Mutational and transcriptional landscape of spontaneous gene duplications and deletions in Caenorhabditis elegans.

Gene duplication and deletion are pivotal processes shaping the structural and functional repertoire of genomes, with implications for disease, adaptation, and evolution. We employed a mutation accumulation (MA) framework partnered with high-throughput genomics to assess the molecular and transcriptional characteristics of newly arisen gene copy-number variants (CNVs) in Caenorhabditis elegans populations subjected to varying intensity of selection. Here, we report a direct spontaneous genome-wide rate of gene duplication of 2.9 × 10-5/gene per generation in C. elegans, the highest for any species to date. The rate of gene deletion is sixfold lower (5 × 10-6/gene per generation). Deletions of highly expressed genes are particularly deleterious, given their paucity in even the N = 1 lines with minimal efficacy of selection. The increase in average transcript abundance of new duplicates arising under minimal selection is significantly greater than twofold compared with single copies of the same gene, suggesting that genes in segmental duplications are frequently overactive at inception. The average increase in transcriptional activity of gene duplicates is greater in the N = 1 MA lines than in MA lines with larger population bottlenecks. There is an inverse relationship between the ancestral transcription levels of new gene duplicates and population size, with duplicate copies of highly expressed genes less likely to accumulate in larger populations. Our results demonstrate a fitness cost of increased transcription following duplication, which results in purifying selection against new gene duplicates. However, on average, duplications also provide a significant increase in gene expression that can facilitate adaptation to novel environmental challenges.

John Sulston (1942-2018): a personal perspective.

John Sulston changed the way we do science, not once, but three times - initially with the complete cell lineage of the nematode Caenorhabditis elegans, next with completion of the genome sequences of the worm and human genomes and finally with his strong and active advocacy for open data sharing. His contributions were widely recognized and in 2002 he received the Nobel Prize in Physiology and Medicine.

Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced Multi-mutation Strains and Using the Sequence Kernel Association Test (SKAT).

Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using C. elegans as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT-1.1 in ciliated sensory neuron function and morphogenesis. BGNT-1.1 functions at the trans-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT-1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy.

Mitochondrial Mutation Rate, Spectrum and Heteroplasmy in Caenorhabditis elegans Spontaneous Mutation Accumulation Lines of Differing Population Size.

Mitochondrial genomes of metazoans, given their elevated rates of evolution, have served as pivotal markers for phylogeographic studies and recent phylogenetic events. In order to determine the dynamics of spontaneous mitochondrial mutations in small populations in the absence and presence of selection, we evolved mutation accumulation (MA) lines of Caenorhabditis elegans in parallel over 409 consecutive generations at three varying population sizes of N = 1, 10, and 100 hermaphrodites. The N =1 populations should have a minimal influence of natural selection to provide the spontaneous mutation rate and the expected rate of neutral evolution, whereas larger population sizes should experience increasing intensity of selection. New mutations were identified by Illumina paired-end sequencing of 86 mtDNA genomes across 35 experimental lines and compared with published genomes of natural isolates. The spontaneous mitochondrial mutation rate was estimated at 1.05 × 10-7/site/generation. A strong G/C→A/T mutational bias was observed in both the MA lines and the natural isolates. This suggests that the low G + C content at synonymous sites is the product of mutation bias rather than selection as previously proposed. The mitochondrial effective population size per worm generation was estimated to be 62. Although it was previously concluded that heteroplasmy was rare in C. elegans, the vast majority of mutations in this study were heteroplasmic despite an experimental regime exceeding 400 generations. The frequencies of frameshift and nonsynonymous mutations were negatively correlated with population size, which suggests their deleterious effects on fitness and a potent role for selection in their eradication.

Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon.

We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

The million mutation project: a new approach to genetics in Caenorhabditis elegans.

We have created a library of 2007 mutagenized Caenorhabditis elegans strains, each sequenced to a target depth of 15-fold coverage, to provide the research community with mutant alleles for each of the worm's more than 20,000 genes. The library contains over 800,000 unique single nucleotide variants (SNVs) with an average of eight nonsynonymous changes per gene and more than 16,000 insertion/deletion (indel) and copy number changes, providing an unprecedented genetic resource for this multicellular organism. To supplement this collection, we also sequenced 40 wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets demonstrates that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. We also find a wide range of rDNA and telomere repeat copy number in both sets. Scanning the mutant collection for molecular phenotypes reveals a nonsense suppressor as well as strains with higher levels of indels that harbor mutations in DNA repair genes and strains with abundant males associated with him mutations. All the strains are available through the Caenorhabditis Genetics Center and all the sequence changes have been deposited in WormBase and are available through an interactive website.

Chromosome movements promoted by the mitochondrial protein SPD-3 are required for homology search during Caenorhabditis elegans meiosis.

Pairing of homologous chromosomes during early meiosis is essential to prevent the formation of aneuploid gametes. Chromosome pairing includes a step of homology search followed by the stabilization of homolog interactions by the synaptonemal complex (SC). These events coincide with dramatic changes in nuclear organization and rapid chromosome movements that depend on cytoskeletal motors and are mediated by SUN-domain proteins on the nuclear envelope, but how chromosome mobility contributes to the pairing process remains poorly understood. We show that defects in the mitochondria-localizing protein SPD-3 cause a defect in homolog pairing without impairing nuclear reorganization or SC assembly, which results in promiscuous installation of the SC between non-homologous chromosomes. Preventing SC assembly in spd-3 mutants does not improve homolog pairing, demonstrating that SPD-3 is required for homology search at the start of meiosis. Pairing center regions localize to SUN-1 aggregates at meiosis onset in spd-3 mutants; and pairing-promoting proteins, including cytoskeletal motors and polo-like kinase 2, are normally recruited to the nuclear envelope. However, quantitative analysis of SUN-1 aggregate movement in spd-3 mutants demonstrates a clear reduction in mobility, although this defect is not as severe as that seen in sun-1(jf18) mutants, which also show a stronger pairing defect, suggesting a correlation between chromosome-end mobility and the efficiency of pairing. SUN-1 aggregate movement is also impaired following inhibition of mitochondrial respiration or dynein knockdown, suggesting that mitochondrial function is required for motor-driven SUN-1 movement. The reduced chromosome-end mobility of spd-3 mutants impairs coupling of SC assembly to homology recognition and causes a delay in meiotic progression mediated by HORMA-domain protein HTP-1. Our work reveals how chromosome mobility impacts the different early meiotic events that promote homolog pairing and suggests that efficient homology search at the onset of meiosis is largely dependent on motor-driven chromosome movement.

Rapid Increase in frequency of gene copy-number variants during experimental evolution in Caenorhabditis elegans.

BACKGROUND: Gene copy-number variation (CNVs), which provides the raw material for the evolution of novel genes, is widespread in natural populations. We investigated whether CNVs constitute a common mechanism of genetic change during adaptation in experimental Caenorhabditis elegans populations. Outcrossing C. elegans populations with low fitness were evolved for >200 generations. The frequencies of CNVs in these populations were analyzed by oligonucleotide array comparative genome hybridization, quantitative PCR, PCR, DNA sequencing across breakpoints, and single-worm PCR. RESULTS: Multiple duplications and deletions rose to intermediate or high frequencies in independent populations. Several lines of evidence suggest that these changes were adaptive: (i) copy-number changes reached high frequency or were fixed in a short time, (ii) many independent populations harbored CNVs spanning the same genes, and (iii) larger average size of CNVs in adapting populations relative to spontaneous CNVs. The latter is expected if larger CNVs are more likely to encompass genes under selection for a change in gene dosage. Several convergent CNVs originated in populations descended from different low fitness ancestors as well as high fitness controls. CONCLUSIONS: We show that gene copy-number changes are a common class of adaptive genetic change. Due to the high rates of origin of spontaneous duplications and deletions, copy-number changes containing the same genes arose readily in independent populations. Duplications that reached high frequencies in these adapting populations were significantly larger in span. Many convergent CNVs may be general adaptations to laboratory conditions. These results demonstrate the great potential borne by CNVs for evolutionary adaptation.

Spectrum of variations in dog-1/FANCJ and mdf-1/MAD1 defective Caenorhabditis elegans strains after long-term propagation.

BACKGROUND: Whole and partial chromosome losses or gains and structural chromosome changes are hallmarks of human tumors. Guanine-rich DNA, which has a potential to form a G-quadruplex (G4) structure, is particularly vulnerable to changes. In Caenorhabditis elegans, faithful transmission of G-rich DNA is ensured by the DOG-1/FANCJ deadbox helicase. RESULTS: To identify a spectrum of mutations, after long-term propagation, we combined whole genome sequencing (WGS) and oligonucleotide array Comparative Genomic Hybridization (oaCGH) analysis of a C. elegans strain that was propagated, in the absence of DOG-1 and MDF-1/MAD1, for a total of 470 generations, with samples taken for long term storage (by freezing) in generations 170 and 270. We compared the genomes of F170 and F470 strains and identified 94 substitutions, 17 InDels, 3 duplications, and 139 deletions larger than 20 bp. These homozygous variants were predicted to impact 101 protein-coding genes. Phenotypic analysis of this strain revealed remarkable fitness recovery indicating that mutations, which have accumulated in the strain, are not only tolerated but also cooperate to achieve long-term population survival in the absence of DOG-1 and MDF-1. Furthermore, deletions larger than 20 bp were the only variants that frequently occurred in G-rich DNA. We showed that 126 of the possible 954 predicted monoG/C tracts, larger than 14 bp, were deleted in unc-46 mdf-1 such-4; dog-1 F470 (JNC170). CONCLUSIONS: Here, we identified variants that accumulated in C. elegans' genome after long-term propagation in the absence of DOG-1 and MDF-1. We showed that DNA sequences, with G4-forming potential, are vulnerable to deletion-formation in this genetic background.

The C. elegans CDK8 Mediator module regulates axon guidance decisions in the ventral nerve cord and during dorsal axon navigation.

Receptors expressed on the growth cone of outgrowing axons detect cues required for proper navigation. The pathway choices available to an axon are in part defined by the set of guidance receptors present on the growth cone. Regulated expression of receptors and genes controlling the localization and activity of receptors ensures that axons respond only to guidance cues relevant for reaching their targets. In genetic screens for axon guidance mutants, we isolated an allele of let-19/mdt-13, a component of the Mediator, a large ~30 subunit protein complex essential for gene transcription by RNA polymerase II. LET-19/MDT-13 is part of the CDK8 module of the Mediator. By testing other Mediator components, we found that all subunits of the CDK8 module as well as some other Mediator components are required for specific axon navigation decisions in a subset of neurons. Expression profiling demonstrated that let-19/mdt-13 regulates the expression of a large number of genes in interneurons. A mutation in the sax-3 gene, encoding a receptor for the repulsive guidance cue SLT-1, suppresses the commissure navigation defects found in cdk-8 mutants. This suggests that the CDK8 module specifically represses the SAX-3/ROBO pathway to ensure proper commissure navigation.

A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (Kindlin) to integrin adhesion sites.

Caenorhabditis elegans muscle cells attach to basement membrane through adhesion plaques. PAT-3 (ß-integrin), UNC-112 (kindlin), and PAT-4 (integrin-linked kinase) are associated with these structures. Genetic analysis indicated that PAT-4 is required for UNC-112 to be properly localized. We investigated the molecular basis of this requirement. We show that the cytoplasmic tail of PAT-3 binds to full-length UNC-112 and that the N- and C-terminal halves of UNC-112 bind to each other. We demonstrate competition between the UNC-112 C-terminal half and PAT-4 for binding to the UNC-112 N-terminal half. The D382V mutation results in lack of binding to PAT-4 and lack of localization to adhesion structures. T346A or E349K mutations, which abolish interaction of the N- and C-terminal halves, permit D382V UNC-112 to localize to adhesion structures. The following model is proposed. UNC-112 exists in closed inactive and open active conformations, and upon binding of PAT-4 to the UNC-112 N-terminal half, UNC-112 is converted into the open state, able to bind to PAT-3.

Targeted gene deletions in C. elegans using transposon excision.

We developed a method, MosDEL, to generate targeted knockouts of genes in Caenorhabditis elegans by injection. We generated a double-strand break by mobilizing a Mos1 transposon adjacent to the region to be deleted; the double-stranded break is repaired using injected DNA as a template. Repair can delete up to 25 kb of DNA and simultaneously insert a positive selection marker.

Membrane extensions are associated with proper anterior migration of muscle cells during Caenorhabditis elegans embryogenesis.

C. elegans body wall muscle is formed after a series of well-orchestrated steps. With the onset of specification embryonic muscle cells accumulate under the hypodermal seam cells at the left and right sides of the embryo. Shortly thereafter they begin to migrate dorsally and ventrally resting beneath the dorsal and ventral hypodermis eventually forming the four muscle quadrants present upon hatching. In this study we describe the plasma membrane dynamics of these migrating cells and observe the extension of filopodia and lamellipodia during dorso-ventral migration but not during the earlier stages of accumulation. We also describe an anterior migration event during embryonic muscle morphogenesis, whereby the anterior-most pair of cells in each of the four muscle quadrants extends long processes to the anterior tip of the developing embryo. Anteriormost muscle cells then follow these extensions into their final positions in the developing embryo. Using RNAi and mutant analysis, we have identified laminin as being involved in mediating the dorsal-ventral muscle migrations. Finally we show that the α-integrin INA-1, the ephrin VAB-2 and its receptor VAB-1 and the Robo receptor SAX-3 indirectly promote the proper extension of the ventral anterior muscle processes by organizing the embryonic neurons so as to provide a clear path for muscle membrane extension.

C. elegans ADAMTS ADT-2 regulates body size by modulating TGFß signaling and cuticle collagen organization.

Organismal growth and body size are influenced by both genetic and environmental factors. We have utilized the strong molecular genetic techniques available in the nematode Caenorhabditis elegans to identify genetic determinants of body size. In C. elegans, DBL-1, a member of the conserved family of secreted growth factors known as the Transforming Growth Factor ß superfamily, is known to play a major role in growth control. The mechanisms by which other determinants of body size function, however, is less well understood. To identify additional genes involved in body size regulation, a genetic screen for small mutants was previously performed. One of the genes identified in that screen was sma-21. We now demonstrate that sma-21 encodes ADT-2, a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases. ADAMTS proteins are believed to remodel the extracellular matrix and may modulate the activity of extracellular signals. Genetic interactions suggest that ADT-2 acts in parallel with or in multiple size regulatory pathways. We demonstrate that ADT-2 is required for normal levels of expression of a DBL-1-responsive transcriptional reporter. We further demonstrate that adt-2 regulatory sequences drive expression in glial-like and vulval cells, and that ADT-2 activity is required for normal cuticle collagen fibril organization. We therefore propose that ADT-2 regulates body size both by modulating TGFß signaling activity and by maintaining normal cuticle structure.

An integrated strategy to study muscle development and myofilament structure in Caenorhabditis elegans.

A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known.

ccd-5, a novel cdk-5 binding partner, regulates pioneer axon guidance in the ventral nerve cord of Caenorhabditis elegans.

During nervous system development, axons navigate complex environments to reach synaptic targets. Early extending axons must interact with guidance cues in the surrounding tissue, while later extending axons can interact directly with earlier "pioneering" axons, "following" their path. In Caenorhabditis elegans, the AVG neuron pioneers the right axon tract of the ventral nerve cord. We previously found that aex-3, a rab-3 guanine nucleotide exchange factor, is essential for AVG axon navigation in a nid-1 mutant background and that aex-3 might be involved in trafficking of UNC-5, a receptor for the guidance cue UNC-6/netrin. Here, we describe a new gene in this pathway: ccd-5, a putative cdk-5 binding partner. ccd-5 mutants exhibit increased navigation defects of AVG pioneer as well as interneuron and motor neuron follower axons in a nid-1 mutant background. We show that ccd-5 acts in a pathway with cdk-5, aex-3, and unc-5. Navigation defects of follower interneuron and motoneuron axons correlate with AVG pioneer axon defects. This suggests that ccd-5 mostly affects pioneer axon navigation and that follower axon defects are largely a secondary consequence of pioneer navigation defects. To determine the consequences for nervous system function, we assessed various behavioral and movement parameters. ccd-5 single mutants have no significant movement defects, and nid-1 ccd-5 double mutants are less responsive to mechanosensory stimuli compared with nid-1 single mutants. These surprisingly minor defects indicate either a high tolerance for axon guidance defects within the motor circuit and/or an ability to maintain synaptic connections among commonly misguided axons.

Sequenced Breakpoints of Crossover Suppressor/Inversion qC1.

We used whole-genome sequencing (WGS) data from a number of balanced lethal strains in Caenorhabditis elegans to show that the crossover suppressor qC1 is an inversion. The rearrangement is complex, with a large primary inversion that contains several other smaller inverted regions. The graphical representation below depicts these various qC1 rearrangements for ease of conceptualization. It is the simplest chromosomal structure compatible with the data currently available, but even then it is worth noting that the complexity of the qC1 chromosome can make the graphical reconstruction difficult to understand, and it may seem a bit like relativity theory or artwork from M.C. Escher (https://moa.byu.edu/m-c-eschers-relativity/).

Whole Genome Sequencing identifies reciprocal translocation hT2(I;III) breakpoints.

We used whole-genome sequencing (WGS) data from a Caenorhabditis elegans strain homozygous for the reciprocal translocation hT2(I;III) to identify its breakpoints molecularly. The translocation structure is fairly straightforward, with only minor secondary rearrangement in addition to the primary breakpoints. The graphical representation below depicts the two hT2 half-translocations for ease of conceptualization.

Identification of essential genes in Caenorhabditis elegans through whole-genome sequencing of legacy mutant collections.

It has been estimated that 15%-30% of the ∼20,000 genes in C. elegans are essential, yet many of these genes remain to be identified or characterized. With the goal of identifying unknown essential genes, we performed whole-genome sequencing on complementation pairs from legacy collections of maternal-effect lethal and sterile mutants. This approach uncovered maternal genes required for embryonic development and genes with apparent sperm-specific functions. In total, 58 putative essential genes were identified on chromosomes III-V, of which 52 genes are represented by novel alleles in this collection. Of these 52 genes, 19 (40 alleles) were selected for further functional characterization. The terminal phenotypes of embryos were examined, revealing defects in cell division, morphogenesis, and osmotic integrity of the eggshell. Mating assays with wild-type males revealed previously unknown male-expressed genes required for fertilization and embryonic development. The result of this study is a catalog of mutant alleles in essential genes that will serve as a resource to guide further study toward a more complete understanding of this important model organism. As many genes and developmental pathways in C. elegans are conserved and essential genes are often linked to human disease, uncovering the function of these genes may also provide insight to further our understanding of human biology.

ELT-2 is the predominant transcription factor controlling differentiation and function of the C. elegans intestine, from embryo to adult.

Starting with SAGE-libraries prepared from C. elegans FAC-sorted embryonic intestine cells (8E-16E cell stage), from total embryos and from purified oocytes, and taking advantage of the NextDB in situ hybridization data base, we define sets of genes highly expressed from the zygotic genome, and expressed either exclusively or preferentially in the embryonic intestine or in the intestine of newly hatched larvae; we had previously defined a similarly expressed set of genes from the adult intestine. We show that an extended TGATAA-like sequence is essentially the only candidate for a cis-acting regulatory motif common to intestine genes expressed at all stages. This sequence is a strong ELT-2 binding site and matches the sequence of GATA-like sites found to be important for the expression of every intestinal gene so far analyzed experimentally. We show that the majority of these three sets of highly expressed intestinal-specific/intestinal-enriched genes respond strongly to ectopic expression of ELT-2 within the embryo. By flow-sorting elt-2(null) larvae from elt-2(+) larvae and then preparing Solexa/Illumina-SAGE libraries, we show that the majority of these genes also respond strongly to loss-of-function of ELT-2. To test the consequences of loss of other transcription factors identified in the embryonic intestine, we develop a strain of worms that is RNAi-sensitive only in the intestine; however, we are unable (with one possible exception) to identify any other transcription factor whose intestinal loss-of-function causes a phenotype of comparable severity to the phenotype caused by loss of ELT-2. Overall, our results support a model in which ELT-2 is the predominant transcription factor in the post-specification C. elegans intestine and participates directly in the transcriptional regulation of the majority (>80%) of intestinal genes. We present evidence that ELT-2 plays a central role in most aspects of C. elegans intestinal physiology: establishing the structure of the enterocyte, regulating enzymes and transporters involved in digestion and nutrition, responding to environmental toxins and pathogenic infections, and regulating the downstream intestinal components of the daf-2/daf-16 pathway influencing aging and longevity.