Burga, Alejandro et al. (2017) International Worm Meeting "Mapping complex traits in C. elegans using a bulk segregant approach."

Kruglyak, Leonid, Ben-David, Eyal, Burga, Alejandro

[International Worm Meeting, 2017]

Most diseases and traits are influenced by multiple genetic factors. However, identifying variants underlying phenotypic differences between individuals is hard and laborious even in model organisms. For instance, Genome-Wide Association Studies (GWAS) require hundreds of C. elegans wild isolates to be individually phenotyped, a process prone to measurement noise, particularly for traits influenced by environmental factors. Alternatively, in Quantitative Trait Loci (QTL) mapping, large recombinant inbred line (RIL) panels need to be generated, maintained and phenotyped, which limits the number of parental genotypes which can be interrogated. Thus, despite the previous success of these strategies in identifying causal loci, a long road still lies ahead for understanding the complex relationship between genotype and phenotype. To gain insights into the genetic basis of complex traits, we developed a novel Bulk Segregant Analysis (BSA) strategy in C. elegans as a versatile and complementary approach to GWAS and panels of recombinant inbred lines (RILs). Our method is based on mating obligate outcrossing strains carrying the fog-2(q71) allele for multiple generations, allowing the accumulation of recombination events, and leading to increased mapping resolution. The resulting population is composed of thousands of genetically unique individuals, which can be used to map traits that vary between the parental strains. Furthermore, the population can be directly interrogated at multiple generations for variants affecting fitness, allowing us to study the dynamics of selection. To guide the choice of parameters for our experiments, we developed a computational framework in R, bulkPop, for simulating the BSA pipeline. This framework takes into account the known genetic map and realistic patterns of linkage disequilibrium, and allows us to optimize the number of individuals and number of generations. We implemented our method to study a cross between the reference strain N2 and the wild isolate CB4856 from Hawaii, and found highly reproducible fitness peaks that were closely matched by our simulations. As a proof of principle, we mapped the known zeel-1/peel-1 genetic incompatibility on Chr. I. and discovered previously unknown loci influencing fitness/growth. In addition, BSA can also be used to map variants affecting gene expression. We used BSA followed by fluorescence sorting of worms (COPAS Biosorter) to map a de novo genetic variant in sti-1 leading to up-regulation of the chaperone daf-21 (HSP90). We will further discuss current limitations of the approach and future directions to improve the mapping resolution.

Tikanova, Polina et al. (2021) International Worm Meeting "Evolution of selfishness from a core tRNA synthetase in C. tropicalis"

Ben-David, Eyal, Tikanova, Polina, Pliota, Pinelopi, Dong, Gang, Ross, Julian, Okweri, Jacqueline, Burga, Alejandro, Krogull, Daniel

[International Worm Meeting, 2021]

Selfish genetic elements maximize their own transmission despite being neutral or even harmful to their host. One of the most extreme examples of this behavior are toxin-antidote elements (TAs). TAs comprise two linked genes: one encodes a toxin and the other its cognate antidote. The toxin is expressed in the parental germline, and the antidote is expressed zygotically. In crosses between individuals that carry the TA and ones that do not, the toxin is delivered to all of the progeny by the egg or sperm, but only embryos that inherit at least one copy of the TA are able to express the antidote and therefore survive. We recently identified numerous TAs in C. elegans, C. tropicalis, and C. briggsae. However, the molecular underpinnings of all known eukaryotic TAs are still largely unknown. A critical barrier remains dissecting the molecular mechanisms of TA elements, which is very challenging given their lack of homology to well-studied proteins. Here we report the fine mapping and in vivo characterization of a C. tropicalis TA that evolved from a core essential gene: klmt-1/kss-1. The toxin, klmt-1 (Killer of L1 and embryos Maternal Toxin), evolved via gene duplication from fars-3, the beta subunit of phenylalanyl-tRNA synthetase (PheRS), which is responsible for charging tRNAPhe. We hypothesize that KLMT-1 can bind FARS-1 and thus interfere with tRNAPhe charging and translation. We found that KLMT-1 can bind FARS-1, the alpha subunit of the PheRS complex in vitro (see poster by Ross et al.). To test it in vivo, we are performing Co-IP experiments in endogenously tagged strains. Interestingly, KLMT-1 levels decrease rapidly during development and are hardly detectable by the time of hatching, suggesting that the toxin is actively being degraded. We hypothesize the degradation of the toxin is driven by its cognate antidote. The antidote KSS-1 (Klmt-rescue by zygotically-expreSSed antidote) has a predicted F-box domain on its N-terminus. F-box proteins act as adaptors between target proteins and the SCF complex, which mediates proteasome-mediated degradation. In support of this model, we found that KSS-1 can bind several SKP-1 orthologs of C. tropicalis. Furthermore, in vivo studies revealed that loss of kss-1 drastically increases the half-life of the toxin. Unlike other eukaryotic TAs, klmt-1/kss-1 originated from a core essential protein. Understanding the molecular mechanism of this TA will provide key insights into the inception of selfish genes.

Ross, James Julian et al. (2021) International Worm Meeting "Biochemical and structural characterization of a tRNA-synthetase-based selfish element in C. tropicalis"

Stefania, Valeria, Ben-David, Eyal, Tikanova, Polina, Burga, Alejandro, Hunold, Manuel, Hagmuller, Andreas, Dong, Gang, Ross, James Julian

[International Worm Meeting, 2021]

Selfish genetic elements have evolved manifold ways to persist in genomes and populations without offering any apparent benefit to the host. In a particularly radical example, toxin-antidote elements (TAs) secure their position in host populations by selectively removing progeny that do not inherit a copy of the respective underlying gene pair. Numerous TAs have been identified and mechanistically dissected in bacteria. However, only few examples have been reported in eukaryotes. Importantly, the underlying molecular mechanisms of eukaryotic TAs remain completely unresolved to date, hindering our understanding of this ongoing arms-race between the host genome and parasitic elements. Initially discovered in C. elegans, we recently greatly expanded the repertoire of known animal TAs with the discovery of multiple TAs in a cross between two isolates of the nematode Caenorhabditis tropicalis (Ben-David, Pliota et al., 2021). Here, we focus on a single TA from this cross, aiming to uncover its mechanism of action (for genetic and in vivo characterization see poster by Tikanova et al.). We identified and validated the novel toxin and antidote genes, which we named klmt-1 (Killer of L1 and embryos Maternal Toxin) and kss-1 (Klmt-rescue by zygotically-expreSSed antidote), respectively. We found that KLMT-1 shares 52% sequence identity with the beta subunit of the phenylalanyl-tRNA-synthetase (PheRS) and both N and C-terminal ends are predicted to be intrinsically disordered. We hypothesized that KLMT-1 kills worms by competing with the beta subunit in the heterotetrameric PheRS complex, thereby interfering with amino acid charging. In line with this hypothesis, we found that KLMT-1 is capable of binding the alpha subunit (FARS-1) of the PheRS in pulldown experiments and that FARS-1/KLMT-1 form an oligomeric complex in vitro. Furthermore, using a metabolic labelling approach, we quantified the activity of the C. tropicalis PheRS in bacteria and found that the presence of KLMT-1 strongly reduces the ability of the enzyme complex to charge its tRNA. Intriguingly, removal of the disordered termini of KLMT-1 further increased its inhibitory effect, indicating potential ways of spatial or temporal regulation of TA activity. Our findings serve as the basis to formulate a model for KLMT-1 toxicity, thereby for the first time providing mechanistic insight into a TA from animals and will be further substantiated by structural investigations of the FARS-1/KLMT-1 complex.

Willis, John et al. (2021) International Worm Meeting "Natural variation in the aldehyde oxidase, gad-3, confers oxidative stress resistance between C.elegans strains"

Phillips, Patrick, Moerdyak-Schauwecker, Megan, Willis, John, Petruccione , Erin, Teterina, Anastasia

[International Worm Meeting, 2021]

In this study we sought to identify allelic variants responsible for mediating paraquat (PQ) resistance between C.elegans strains N2 and MY16. We observed that MY16 is two to three-fold more resistant then N2 to oxidative stress from PQ exposure. To identify causative alleles, we developed an experimental evolution approach which relies on multiple rounds of strong selection on standing genetic variation. Genetic variation is created by making strains obligate outcrossing (by introducing a fog-2 mutation) and forcing random mating for twelve generations to create starting pools (n=3). A similar approach was taken by Burga et al. 2019. In our method, which we call Evolutionary Segregant analysis (ESA), we start with a pool of 30k worms from the F12 generation. We performed a strong selection by recovering only ~ 0.3% of the starting population resistant to PQ. This protocol was performed for five rounds with appropriate controls. All samples were sequenced using RAD-seq. The mapping data pointed to a ~150kb region on LGV. To identify causative alleles, we focused on candidate genes with a role in oxidative stress. From this group, gad-3 seemed like a solid candidate. Gad-3 (B0222.9) is an evolutionarily conserved aldehyde oxidase downstream of the sirtuin (Sir-2.1) pathway and has a role in oxidative stress response and longevity (Park et al., 2009). GAD-3 dependent production of hydrogen peroxide is responsible for longevity increases from sirtuin (sir-2.1) pathway over-expression (Schmeisser et al., 2013). GAD-3 protein differs by three mutations between strains. We focused our efforts on testing a mutation in a widely conserved Iron-sulfur cluster domain of GAD-3. Using CRISPR, we performed congenic allele swaps for both MY16 and N2 strains. In both cases, the MY16 allele conferred increased resistance to PQ exposure. In addition, we found that hydrogen peroxide production is reduced with the gad-3 N2 allele in an MY16 background. Ongoing experiments are testing the reverse allele swap. Our results suggest that GAD-3 dependent production of hydrogen peroxide is mainly responsible for oxidative stress resistance between these natural isolates.