In vivo analysis of FANCD2 recruitment at meiotic DNA breaks in Caenorhabditis elegans

AbstractFanconi Anemia is a rare genetic disease associated with DNA repair defects, congenital abnormalities and infertility. Most of FA pathway is evolutionary conserved, allowing dissection and mechanistic studies in simpler model systems such as Caenorhabditis elegans. In the present study, we employed C. elegans to better understand the role of FA group D2 (FANCD2) protein in vivo, a key player in promoting genome stability. We report that localization of FCD-2/FANCD2 is dynamic during meiotic prophase I and requires its heterodimeric partner FNCI-1/FANCI. Strikingly, we found that FCD-2 recruitment depends on SPO-11-induced double-strand breaks (DSBs) but not RAD-51-mediated strand invasion. Furthermore, exposure to DNA damage-inducing agents boosts FCD-2 recruitment on the chromatin. Finally, analysis of genetic interaction between FCD-2 and BRC-1 (the C. elegans orthologue of mammalian BRCA1) supports a role for these proteins in different DSB repair pathways. Collectively, we showed a direct involvement of FCD-2 at DSBs and speculate on its function in driving meiotic DNA repair.

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Histone marks: repairing DNA breaks within the context of chromatin

Biochemical Society Transactions ◽

10.1042/bst20110747 ◽

2012 ◽

Vol 40 (2) ◽

pp. 370-376 ◽

Cited By ~ 62

Author(s):

Kyle M. Miller ◽

Stephen P. Jackson

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Genome Stability ◽

Nuclear Dna ◽

Dna Lesions ◽

Dna Breaks ◽

Strand Breaks ◽

Dna Dsbs

Inherited or acquired defects in detecting, signalling or repairing DNA damage are associated with various human pathologies, including immunodeficiencies, neurodegenerative diseases and various forms of cancer. Nuclear DNA is packaged into chromatin and therefore the true in vivo substrate of damaged DNA occurs within the context of chromatin. Our work aims to decipher the mechanisms by which cells detect DNA damage and signal its presence to the DNA-repair and cell-cycle machineries. In particular, much of our work has focused on DNA DSBs (double-strand breaks) that are generated by ionizing radiation and radiomimetic chemicals, and which can also arise when the DNA replication apparatus encounters other DNA lesions. In the present review, we describe some of our recent work, as well as the work of other laboratories, that has identified new chromatin proteins that mediate DSB responses, control SDB processing or modulate chromatin structure at DNA-damage sites. We also aim to survey several recent advances in the field that have contributed to our understanding of how particular histone modifications and involved in DNA repair. It is our hope that by understanding the role of chromatin and its modifications in promoting DNA repair and genome stability, this knowledge will provide opportunities for developing novel classes of drugs to treat human diseases, including cancer.

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Transfer and tissue-specific accumulation of components in embryos of Caenorhabditis elegans and dolichura: in vivo analysis with a low-cost signal

Development ◽

10.1242/dev.114.2.317 ◽

1992 ◽

Vol 114 (2) ◽

pp. 317-330 ◽

Cited By ~ 4

Author(s):

O. Bossinger ◽

E. Schierenberg

Keyword(s):

Caenorhabditis Elegans ◽

Low Cost ◽

Free Living ◽

Tissue Specific ◽

C Elegans ◽

Specific Accumulation ◽

In Vivo Analysis

The pattern of autofluorescence in the two free-living namatodes Rhabditis dolichura and Caenorhabditis compared. In C. elegans, during later embryogenesis cells develop a typical bluish autofluorescence as illumination, while in Rh. dolichura a strong already present in the unfertilized egg. Using a new,

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A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

Nature Communications ◽

10.1038/s41467-019-12802-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 21

Author(s):

William H. Gittens ◽

Dominic J. Johnson ◽

Rachal M. Allison ◽

Tim J. Cooper ◽

Holly Thomas ◽

...

Keyword(s):

Dna Cleavage ◽

Genome Stability ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Strand Breaks ◽

Single Strand Breaks ◽

Human Genomes ◽

Nucleotide Resolution

Abstract DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

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A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

10.1101/530667 ◽

2019 ◽

Cited By ~ 2

Author(s):

William Gittens ◽

Dominic J. Johnson ◽

Rachal M. Allison ◽

Tim J. Cooper ◽

Holly Thomas ◽

...

Keyword(s):

Dna Cleavage ◽

Genome Stability ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Strand Breaks ◽

Single Strand Breaks ◽

Human Genomes ◽

Nucleotide Resolution

AbstractDNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy enables us to reveal the influence primary DNA sequence has upon Top2 cleavage—distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

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Relevance of bioassay of biologically active substances (BAS) with geroprotective properties in the model of the nematode Caenorhabditis elegans in experiments in vivo

Current Aging Science ◽

10.2174/1874609814666211202144911 ◽

2021 ◽

Vol 14 ◽

Author(s):

Lyubov S. Dyshlyuk ◽

Anastasiya I. Dmitrieva ◽

Margarita Yu. Drozdova ◽

Irina S. Milentyeva ◽

Alexander Yu. Prosekov

Keyword(s):

Caenorhabditis Elegans ◽

Plant Extracts ◽

Aging Process ◽

Model Organism ◽

Biologically Active ◽

Model Systems ◽

C Elegans ◽

Living Organisms ◽

Active Substances

: Aging is a process global in nature. The age of living organisms contributes to the appearance of chronic diseases, which not only reduce the quality of life, but also significantly damage it. Modern medicines can successfully fight multiple diseases and prolong life. At the same time, medications have a large number of side effects. New research indicates that bioactive phytochemicals have great potential for treating even the most severe diseases and can become an alternative to medicines. Despite many studies in this area, the effects of many plant ingredients on living organisms are poorly understood. Analysis of the mechanisms through which herbal preparations influence the aging process helps to select the right active substances, determine the optimal doses to obtain the maximum positive effect. It is preferable to check the effectiveness of plant extracts and biologically active components with geroprotective properties in vivo. For these purposes, live model systems such as Rattus rattus, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans are used. These models help to comprehensively study the impact of the developed new drugs on the aging process. The model organism C. elegans is gaining increasing popularity in these studies because of its many advantages. This review article discusses the advantages of the nematode C. elegans as a model organism for studying the processes associated with aging. The influence of various BAS and plant extracts on the increase in the life span of the nematode, on the increase in its stress resistance and on other markers of aging is also considered. The review showed that the nematode C. elegans has a number of advantages over other organisms and is a promising model system for studying the geroprotective properties of BAS.

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Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e17-06-0412 ◽

2018 ◽

Vol 29 (6) ◽

pp. 722-735 ◽

Cited By ~ 6

Author(s):

Sarah Bianchi ◽

Kacper B. Rogala ◽

Nicola J. Dynes ◽

Manuel Hilbert ◽

Sebastian A. Leidel ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Microtubule Network ◽

Microtubule Binding ◽

C Elegans ◽

Evolutionarily Conserved ◽

Organelle Assembly ◽

In Vivo Analysis ◽

Cilia And Flagella

Centrioles are microtubule-based organelles that organize the microtubule network and seed the formation of cilia and flagella. New centrioles assemble through a stepwise process dependent notably on the centriolar protein SAS-5 in Caenorhabditis elegans. SAS-5 and its functional homologues in other species form oligomers that bind the centriolar proteins SAS-6 and SAS-4, thereby forming an evolutionarily conserved structural core at the onset of organelle assembly. Here, we report a novel interaction of SAS-5 with microtubules. Microtubule binding requires SAS-5 oligomerization and a disordered protein segment that overlaps with the SAS-4 binding site. Combined in vitro and in vivo analysis of select mutants reveals that the SAS-5–microtubule interaction facilitates centriole assembly in C. elegans embryos. Our findings lead us to propose that the interdependence of SAS-5 oligomerization and microtubule binding reflects an avidity mechanism, which also strengthens SAS-5 associations with other centriole components and, thus, promotes organelle assembly.

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A Region of the Myosin Rod Important for Interaction With Paramyosin in Caenorhabditis elegans Striated Muscle

Genetics ◽

10.1093/genetics/156.2.631 ◽

2000 ◽

Vol 156 (2) ◽

pp. 631-643

Author(s):

Pamela E Hoppe ◽

Robert H Waterston

Keyword(s):

Caenorhabditis Elegans ◽

Heavy Chain ◽

Molecular Interaction ◽

Striated Muscle ◽

Genetic Interaction ◽

Specific Interaction ◽

Thick Filament ◽

Parallel Arrangement ◽

Myosin Rod

Abstract The precise arrangement of molecules within the thick filament, as well as the mechanisms by which this arrangement is specified, remains unclear. In this article, we have exploited a unique genetic interaction between one isoform of myosin heavy chain (MHC) and paramyosin in Caenorhabditis elegans to probe the molecular interaction between MHC and paramyosin in vivo. Using chimeric myosin constructs, we have defined a 322-residue region of the MHC A rod critical for suppression of the structural and motility defects associated with the unc-15(e73) allele. Chimeric constructs lacking this region of MHC A either fail to suppress, or act as dominant enhancers of, the e73 phenotype. Although the 322-residue region is required for suppression activity, our data suggest that sequences along the length of the rod also play a role in the isoform-specific interaction between MHC A and paramyosin. Our genetic and cell biological analyses of construct behavior suggest that the 322-residue region of MHC A is important for thick filament stability. We present a model in which this region mediates an avid interaction between MHC A and paramyosin in parallel arrangement in formation of the filament arms.

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Crosstalk between Different DNA Repair Pathways Contributes to Neurodegenerative Diseases

Biology ◽

10.3390/biology10020163 ◽

2021 ◽

Vol 10 (2) ◽

pp. 163

Author(s):

Swapnil Gupta ◽

Panpan You ◽

Tanima SenGupta ◽

Hilde Nilsen ◽

Kulbhushan Sharma

Keyword(s):

Dna Repair ◽

Neurodegenerative Diseases ◽

3D Models ◽

Model Systems ◽

Spinocerebellar Ataxias ◽

C Elegans ◽

Cellular Processes ◽

Age Related ◽

Damage Response ◽

Lateral Sclerosis

Genomic integrity is maintained by DNA repair and the DNA damage response (DDR). Defects in certain DNA repair genes give rise to many rare progressive neurodegenerative diseases (NDDs), such as ocular motor ataxia, Huntington disease (HD), and spinocerebellar ataxias (SCA). Dysregulation or dysfunction of DDR is also proposed to contribute to more common NDDs, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Amyotrophic Lateral Sclerosis (ALS). Here, we present mechanisms that link DDR with neurodegeneration in rare NDDs caused by defects in the DDR and discuss the relevance for more common age-related neurodegenerative diseases. Moreover, we highlight recent insight into the crosstalk between the DDR and other cellular processes known to be disturbed during NDDs. We compare the strengths and limitations of established model systems to model human NDDs, ranging from C. elegans and mouse models towards advanced stem cell-based 3D models.

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Caenorhabditis elegans as an in vivo Model to Assess Amphetamine Tolerance

Brain Behavior and Evolution ◽

10.1159/000514858 ◽

2021 ◽

pp. 1-9

Author(s):

Dayana Torres Valladares ◽

Sirisha Kudumala ◽

Murad Hossain ◽

Lucia Carvelli

Keyword(s):

Caenorhabditis Elegans ◽

Molecular Mechanisms ◽

Drugs Of Abuse ◽

Genetic Model ◽

In Vivo Model ◽

C Elegans ◽

Hyperactivity Disorder ◽

In Vitro Data

Amphetamine is a potent psychostimulant also used to treat attention deficit/hyperactivity disorder and narcolepsy. In vivo and in vitro data have demonstrated that amphetamine increases the amount of extra synaptic dopamine by both inhibiting reuptake and promoting efflux of dopamine through the dopamine transporter. Previous studies have shown that chronic use of amphetamine causes tolerance to the drug. Thus, since the molecular mechanisms underlying tolerance to amphetamine are still unknown, an animal model to identify the neurochemical mechanisms associated with drug tolerance is greatly needed. Here we took advantage of a unique behavior caused by amphetamine in <i>Caenorhabditis elegans</i> to investigate whether this simple, but powerful, genetic model develops tolerance following repeated exposure to amphetamine. We found that at least 3 treatments with 0.5 mM amphetamine were necessary to see a reduction in the amphetamine-induced behavior and, thus, to promote tolerance. Moreover, we found that, after intervals of 60/90 minutes between treatments, animals were more likely to exhibit tolerance than animals that underwent 10-minute intervals between treatments. Taken together, our results show that <i>C. elegans</i> is a suitable system to study tolerance to drugs of abuse such as amphetamines.

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Automated and customizable quantitative image analysis of whole Caenorhabditis elegans germlines

Genetics ◽

10.1093/genetics/iyab010 ◽

2021 ◽

Author(s):

Erik Toraason ◽

Victoria L Adler ◽

Nicole A Kurhanewicz ◽

Acadia DiNardo ◽

Adam M Saunders ◽

...

Keyword(s):

Image Analysis ◽

Caenorhabditis Elegans ◽

Germ Cell ◽

Meiotic Prophase ◽

Quantitative Image Analysis ◽

Dna Breaks ◽

C Elegans ◽

Quantitative Image ◽

Cytological Features ◽

Single Nucleus

Abstract Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-content image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: (1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and (2) assessment of these individual nuclei based on their position within the germline. We show the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double-strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields using the C. elegans germline as a model system.

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