Ding, Wei et al. (2015) International Worm Meeting "s-adenosylmethione levels govern innate immunity through distinct methylation-dependent pathways ."

Ding, Wei, Walker, Amy, Hou, Nicole, Smulan, Lorissa, Bozonnet, Noemie, Taubert, Stefan, Watts, Jennifer

[International Worm Meeting, 2015]

s-adenosylmethionine (SAMe) modulates cellular functions as the sole methyl donor modifying histones, nucleic acids and phospholipids. Fluctuation in SAMe impacts phosphatidylcholine (PC) synthesis in the liver and several mammalian studies correlate SAMe levels with variations in DNA or histone methylation. In addition, low SAMe is associated with with lipid accumulation, increased tissue injury and immune responses in mammalian metabolic diseases, such as fatty liver disease. However, molecular connections between SAMe limitation, individual methyltransferases and these phenotypes are unclear. We had previously found that knockdown or mutation in the C. elegans SAMe synthase sams-1 exhibited lipid accumulation and now we find that the low SAMe can also associated with activation or attenuation Caenorhabditis elegans immune responses depending on bacterial food source. On non-pathogenic Eschericia coli diets, innate immune pathways are stimulated with increased expression of genes associated with defense against bacterial pathogens and constituative phosphorylation of PMK-1, a p38 MAP kinase essential for innate immune responses. Dietary rescue experiments show that this activation of innate immunity is downstream of methylation-dependent PC production.However, when challenged with pathogenic Pseudomonas aeruginosa, sams-1(lof) animals die rapidly, despite activation of protective MAPK pathways. We find that distinct SAMe-dependent mechanisms are linked to Pseudomonas-specific responses in sams-1 animals. C. elegans undertakes a broad transcriptional response to pathogenic bacteria and we find that low SAMe restricts an activating histone methylation mark, H3K4me3, on promoters of Pseudomonas-responsive genes and limits induction of these pathogen responsive genes. Furthermore, we find the H3K4 methyltransferase set-16/MLL is critical for transcriptional responses to bacterial stress. Thus, our studies provide molecular links between SAMe levels and innate immune functions and suggest that a primary effect of SAMe depletion is limitation of stress-induced gene expression.

Ding, Wei et al. (2013) International Worm Meeting "Genome wide responses to methyl donor availability reveal effects on metabolic, stress response and germline function genes."

Ding, Wei, Irwin, Michael, Hansen, Malene, Walker, Amy K.

[International Worm Meeting, 2013]

Animals must coordinate nutritional input and metabolism with growth and reproduction. Small molecules, such as s-adenosylmethionine (SAMe) produced by the 1-carbon/folate cycle (1CC) and used as the major methyl donor, can modify protein activity and contribute to phospholipid biosynthesis. In C. elegans, reducing function of sams-1, a SAMe synthase, increases lifespan and lipid droplet accumulation while reducing fertility. We hypothesize that SAMe levels provide signals reseting physiology to compensate for altered nutritional inputs. SAMe is used for methylation of proteins, such as histones in epigenetic modification, but is also critical for production of phosphatidylcholine (PC), a major membrane phospholipid. Indeed sams-1(RNAi) animals have low levels of PC and lipid accumulation, fertility and lifespan return to wild-type levels when methylation-independent pathways of PC production are supported through dietary addition of choline. However, links between SAMe levels and direct changes of gene expression are unknown. To separate SAMe-specific effects from combined SAMe/PC functions, we compared global gene expression levels in sams-1(RNAi) animals with sams-1(RNAi) rescued by choline. Major functional groups of genes increasing with sams-1(RNAi) included metabolism (1CC, lipid biosynthesis, carbohydrate modification) and stress responses. However, decreased genes grouped in distinct subsets of metabolic pathways (carbohydrate catabolism, amino sugar metabolism and lipid binding), as well as genes important for germline function and RNA processing. We found that more than 90% of sams-1(RNAi) up- or downregulated genes returned to wild-type levels with choline supplementation. Interestingly, genes not returning to wild-type levels clustered in metabolic pathways such as the 1CC, suggesting potential for PC-independent SAMe effects. However, essentially all germline function genes were rescued. This correlates with the near complete rescue of fertility by choline, and suggests coordination of brood size with metabolic function acts through SAMe/PC pathways in these animals.

Green, James W.M. et al. (2013) International Worm Meeting "Comparative mapping of dauer larvae development in growing populations of C. elegans and C. briggsae."

Harvey, Simon C., Green, James W.M.

[International Worm Meeting, 2013]

Dauer larvae in Caenorhabditis elegans are long-lived and resistant to environmental stress. Outside of growing populations they are also the only life cycle stage that can be routinely isolated from the environment. The appropriate induction of dauer larvae development is therefore likely to be critical to genotype fitness in C. elegans. Given this, the extensive variation observed between wild isolates requires explanation. Does it represent adaptation to different environments or does it imply that there are multiple different ways to maximise fitness within the same environment? To investigate this question we have analysed dauer larvae formation and population growth in growing populations using different recombinant inbred lines (RILs) of C. elegans. These analyses allow direct comparisons between: (1) different RIL panels produced from distinct parental isolates analysed for the same dauer development same trait; (2) RILs and nearly isogenic lines (NILs) produced from the same parental isolates and analysed for the same dauer development same trait; and (3) different dauer development traits mapped using the same RIL panel. These results identify common QTL regions and both genotype and trait specific QTLs. Comparison with the results of analysis of variation in dauer larvae development within growing populations of C. briggsae RILs allows a more general picture of the control of variation of dauer larvae development in growing populations.

Yang J et al. (1991) International C. elegans Meeting "SITE-SPECIFIC MUTAGENESIS OF CUTICLE COLLAGENS"

Kramer JM, Yang J

[International C. elegans Meeting, 1991]

Sequence comparisons of fourteen characterized C. elegans cuticle collagens have identified some highly conserved amino acids. These conserved amino acids include the cysteines flanking the Gly-X-Y regions and four short sequences termed homology boxes A-D. Alterations of some of these conseNed residues have been shown to cause morphological changes in the animal. For example, a Cys substitution for the third Arg in homology box A in sqt-l and rol-6 causes a right roller phenotype while the same mutation in dpy-10 leads to a left roller phenotype. To investigate the importance of the conseNed amino acids in the cuticle collagens, we have made site- specific mutations of the sqt-l and rol-6 collagen genes and subsequently introduced them into animals by microinjection. To demonstrate that altered collagens in transgenic arrays produce appropriate phenotypes we have tested cloned mutant alleles of sqt-l and rol-6. We have found that the recessive alleles produce roller phenotypes in null backgrounds but not in WT, while the dominant alleles produce phenotypes even in the WT background. Thus the transgenic system produces appropriate phenotypes. So far we have made several site-specific mutants. A Cys-to-Ser substitution at the carboxy end of the Gly-X-Y of rol-6 causes a recessive-like left roller phenotype. The same alteration in sqt-l also causes a recessive left roller phenotype. This is the first left roller mutant of rol-6. An Arg-to-Cys substitution of the first Arg in homology box A of rol-6 was found to cause a dominant-like right roller phenotype, again identical to the same mutation in sqt-l. When the second Arg in homology box A of sqt-l was changed to Cys (box A consensus:RxxrRQ, in sqt-l and rol-6, cys-to-arg changes on 1st and 3rd R are dom. right roller) no abnormal phenotype was detected in the transformants. These results indicate that the presence of a Cys anywhere in Box A is not sufficient to cause a dominant right roller phenotype. In general, the same alterations in sqt-l and rol-6 produce the same phenotypes indicating that conserved amino acids in these two collagens share similar functions during cuticle construction.

Walker, Amy K. et al. (2011) International Worm Meeting "Control of one-carbon cycle genes by SBP-1 links SAMe production, PC biosynthesis and lipogenesis."

Watts, Jennifer L., Jacobs, Rene, Walker, Amy K., Rottiers, Veerle, Hart, Anne, Naar, Anders, Hansen, Malene

[International Worm Meeting, 2011]

To understand regulation of lipogenesis, we have examined the SBP-1/SREBP transcription factors necessary for fatty acid, phospholipid and cholesterol biosynthetic gene expression and lipid accumulation in vivo. Inactive SREBPs reside cytoplasmically in the ER and are activated by transit to the Golgi, where proteases release transcriptionally active forms to the nucleus. We have found a new set of SBP target genes in the 1-carbon cycle (1CC), including sams-1, an enzyme which produces s-adenosylmethionine (SAMe), the major methyl donor. We also found a feedback loop increasing SBP-1-dependent transcription and lipogenesis in C. elegans, murine liver and human cells when SAMe is limited. Deficiencies folate or other 1C metabolites predispose human fatty liver disease; thus, our studies linking SAMe production to SBP-1-dependent lipogenesis in C. elegans are likely relevant to metabolic disorders common in Western populations. sbp-1(RNAi) animals are lean and several SBP-1 targets, such as fat-7 are also required for lipogenesis. However, sams-1(RNAi) animals have increased lipid accumulation and lipogenic gene expression which depend on sbp-1, suggesting a feedback loop activating SBP-1 when SAMe is limiting. Metabolomic profiling after sams-1 RNAi revealed defects in phosphatidylcholine (PC) biosynthesis, which can require multiple methylation steps. Genetic ablation of PC biosynthesis coupled with dietary rescue experiments show that SBP-1 activity is sensitive to PC levels. We also found that murine liver or human cells with diminished SAMe or PC synthesis had increases in lipogenesis and SREBP-1 activity. Because PC is a major membrane component, we reasoned that SBP-1 activation was mechanistically linked to ER or Golgi function. We found that ER stress responses are strongly activated after sams-1 RNAi, but are not contributors to SBP-1 activation. However, Golgi markers were disrupted. Therefore we examined localization of Golgi-resident, SREBP-activating proteases in human cells and found that limiting SAMe or PC caused these proteases to relocalize to the ER, allowing processing of SREBP-1 and activation. Our observations show SBP-1/SREBP-1 may be over activated when SAMe and PC are low and may also reveal how methyl-deficient diets contribute to human fatty liver disease.

Rock Pulak et al. (2003) International Worm Meeting "Measurement and analysis of stress induced responses in transgenic Caenorhabditis elegans"

Rock Pulak, Britta Moellers, Julia Thompson

[International Worm Meeting, 2003]

We have constructed multiple transgenic Caenorhabditis elegans strains for the multiplex analysis of stress induced responses. Our analysis includes the inducible response from a heat shock protein promoter and a metallothionein promoter together in the same animal at the same time. We constructed two different strains expressing fluorescent reef coral protein DsRed and ZsYellow (both from Clontech) from the inducible promoter mtl-2. We crossed these transgenes into a transgenic strain that expresses jellyfish Green Fluorescent Protein under the control of the promoter for the inducible small heat shock protein gene hsp-16-2 (Link, CD et al., 1999). All analyses were performed on COPAS instrumentation, an instrument for optical analysis in flow. Transgene expression allows direct visualization and quantitation of the corresponding reporter gene expression in living animals. Our analysis includes the response to heat shock, certain chemicals and heavy metals. Both promoters are monitored in the same animal. The promoters respond differently to the same inducing agents both in terms of dose response and the kinetics of the response. The ability to directly observe the stress response in living animals significantly simplifies the identification of both exogenous physical and chemical insults, as well as, genetic alterations that modulate the stress response in C. elegans. The COPAS instrumentation allows for the automation of this process

Shugo Hamahashi et al. (2001) International C. elegans Meeting "Cell lineage acquiring system for early embryogenesis"

Shuichi Onami, Shugo Hamahashi, Hiroaki Kitano

[International C. elegans Meeting, 2001]

We have developed a computerized system that automatically acquires a cell lineage of early C. elegans embryo. At first, the system prepared a 4D Nomarski DIC microscope image of a target embryo that consists of 3D images taken every minute for 2 hours. A 3D image consists of more than 50 focal plane images at 0.5-micrometer intervals. The system then detected the regions of cell nucleus in each focal plane image. We found that the image entropy efficiently distinguishes nucleus regions from cytoplasm regions because it measures the roughness of images. All the focal plane images, whose size was about 600x600 pixels, were scanned by a small square window of 10x10 pixels measuring the image entropy of the inside regions, in order to convert the original images into entropy-value images. The regions of lower entropy are the nucleus regions. Finally, the system created a cell lineage based on the nucleus regions. Nucleus regions that represent the same nucleus at the same time point were grouped into a 3D nucleus region, then each pair of them were connected if they represent the same nucleus at two consecutive time points. The connections generated the lineage of 3D nucleus regions, which is the cell lineage. The system output three-dimensional positions of nuclei at each time point and their lineage. Currently, our system can generate the cell lineage from the 1-cell stage to about the 20-cell stage.

Tanja Starc et al. (2002) East Coast Worm Meeting "Structure function analysis of glutamate-gated chloride channels"

Joseph A Dent, Tanja Starc

[East Coast Worm Meeting, 2002]

Glutamate-gated chloride channels (GluCl) belong to the family of ligand-gated ion channels that includes nicotinic acetylcholine, serotonin, GABA and glycine receptors. The subunit composition of GluCl in vivo is not clear. By analogy with other ligand-gated channels of the same family we assume that they possess a pentameric structure probably consisting of at least a and b subunits. Caenorhabditis elegans has different GluCl channels formed by different a subunits. Five GluCl subunits genes have been characterized so far. They encode the subunit proteins a1, a2, a3, a4 and b. The subunits share the same structure; a large extracellular amino-terminal domain, four membranespanning domains (TM1-TM4), a cytoplasmic loop between TM3 and TM4, and an extracellular carboxy terminal. The best understood GluCl channel, which consists of a2 and b subunit, is expressed in the muscle cells of the pharynx. Here the channels mediate the response to the motor neuron M3.

Melissa Ann Browning et al. (2002) Mid-west Worm Meeting "POP-1 function within the vulval secondary lineage."

Melissa Ann Browning, Russell J Hill

[Mid-west Worm Meeting, 2002]

The C. elegans vulva is produced from the descendants of the primary and secondary vulval lineages. These descendant cells differentiate into distinct vulval cell types in a precise pattern. The mechanisms which regulate the cell type decisions occurring among these descendant cells is currently not well understood. The transcription factor POP-1 is a component of the C. elegans Wnt pathway and is expressed during vulval development in a pattern that correlates with the cell type decisions made by these descendants (Hill et al., I.W.M., 1999). For example, after the precursor P7.p has undergone two rounds of cell division (P7.pxx), its descendants are of cell types D, C, B, and A (from anterior to posterior). These descendant cells express POP-1 in the pattern Low, High, Low, High (from anterior to posterior). Thus, each P7.pxx cell expresses a different level of POP-1 than its sister cell and adopts a cell type distinct from its sister. Therefore, we tested whether sister P7.pxx cells would produce the same cell type if they had the same level of POP-1 activity.

Matai, Latika et al. (2021) International Worm Meeting "MicroRNA cluster 229-66 promotes longevity through interaction with SKN-1 and DAF-16 in C. elegans"

Parsons, Christine, Lee, Jonathan D, Slack, Frank J., Smith-Vikos, Thalyana, Matai, Latika, Shlomchik, Mariel, Saxena, Tanvi

[International Worm Meeting, 2021]

Recently, several microRNAs have emerged as potential contributors towards pathways that regulate aging. We have previously shown that miR-228 is required for dietary restriction mediated longevity through interaction with PHA-4 and SKN-1 transcription factors. mir-229,64,65,66 is a cluster of microRNAs that are in the same family as mir-228 and share the same human homologs. They are present immediately adjacent to each other on the same chromosome and are thought to share the same promoter and identical seed sequences. Interestingly, we found that in contrast to the anti-longevity role of miR-228, miR-229-66 cluster is required for C. elegans lifespan and longevity observed in mir-228 mutants. Dietary restriction and reduced insulin signaling up-regulates the expression of miR-229-66 cluster which in turn is critical for the complete extension of lifespan observed under these longevity perturbations. We also show that expression of transcription factors SKN-1 and DAF-16, which are known for their vital role in DR and low IIS mediated longevity, is positively regulated by miR-229-66 cluster. Conversely, these TFs also positively regulate expression of this miRNA cluster, indicating a positive feedback loop essential for longevity. Infact, we observed that miR-229-66 is a requisite for lifespan extension conferred by SKN-1 over expression. Given the conservation of these miRNAs and TFs across species, these interactions are likely to be conserved for longevity in more complex organisms as well.