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We focus on characterizing NHR-49, which regulates fat metabolism, and lifespan (Marc van Gilst and Keith R. Yamamoto, unpublished).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.

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.

Yuriy Shostak et al. (2001) International C. elegans Meeting "From Binding Sites to Target"

Yuriy Shostak, Keith R Yamamoto

[International C. elegans Meeting, 2001]

The intracellular receptor daf-12 regulates dauer larva formation, developmental age, and affects C. elegans lifespan. Daf-12 alleles with distinct protein sequence alterations partially uncouple its phenotypic effects. We would like to understand the molecular mechanisms of Daf-12 action, including the basis for differential phenotypic outcomes of specific daf-12 mutants. We developed an In Vitro Genomic Selection method that yielded a series of specific C. elegans genomic DNA fragments containing Daf-12 binding sites. We identified DNA sequences within these fragments that were required for specific Daf-12 binding in vitro . Daf-12 selectively activated transcription of the reporter gene in yeast from these C. elegans genomic fragments. The specific Daf-12 binding sites within these fragments were necessary and sufficient for Daf-12-dependent transcriptional activation in Saccharomyces cerevisiae . We inserted some of these fragments into C. elegans reporter plasmids and showed tissue-specific activity of the reporter gene that depended on Daf-12 binding site sequences within the fragments. Hence, C. elegans genomic DNA fragments bound specifically by Daf-12 in vitro display Daf-12 response element activity in vivo in both yeast and C. elegans . In principle, In Vitro Genomic Selection should also enable the identification of potential target genes. We are currently investigating whether Daf-12 binding site-containing C. elegans genomic fragments are linked to daf-12 -regulated target genes. We are furthermore investigating tissue specificity and activities from the C. elegans reporter gene with Daf-12 binding sites in various distinct daf-12 mutant strains. During the course of our studies, we found that a C-terminal truncated Daf-12 derivative was a much stronger transcriptional activator than full length Daf-12 in the yeast reporter system. The transcriptional activity of this truncated Daf-12, which lacks its putative ligand-binding domain, implies that a small molecule ligand may control Daf-12 activity in C. elegans .

Jen-Chywan Wang et al. (2001) International C. elegans Meeting "Isolation of Genes involved in the Regulation of Sterol Metabolism"

Jen-Chywan Wang, Keith R Yamamoto

[International C. elegans Meeting, 2001]

Steroids are required for normal growth and reproduction in C. elegans. In order to investigate the mechanisms that underly the steroid function in worm physiology, we tested the effects of three mammalian steroid biosynthesis inhibitors on worm growth and reproduction. Aminoglutethimide (AMG), an inhibitor for cytochrome p450 side-chain cleavage enzyme (cyp-11) and aromatase (cyp-19), used to treat breast cancer, has no significant effect on worm growth and reproduction. Nor did metyrapone, a cyp11b-1 inhibitor used to treat Cushing's disease. In contrast, adding ketoconazole, an anti-prostate cancer and anti-fungal drug that inhibits several steps of steroid biosynthesis in mammals and cytochrome P450-dependent 14 alpha-demethylase in fungii, to culture plates significantly inhibits growth and reproduction. Worms treated with low conc. (100mM) of ketoconazole grow slightly slower than worms that were grown in normal plates. The average brood size of ketconazole-treated worms is significantly smaller than worms grown in normal condition. These phenotypes are similar to phenotypes found when worms grown in cholesterol-deprived media. When worms were treated with high conc. (250 mM) of ketoconazole, a severe growth defect was found. Some worms arrested at early larval stage, and worms that survived remained at L1 or L2 stage for at least 7 days. Based on this phenotype, we have set up a screen to isolate the suppressors for the ketoconazole effects. From a pilot screen, we have isolated several homozygous mutants that can suppress the ketoconazole effect. We are currently conducting a large scale screen and will report our results in the meeting.

Jen-Chywan Wang et al. (2001) International C. elegans Meeting "Characterization of CeTRAP240, a C. elegans Homolog of a Component in Multi-protein Transcriptional coactivator Complexes"

Jen-Chywan Wang, Keith R Yamamoto

[International C. elegans Meeting, 2001]

Recent studies have identified several mammalian multi-protein complexes, such as TRAP, DRIP and ARC, that function as coregulators for the transcriptional control of protein-encoding genes by RNA polymerase II. It is well established that these complexes can enhance the activator-stimulated gene transcription in vitro. However, the functions, including the physiological roles, of individual component in these complexes are mostly unknown. We are employing C. elegans as a model system to dissect the roles of CeTRAP240, a worm homolog of a component of TRAP/DRIP/ARC complexes, in the worm development. Elimination or reduction of CeTRAP240 expression in worms by RNA-mediated interference results in a high degree of embryonic arrest. Interestingly, although CeTRAP240 is ubiquitously expressed in embryos, RNAi of CeTRAP240 shows the specific reduction of HLH-1 (muscle cell differentiation marker) expression, but not PHA-4 (digestive tract marker), SCM (seam cell marker), UNC-47 (GABA neuron marker) or JAM-1 (epithelial cell marker). Thus, CeTRAP240 may play a specific role in muscle differentiation. In contrast to CeTRAP240, RNA-mediated interference of another worm homolog of a component of TRAP/DRIP/ARC complexes, CeTRAP230 (sop-1), only causes a small degree of embryonic arrest. However, many adult animals develop burst vulva and/or dumpy phenotypes, which are often associated with defects in hypodermal development. The mutation in sop-1 gene is able to suppress the pal-1(e2091) mutant male tail phenotype. The pal-1(e2091) mutants specifically lose pal-1 expression in the postembryonic neuroblast cell V6, which results in failure of the generation of V6 rays in adult male tail. We found that RNAi of CeTRAP240 also can suppress the pal-1(e2091) phenotype. Thus, CeTRAP240 may function in the same pathway with sop-1 to regulate pal-1 gene expression. Future experiments will take advantage of the C. elegans genetic system for developing screens designed to identify genes that specifically interact with these coregulators.

Marc R Van Gilst et al. (2001) International C. elegans Meeting "Functional Analysis of C. elegans Nuclear Hormone Receptors"

Keith R Yamamoto, Marc R Van Gilst

[International C. elegans Meeting, 2001]

Marc R Van Gilst et al. (2002) West Coast Worm Meeting "Functional Evolution of Diverged Nuclear Hormone Receptors in C. elegans"

Keith R Yamamoto, Marc R Van Gilst

[West Coast Worm Meeting, 2002]

A major focus of our work is to understand the evolution of the NHR expansion and diversification in nematodes. Our goals are to retrace the ancestral origin of these diverged NHRs, to pinpoint the genomic events that led to the expansion, and to understand the evolution of their biological function and structural mechanisms. In order to carry out these studies, we have developed a classification scheme defined by the degree of divergence of the P-box element from the "conserved" worm receptors. The P-box is the fundamental structural unit of NHR DNA binding, and is the most conserved structural element across the NHR family. We are addressing NHR evolution both physiologically and structurally. Here I will present our physiological and functional investigations of the most homologous set of the divergent NHRs. RT PCR of a mixed-stage library indicates that all of the NHRs we have investigated in this work are expressed at comparable levels. Simple RNAi studies reveal no distinct physiological role for the majority of these NHRs, however some receptors, including NHR-31 and NHR-49 do have interesting biological functions. We are currently characterizing NHR-31 and NHR-49 activity and working to reveal the physiological roles of the other NHRs that have no apparent RNAi phenotype. In order to characterize worm NHR functional evolution, we have focused on the DNA binding mechanism, as the NHR-DNA binding interaction is the most well conserved of C. elegans NHR properties. Our data reveal that some of the "divergent" NHRs employ similar mechanisms to that of their "conserved" NHR relatives, while others do not, suggesting that they either have evolved new DNA reponse element specificity, employ a different type of DNA binding mechanism, or do not bind DNA at all. Interestingly, the level of functional conservation does not necessarily correlate with the level of sequence identity. Further investigation of these NHRs requires the identification of the novel DNA binding sites for these functionally "diverged" worm NHRs.

Marc R Van Gilst et al. (2002) West Coast Worm Meeting "Functional Characterization of the Conserved Set of C. elegans Nuclear Hormone Receptors"

Marc R Van Gilst, Keith R Yamamoto

[West Coast Worm Meeting, 2002]

Nuclear Hormone Receptors comprise a large class of ligand-regulated transcription factors found in metazoans. NHRs are important regulatory factors that "sense" their environment through interaction with small lipophilic molecules and translate these signals into precise transcriptional regulation of target genes. The activity of an NHR is also modulated by the type of DNA response element to which it binds and by the composition of its protein cofactors in the cell. This complex regulatory system enables NHR regulation to be important for numerous biological processes including development, sexual differentiation, lipid and fatty-acid metabolism and xenobiotic resistance.

Neal Freedman et al. (2002) West Coast Worm Meeting "Identification of Genes Involved in Sterol Metabolism in C. elegans"

Neal Freedman, Jen-Chywan Wang, Keith R Yamamoto

[West Coast Worm Meeting, 2002]

Cholesterol is required for normal growth and reproduction in C. elegans. To investigate whether sterols might regulate worm physiology, we tested the effects of mammalian sterol biosynthesis inhibitors on worm growth, reproduction, and lifespan. Among those studied, a class of imidazole based inhibitors: ketoconazole, miconazole, sulconazole, and clotrimazole, each of which inhibits a broad yet unique spectrum of P450 enzymes, significantly affected C. elegans growth and reproduction. At low concentrations (100 M ketoconazole), the inhibitors reduced growth rates and brood size, similar phenotypes to those observed in cholesterol-deprived media. At intermediate concentrations (200 M ketoconazole), worms displayed a severe growth defect, remaining at L1 or L2 for at least seven days. At high concentrations (300 M ketoconazole), worms died at L1 or L2. In addition, the imidazole based inhibitors significantly reduced adult lifespan. Based on these phenotypes, we have screened for mutants that are resistant to the imidazole compounds. We have isolated several such mutants and are currently mapping and characterizing them.

Stefan Taubert et al. (2005) International Worm Meeting "Characterization of a novel NHR-cofactor interaction pair regulating fatty acid desaturation, starvation response, and normal longevity in C. elegans"

Marc Van Gilst, Stefan Taubert, Keith R Yamamoto

[International Worm Meeting, 2005]

Nuclear Hormone Receptors (NHRs) are ligand-regulated transcription factors (TFs) that govern processes such as organism development, sexual differentiation, and energy homeostasis in metazoans. The genome of C. elegans encodes 284 putative NHRs, the function of most of which is elusive. One of these, NHR-49, regulates transcription of genes involved in fat metabolism. Interestingly, NHR-49 is required for gene expression during periods of feeding, e.g. the fatty acid (FA) desaturase fat-7, as well as for upregulation of certain metabolic genes during periods of starvation (e.g. acs-2). Thus, NHR-49 function strikingly resembles the role of the mammalian NHR Peroxisome Proliferator-Activated-Receptor . To investigate molecular mechanisms underlying NHR-49 regulation of fat homeostasis we aimed to isolate transcriptional cofactors of NHR-49. We performed a yeast-two-hybrid-screen using the NHR-49 ligand-binding-domain as bait. One positive clone expresses a cDNA orthologous to a subunit of yeast Mediator. This multi-subunit-protein-complex acts as a transcriptional coactivator for many distinct TFs, including several human NHRs. However, the Mediator subunit we identified has not previously been reported to interact with NHRs in any organism, therefore representing a novel NHR-cofactor interaction pair. To address the role of this potential NHR-49 cofactor in vivo we utilized feeding RNAi. In N2 L4 worms, RNAi specific to this Mediator subunit prevented starvation-induced mRNA upregulation of NHR-49 targets (determined by qRT-PCR). Similarly, starvation-independent expression of all three FA 9-desaturases (fat-5, -6 and 7) was reduced in worms feeding on Mediator-subunit RNAi. Interestingly, fat-6 is regulated in an NHR-49 independent fashion, suggesting that another TF controls its expression through the same Mediator-subunit. The coordinate regulation of the 9-desaturases suggests that this Mediator-subunit governs FA-desaturation in C. elegans. To verify this hypothesis we analyzed the FA-composition of N2 worms by GC-MS. We found that Mediator-subunit RNAi significantly reduced levels of C18 and C20 mono-or poly-unsaturated FAs. Curiously, prolonged Mediator-subunit RNAi treatment caused larval developmental defects, adult sterility, uncoordinated locomotory behavior, and premature death. Thus, we propose that proper regulation of FA-desaturation by distinct TF-Mediator complexes is important for normal nematode viability. Interestingly, this Mediator subunit is conserved in mammals, suggesting that a similar regulatory loop may be important in higher metazoans.