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[
European Worm Meeting,
2008]
Transposable elements (TE) are widely acknowledged as drivers of genome. evolution. To analyze the potential mutagenicity of TE excision, we. investigated the nature of the modifications that the C. elegans genome. undergoes during the repair of a DNA double-strand break (DSB) triggered by. TE mobilization.. Mos1 is a Drosophila transposon which can be experimentally mobilized. in C. elegans. We took advantage of this system to trigger single DSBs at. defined loci and study their repair. We demonstrated that DSB are repaired. by pathways relying on both end-joining and homologous recombination.. Specifically, detailed analysis of germ line repair events showed that most. of the Mos1-induced DSB are repaired by recombination using the homologous. chromosome as a repair template and that these events can be associated. with crossovers. Recombination at homologous ectopic sites was also. observed at a low but significant frequency. In addition, end-joining. events and intrachromosomal recombination were also detected. Together,. these results demonstrate that TE can induce heritable gross chromosomal. rearrangements as well as point mutations and small deletions or insertions. when they transpose in the germ line of wild-type C. elegans.. These data were used to develop MosTIC (which stands for "Mos1 induced. transgene instructed gene conversion"), a technique based on homologous. recombination that enables genomic engineering in C. elegans. This. technique will be presented in details.
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Cerrato, C., Dong, Y., Dernburg, A.F., Ahringer, J., Janes, J., Carelli, F.N., Appert, A.
[
International Worm Meeting,
2019]
Transposable elements (TEs) are DNA sequences capable of inserting into new genomic locations. Although new TE integrations are usually neutral or deleterious for the host, TE amplification can disperse protein binding sites in the genome, and thus generate new regulatory elements. Distinct classes of TEs have been shown to be enriched at promoters or enhancers active in specific tissues or cell types. TE expansion has, therefore, the potential to drive the evolution of gene regulatory networks. To date, nonetheless, these observations have been mostly limited to correlation analyses or tested in cell lines. Here we provide evidence that two classes of DNA transposons have been co-opted as new regulatory elements in C. elegans. We found that a pair of DNA motifs, strongly enriched in germline-specific promoters, are part of the inverted repeat sequences of CERP2 and CELE2, two inactive DNA transposons. Through reporter assays, we validated the activity of the two motifs, confirming their role in driving germline-specific regulatory elements in vivo. Comparative analyses revealed that CERP2-associated motifs are found across the whole Caenorhabditis clade but not in other nematodes, whereas the CELE2-expanded motifs are C. elegans-specific. By annotating regulatory elements in the sister species C. briggsae, we confirmed that C. elegans-specific repeat expansions led to the emergence of a number of germline promoters which could not be identified at the orthologous locations in C. briggsae. Finally, using chromatin immunoprecipitation assays, we found that two fast evolving proteins important for germ line function have binding associated with the CERP2/CELE2 motifs. Overall, our work provides strongly suggests that distinct waves of TE expansion have shaped the germline regulatory network in the Caenorhabditis genus.
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[
International Worm Meeting,
2021]
Breeding systems determine the way genes are transmitted to the next generation, consequently driving genetic variation and genome evolution. Breeding systems may also play a role in the activity and prevalence of transposable elements. Selfing should increase the selection efficacy against TEs as a result of high homozygosity, with the majority of TEs present having reached fixation through genetic drift. Conversely, outcrossing should facilitate the spread of TE insertions throughout populations, and result in a higher number of moderately deleterious mutations. In the Caenorhabditis group Elegans, there doesn't appear to be a clear pattern of TE dynamics across partially-selfing species and obligately outcrossing species. Most species in the Elegans group, appear to have chromosomal heterogeneity in repeat density, with chromosome arms being more repeat rich, and gene poor, than the centers. This association is not always consistent, as is the case for the outcrossing species C. inopinata and C. bovis, where some, but not all, repeat types have a more uniform distribution across the chromosome, with little divergence between copies, which may indicate recent TE activity. It is currently unknown whether these observations are also found across species from the obligately outcrossing Caenorhabditis group Japonica. Here we present the first chromosome-scale assembly for a species belonging to the Japonica group, Caenorhabditis becei. This high-quality assembly was generated from PacBio long read data, Hi-C data, and a genetic map of an advanced intercross panel. The gene annotation for this genome was generated using Iso-Seq data and RNA-seq data from C. becei, as well as orthologous protein-coding and protein sequences from other Caenorhabditis species. We then characterized features such as synteny to other Caenorhabditis species, genome size, recombination rate domain structure, gene content, orthology, and distribution. We created a classified library of repetitive elements identified through de novo approaches. We generated TE annotations for the C. becei genome, and we reveal the patterns of repeat density, prevalence, distribution, and divergence.
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[
International C. elegans Meeting,
1997]
gld-1 is a female germ cell specific tumor suppressor gene that is essential for normal oocyte differentiation and meiotic prophase progression (Francis et al., 1995a,b). GLD-1 is a member of a small family of proteins, including the mouse/human Sam68 and Quaking proteins, that are highly related over an ~ 200 amino acid region which contains a 70 to 100 amino acid RNA binding domain called the KH motif (Jones and Schedl, 1995). Extensive mutational analysis of
gld-1 has demonstrated the importance of conserved sequences for its in vivo function. GLD-1 is a cytoplasmic protein and therefore is likely to control mRNA translation or RNA stability in the cytoplasm (Jones et al., 1996). Currently, no obvious RNA targets have been identified from genetic analysis. We have employed a biochemical approach to identify in vivo RNA targets of GLD-1. The strategy is based on the ability of anti GLD-1 antibodies to immunoprecipitate (IP) GLD-1 from a worm lysate and the strong likelihood that GLD-1 present in the lysate is functional and bound to RNAs. GLD-1 was IPed with affinity purified polyclonal antibodies from a cytosol extract of adult hermaphrodites or with rabbit IgG as a control. RNAs co-IP with GLD-1, as well as with rabbit IgG, were converted into cDNAs. Non-specifically trapped RNAs from the GLD-1 IP were eliminated by subtracting cDNAs from the GLD-1 IP with cDNAs from the control IP. The difference product after 4 rounds of subtraction (DP4) was used to screen a Lambda ZAP II cDNA library constructed with the cDNAs from the GLD-1 IP. Duplicate filters were also screened with a probe made from control IP cDNA. Clones that are positive only with the DP4 probe are currently being characterized. Francis et al., 1995a Genetics 139, 579-606; Francis et al., 1995b Genetics 139, 607-630; Jones and Schedl, 1995 Genes Dev. 9, 1491-1504; Jones et al., 1996 Dev. Biol. 180, 165-183. This work was supported by NIH Grant HD25614.
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[
International Worm Meeting,
2009]
The C. elegans genome is around 100 Mb long, and about 12% of it is derived from transposable elements (TEs). There has been a recent growing interest in the possible role of mobile elements in genome regulation and evolutionary plasticity; however, there has been no comprehensive, detailed analysis of the overall C. elegans TE distribution and their possible functional roles. In this study, we analyzed the intra and inter-chromosomal distribution pattern of all 156 TE families known in C. elegans. We found that more than 12% of these families are over-represented on the X chromosome. The many unique features of the X chromosome, such as dosage compensation, inactivation in the germline and low gene density make it an interesting case study for possible biological relevance of the skewed distribution of these families. Cele45, for example, is a family of Short Interspersed Elements (SINEs) which has 3 times more insertions on the X chromosome than expected. Conversely, we found that 10% of the TE families are completely absent in this chromosome, despite their high copy number (more than 500 copies) in the autosomes. Cele4, a DNA transposon, has 831 copies in the C. elegans genome and none of those are found on the X chromosome. It has been previously shown that the distribution pattern of DNA transposons is positively correlated with high recombination rates, which usually occur in the chromosomal arms. In the X chromosome, however, recombination rates are uniform across its length. Intriguingly, we found that the distribution pattern of most of the families (51%) of both DNA and RNA transposons does not correlate with recombination rate on the X chromosome. Thus, other factors must account for the preferential accumulation of these TEs on the arms of the X chromosome.
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[
International Worm Meeting,
2021]
Transposable elements (TEs) are powerful agents of evolution that can rewire transcriptional programs by mobilizing and distributing transcription factor (TF) DNA-binding motifs throughout genomes. To investigate the extent that TEs provide TF-binding motifs in C. elegans, we determined the genomic positions of DNA-binding motifs for over 200 different TFs (1). Surprisingly, we found that almost all of the examined TFs have binding motifs that reside within TEs, and all types of TEs have at least one instance of a TF motif, demonstrating that TEs provide previously unappreciated numbers of TF-binding motifs to the C. elegans genome. After determining the occurrence of TF motifs in TEs relative to the rest of the genome, we identified numerous TF-binding motifs that are highly enriched within TEs compared to what would be expected by chance. Consistent with potential functional roles for these TE-enriched TF-binding sequences, we found that significantly more TEs with TF motifs display evidence for selection compared to those lacking motifs through the use of publicly available genome variation data (2). We also compared the locations of TE-residing TF motifs to published ATAC-seq (3) and ChIP-seq (4) data, which identify regions of open chromatin associated with TF DNA binding and regions bound by TFs of interest, respectively. Strikingly, we found that all of the TF motif types that occur in TEs have instances of residing within accessible chromatin, and the overwhelming majority of TF-binding motifs located within TEs associate with their cognate TFs, suggesting extensive binding of TFs to sequences within TEs. Additionally, TEs with accessible or TF-bound motifs reside in the putative promoter regions of ~14% of all protein-coding genes, providing widespread possibilities for influencing gene expression. Taken together, our work shows that TE-provided TF-binding sites are ubiquitous in C. elegans and have broad potential to rewire gene expression. 1. Weirauch et al. (2014) Cell 158:1431-1443. 2. Cook et al. (2016) Nucleic Acids Research 45:D650-D657. 3. Daugherty et al. (2017) Genome Research 27:2096-2107. 4. Kudron et al. (2018) Genetics 208:937-949.
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[
West Coast Worm Meeting,
2004]
Tobacco use is the second major cause of death worldwide. The acquisition of tolerance to nicotine is a key step in the development of nicotine addiction. Twenty seven nicotinic acetylcholine receptor subunits have been identified in C. elegans (reviewed in Jones and Sattelle, 2003). However, few of the molecules which modify nAChR function, abundance, or subcellular localization in response to nicotine exposure are known. We are performing a RNAi-based screen of the first chromosome using the Ahringer lab RNAi feeding library in a
rrf-3 background. In preliminary experiments, worms fed RNAi against genes already known to be involved in nicotine responces (including
unc-63 and
unc-50) have shown resistance in our nicotine-induced paralysis assay. Results of this screen should provide insight into the mechanisms of nicotine tolerance.
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[
International Worm Meeting,
2013]
The nematode Caenorhabditis elegans has emerged as an important tool in pharmacology and toxicology because of its invariant and fully described developmental program, well-characterized genome, ease of maintenance and genetic manipulation, short and prolific life cycle, and small body size. This model has shown to be useful in the evaluation of the biological activity and the mechanisms of action of new synthetic compounds such as organoselenium and organotellurium compounds. Most of these compounds depict antioxidant potential in other models, however little is known regarding their mechanisms. Antioxidant response can be modulated by intracellular signaling, such as FOXO pathway, which is DAF-16 pathway in worms. This work was based on the hypothesis that depending on the chemical structure of the compounds, they would modulate the insulin-like pathway DAF-16 and consequently depict higher antioxidant potential against pro-oxidants. N2, TJ356 and CF1553 strains were handled and maintained at 20 deg C on E. coli OP50/ NGM plates. The lethal dose 50% (LD50) of the compounds was determined with doses ranging from 0.1 to 2250 mM. Synchronized L1 worms were treated with the compounds for 30 min. For stress- resistance assays, worms were pre-treated for 30 min with compounds and then exposed for 30 min to the prooxidant paraquat. We determined the LD50 using a sigmoidal dose-reponse curve and for the resistance assays we used one-way ANOVA to compare groups. Our results showed that xylofuranosides and some quinolines containing Se or Te have very low toxicity. We have found that Te-containing compounds can modulate DAF-16 pathway at lower concentrations, with consequent increase in SOD-3::GFP expression. Consequently, these compounds presented higher antioxidant potential at sublethal concentrations in wild type worms, as observed by protection against paraquat. We also observed that quinolines with a donator group have higher antioxidant activity. Furthermore, these studies show that C. elegans can be contribute to the rational drug synthesis field.
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[
International Worm Meeting,
2005]
Despite more than fifty years of knowledge of the link between smoking and lung cancer, tobacco use remains the leading preventable cause of disease and premature death in the United States. The acquisition of tolerance to nicotine is a key step in the development of nicotine addiction. Twenty seven nicotinic acetylcholine receptor (nAChR) sub-units have been identified in C. elegans (reviewed in Jones and Sattelle, 2003). However, few of the molecules which modify nAChR function, abundance, or sub-cellular localization in response to nicotine exposure are known. We have performed a RNAi-based screen of the first chromosome, as well as a candidate gene screen of predicted neuropeptides and G-protein coupled receptors, using the Ahringer lab RNAi feeding library in a
rrf-3 background. We have identified 47 candidate genes which might affect cholinergic signaling. These fall into multiple classes, including molecules involved in protein trafficking and turnover, cell structural components, kinases and phosphatases, as well as transcriptional regulators. Results of this screen should provide insight into the mechanisms of nicotine tolerance.
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[
Midwest Worm Meeting,
1996]
The
gld-1 gene is involved in multiple aspects of C. elegans germline development. The primary, essential function of
gld-1 is to direct oogenesis. In the absence of
gld-1 activity, germ cells that would otherwise develop into oocytes enter meiosis normally but exit early pachytene to re-enter a mitotic cell cycle and form a tumor. The gene also plays nonessential roles in inhibiting premeiotic germ cell proliferation and promoting germ cells to adopt the male sexual fate [1,2].
gld-1 encodes a 463 amino acid protein that contains an evolutionarily conserved domain of ~200 amino acids that is also found in the mammalian Quaking and Sam68 proteins. A KH RNA binding motif lies within this domain [3]. Since GLD-1 appears to be localized exclusively to the cytoplasm, the protein is likely to regulate mRNA translation or stability [4]. To better understand how
gld-1 acts in the control of germline development, we are using the yeast two-hybrid system [5] to isolate proteins that physically associate with the gene product. Through this approach we hope to identify regulators and cofactors of GLD-1. By screening an oligo(dT)-primed cDNA library (gift of R. Barstead) with a fragment of GLD-1 that includes the amino terminal half of the conserved domain, we identified the F28E10.1 gene product as a protein that specifically binds GLD-1. The F28E10.1 locus, which was identified by the C. elegans genome sequencing consortium, is predicted to encode a novel, hydrophilic protein of 118 kD. This gene maps to the region of chromosome IV that is deleted by mDf4 and may correspond to one of the lethal or sterile loci uncovered by the deficiency [6,7]. We are currently testing the biological relevance of the interaction between GLD-1 and F28E10.1 through in vivo assays. Progress will be reported. [1] Francis et al. 1995a. Genetics 139: 579-606; [2] Francis et al. 1995b. Genetics 139: 607-630; [3] Jones and Schedl. 1995. Genes Dev 9: 1491-1504; [4] Jones et al., in preparation; [5] Chien et al. 1991. PNAS 88: 9578-9582; [6] Rogalski and Riddle. 1988. Genetics 118: 61-74; [7] Cassada et al. 1981. Dev Biol 84: 193-205.