Predicting gene essentiality in Caenorhabditis elegans by feature engineering and machine-learning.

Defining genes that are essential for life has major implications for understanding critical biological processes and mechanisms. Although essential genes have been identified and characterised experimentally using functional genomic tools, it is challenging to predict with confidence such genes from molecular and phenomic data sets using computational methods. Using extensive data sets available for the model organism Caenorhabditis elegans, we constructed here a machine-learning (ML)-based workflow for the prediction of essential genes on a genome-wide scale. We identified strong predictors for such genes and showed that trained ML models consistently achieve highly-accurate classifications. Complementary analyses revealed an association between essential genes and chromosomal location. Our findings reveal that essential genes in C. elegans tend to be located in or near the centre of autosomal chromosomes; are positively correlated with low single nucleotide polymorphim (SNP) densities and epigenetic markers in promoter regions; are involved in protein and nucleotide processing; are transcribed in most cells; are enriched in reproductive tissues or are targets for small RNAs bound to the argonaut CSR-1. Based on these results, we hypothesise an interplay between epigenetic markers and small RNA pathways in the germline, with transcription-based memory; this hypothesis warrants testing. From a technical perspective, further work is needed to evaluate whether the present ML-based approach will be applicable to other metazoans (including Drosophila melanogaster) for which comprehensive data sets (i.e. genomic, transcriptomic, proteomic, variomic, epigenetic and phenomic) are available.

A Novel Mechanism To Prevent H2S Toxicity in Caenorhabditis elegans.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that can be cytoprotective, especially in conditions of ischemia/reperfusion injury. However, H2S is also toxic, and unregulated accumulation or exposure to environmental H2S can be lethal. In Caenorhabditis elegans, the hypoxia inducible factor (hif-1) coordinates the initial transcriptional response to H2S, and is essential to survive exposure to low concentrations of H2S. We performed a forward genetic screen to identify mutations that suppress the lethality of hif-1 mutant animals in H2S. The mutations we recovered are specific for H2S, as they do not suppress embryonic lethality or reproductive arrest of hif-1 mutant animals in hypoxia, nor can they prevent the death of hif-1 mutant animals exposed to hydrogen cyanide. The majority of hif-1 suppressor mutations we recovered activate the skn-1/Nrf2 transcription factor. Activation of SKN-1 by hif-1 suppressor mutations increased the expression of a subset of H2S-responsive genes, consistent with previous findings that skn-1 plays a role in the transcriptional response to H2S. Using transgenic rescue, we show that overexpression of a single gene, rhy-1, is sufficient to protect hif-1 mutant animals in H2S. The rhy-1 gene encodes a predicated O-acyltransferase enzyme that has previously been shown to negatively regulate HIF-1 activity. Our data indicate that RHY-1 has novel, hif-1 independent, function that promotes survival in H2S.

WormBase: A Model Organism Database.

WormBase is an open-access model-organism database that provides current and accurate genetic information of C. elegans and related nematodes. Users can search WormBase by several fields, including a gene or human disease. A special feature of the database is the inclusion of micropublications, peer-reviewed data that may go unpublished in traditional venues.

Endoplasmic Reticulum Homeostasis Is Modulated by the Forkhead Transcription Factor FKH-9 During Infection of Caenorhabditis elegans.

Animals have evolved critical mechanisms to maintain cellular and organismal proteostasis during development, disease, and exposure to environmental stressors. The Unfolded Protein Response (UPR) is a conserved pathway that senses and responds to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen. We have previously demonstrated that the IRE-1-XBP-1 branch of the UPR is required to maintain Caenorhabditis elegans ER homeostasis during larval development in the presence of pathogenic Pseudomonas aeruginosa In this study, we identify loss-of-function mutations in four conserved transcriptional regulators that suppress the larval lethality of xbp-1 mutant animals caused by immune activation in response to infection by pathogenic bacteria: FKH-9, a forkhead family transcription factor; ARID-1, an ARID/Bright domain-containing transcription factor; HCF-1, a transcriptional regulator that associates with histone modifying enzymes; and SIN-3, a subunit of a histone deacetylase complex. Further characterization of FKH-9 suggests that loss of FKH-9 enhances resistance to the ER toxin tunicamycin and results in enhanced ER-associated degradation (ERAD). Increased ERAD activity of fkh-9 loss-of-function mutants is accompanied by a diminished capacity to degrade cytosolic proteasomal substrates and a corresponding increased sensitivity to the proteasomal inhibitor bortezomib. Our data underscore how the balance between ER and cytosolic proteostasis can be influenced by compensatory activation of ERAD during the physiological ER stress of infection and immune activation.

Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans.

CRISPR-based genome editing using ribonucleoprotein complexes and synthetic single-stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs-such as green fluorescent protein tags remains challenging in many systems. Here, we describe a streamlined and optimized editing protocol for the nematode Caenorhabditis elegans We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging 14 Argonaute proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based, partially single-stranded, "hybrid" donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci, achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems, and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

Distinct Roles of Two Histone Methyltransferases in Transmitting H3K36me3-Based Epigenetic Memory Across Generations in Caenorhabditis elegans.

Epigenetic information contributes to proper gene expression and development, and can be transmitted not only through mitotic divisions but also from parents to progeny. We investigated the roles in epigenetic inheritance of MES-4 and MET-1, the two Caenorhabditis elegans enzymes that methylate H3K36 (histone H3 Lys 36). Mass spectrometry analysis confirmed immunostaining results showing that both MES-4 and MET-1 catalyze H3K36me3. In the adult germline, MES-4 is enriched in the distal mitotic zone and MET-1 is enriched in the meiotic pachytene zone. Embryos inherit H3K36me3-marked chromosomes from both the oocyte and sperm, and a maternal load of MES-4 and MET-1 Maternal MES-4 quickly associates with sperm chromosomes; that association requires that the sperm chromosomes bear H3K36me3, suggesting that MES-4 is recruited to chromosomes by preexisting H3K36me3. In embryos that inherit H3K36me3-positive oocyte chromosomes and H3K36me3-negative sperm chromosomes, MES-4 and H3K36me3 are maintained on only a subset of chromosomes until at least the 32-cell stage, likely because MES-4 propagates H3K36me3 on regions of the genome with preexisting H3K36me3. In embryos lacking MES-4, H3K36me3 levels on chromosomes drop precipitously postfertilization. In contrast to the relatively high levels of MES-4 in early-stage embryos, MET-1 levels are low at early stages and start increasing by the ∼26-cell stage, consistent with expression from the zygotic genome. Our findings support the model that MET-1 mediates transcription-coupled H3K36me3 in the parental germline and transcriptionally active embryos, and that MES-4 transmits an epigenetic memory of H3K36me3 across generations and through early embryo cell divisions by maintaining inherited patterns of H3K36me3.

Meiotic Double-Strand Break Proteins Influence Repair Pathway Utilization.

Double-strand breaks (DSBs) are among the most deleterious lesions DNA can endure. Yet, DSBs are programmed at the onset of meiosis, and are required to facilitate appropriate reduction of ploidy in daughter cells. Repair of these breaks is tightly controlled to favor homologous recombination (HR)-the only repair pathway that can form crossovers. However, little is known about how the activities of alternative repair pathways are regulated at these stages. We discovered an unexpected synthetic interaction between the DSB machinery and strand-exchange proteins. Depleting the Caenorhabditis elegans DSB-promoting factors HIM-5 and DSB-2 suppresses the formation of chromosome fusions that arise in the absence of RAD-51 or other strand-exchange mediators. Our investigations reveal that nonhomologous and theta-mediated end joining (c-NHEJ and TMEJ, respectively) and single strand annealing (SSA) function redundantly to repair DSBs when HR is compromised, and that HIM-5 influences the utilization of TMEJ and SSA.

The mTOR Target S6 Kinase Arrests Development in Caenorhabditis elegans When the Heat-Shock Transcription Factor Is Impaired.

The widely conserved heat-shock response, regulated by heat-shock transcription factors, is not only essential for cellular stress resistance and adult longevity, but also for proper development. However, the genetic mechanisms by which heat-shock transcription factors regulate development are not well understood. In Caenorhabditis elegans, we conducted an unbiased genetic screen to identify mutations that could ameliorate the developmental-arrest phenotype of a heat-shock factor mutant. Here, we show that loss of the conserved translational activator rsks-1/S6 kinase, a downstream effector of mechanistic Target of Rapamycin (mTOR) kinase, can rescue the developmental-arrest phenotype of hsf-1 partial loss-of-function mutants. Unexpectedly, we show that the rescue is not likely caused by reduced translation, nor by activation of any of a variety of stress-protective genes and pathways. Our findings identify an as-yet unexplained regulatory relationship between the heat-shock transcription factor and the mTOR pathway during C. elegans development.

Using WormBase ParaSite: An Integrated Platform for Exploring Helminth Genomic Data.

WormBase ParaSite ( parasite.wormbase.org ) is a comprehensive resource for the genomes of parasitic nematodes and flatworms (helminths). It currently includes genomic data for over 100 helminth species, adding value by way of consistent functional annotation, gene comparative analysis and gene expression analysis. We provide several ways of exploring the data including a choice of genome browsers, genome and gene summary pages, text and sequence searching, a query wizard, bulk downloads, and programmatic interfaces. WormBase ParaSite is released three to six times per year, and is developed in collaboration with WormBase ( www.wormbase.org ) and Ensembl Genomes ( www.ensemblgenomes.org ).

WormBase ParaSite - a comprehensive resource for helminth genomics.

The number of publicly available parasitic worm genome sequences has increased dramatically in the past three years, and research interest in helminth functional genomics is now quickly gathering pace in response to the foundation that has been laid by these collective efforts. A systematic approach to the organisation, curation, analysis and presentation of these data is clearly vital for maximising the utility of these data to researchers. We have developed a portal called WormBase ParaSite (http://parasite.wormbase.org) for interrogating helminth genomes on a large scale. Data from over 100 nematode and platyhelminth species are integrated, adding value by way of systematic and consistent functional annotation (e.g. protein domains and Gene Ontology terms), gene expression analysis (e.g. alignment of life-stage specific transcriptome data sets), and comparative analysis (e.g. orthologues and paralogues). We provide several ways of exploring the data, including genome browsers, genome and gene summary pages, text search, sequence search, a query wizard, bulk downloads, and programmatic interfaces. In this review, we provide an overview of the back-end infrastructure and analysis behind WormBase ParaSite, and the displays and tools available to users for interrogating helminth genomic data.

Tissue enrichment analysis for C. elegans genomics.

BACKGROUND: Over the last ten years, there has been explosive development in methods for measuring gene expression. These methods can identify thousands of genes altered between conditions, but understanding these datasets and forming hypotheses based on them remains challenging. One way to analyze these datasets is to associate ontologies (hierarchical, descriptive vocabularies with controlled relations between terms) with genes and to look for enrichment of specific terms. Although Gene Ontology (GO) is available for Caenorhabditis elegans, it does not include anatomical information. RESULTS: We have developed a tool for identifying enrichment of C. elegans tissues among gene sets and generated a website GUI where users can access this tool. Since a common drawback to ontology enrichment analyses is its verbosity, we developed a very simple filtering algorithm to reduce the ontology size by an order of magnitude. We adjusted these filters and validated our tool using a set of 30 gold standards from Expression Cluster data in WormBase. We show our tool can even discriminate between embryonic and larval tissues and can even identify tissues down to the single-cell level. We used our tool to identify multiple neuronal tissues that are down-regulated due to pathogen infection in C. elegans. CONCLUSIONS: Our Tissue Enrichment Analysis (TEA) can be found within WormBase, and can be downloaded using Python's standard pip installer. It tests a slimmed-down C. elegans tissue ontology for enrichment of specific terms and provides users with a text and graphic representation of the results.

Wormpath: searching for molecular interaction networks in Caenorhabditis elegans.

BACKGROUND: High-throughput transcriptional profiling using Next-Generation Sequencing (RNA-Seq) or microarray technology have become standard tools in molecular biology. Successful investigations of gene regulatory mechanisms from these data typically employ mathematical models of biological networks. RESULTS: We have developed Wormpath, a software for molecular network discovery which operates on the genetic and physical interaction data of the Wormbase, a comprehensive resource of molecular data on Caenorhabditis elegans. We use Wormpath to show that the insulin/insulin-like growth factor signalling (IIS) pathway responds to UV-induced DNA damage during development. CONCLUSIONS: Our software provides highly facilitated access to C. elegans interaction data and is capable of identifying essential molecular networks within a list of differentially expressed genes.