A metabolic regulatory network for the Caenorhabditis elegans intestine.

Metabolic perturbations can affect gene expression, for instance to rewire metabolism. While numerous efforts have measured gene expression in response to individual metabolic perturbations, methods that determine all metabolic perturbations that affect the expression for a given gene or set of genes have not been available. Here, we use a gene-centered approach to derive a first-pass metabolic regulatory network for Caenorhabditis elegans by performing RNAi of more than 1,400 metabolic genes with a set of 19 promoter reporter strains that express a fluorescent protein in the animal's intestine. We find that metabolic perturbations generally increase promoter activity, which contrasts with transcription factor (TF) RNAi, which tends to repress promoter activity. We identify several TFs that modulate promoter activity in response to perturbations of the electron transport chain and explore complex genetic interactions among metabolic pathways. This work provides a blueprint for a systems-level understanding of how metabolism affects gene expression.

WormPaths: Caenorhabditis elegans metabolic pathway annotation and visualization.

In our group, we aim to understand metabolism in the nematode Caenorhabditis elegans and its relationships with gene expression, physiology, and the response to therapeutic drugs. Visualization of the metabolic pathways that comprise the metabolic network is extremely useful for interpreting a wide variety of experiments. Detailed annotated metabolic pathway maps for C. elegans are mostly limited to pan-organismal maps, many with incomplete or inaccurate pathway and enzyme annotations. Here, we present WormPaths, which is composed of two parts: (1) the careful manual annotation of metabolic genes into pathways, categories, and levels, and (2) 62 pathway maps that include metabolites, metabolite structures, genes, reactions, and pathway connections between maps. These maps are available on the WormFlux website. We show that WormPaths provides easy-to-navigate maps and that the different levels in WormPaths can be used for metabolic pathway enrichment analysis of transcriptomic data. In the future, we envision further developing these maps to be more interactive, analogous to road maps that are available on mobile devices.

WormPaths: Caenorhabditis elegans metabolic pathway annotation and visualization.

In our group, we aim to understand metabolism in the nematode Caenorhabditis elegans and its relationships with gene expression, physiology and the response to therapeutic drugs. On March 15, 2020, a stay-at-home order was put into effect in the state of Massachusetts, USA, to flatten the curve of the spread of the novel SARS-CoV2 virus that causes COVID-19. For biomedical researchers in our state, this meant putting a hold on experiments for nine weeks until May 18, 2020. To keep the lab engaged and productive, and to enhance communication and collaboration, we embarked on an in-lab project that we all found important but that we never had the time for: the detailed annotation and drawing of C. elegans metabolic pathways. As a result, we present WormPaths, which is composed of two parts: 1) the careful manual annotation of metabolic genes into pathways, categories and levels, and 2) 66 pathway maps that include metabolites, metabolite structures, genes, reactions, and pathway connections between maps. These maps are available on our WormFlux website. We show that WormPaths provides easy-to-navigate maps and that the different levels in WormPaths can be used for metabolic pathway enrichment analysis of transcriptomic data. In the unfortunate event of additional lockdowns, we envision further developing these maps to be more interactive, with an analogy of road maps that are available on mobile devices.

WormCat: An Online Tool for Annotation and Visualization of Caenorhabditis elegans Genome-Scale Data.

The emergence of large gene expression datasets has revealed the need for improved tools to identify enriched gene categories and visualize enrichment patterns. While gene ontogeny (GO) provides a valuable tool for gene set enrichment analysis, it has several limitations. First, it is difficult to graph multiple GO analyses for comparison. Second, genes from some model systems are not well represented. For example, ∼30% of Caenorhabditis elegans genes are missing from the analysis in commonly used databases. To allow categorization and visualization of enriched C. elegans gene sets in different types of genome-scale data, we developed WormCat, a web-based tool that uses a near-complete annotation of the C. elegans genome to identify coexpressed gene sets and scaled heat map for enrichment visualization. We tested the performance of WormCat using a variety of published transcriptomic datasets, and show that it reproduces major categories identified by GO. Importantly, we also found previously unidentified categories that are informative for interpreting phenotypes or predicting biological function. For example, we analyzed published RNA-seq data from C. elegans treated with combinations of lifespan-extending drugs, where one combination paradoxically shortened lifespan. Using WormCat, we identified sterol metabolism as a category that was not enriched in the single or double combinations, but emerged in a triple combination along with the lifespan shortening. Thus, WormCat identified a gene set with potential. phenotypic relevance not found with previous GO analysis. In conclusion, WormCat provides a powerful tool for the analysis and visualization of gene set enrichment in different types of C. elegans datasets.

A Delicate Balance between Bacterial Iron and Reactive Oxygen Species Supports Optimal C. elegans Development.

Iron is an essential micronutrient for all forms of life; low levels of iron cause human disease, while too much iron is toxic. Low iron levels induce reactive oxygen species (ROS) by disruption of the heme and iron-sulfur cluster-dependent electron transport chain (ETC). To identify bacterial metabolites that affect development, we screened the Keio Escherichia coli collection and uncovered 244 gene deletion mutants that slow Caenorhabditis elegans development. Several of these genes encode members of the ETC cytochrome bo oxidase complex, as well as iron importers. Surprisingly, either iron or anti-oxidant supplementation reversed the developmental delay. This suggests that low bacterial iron results in high bacterial ROS and vice versa, which causes oxidative stress in C. elegans that subsequently impairs mitochondrial function and delays development. Our data indicate that the bacterial diets of C. elegans provide precisely tailored amounts of iron to support proper development.

A Persistence Detector for Metabolic Network Rewiring in an Animal.

Biological systems must possess mechanisms that prevent inappropriate responses to spurious environmental inputs. Caenorhabditis elegans has two breakdown pathways for the short-chain fatty acid propionate: a canonical, vitamin B12-dependent pathway and a propionate shunt that is used when vitamin B12 levels are low. The shunt pathway is kept off when there is sufficient flux through the canonical pathway, likely to avoid generating shunt-specific toxic intermediates. Here, we discovered a transcriptional regulatory circuit that activates shunt gene expression upon propionate buildup. Nuclear hormone receptor 10 (NHR-10) and NHR-68 function together as a "persistence detector" in a type 1, coherent feed-forward loop with an AND-logic gate to delay shunt activation upon propionate accumulation and to avoid spurious shunt activation in response to a non-sustained pulse of propionate. Together, our findings identify a persistence detector in an animal, which transcriptionally rewires propionate metabolism to maintain homeostasis.

Many transcription factors contribute to C. elegans growth and fat storage.

Reverse genetic screens by RNA interference (RNAi) in model organisms such as the nematode Caenorhabditis elegans have provided numerous insights into gene function, thereby connecting genotype to phenotype. However, genes that contribute only subtly are often missed because relatively large numbers of measurements and reliable quantification are required to overcome experimental and biological noise that may mask subtle phenotypic effects. Here, we address this challenge by focusing on two phenotypes in C. elegans: growth and fat storage. We carried out comprehensive RNAi knockdown of transcription factors (TFs), as these are known important regulators of biological processes during development and the maintenance of homeostasis. Microscopy images of TF knockdown animals stained with Oil Red O (ORO) were captured, and body size (proxy for growth) and ORO staining intensity (proxy for fat storage) were precisely quantified using a newly developed imaging tool we named IPPOME (Image Processing for Precise and Objective MEasurement). We found that a surprisingly large proportion of TFs contribute to growth and fat storage, but that most TFs have only subtle, yet significant effects. This study provides a blueprint for studies of other genes and phenotypes in C. elegans.

PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions.

Interactions between RNA binding proteins (RBPs) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using 'protein-centered' (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest-in a 'gene-centered' manner-yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA 'bait' can be tested versus multiple RBP 'preys' in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3' UTRs. PRIMA faithfully recapitulated numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

C. elegans and its bacterial diet as a model for systems-level understanding of host-microbiota interactions.

Resident microbes of the human body, particularly the gut microbiota, provide essential functions for the host, and, therefore, have important roles in human health as well as mitigating disease. It is difficult to study the mechanisms by which the microbiota affect human health, especially at a systems-level, due to heterogeneity of human genomes, the complexity and heterogeneity of the gut microbiota, the challenge of growing these bacteria in the laboratory, and the lack of bacterial genetics in most microbiotal species. In the last few years, the interspecies model of the nematode Caenorhabditis elegans and its bacterial diet has proven powerful for studying host-microbiota interactions, as both the animal and its bacterial diet can be subjected to large-scale and high-throughput genetic screening. The high level of homology between many C. elegans and human genes, as well as extensive similarities between human and C. elegans metabolism, indicates that the findings obtained from this interspecies model may be broadly relevant to understanding how the human microbiota affects physiology and disease. In this review, we summarize recent systems studies on how bacteria interact with C. elegans and affect life history traits.

A gene-centered C. elegans protein-DNA interaction network provides a framework for functional predictions.

Transcription factors (TFs) play a central role in controlling spatiotemporal gene expression and the response to environmental cues. A comprehensive understanding of gene regulation requires integrating physical protein-DNA interactions (PDIs) with TF regulatory activity, expression patterns, and phenotypic data. Although great progress has been made in mapping PDIs using chromatin immunoprecipitation, these studies have only characterized ~10% of TFs in any metazoan species. The nematode C. elegans has been widely used to study gene regulation due to its compact genome with short regulatory sequences. Here, we delineated the largest gene-centered metazoan PDI network to date by examining interactions between 90% of C. elegans TFs and 15% of gene promoters. We used this network as a backbone to predict TF binding sites for 77 TFs, two-thirds of which are novel, as well as integrate gene expression, protein-protein interaction, and phenotypic data to predict regulatory and biological functions for multiple genes and TFs.

Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans.

Metabolic network rewiring is the rerouting of metabolism through the use of alternate enzymes to adjust pathway flux and accomplish specific anabolic or catabolic objectives. Here, we report the first characterization of two parallel pathways for the breakdown of the short chain fatty acid propionate in Caenorhabditis elegans. Using genetic interaction mapping, gene co-expression analysis, pathway intermediate quantification and carbon tracing, we uncover a vitamin B12-independent propionate breakdown shunt that is transcriptionally activated on vitamin B12 deficient diets, or under genetic conditions mimicking the human diseases propionic- and methylmalonic acidemia, in which the canonical B12-dependent propionate breakdown pathway is blocked. Our study presents the first example of transcriptional vitamin-directed metabolic network rewiring to promote survival under vitamin deficiency. The ability to reroute propionate breakdown according to B12 availability may provide C. elegans with metabolic plasticity and thus a selective advantage on different diets in the wild.

AVX-470: a novel oral anti-TNF antibody with therapeutic potential in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, which is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF). We developed and characterized AVX-470, a novel polyclonal antibody specific for human TNF. We evaluated the oral activity of AVX-470m, a surrogate antibody specific for murine TNF, in several well-accepted mouse models of IBD. METHODS: AVX-470 and AVX-470m were isolated from the colostrum of dairy cows that had been immunized with TNF. The potency, specificity, and affinity of both AVX-470 and AVX-470m were evaluated in vitro and compared with infliximab. AVX-470m was orally administered to mice either before or after induction of colitis, and activity was measured by endoscopy, histopathology, immunohistochemistry, and quantitative measurement of messenger RNA levels. Colitis was induced using either 2,4,6-trinitrobenzene sulfonate or dextran sodium sulfate. RESULTS: AVX-470 and AVX-470m were shown to be functionally comparable in vitro. Moreover, the specificity, neutralizing potency, and affinity of AVX-470 were comparable with infliximab. Orally administered AVX-470m effectively reduced disease severity in several mouse models of IBD. Activity was comparable with that of oral prednisolone or parenteral etanercept. The antibody penetrated the colonic mucosa and inhibited TNF-driven mucosal inflammation with minimal systemic exposure. CONCLUSIONS: AVX-470 is a novel polyclonal anti-TNF antibody with an in vitro activity profile comparable to that of infliximab. Oral administration of a surrogate antibody specific for mouse TNF is effective in treating mouse models of IBD, delivering the anti-TNF to the site of inflammation with minimal systemic exposure.

Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability.

A polarity complex of mPar-6 and atypical PKC binds, phosphorylates and regulates mammalian Lgl.

The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.

Cutting edge: quantitative imaging of raft accumulation in the immunological synapse.

Although the accumulation of lipid rafts at the immunological synapse is now well accepted, the degree of the accumulation, the localization within the fine structure of the immunological synapse, and the region from which lipid rafts are recruited have not been defined. In this work we show that lipid rafts preferentially accumulate in the central zone of the immunological synapse, the central supramolecular activation complex (C-SMAC). However, quantitative analyses indicate that the level of recruitment of lipid rafts to the C-SMAC is relatively small and suggests that rearrangement of lipid rafts from the peripheral zone of the synapse into the C-SMAC can account for this accumulation. We also assessed the effects of CD28 deficiency on lipid raft recruitment to the immunological synapse. The accumulation of lipid occurred independently of the CD28/B7 system and was not measurably altered by CD28.

T cell receptor signaling precedes immunological synapse formation.

The area of contact between a T cell and an antigen-presenting cell (APC) is known as the immunological synapse. Although its exact function is unknown, one model suggests that it allows for T cell receptor (TCR) clustering and for sustained signaling in T cells for many hours. Here we demonstrate that TCR-mediated tyrosine kinase signaling in naïve T cells occurred primarily at the periphery of the synapse and was largely abated before mature immunological synapses had formed. These data suggest that many hours of TCR signaling are not required for T cell activation. These observations challenge current ideas about the role of immunological synapses in T cell activation.

Regulation of Lck activity by CD4 and CD28 in the immunological synapse.

Although the Src family tyrosine kinase Lck is essential for T cell receptor (TCR) signaling, whether or how Lck is activated is unknown. Using a phosphospecific antiserum to Lck, we show here that Lck becomes autophosphorylated when T cells are stimulated by antigen-presenting cells (APCs). We found that TCR cross-linking alone could not stimulate Lck autophosphorylation and CD45 was not required for this process. Instead, the T cell accessory molecules CD4 and CD28 cooperated to induce autophosphorylation of Lck. CD4 recruited Lck to the T cell--APC interface, whereas CD28 sustained Lck activation. These data show how the multiple interactions afforded by the immunological synapse drive efficient and highly specific signaling.