sinaptonêmico/genética , Animais , Humanos , Modelos Genéticos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Complexo Sinaptonêmico/metabolismoRESUMO
Epidemiological studies continue to reveal the enduring impact of exposures to environmental chemicals on human physiology, including our reproductive health. Phthalates, a well characterized class of endocrine disrupting chemicals and commonly utilized plasticizers, are among one of the many toxicants ubiquitously present in our environment. Phthalate exposure has been linked to increases in the rate of human aneuploidy, a phenomenon that is detected in 0.3% of livebirths resulting in genetic disorders including trisomy 21, approximately 4% of stillbirths, and over 35% of miscarriages. Here we review recent epidemiological and experimental studies that have examined the role that phthalates play in germline dysfunction, including increases in apoptosis, oxidative stress, DNA damage, and impaired genomic integrity, resulting in aneuploidy. We will further discuss subject variability, as it relates to diet and polymorphisms, and the sexual dimorphic effects of phthalate exposure, as it relates to sex-specific targets. Lastly, we discuss some of the conserved effects of phthalate exposure across humans, mammalian models and nonmammalian model organisms, highlighting the importance of using model organisms to our advantage for chemical risk assessment and unveiling potential mechanisms that underlie phthalate-induced reproductive health issues across species.
Assuntos
Aneuploidia , Exposição Ambiental/efeitos adversos , Doenças Genéticas Inatas/etiologia , Ácidos Ftálicos/efeitos adversos , Adulto , Exposição Ambiental/análise , Exposição Ambiental/estatística & dados numéricos , Feminino , Doenças Genéticas Inatas/epidemiologia , Humanos , GravidezRESUMO
Breast cancer susceptibility gene 1 (BRCA1) and binding partner BRCA1-associated RING domain protein 1 (BARD1) form an essential E3 ubiquitin ligase important for DNA damage repair and homologous recombination. The Caenorhabditis elegans orthologs, BRC-1 and BRD-1, also function in DNA damage repair, homologous recombination, as well as in meiosis. Using functional GFP fusions we show that in mitotically-dividing germ cells BRC-1 and BRD-1 are nucleoplasmic with enrichment at foci that partially overlap with the recombinase RAD-51. Co-localization with RAD-51 is enhanced under replication stress. As cells enter meiosis, BRC-1-BRD-1 remains nucleoplasmic and in foci, and beginning in mid-pachytene the complex co-localizes with the synaptonemal complex. Following establishment of the single asymmetrically positioned crossover on each chromosome pair, BRC-1-BRD-1 concentrates to the short arm of the bivalent. Localization dependencies reveal that BRC-1 and BRD-1 are interdependent and the complex fails to properly localize in both meiotic recombination and chromosome synapsis mutants. Consistent with a role for BRC-1-BRD-1 in meiotic recombination in the context of the synaptonemal complex, inactivation of BRC-1 or BRD-1 enhances the embryonic lethality of mutants defective in chromosome synapsis. Our data suggest that under meiotic dysfunction, BRC-1-BRD-1 stabilizes the RAD-51 filament and alters the recombination landscape; these two functions can be genetically separated from BRC-1-BRD-1's role in the DNA damage response. Together, we propose that BRC-1-BRD-1 serves a checkpoint function at the synaptonemal complex where it monitors and modulates meiotic recombination.
Assuntos
Proteína BRCA1/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Meiose/genética , Recombinação Genética , Complexo Sinaptonêmico/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Alelos , Animais , Proteína BRCA1/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Replicação do DNA , Embrião não Mamífero , Genes Reporter , Células Germinativas , Transporte Proteico , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Human neurons are functional over an entire lifetime, yet the mechanisms that preserve function and protect against neurodegeneration during ageing are unknown. Here we show that induction of the repressor element 1-silencing transcription factor (REST; also known as neuron-restrictive silencer factor, NRSF) is a universal feature of normal ageing in human cortical and hippocampal neurons. REST is lost, however, in mild cognitive impairment and Alzheimer's disease. Chromatin immunoprecipitation with deep sequencing and expression analysis show that REST represses genes that promote cell death and Alzheimer's disease pathology, and induces the expression of stress response genes. Moreover, REST potently protects neurons from oxidative stress and amyloid ß-protein toxicity, and conditional deletion of REST in the mouse brain leads to age-related neurodegeneration. A functional orthologue of REST, Caenorhabditis elegans SPR-4, also protects against oxidative stress and amyloid ß-protein toxicity. During normal ageing, REST is induced in part by cell non-autonomous Wnt signalling. However, in Alzheimer's disease, frontotemporal dementia and dementia with Lewy bodies, REST is lost from the nucleus and appears in autophagosomes together with pathological misfolded proteins. Finally, REST levels during ageing are closely correlated with cognitive preservation and longevity. Thus, the activation state of REST may distinguish neuroprotection from neurodegeneration in the ageing brain.
Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Estresse Oxidativo , Proteínas Repressoras/metabolismo , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Animais , Autofagia , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Caenorhabditis elegans/metabolismo , Morte Celular/genética , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Cognição , Disfunção Cognitiva/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Regulação da Expressão Gênica , Humanos , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , Longevidade , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Fagossomos , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Regulação para Cima , Via de Sinalização Wnt , Adulto JovemRESUMO
The Fanconi anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 (KIAA1018) and EXDL2. FAN1 colocalizes at sites of DNA damage with the ID complex in a manner dependent on FAN1's ubiquitin-binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5'-3' exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Exonucleases/metabolismo , Anemia de Fanconi/enzimologia , Testes Genéticos/métodos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Linhagem Celular , Dano ao DNA , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Endodesoxirribonucleases , Endonucleases/metabolismo , Exodesoxirribonucleases/química , Exonucleases/química , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Genoma Humano/genética , Humanos , Mitomicina/farmacologia , Dados de Sequência Molecular , Enzimas Multifuncionais , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismoRESUMO
The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.
Assuntos
Acetilcoenzima A/metabolismo , Acetil-CoA Hidrolase/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Recombinação Genética , Acetilação , Animais , Quebras de DNA de Cadeia Dupla , Histonas/metabolismo , Cromossomo X/metabolismoRESUMO
Meiosis is a specialized form of cell division required for the formation of haploid gametes and therefore is essential for successful sexual reproduction. Various steps are exquisitely coordinated to ensure accurate chromosome segregation during meiosis, thereby promoting the formation of haploid gametes from diploid cells. Recent studies are demonstrating that an important form of regulation during meiosis is exerted by the post-translational protein modification known as sumoylation. Here, we review and discuss the various critical steps of meiosis in which SUMO-mediated regulation has been implicated thus far. These include the maintenance of meiotic centromeric heterochromatin , meiotic DNA double-strand break repair and homologous recombination, centromeric coupling, and the assembly of a proteinaceous scaffold between homologous chromosomes known as the synaptonemal complex.
Assuntos
Cromossomos/metabolismo , Meiose , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Animais , Cromossomos/química , Cromossomos/genética , Heterocromatina/metabolismo , Humanos , Conformação de Ácido NucleicoRESUMO
Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs) can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR) and DSB repair (DSBR) within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Quinase 1 do Ponto de Checagem , Segregação de Cromossomos , Sequência Conservada , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Feminino , Raios gama/efeitos adversos , Células Germinativas/efeitos dos fármacos , Células Germinativas/fisiologia , Células Germinativas/efeitos da radiação , Masculino , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.