Sterken, Mark et al. (2021) International Worm Meeting "Genetic background-dependent expression of clec-62 in Caenorhabditis elegans"

Sterken, Mark, Kammenga, Jan, Steeghs, Maarten, Riksen, Joost

[International Worm Meeting, 2021]

The pursuit of genes underlying quantitative traits is often accompanied by creating introgression lines (ILs). ILs consist of a single genetic locus introgressed in an otherwise homogeneous background. We previously generated a genome-covering IL panel by introgression of segments of the strain N2 in a CB4856 genetic background. This population complements a previously created IL panel with CB4856 segments in an N2 genetic background. Combining both panels allow the study of the effect of the genetic background at a higher resolution than previously possible with chromosome substitution strains. We exposed 150 strains of a N2xCB4856 recombinant inbred line population and the two IL panels to a normal growth regime (20oC on standard NGM plates). After 48 hours of growth, we collected the animals, flash froze them and proceeded to isolate RNA. Via qPCR, we measured the expression of clec-62, finding that only in the N2-background population QTL were detected. In other words, no QTL were detected in the RIL and reciprocal IL population. Thereby we show the strong role of the genetic background on trait variation. Here we present a case of either closely-linked loci that were only segregated in one population, or an epistatic interaction depending on the genetic background. We are currently in the process of building a double introgression panel to experimentally test the effect of two combined backgrounds.

Sterken, Mark G. et al. (2015) International Worm Meeting "Transcriptional regulation in stress recovery of Caenorhabditis elegans ."

Sterken, Mark G., Bevers, Roel P.J., Kammenga, Jan E., Riksen, Joost A.G., Snoek, L. Basten

[International Worm Meeting, 2015]

C. elegans serves as an important model for integrative analysis of stress response. Analyzing the transcriptional regulation of stress response and subsequent recovery provides insight into the mechanisms of fitness optimization. Although C. elegans stress responses are well studied, the recovery from stress is poorly understood. We investigated the transcriptional response over the course of a heat-shock experiment in fully sequenced recombinant inbred lines (RILs). This allowed us to identify regulatory loci for stress recovery.The RILs, derived from a cross between N2 and CB4856, were exposed to heat-shock after which they were allowed to recover. In both environments, and in a control environment (no heat stress), genome-wide expression levels were measured. We then performed expression quantitative trait loci (eQTL) mapping across these three environments to identify regulatory loci that contribute to the recovery response.Across all RILs the largest difference in gene expression was found between control and heat-shock. Recovering populations showed patterns of gene expression that were in-between the control and heat-shock. Some RILs had a more control-like gene expression phenotype while other RILs were more heat-shock-like, thus allowing for mapping of loci regulating genetic variation in recovery. We found that genetic variation in gene expression was largely independent of the environment. More specifically, approximately 50% of the eQTL were similar in location and effect over the three environments; therefore did not display interaction with the environment. Only 1% of the genes with an eQTL in all environments displayed environment-specific interaction. The remainder of the eQTL with an environment-specific interaction were specifically detected in one environment.In conclusion, the combination of natural variation and gene expression over the course of a stress response allows for mapping loci underlying regulation in this response. The genes with an eQTL displaying environment interaction are specific for one environment. Incorporation of environment interactions facilitates specifying groups of related eQTL and thereby allows for detailed eQTL network analysis.

Sterken, Mark et al. (2021) International Worm Meeting "Transcriptional analysis of the response of C. elegans to ethanol exposure"

van Wijk, Marijke, Davies, Andrew, Sterken, Mark, Kammenga, Jan, Bettinger, Jill, Carnell, Lucinda, Quamme, Elizabeth, Mathies, Laura, Riksen, Joost

[International Worm Meeting, 2021]

Ethanol consumption induces differential gene expression that are likely to at least partially underlie the progression to alcohol use disorder. We use C. elegans to study the effects of ethanol on nervous system function. To understand the gene expression dynamics that occur upon ethanol exposure, we conducted a time series experiment exposing C. elegans to a behaviorally intoxicating dose of ethanol for 30, 60, 120, and 480 minutes. The number of differentially expressed genes was positively correlated with exposure duration. We used K- means clustering to cluster genes based on expression over time and divided them in 5 distinct clusters. Gene ontology analysis showed enrichment for processes known to be involved in ethanol responses, for example: drug metabolism - cytochrome P450 and metabolic pathways. Furthermore, at eight hours of exposure several genes that are known to be involved in physiological response to ethanol were down regulated; some of those genes have been previously identified as direct ethanol targets, including the gene encoding the BK SLO-1 channel, the synaptic protein-encoding unc-13 gene, as well as genes involved in GABA signaling, suggesting a simple model in which downregulation of direct ethanol targets could decrease the depressing effects of ethanol and could contribute to the development of tolerance to ethanol. Our exposure paradigm delivered ethanol in the absence of food, and we found that eight hours of food deprivation had a significant impact on overall gene expression as assessed by principal component analysis and transcriptional development analysis. Interestingly, ethanol was able to partially rescue the effect of food deprivation at eight hours, extending previous observations that C. elegans can use ethanol as a calorie source. This timeseries captured gene expression dynamics in response to ethanol over time and will be used as a resource for our follow up studies, including investigating natural variation in gene expression in response to ethanol.

Sterken, Mark G. et al. (2013) International Worm Meeting "Orsay virus replication kinetics in C. elegans strain Bristol N2."

Snoek, L. Basten, Bosman, Kobus J., Bakker, Jaap, Pijlman, Gorben P., Kammenga, Jan E., Sterken, Mark G., Riksen, Joost A.G., Daamen, Jikke

[International Worm Meeting, 2013]

The recently discovered Orsay virus (OrV) is the first virus able to complete a full infection cycle in C. elegans. This discovery creates the opportunity to study host-virus interactions in a genetically tractable host system. OrV can be transmitted horizontally (from one individual worm to the next) and the infection process is affected by the RNAi response machinery. OrV gives us the opportunity to investigate viral replication dynamics and anti-viral mechanisms in the host, including RNAi. By infecting worm cohorts at different time points, and monitoring through qPCR, the influence of worm age and genotype on viral replication was determined. Next, several subsequent generations exposed to virus were re-infected to determine if trans-generational RNAi plays a role in infections in C. elegans populations. We found an age-related resistance to OrV infection and faster replication in the isolate JU1580 (in which OrV was discovered) than in Bristol N2. Nonetheless, N2 is perfectly able to sustain viral replication. However, an inherited anti-viral response makes offspring of infected N2 less susceptible to viral replication. Consequently, N2 populations can lose the infection after a limited number of generations, whereas JU1580 populations remain infected. Experiments with two RNAi deficient mutants confirm that the RNAi response is responsible for this inherited anti-viral response. We present a quantitative and reproducible method for studying OrV infection in synchronized populations and consider some important parameters for experimental settings. Using this method, we found that the canonical Bristol N2 strain can be used to study OrV, which enables the use of all the tools that are available for this genotype. The presence of a heritable RNAi response establishes the importance of this pathway in clearance of viral infections in C. elegans.

Sterken, Mark G. et al. (2015) International Worm Meeting "Stress tolerance and lifespan in C. elegans are modulated by natural allelic variation in cmk-1 ."

Sterken, Mark G., Lehner, Ben, Van 't Hof, Arjen, Brenchley, Rachel, Snoek, L. Basten, Kammenga, Jan E., Bevers, Roel P.J., Cossins, Andrew, Volkers, Rita J.M.

[International Worm Meeting, 2015]

The genetic control of aging has primarily been studied using laboratory-derived mutants of Bristol N2.These mutants often display extreme lifespan accompanied by increased stress resistance. Yet, the identification of genes that underlie variation in lifespan and stress tolerance in natural populations remains a major challenge. We investigated gene expression architecture and longevity and found that that stress tolerance and lifespan in C. elegans are modulated by natural allelic variation in cmk-1. Variation in gene expression levels was linked to genomic loci (eQTL) using fully sequenced recombinant inbred populations derived from wild types Bristol N2 and CB4856. These strains were grown at 20oC for the entire experiment, or exposed to a 35oC heat-shock. This allowed us to identify regulatory loci affecting multiple transcript levels under heat-stress conditions. These loci were verified using introgression lines and the causal regulator was identified by screening knock-out mutants of candidate genes. Moreover, lifespan was measured under control conditions (grown at 20oC) and in strains that received a heat shock treatment. We identified a major stress-related regulatory locus on the top left arm of chromosome IV. The locus affects expression variation in more than 100 genes and one of the causal genes underlying the variation was found to be cmk-1. The effects of this variation include modulation of insulin-like signaling targets supporting a model in which allelic variation in cmk-1 regulates a subset of daf-16 targets under stress conditions, leading to increased stress resistance and prolonged lifespan.We conclude that genes affecting lifespan variation in nature may not be those identified as having large effects when mutated in Bristol N2. Taken together our data eliminate the standing view that death is genetically programmed and suggests that older age is a correlated trait of combined stress tolerance effects.

Sterken, Mark G. et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "Dissecting the genetic architecture of resistance to Orsay virus infection in C. elegans"

Snoek, L. Basten, Sterken, Mark G., Volkers, Rita J.M., Bosman, Kobus J., Kammenga, Jan E., Wang, Yiru, Pijlman, Gorben P., Riksen, Joost A.G.

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Host-pathogen interactions play a major role in evolutionary selection and in shaping natural genetic variation. Recent identification of viral infection in C. elegans has prompted research into understanding the underlying pathways of Orsay virus (OrV) infection in natural populations. Here we report the dissection of the genetic architecture of OrV infection.We found that wild type Hawaii CB4856 strain was more resistant to OrV than the canonical Bristol N2 strain. To gain insight into the genetic architecture of resistance, 52 fully sequenced recombinant inbred lines (CB4856 x N2 RILs) were exposed to OrV using our recently developed quantitative assay. This led to the identification of two distinct loci on chromosome IV associated with OrV resistance. These loci were both associated with a lower viral load in the CB4856 genotype. Strikingly, these loci do not harbour the recently found drh-1 locus, which encodes a RIG-I like helicase that plays an important role in antiviral RNAi. To verify our results and gain additional insight into the genetic architecture, a panel of 18 introgression lines (ILs) (together covering chromosome IV entirely) was exposed to OrV. Both loci could be verified by ILs, also showing more resistance against OrV infection with the CB4856 locus.Our results provide insight in the loci underlying the higher viral resistance in CB4856. They also form an important step toward identifying polymorphic genes underlying resistance to viral infection in C. elegans.

David J Katz et al. (2005) International Worm Meeting "Investigating the link between NANOS and chromatin for germ cell maintenance in C. elegans"

William G Kelly, David J Katz

[International Worm Meeting, 2005]

In Drosophila, nanos is required for primordial germ cells (PGCs) to differentiate into a functional germ line. Lack of NANOS function causes defects in germ cell migration, differentiation, and quiescence and ultimately leads to sterility in both males and females. Similarly, in mice a lack of nanos3 results in the complete loss of germ cells in both sexes (Tsuda et al. 2003) and functional loss of two of the three nanos genes in C. elegans, nos1 and nos2, also results in sterility (Subramaniam and Seydoux, 1999). It has therefore been proposed that nanos is a conserved master regulator of the germ cell fate. In the C. elegans early embryo, the chromatin in all cells contain the active chromatin mark, histone H3 methylated on lysine 4 (H3meK4). However upon their birth, the primordial germ cells Z2 and Z3 dramatically lose this mark. This loss of the active H3meK4 mark has been suggested to be part of a chromatin-based transcriptional repression mechanism. Remarkably, absence of the H3meK4 mark has been shown to be dependent upon nanos activity (Schaner et al. 2003). Thus it is possible that nanos may be partially affecting its role in maintaining the undifferentiated germ cell fate through a chromatin-based mechanism. We aim to explore this potential link by further characterizing the role of C. elegans nanos in germ cell fate. To this end, we are taking both forward and reverse genetic approaches as well as conducting ectopic expression studies.

Snyder, Matthew P. et al. (2013) International Worm Meeting "Turnover of the H3K9me2 Mark During Late Spermatogenesis."

Snyder, Matthew P., Xu, Xia, Maine, Eleanor

[International Worm Meeting, 2013]

Meiotic silencing is a conserved phenomenon targeting unpaired chromosomes and chromosomal regions during prophase of meiosis I. Meiotic silencing in animals typically occurs at the chromatin level and involves accumulation of histone modifications thought to promote a closed chromatin configuration. This chromatin structure may contribute to transcriptional repression and meiotic chromosomal events such as chromosome disjunction (Bean et al 2004, Jaramillo-Lambert and Engebrecht 2010). During meiosis in C. elegans, non-synapsed chromosomes are enriched for H3K9me2 relative to synapsed chromosomes (Kelly et al. 2002; Bean et al. 2004). Such non-synapsed chromosomes include the male X, homologous chromosomes that fail to synapse due to mutation, and chromosomal translocations/duplications. The pattern of H3K9me2 accumulation during meiosis depends on activity of the small RNA machinery, which may have a role in targeting the mark to unpaired chromosomes and ensuring that the mark does not persist abnormally (Maine et al. 2005; She et al. 2009). Taking a combined biochemical/genetic approach, we are identifying additional factors important for regulating H3K9me2 distribution during meiosis. Through this approach, we are identifying genes that appear to be important for turnover of the H3K9me2 mark during late spermatogenesis.

Gyoergyi Csankovszki et al. (2002) West Coast Worm Meeting "Mapping chromosomal targets of the dosage compensation complex"

Barbara J Meyer, Gyoergyi Csankovszki

[West Coast Worm Meeting, 2002]

The worm C. elegans compensates for the difference in X-linked gene dosage between XX hermaphrodites and XO males by reducing gene expression two-fold in XX animals. Downregulation of gene expression is achieved by the dosage compensation complex (DCC), a large multiprotein complex that binds the two X chromosomes in hermaphrodites. However, the cis-acting chromosomal elements that recruit and bind the complex are not known. Using free and attached X chromosomal duplications we are attempting to identify regions of the X chromosome that are sufficient to recruit the complex. By analyzing the volume and number of DCC-bound chromosomal bodies, a large duplication of the right 30% of X, mnDp10, was shown earlier to be competent to bind the DCC (1). Using combined fluorescent in situ hybridization (to mark X chromosomal territories) and immunofluorescence (to mark chromosomes that bind the dosage compensation complex) we confirmed these results. By analyzing smaller duplications that span the entire chromosome we hope to map chromosomal targets of the DCC.

Huang, Yuqing et al. (2021) International Worm Meeting "Polymorphic modifiers of human alpha-synuclein in Caenorhabditis elegans"

Vogels, Demi, Chen, Yuzhi, Riksen, Joost, Tegelbeckers, Vivian, Huang, Yuqing, Sterken, Mark, Wang, Yiru, Kammenga, Jan

[International Worm Meeting, 2021]

Parkinson's Disease (PD) is a common neurodegenerative disorder in elderly humans. PD is characterized by neuronal loss in the substantia nigra and an aggregation of alpha-synuclein into Lewy bodies. The morbidity in humans is dependent on the genetic background, although the exact mechanisms of background effects of genetic variation are unknown. We turned to an alpha-synuclein aggregation model in the model nematode Caenorhabditis elegans to investigate the role of genetic variation on alpha-synuclein aggregation. We mapped regulators of gene expression (eQTL) in Recombinant Inbred Lines (RILs) constructed by crossing C. elegans strains NL5901 and SCH4856 which harbor the human alpha-synuclein. We found three regulatory hot-spots on Chromosome V. To pin-point the causal locus, three Introgression Lines (ILs) with wild type CB4856 introgressions in an N2 background on Chromosome V were crossed with NL5901. From each cross we obtained four genotypes: i) no alpha-synuclein and no introgression, ii) no alpha-synuclein but with introgression, iii) with alpha-synuclein but no introgression, iv) with alpha-synuclein and introgression. These verified the regulatory hotspot and through mining WormNet and CeNDR we found hcp-1 as potential polymorphic modifier influencing human alpha-synuclein expression in C. elegans. We will follow up with updated RILs sequence and confirm the candidate modifier further with CRISPR/CAS-9 for gene function detection.