Counteracting Environmental Chemicals with Coenzyme Q10: An Educational Primer for Use with "Antioxidant CoQ10 Restores Fertility by Rescuing Bisphenol A-Induced Oxidative DNA Damage in the Caenorhabditis elegans Germline".

Environmental toxicants are chemicals that negatively affect human health. Although there are numerous ways to limit exposure, the ubiquitous nature of certain environmental toxicants makes it impossible to avoid them entirely. Consequently, scientists are continuously working toward developing strategies for combating their harmful effects. Using the nematode Caenorhabditis elegans, a model with many genetic and physiological similarities to humans, researchers in the Colaiácovo laboratory have identified several molecular mechanisms by which the toxic agent bisphenol A (BPA) interferes with reproduction. Here, we address their recent discovery that a widely available compound, Coenzyme Q10 (CoQ10), can rescue BPA-induced damage. This work is significant in that it poses a low-cost method for improving reproductive success in humans. The goal of this primer is to assist educators and students with navigating the paper entitled "Antioxidant CoQ10 Restores Fertility by Rescuing Bisphenol A-Induced Oxidative DNA Damage in the Caenorhabditis elegans Germline." It is ideally suited for integration into an upper-level undergraduate course such as Genetics, Cell and Molecular Biology, Developmental Biology, or Toxicology. The primer provides background information on the history of BPA, the utility of the C. elegans germ line as a model for studying reproductive toxicity, and research methods including assessment of programmed cell death, fluorescent microscopy applications, and assays to quantify gene expression. Questions for deeper exploration in-class or online are provided.Related article in GENETICS: Hornos Carneiro MF, Shin N, Karthikraj R, Barbosa F Jr, Kannan K, Colaiácovo MP. Antioxidant CoQ10 restores fertility by rescuing bisphenol A-induced oxidative DNA damage in the Caenorhabditis elegans Germline. Genetics 214:381-395.

Neonicotinoid-containing insecticide disruption of growth, locomotion, and fertility in Caenorhabditis elegans.

Neonicotinoids, a class of insecticides structurally similar to nicotine that target biting and sucking insects, are the most widely used insecticides today, in part due to their supposed low toxicity in other organisms. However, a growing body of research has found that even low doses of neonicotinoids can induce unexpected negative effects on the physiology and survival of a wide range of non-target organisms. Importantly, no work has been done on the commercial formulations of pesticides that include imidacloprid as the active ingredient, but that also contain many other components. The present study examines the sublethal effects of "Tree and Shrub"™ ("T+S"), a commercial insecticide containing the neonicotinoid imidacloprid as its active ingredient, on Caenorhabditis elegans. We discovered that "T+S" significantly stunted the overall growth in wildtype nematodes, an effect that was exacerbated by concurrent exposure to heat stress. "T+S" also negatively impacted fecundity as measured by increased germline apoptosis, a decrease in egg-laying, and fewer viable offspring. Lastly, exposure to "T+S" resulted in degenerative changes in nicotinic cholinergic neurons in wildtype nematodes. As a whole, these findings demonstrate widespread toxic effects of neonicotinoids to critical functions in nematodes.

The tumor suppressor BRCA1-BARD1 complex localizes to the synaptonemal complex and regulates recombination under meiotic dysfunction in Caenorhabditis elegans.

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.

Maintenance of Genome Integrity by Mi2 Homologs CHD-3 and LET-418 in Caenorhabditis elegans.

Meiotic recombination depends upon the tightly coordinated regulation of chromosome dynamics and is essential for the production of haploid gametes. Central to this process is the formation and repair of meiotic double-stranded breaks (DSBs), which must take place within the constraints of a specialized chromatin architecture. Here, we demonstrate a role for the nucleosome remodeling and deacetylase (NuRD) complex in orchestrating meiotic chromosome dynamics in Caenorhabditis elegans Our data reveal that the conserved Mi2 homologs Chromodomain helicase DNA-binding protein (CHD-3) and its paralog LET-418 facilitate meiotic progression by ensuring faithful repair of DSBs through homologous recombination. We discovered that loss of either CHD-3 or LET-418 results in elevated p53-dependent germ line apoptosis, which relies on the activation of the conserved checkpoint kinase CHK-1 Consistent with these findings, chd-3 and let-418 mutants produce a reduced number of offspring, indicating a role for Mi2 in forming viable gametes. When Mi2 function is compromised, persisting recombination intermediates are detected in late pachytene nuclei, indicating a failure in the timely repair of DSBs. Intriguingly, our data indicate that in Mi2 mutant germ lines, a subset of DSBs are repaired by nonhomologous end joining, which manifests as chromosomal fusions. We find that meiotic defects are exacerbated in Mi2 mutants lacking CKU-80, as evidenced by increased recombination intermediates, corpses, and defects in chromosomal integrity. Taken together, our findings support a model wherein the C. elegans Mi2 complex maintains genomic integrity through reinforcement of a chromatin landscape suitable for homology-driven repair mechanisms.

CRISPR Technology Reveals RAD(51)-ical Mechanisms of Repair in Roundworms: An Educational Primer for Use with "Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans".

The mechanisms cells use to maintain genetic fidelity via DNA repair and the accuracy of these processes have garnered interest from scientists engaged in basic research to clinicians seeking improved treatment for cancer patients. Despite the continued advances, many details of DNA repair are still incompletely understood. In addition, the inherent complexity of DNA repair processes, even at the most fundamental level, makes it a challenging topic. This primer is meant to assist both educators and students in using a recent paper, "Promotion of homologous recombination by SWS-1 in complex with RAD-51 paralogs in Caenorhabditis elegans," to understand mechanisms of DNA repair. The goals of this primer are to highlight and clarify several key techniques utilized, with special emphasis on the clustered, regularly interspaced, short palindromic repeats technique and the ways in which it has revolutionized genetics research, as well as to provide questions for deeper in-class discussion.

Pseudosynapsis and decreased stringency of meiotic repair pathway choice on the hemizygous sex chromosome of Caenorhabditis elegans males.

During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes.

Heteromorphic sex chromosomes: navigating meiosis without a homologous partner.

Accurate chromosome segregation during meiosis relies on homology between the maternal and paternal chromosomes. Yet by definition, sex chromosomes of the heterogametic sex lack a homologous partner. Recent studies in a number of systems have shed light on the unique meiotic behavior of heteromorphic sex chromosomes, and highlight both the commonalities and differences in divergent species. During meiotic prophase, the homology-dependent processes of pairing, synapsis, and recombination have been modified in many different ways to ensure segregation of heteromorphic sex chromosomes at the first meiotic division. Additionally, an almost universal feature of heteromorphic sex chromosomes during meiosis is transcriptional silencing, or meiotic sex chromosome inactivation, an essential process proposed to prevent expression of genes deleterious to meiosis in the heterogametic sex as well as to shield unpaired sex chromosomes from recognition by meiotic checkpoints. Comparative analyses of the meiotic behavior of sex chromosomes in nematodes, mammals, and birds reveal important conserved features as well as provide insight into sex chromosome evolution.

Caenorhabditis elegans histone methyltransferase MET-2 shields the male X chromosome from checkpoint machinery and mediates meiotic sex chromosome inactivation.

Meiosis is a specialized form of cellular division that results in the precise halving of the genome to produce gametes for sexual reproduction. Checkpoints function during meiosis to detect errors and subsequently to activate a signaling cascade that prevents the formation of aneuploid gametes. Indeed, asynapsis of a homologous chromosome pair elicits a checkpoint response that can in turn trigger germline apoptosis. In a heterogametic germ line, however, sex chromosomes proceed through meiosis with unsynapsed regions and are not recognized by checkpoint machinery. We conducted a directed RNAi screen in Caenorhabditis elegans to identify regulatory factors that prevent recognition of heteromorphic sex chromosomes as unpaired and uncovered a role for the SET domain histone H3 lysine 9 histone methyltransferase (HMTase) MET-2 and two additional HMTases in shielding the male X from checkpoint machinery. We found that MET-2 also mediates the transcriptional silencing program of meiotic sex chromosome inactivation (MSCI) but not meiotic silencing of unsynapsed chromatin (MSUC), suggesting that these processes are distinct. Further, MSCI and checkpoint shielding can be uncoupled, as double-strand breaks targeted to an unpaired, transcriptionally silenced extra-chromosomal array induce checkpoint activation in germ lines depleted for met-2. In summary, our data uncover a mechanism by which repressive chromatin architecture enables checkpoint proteins to distinguish between the partnerless male X chromosome and asynapsed chromosomes thereby shielding the lone X from inappropriate activation of an apoptotic program.

Trans-generational epigenetic regulation of C. elegans primordial germ cells.

BACKGROUND: The processes through which the germline maintains its continuity across generations has long been the focus of biological research. Recent studies have suggested that germline continuity can involve epigenetic regulation, including regulation of histone modifications. However, it is not clear how histone modifications generated in one generation can influence the transcription program and development of germ cells of the next. RESULTS: We show that the histone H3K36 methyltransferase maternal effect sterile (MES)-4 is an epigenetic modifier that prevents aberrant transcription activity in Caenorhabditis elegans primordial germ cells (PGCs). In mes-4 mutant PGCs, RNA Pol II activation is abnormally regulated and the PGCs degenerate. Genetic and genomewide analyses of MES-4-mediated H3K36 methylation suggest that MES-4 activity can operate independently of ongoing transcription, and may be predominantly responsible for maintenance methylation of H3K36 in germline-expressed loci. CONCLUSIONS: Our data suggest a model in which MES-4 helps to maintain an 'epigenetic memory' of transcription that occurred in germ cells of previous generations, and that MES-4 and its epigenetic product are essential for normal germ cell development.

emb-4 is a conserved gene required for efficient germline-specific chromatin remodeling during Caenorhabditis elegans embryogenesis.

In C. elegans, germline blastomeres are initially kept transcriptionally quiescent by the maternally loaded CCCH zinc-finger protein PIE-1. PIE-1 disappears upon the birth of the primordial germ cells Z2 and Z3, yet these cells appear to remain quiescent. We have previously demonstrated that there is a chromatin-based repression that succeeds PIE-1 degradation. The chromatin in Z2/Z3 loses certain histone modifications, including histone H3 lysine 4 dimethylation (H3K4me2), a conserved marker for transcriptionally competent chromatin. We find that mutations in the maternal-effect gene emb-4 cause defects in both PIE-1 degradation and germline-specific chromatin remodeling. emb-4 encodes a highly conserved protein with orthologs in fly, mouse, and human and has a subtle role in Notch signaling. The embryonic phenotype of emb-4 is consistent with a defect in the efficient and timely activation of developmental programs, including germline chromatin remodeling. We also find that, as in early somatic blastomeres, the degradation of PIE-1 in Z2/Z3 is facilitated by zinc-finger-interacting protein ZIF-1, and in the absence of either zif-1 or emb-4, PIE-1 is abnormally retained in Z2/Z3.