Yamamoto KR et al. (1995) International C. elegans Meeting "Properties of an oxygen-sensitive mutant mev-3"

Yamamoto KR, Honda S, Ishii N, Suzuki K

[International C. elegans Meeting, 1995]

A mutant of mev-3, spontaneously isolated on the basis of its resistance to methyl viologen, was hypersensitive to oxygen. Oxygen retarded development and reduced fecundity in a concentration-dependent fashion in mev-3 but not in wild type. In addition, the life spans of mev-3 was progressively shortened when animals were incubated under 70% and more oxygen.

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.

Fu, Donald et al. (2011) International Worm Meeting "In situ study of oxidative stress using a new optogenetic tool, KillerRed."

Fu, Donald, Kotera, Ippei, Suzuki, Hiroshi, Su, Po-An

[International Worm Meeting, 2011]

Parkinson's Disease (PD), characterized by the progressive degeneration of dopaminergic (DA) neurons, leads to locomotive deficits. Genetic studies suggests a role for oxidative stress in impairing activity and viability of DA neurons. Conventional methods induced stress in C. elegans by administering mitochondrial respiratory chain inhibitors (such as rotenone and paraquat) to the growth medium. However, these drugs diffuse and affect all cells in animals, and the effects have mostly been scored by the animals' death. It is difficult to assess how drugs affect only DA neurons and locomotive deficits. To overcome these limitations, we adopt a new optogenetic tool, KillerRed: a red fluorescent protein engineered to generate oxygen radicals on photoactivation. KR can be genetically targeted to cells by cell-specific promoters, and to specific subcellular compartments by site-localization motifs. We can also control irradiation time and intensity to manipulate timing and duration of stress. Studies have shown its potency in inducing cell death in mammalian cultured cells and Zebrafish. To test this tool in C. elegans, we investigated its effects on the mechanosensory system by expressing KR solely in touch neurons using mec-4p and scoring morphology and behavior (forward escape mediated by PLM neurons). We established a method to confirm activation: after photoactivation, KR fluorescence diminished to <1% of its initial levels, with a decay half-life of ~5.43s. Irradiation of PLM neurons for 10mins lead to profound changes in cell shape or their disappearance within 2h, without affecting neighboring KR(-) cells. Behavioral analysis revealed 73% reduction in touch sensitivity. We are now applying this tool to study oxidative stress in DA neurons; we targeted KR to the mitochondria using dat-1p and cytochrome-c oxidase motif. To quantitatively examine locomotion dependence on DA neurons, we employ a computer behavior analysis system: Our new worm tracker allows comparison of stressed worms to wild-type, to DA-deficient cat-2 mutants, and directly address DA-activity suppression via optogenetic tools (dat-1p::NpHR). We are also expressing KR in other subcellular compartments and crossing them with mutants of PD genes (such as pdr-1, djr-1.1/2, pink-1) to examine how they protect against stress in DA neurons. Thus, we propose that KR is a useful tool to study oxidative stress and for in vivo ablation of neurons in C. elegans.

Pranali P Pathare et al. (2007) International Worm Meeting "Coordinate regulation of sphingolipid metabolism by a novel nuclear receptor partnership."

Marc Van Gilst, Pranali P Pathare

[International Worm Meeting, 2007]

The C. elegans NHR-49 is a lipid sensing nuclear receptor that has been shown to orchestrate multiple aspects of fat homeostasis, including mitochondrial <font face=symbol>b</font>-oxidation and fatty acid desaturation. To more comprehensively define the targets of NHR-49, we exploited global gene expression profiling. These experiments revealed a previously uncharacterized role for NHR-49 in the regulation of genes involved in membrane lipid metabolism, particularly glycosphingolipid processing. Indeed, deletion of nhr-49 resulted in dramatically increased expression of several sphingolipid (SL) metabolism genes, as well as other factors likely to influence phospholipid processing. Sphingolipids are important structural components of cell membranes as well as potent signaling molecules implicated in apoptosis, cell division and differentiation. However, little is known about the transcription factors that control SL gene expression. Consistent with NHR-49s impact on SL metabolism genes, biochemical analysis revealed a significantly altered sphingolipid profile in nhr-49 knockout mutants. Therefore, our findings reveal a new role for NHR-49 in the control of sphingolipid composition. Two hybrid studies have found that NHR-49 can bind to many different C. elegans NHRs1. Since nuclear receptors often regulate gene expression by forming heterodimers, we examined these potential binding partners to determine if they impacted NHR-49 gene regulation. Notably, we found that knockout of nhr-66 affected only a subset of NHR-49 targets. In particular, nhr-66 was exclusively required for the repression of NHR-49s sphingolipid and phospholipid metabolism targets. In contrast, deletion of nhr-66 did not impact NHR-49 activation of <font face=symbol>b</font>-oxidation and fatty acid desaturation genes. Thus, our findings support a model whereby NHR-49 and NHR-66 heterodimerize to mediate the repression of genes involved in membrane lipid composition. Therefore, NHR-49 independently regulates multiple lipid metabolism pathways, and the distinct lipid metabolism networks targeted by NHR-49 are determined by dimerization with different NHR partners. (1)Taubert S, Van Gilst M, Hansen M, and Yamamoto KR. (2006) Genes Dev 20, 1137-1149.

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.

Noriko SASAKAWA et al. (2010) East Asia Worm Meeting "RNAi screening of genes required for normal production of seam cells and DTCs"

Noriko SASAKAWA, Tomomi TAKANO, Hitoshi Sawa, Mayumi Shibuya

[East Asia Worm Meeting, 2010]

Asymmetric cell division is an important mechanism to produce cellular diversity during development. In C. elegans, the Wnt signaling pathway regulates asymmetries of most cell division. For example, hypodermal stem cells called seam cells undergo self-renewing asymmetric divisions in each larval stage. The polarities of all seam cells are regulated redundantly by multiple Wnt genes. In contrast, divisions of the somatic gonadal precursor cells (SGPs) which generate distal tip cells (DTCs) are Wnt-independent (Abstract by Yamamoto et al.). However, how Wnts polarize seam cells and how the polarities of SGPs are determined are not understood. To identify genes that are required for Wnt-dependent or Wnt-independent asymmetric cell divisions, we screened for genes that affect production of seam cells or DTCs by feeding RNAi using an RNAi library that includes 86% of all the genes. We have identified 58 and 39 genes whose inhibition altered the numbers of seam cells and DTCs, respectively. We further observed lineages of seam cells at the L2 stage and identified some genes that caused reversal or symmetric divisions by RNAi, suggesting that they are involved in the asymmetric divisions of seam cells. We will analyze asymmetric localization of POP-1/TCF after inhibition of these genes by RNAi.

Stefan Taubert et al. (2004) West Coast Worm Meeting "Biochemical Characterization of the C. elegans Nuclear Hormone Receptor NHR-49"

Keith R Yamamoto, Stefan Taubert, Marc Van Gilst

[West Coast Worm Meeting, 2004]

Nuclear Hormone receptors (NHRs) are a family of ligand-regulated transcription factors involved in diverse metazoan functions, such as sexual differentiation, organ development, and regulation of metabolism. The C. elegans genome contains over 270 NHR genes, approximately five times as many as the human genome. For most of these no function has been described yet. We focus on characterizing NHR-49, which regulates fat metabolism, and lifespan (Marc van Gilst and Keith R. Yamamoto, unpublished). Interestingly, this mirrors the functions of human Peroxisome-proliferator-activated-receptor a (PPARalpha), while the sequence of NHR-49 suggests similarity to another human receptor, Hepatocye Nuclear Receptor 4alpha (HNF4alpha). Since HNF4alpha dimerizes via its ligand-binding-domain (LBD), we utilized yeast two-hybrid based technology to assess whether the LBDs of NHR-49 and 12 related HNF4alpha-like NHRs, can also homodimerize. While several of these LBDs readily dimerized (including NHR-49), some others did not (such as DAF-12). Since the homodimerization of NHR-49's LBD is predicted to occur via two conserved salt-bridges (E5/K55 and E50/R93), we mutated the E50 and K55 residues to alanines. We will present the effect of these mutations on LBD homodimerization and activation of a transcriptional reporter in yeast. In an attempt to identify ligands of NHR-49 we treated worms with clofibrate, a synthetic PPARa ligand used to treat hyperlipidemia. This treatment caused a strong reduction of worm body fat levels, and was accompanied by lethality at higher concentrations. However, similar effects were observed in a strain harboring an NHR-49 deletion, suggesting that the effects of clofibrate are independent of NHR-49. We are currently characterizing changes in the transcriptional program in response to clofibrate treatment.

Axel Bethke et al. (2002) European Worm Meeting "Identifying transcriptional targets of the DAF-12 nuclear hormone receptor"

Axel Bethke, Adam Antebi

[European Worm Meeting, 2002]

The daf-12 gene encodes a nuclear hormone receptor that integrates inputs from insulin/IGF, TGF- and cGMP signalling pathways and influences dauer formation, the heterochronic circuit and life span. Therefore, the set of daf-12 target genes should be highly interesting. Shostak and Yamamoto (IWM, 2001) defined several putative DAF-12 DNA target sequences in the lit-1 promotor by in vitro and in vivo experiments. We are searching for additional physiological candidate genes in identified signalling pathways. Surprisingly, one is daf-9, a cytochrome P450 related to vertebrate steroidogenic hydroxylases. DAF-9 is thought to metabolize a DAF-12 hormone and acts by genetic epistasis experiments upstream of DAF-12. In contrast to this, we find that hypodermal daf-9::gfp expression does not occur in daf-12 null mutants, suggesting feedback regulation by DAF-12 (See abstract by Gerisch et al). Another candidate target is let-7, a heterochronic gene coding for a small temporal RNA (stRNA) that regulates the larval to adult switch. Slack and Johnson (IWM, 2001) showed that a let-7::gfp construct is temporally upregulated by the L4 stage in the same cell type as daf-12::gfp. In addition, daf-12 alleles with delayed heterochronic phenotypes, such as rh61, also result in delayed or inhibited let-7 expression (personal com. Slack et al). To investigate whether these candidates are directly regulated by DAF-12 in vivo, we are beginning chromatin immunoprecipitation (X-ChIP) experiments to define DNA regions to which the nuclear receptor binds. We will also dovetail these experiments with gel mobility shift assays using protein from nuclear extracts to narrow down the region of receptor binding and to identify corresponding response elements.

S Taubert et al. (2004) European Worm Meeting "CHARACTERIZATION OF THE C. ELEGANS NUCLEAR HORMONE RECEPTOR NHR-49"

S Taubert, K Yamamoto, M Van Gilst

[European Worm Meeting, 2004]

Nuclear Hormone receptors (NHRs) are a family of ligand-regulated transcription factors involved in diverse metazoan functions, such as sexual differentiation, organ development, and regulation of metabolism. The C. elegans genome contains over 270 NHR genes, approximately five times as many as the human genome. For most of these no function has been described yet. We focus on characterizing NHR-49, which regulates fat metabolism, and lifespan (Marc van Gilst and Keith R. Yamamoto, unpublished). Interestingly, this mirrors the functions of human Peroxisome-proliferator-activated-receptor a (PPARa), while the sequence of NHR-49 suggests similarity to another human receptor, Hepatocye Nuclear Receptor 4a (HNF4a). Since HNF4a dimerizes via its ligand-binding-domain (LBD), we utilized yeast two-hybrid based technology to assess whether the LBDs of NHR-49 and 12 related HNF4a-like NHRs, can also homodimerize. While several of these LBDs readily dimerized (including NHR-49), some others did not (such as DAF-12). Since the homodimerization of NHR-49's LBD is predicted to occur via two conserved salt-bridges (E5/K55 and E50/R93), we mutated the E50 and K55 residues to alanines. We will present the effect of these mutations on LBD homodimerization and activation of a transcriptional reporter in yeast. In an attempt to identify ligands of NHR-49 we treated worms with clofibrate, a synthetic PPARa ligand used to treat hyperlipidemia. This treatment caused a strong reduction of worm body fat levels, and was accompanied by lethality at higher concentrations. However, similar effects were observed in a strain harboring an NHR-49 deletion, suggesting that the effects of clofibrate are independent of NHR-49. We are currently characterizing changes in the transcriptional program in response to clofibrate treatment.

K Oshio et al. (1999) International C. elegans Meeting "Analysis of neuronal connectivity of C. elegans using random walker"

Y Osana, K Kawamura, S Morita, Y Funabashi, K Oshio, K Oka

[International C. elegans Meeting, 1999]

From the detailed report of White et al. and Albertson et al. , we have almost complete knowledge about the synaptic connectivity of C. elegans with the type of synapse (electric junction or chemical synapse). However, the type of each chemical synapse (excitatory or inhibitory one) is not described. Conventional electrophysiological methods for C. elegans is difficult because the size of the neurons is too small to penetrate the intracellular electrode. On the other hand, computational studies of neuronal circuit are now possible by virtue of the above mentioned elaborate experimental studies of neuroanatomists. We have built a new data base of the whole neuronal circuit including pharyngeal neurons only from the article of White et al. and Albertson et al. There exist two other data bases to the knowledge of the present authors. The first data base was constructed by Achacoso and Yamamoto who also analyzed the properties of the network. Another data base was constructed from the article of White et al. by Durbin, which is available on the internet homepage. To begin understanding signal processing on the nervous system, we have investigated the neuronal connectivity by putting random walkers on certain neurons of the network. Here we ignore internal structure of neurons. Random walker is a particle which moves among neurons randomly, and can be considered to be transmitted signal. In our simulation, random walkers are put on certain sensory neurons at each time step, this corresponds to stimulation which sensory neurons accept. We removed random walkers at certain motor neurons, this means, for instance, signals are transmitted to muscles which cause normal action. As a result, we have found that the degree of relation of each neuron for input neurons can be known from the probability to find random walker, although the difference between excitatory and inhibitory of chemical synapses is not taken into account. The simulational results will be shown comparing with real function such as the touch sensitivity. E-mail: oshio@future.st.keio.ac.jp