van Gilst MR et al. (2000) West Coast Worm Meeting "Molecular Mechanisms of Daf-12"

Yamamoto KR, van Gilst MR, Shostak Y, Antebi A

[West Coast Worm Meeting, 2000]

Dauer larva formation in Caenorhabditis elegans is regulated by the intracellular receptor daf-12, which functions at the convergence of TGF-b, cGMP, and insulin-like signaling pathways. daf-12 has also been implicated in the regulation of C. elegans lifespan and developmental age. Alleles of daf-12 with distinct protein sequence alteration spartially uncouple its phenotypic effects. As a step toward understanding the molecular biology of Daf-12 function, we identified specific DNA sites in the C. elegans genome bound by the protein in vitro. We developed an in vitro selection and amplification method that, using immobilized recombinant daf-12 DNA binding domain, yielded a series of specific C. elegans genomic DNA fragments. We inserted some of these fragments into yeast reporter plasmids and showed that Daf-12 selectively activated transcription of the reporter genes in Saccharomyces cerevisiae. Hence, C. elegans DNA fragments bound specifically by Daf-12 in vitro display Daf-12 response element activity in vivo. Intracellular receptors are multidomain proteins with characteristic DNA binding, putative ligand binding, and potentially ligand-controlled transcriptional regulatory domains. We used the heterologous yeast expression system to investigate contributions of different Daf-12 regions to transcriptional regulation. We found thatDaf-12 derivatives with truncated ligand binding domains are potent transcriptional activators, and mapped an activation domain to the "hinge region" just downstream of the DNA binding domain. In future work, we shall determine whether the identified response elements are linked to Daf-12regulated target genes in C. elegans. Similarly, we shall examine the roles of the activation domain in Daf-12 biology and molecular function in the animal.

Van Gilst, M.R. et al. (2019) International Worm Meeting "Reduced protein translation leads to hypoxia resistance in germline ablated animals."

Itani, O, Van Gilst, M.R., Crowder, M.C.

[International Worm Meeting, 2019]

Cardiovascular disease is one of the leading killers in advanced societies. Cardiovascular disease can lead to ischemic events, such as stroke or heart attack, which disrupt the supply of oxygen to critical tissues. Therefore, understanding the mechanisms by which oxygen deprivation (hypoxia) damages tissues is of considerable interest to modern medicine. Previous studies in C. elegans have found that various sterile mutants of C. elegans are highly resistant to hypoxic injury, therefore we set out to understand the mechanism by which inhibition of germline functions leads to hypoxia resistance. First, we found that germline ablated animals (glp-1(ts)or glp-4(ts)mutants) are not resistant to hypoxia as larvae (up to L4), but quickly transition into highly resistant as they develop into adults. Furthermore, germline ablated animals become even more resistant to hypoxia as they get older (up to day 5-7 of adulthood). Interestingly, although daf-16is not required for the hypoxia resistance of young adults, daf-16isrequired for the aging dependent increase in hypoxia resistance that occurs in germline mutants. We next used RNAseq to identify changes in gene expression that occur between the hypoxia sensitiveL4 stage and the hypoxia resistantadult stage of glp-1mutants. This analysis revealed two predominant sets of genes impacted by the L4 to adult transition, genes involved in fatty acid metabolism were induced in young adults, and genes involved in translation were repressed in young adults. Consistent for a role for reduced translation in the resistance of glp-1 or glp-4mutants, we found that suppressors of reduced translation (ncl-1and lrp-1) are able to suppress the hypoxia resistance of germline ablated animals. Finally, we found that ablation of the germline also prevented the hypoxia dependent formation of mitochondrial protein aggregates, further implicating translation as a key factor in hypoxic injury.

van Gilst MR et al. (2000) West Coast Worm Meeting "Characterization of transcriptional regulation by a class of monomeric nuclear receptors found in C. elegans"

Yamamoto KR, van Gilst MR

[West Coast Worm Meeting, 2000]

Nuclear hormone receptors (NHRs) comprise a large family of metazoan transcription factors that participate in numerous developmental and metabolic processes. NHRs commonly target their transcriptional regulation to particular genes through interaction with DNA sequences called hormone response elements (HREs) and the nature of the transcriptional regulation carried out by a NHR at these genes is often determined by interaction with a small hydrophobic ligand. C. elegans provides unique advantages over other animal models for studying the physiological significance of several aspects of NHR function (i). Targeting of NHRs to promoters by HREs (ii) Characterization of important regulatory domains of NHRs (iii) Interaction of NHRs with protein cofactors and (iv) Interaction of NHRs with potential ligands. We have focused our studies on the C. elegans homologs of a class of NHRs that can activate transcription from AGGTCA DNA half-sites, a common binding motif for a mammalian sub-family of monomeric nuclear receptors that includes ROR, TLX, ERR and SF-1. To this end, we have started our investigations by utilizing the C. elegans receptor CHR3 (nhr-23). CHR3 is closely related to the mammalian receptor ROR, with nearly 100% conservation in the DNA binding domain, and would be expected to fall into the class of monomeric NHRs that we wish to study. Using yeast and worm reporter assays, we have characterized the transcriptional regulatory properties of CHR3. We found that CHR3 can activate transcription from a single AGGTCA half-site and activates synergistically from multimerized half-sites. Transcriptional activation occurs in yeast in the absence of any exogenous ligand. Similar to mammalian receptors, both the N and C terminal domains of CHR3 contain activation functions. Furthermore, CHR3 requires ATP-dependent chromatin remodeling complexes for activation and can interact with a mammalian coactivator GRIP through a conserved structural mechanism. We are currently expanding these systems to find and study other C. elegans NHRs and to set up screens for the identification of potential protein cofactors and small-molecule ligands.

van Gilst MR et al. (2005) PLoS Biol "Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans."

Jolly A, Yamamoto KR, Hadjivassiliou H, van Gilst MR

[PLoS Biol, 2005]

Mammalian nuclear hormone receptors (NHRs), such as liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs), precisely control energy metabolism. Consequently, these receptors are important targets for the treatment of metabolic diseases, including diabetes and obesity. A thorough understanding of NHR fat regulatory networks has been limited, however, by a lack of genetically tractable experimental systems. Here we show that deletion of the Caenorhabditis elegans NHR gene nhr-49 yielded worms with elevated fat content and shortened life span. Employing a quantitative RT-PCR screen, we found that nhr-49 influenced the expression of 13 genes involved in energy metabolism. Indeed, nhr-49 served as a key regulator of fat usage, modulating pathways that control the consumption of fat and maintain a normal balance of fatty acid saturation. We found that the two phenotypes of the nhr-49 knockout were linked to distinct pathways and were separable: The high-fat phenotype was due to reduced expression of enzymes in fatty acid beta-oxidation, and the shortened adult life span resulted from impaired expression of a stearoyl-CoA desaturase. Despite its sequence relationship with the mammalian hepatocyte nuclear factor 4 receptor, the biological activities of nhr-49 were most similar to those of the mammalian PPARs, implying an evolutionarily conserved role for NHRs in modulating fat consumption and composition. Our findings in C. elegans provide novel insights into how NHR regulatory networks are coordinated to govern fat

Van Gilst MR et al. (2005) Proc Natl Acad Sci U S A "A Caenorhabditis elegans nutrient response system partially dependent on nuclear receptor NHR-49."

Hadjivassiliou H, van Gilst MR, Yamamoto KR

[Proc Natl Acad Sci U S A, 2005]

Appropriate response to nutritional stress is critical for animal survival and metabolic health. To better understand regulatory networks that sense and respond to nutritional availability, we developed a quantitative RT-PCR strategy to monitor changes in metabolic gene expression resulting from short-term food deprivation (fasting) in Caenorhabditis elegans. Examining 97 fat and glucose metabolism genes in fed and fasted animals, we identified 18 genes significantly influenced by food withdrawal in all developmental stages. Fasting response genes fell into multiple kinetic classes, with some genes showing significant activation or repression just 1 h after food was removed. As expected, fasting stimulated the expression of genes involved in mobilizing fats for energy production, including mitochondrial beta-oxidation genes. Surprisingly, however, we found that other mitochondrial beta-oxidation genes were repressed by food deprivation. Fasting also affected genes involved in mono- and polyunsaturated fatty acid synthesis: four desaturases were induced, and one stearoyl-CoA desaturase (SCD) was strongly repressed. Accordingly, fasted animals displayed considerable changes in fatty acid composition. Finally, nuclear receptor nhr-49 played a key role in nutritional response, enabling induction of beta-oxidation genes upon food deprivation and facilitating activation of SCD in fed animals. Our characterization of a fasting response system and our finding that nhr-49 regulates a sector within this system provide insight into the mechanisms by which animals respond to nutritional signals.

Sharma S et al. (2019) Int Microbiol "Lipidomic insights to understand membrane dynamics in response to vanillin in Mycobacterium smegmatis."

Sharma S, Fatima Z, Hameed S

[Int Microbiol, 2019]

Considering the emergence of multidrug resistance (MDR) in prevalent human pathogen, Mycobacterium tuberculosis (MTB), there is parallel spurt in development of novel strategies aimed to disrupt MDR. The cell envelope of MTB comprises a wealth of lipid moieties contributing towards long-term survival of pathogen that could be exploited as efficient antitubercular target owing to advancements made in mass spectrometry-based lipidomics technology. This study aimed to utilize the lipidomics approach to unveil several lipid associated changes in response to natural antimycobacterial compound vanillin (Van) in Mycobacterium smegmatis, a surrogate for MTB. Lipidomic analyses revealed that that Van alters the composition of fatty acid (FA), glycerolipid (GL), glycerophospholipid (GP), and saccharolipids (SL). Furthermore, Van leads to potentiation of ampicillin and displayed additive effect. The differential expressions of various lipid biosynthetic pathway genes by RT-PCR corroborated with the lipidomics data. Lastly, we demonstrated enhanced survival of Mycobacterium-infected Caenorhabditis elegans model in presence of Van. Thus, lipidomics approach provided detailed insight into mechanisms of membrane disruption by Van in Mycobacterium smegmatis. Our work offers the basis of further understanding the regulation of lipid homeostasis in MTB so that better therapeutic targets could be identified to combat MDR.

Seidel, Hannah et al. (2011) International Worm Meeting "Questioning Adult Reproductive Diapause."

Kimble, Judith, Seidel, Hannah

[International Worm Meeting, 2011]

Upon starvation, some C. elegans L4 hermaphrodites do not 'bag' as adults but instead survive long-term, reportedly via a regulatory program termed adult reproductive diapause (ARD) (Angelo and Van Gilst, 2009). Key features of ARD include degeneration of most germ cells, embryonic arrest in utero (as evidenced by early-stage embryos visible 3-5 days after starvation), and the ability to regenerate a functional germline upon refeeding (Angelo and Van Gilst, 2009). We have confirmed most but not all of these observations. We removed food at ~2hr intervals from early- to late-stage L4 hermaphrodites, and over the next several days, we scored these animals for germline shrinkage, oocyte production, and embryo viability. After starvation of early L4s, germlines shrank and a fraction of animals survived long-term, as expected for ARD; also as expected, the depleted germlines regenerated after refeeding. However, two observations lead us to question the proposed embryonic arrest associated with ARD. First, in animals starved from early L4, oogenesis was severely delayed, with many animals producing their first oocyte only 3-5 days into adulthood. This delay in oogenesis suggests that the early-stage embryos visible 3-5 days after starvation in putative ARD animals may reflect recent fertilization events, rather than embryonic arrest. Second, the delayed oocytes made viable embryos in some cases but dead embryos in others. This increase in embryo lethality provides an alternate explanation for the ability of some animals to avoid the bagging fate and survive long-term. Furthermore, we found that germline shrinkage was not unique to animals in putative ARD: hermaphrodites starved from late L4 uniformly bagged as adults, yet prior to bagging, these animals nevertheless possessed shrunken germlines much like the germlines reported for ARD. From these observations, we propose that all L4 hermaphrodites respond to starvation equivalently: the oogenic germline degenerates and shrinks; the animal produces as many embryos as possible, given the nutrients available; and the viability of these embryos determines long-term survival. We propose that when starvation begins in late L4, embryos are typically viable, and the animal dies via bagging; when starvation begins earlier in L4, embryos are often inviable, and this inviability allows some animals to survive long-term. We are now performing additional experiments to test this possibility. [Angelo G, Van Gilst M (2009) Starvation protects germline stem cells and extends reproductive longevity in C. elegans. Science 326: 954-958.].

Braeckman BP et al. (2002) Aging Cell "Rebuttal to Van Voorhies: 'the influence of metabolic rate on longevity in the nematode Caenorhabditis elegans'."

Braeckman BP, Houthoofd K, Vanfleteren JR

[Aging Cell, 2002]

The papers by Van Voorhies in Free Radical Biology & Medicine (33, 587-596, 2002) and in this journal claim that the major longevity-extending mutations in C. elegans essentially act by reducing metabolic rate as predicted by the rate-of-living theory, and do not alter any metabolically independent mechanism specific to aging. In contrast, we found no evidence of a reduction in metabolic rate in these mutants using different experimental approaches. Now, Van Voorhies challenges the accuracy of our experimental results.

Halic M et al. (2009) Mol Cell "22G-RNAs in transposon silencing and centromere function."

Moazed D, Halic M

[Mol Cell, 2009]

Three recent papers (Gu et al., 2009; Claycomb et al., 2009; van Wolfswinkel et al., 2009) provide evidence that links a new class of small RNAs and Argonaute-associated complexes to centromere function and genome surveillance.

Kenny, Isabel et al. (2015) International Worm Meeting "Adult Reproductive Diapause and Embryo Arrest in C. elegans."

Gerisch, Bergit, Kenny, Isabel, Heestand, Bree, Antebi, Adam, Ahmed, Shawn

[International Worm Meeting, 2015]

C. elegans have several strategies for surviving starvation conditions. If starved during the last stage of larval development, C. elegans can enter adult reproductive diapause (ARD), resulting in shrinkage of the germline cells, morphological changes to the gonad arms, and reversible sterility. Separate investigations of the ARD phenotype have resulted in different conclusions about the fate of embryos during ARD. Angelo and Van Gilst characterized adult worms in ARD to have shrunken germlines as well as 1-2 arrested embryos (1). Kimble, whilst getting adult worms to successfully arrest reproduction, did not observe arrested embryos in the ARD animals (2).Our initial focus for investigating ARD was to establish a protocol for reliably inducing worms in to the arrested state. The key aspect of this protocol is the timing at which worms are introduced on to starvation plates. Once the protocol was developed, we obtained evidence that all wild-type adults that enter ARD contain embryos. We demonstrate that these embryos are not dead but are instead in a state of developmental arrest. Whilst testing different genotypes involved in stress response and insulin signaling, we found daf-16 mutant worms did not show the embryo arrest characteristic of wild-type ARD animals. These findings that reduced insulin signaling promotes arrest of embryos in ARD. We speculate that the discordant results previously reported regarding the presence of embryos in adults that are in starvation-induced ARD (1,2) may be explained by variations in experimental protocol.1) Angelo, G. and M. Van Gilst. 2009. Starvation Protects Germline Stem Cells and Extends Reproductive Longevity in C. elegans. Science 326: 954-958.2) Kimble, J. and H. Seidel. 2011. The Oogenic Germline Starvation Response in C. elegans. PLoS ONE 6(12): e28074.