Genome sequencing of C. elegans balancer strains reveals previously unappreciated complex genomic rearrangements.

Genetic balancers in Caenorhabditis elegans are complex variants that allow lethal or sterile mutations to be stably maintained in a heterozygous state by suppressing crossover events. Balancers constitute an invaluable tool in the C. elegans scientific community and have been widely used for decades. The first/traditional balancers were created by applying X-rays, UV, or gamma radiation on C. elegans strains, generating random genomic rearrangements. Their structures have been mostly explored with low-resolution genetic techniques (e.g., fluorescence in situ hybridization or PCR), before genomic mapping and molecular characterization through sequencing became feasible. As a result, the precise nature of most chromosomal rearrangements remains unknown, whereas, more recently, balancers have been engineered using the CRISPR-Cas9 technique for which the structure of the chromosomal rearrangement has been predesigned. Using short-read whole-genome sequencing (srWGS) and tailored bioinformatic analyses, we previously interpreted the structure of four chromosomal balancers randomly created by mutagenesis processes. Here, we have extended our analyses to five CRISPR-Cas9 balancers and 17 additional traditional balancing rearrangements. We detected and experimentally validated their breakpoints and have interpreted the balancer structures. Many of the balancers were found to be more intricate than previously described, being composed of complex genomic rearrangements (CGRs) such as chromoanagenesis-like events. Furthermore, srWGS revealed additional structural variants and CGRs not known to be part of the balancer genomes. Altogether, our study provides a comprehensive resource of complex genomic variations in C. elegans and highlights the power of srWGS to study the complexity of genomes by applying tailored analyses.

eEF2K inhibition blocks Aß42 neurotoxicity by promoting an NRF2 antioxidant response.

Soluble oligomers of amyloid-ß (Aß) impair synaptic plasticity, perturb neuronal energy homeostasis, and are implicated in Alzheimer's disease (AD) pathogenesis. Therefore, significant efforts in AD drug discovery research aim to prevent the formation of Aß oligomers or block their neurotoxicity. The eukaryotic elongation factor-2 kinase (eEF2K) plays a critical role in synaptic plasticity, and couples neurotransmission to local dendritic mRNA translation. Recent evidence indicates that Aß oligomers activate neuronal eEF2K, suggesting a potential link to Aß induced synaptic dysfunction. However, a detailed understanding of the role of eEF2K in AD pathogenesis, and therapeutic potential of eEF2K inhibition in AD, remain to be determined. Here, we show that eEF2K activity is increased in postmortem AD patient cortex and hippocampus, and in the hippocampus of aged transgenic AD mice. Furthermore, eEF2K inhibition using pharmacological or genetic approaches prevented the toxic effects of Aß42 oligomers on neuronal viability and dendrite formation in vitro. We also report that eEF2K inhibition promotes the nuclear factor erythroid 2-related factor (NRF2) antioxidant response in neuronal cells, which was crucial for the beneficial effects of eEF2K inhibition in neurons exposed to Aß42 oligomers. Accordingly, NRF2 knockdown or overexpression of the NRF2 inhibitor, Kelch-Like ECH-Associated Protein-1 (Keap1), significantly attenuated the neuroprotection associated with eEF2K inhibition. Finally, genetic deletion of the eEF2K ortholog efk-1 reduced oxidative stress, and improved chemotaxis and serotonin sensitivity in C. elegans expressing human Aß42 in neurons. Taken together, these findings highlight the potential utility of eEF2K inhibition to reduce Aß-mediated oxidative stress in AD.

Prepublication data sharing.

Rapid release of prepublication data has served the field of genomics well. Attendees at a workshop in Toronto recommend extending the practice to other biological data sets.

Multiple UDP glycosyltransferases modulate benzimidazole drug sensitivity in the nematode Caenorhabditis elegans in an additive manner.

Xenobiotic biotransformation is an important modulator of anthelmintic drug potency and a potential mechanism of anthelmintic resistance. Both the free-living nematode Caenorhabditis elegans and the ruminant parasite Haemonchus contortus biotransform benzimidazole drugs by glucose conjugation, likely catalysed by UDP-glycosyltransferase (UGT) enzymes. To identify C. elegans genes involved in benzimidazole drug detoxification, we first used a comparative phylogenetic analysis of UGTs from humans, C. elegans and H. contortus, combined with available RNAseq datasets to identify which of the 63 C. elegans ugt genes are most likely to be involved in benzimidazole drug biotransformation. RNA interference knockdown of 15 prioritized C. elegans genes identified those that sensitized animals to the benzimidazole derivative albendazole (ABZ). Genetic mutations subsequently revealed that loss of ugt-9 and ugt-11 had the strongest effects. The "ugt-9 cluster" includes these genes, together with six other closely related ugts. A CRISPR-Cas-9 deletion that removed seven of the eight ugt-9 cluster genes had greater ABZ sensitivity than the single largest-effect mutation. Furthermore, a double mutant of ugt-22 (which is not a member of the ugt-9 cluster) with the ugt-9 cluster deletion further increased ABZ sensitivity. This additivity of mutant phenotypes suggest that ugt genes act in parallel, which could have several, not mutually exclusive, explanations. ugt mutations have different effects with different benzimidazole derivatives, suggesting that enzymes with different specificities could together more efficiently detoxify drugs. Expression patterns of ugt-9, ugt-11 and ugt-22 gfp reporters differ and so likely act in different tissues which may, at least in part, explain their additive effects on drug potency. Overexpression of ugt-9 alone was sufficient to confer partial ABZ resistance, indicating increasing total UGT activity protects animals. In summary, our results suggest that the multiple UGT enzymes have overlapping but not completely redundant functions in benzimidazole drug detoxification and may represent "druggable" targets to improve benzimidazole drug potency.

A Strategy To Isolate Modifiers of Caenorhabditis elegans Lethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2.

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen's design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10-4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations.