Marta Kostrouchová

Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans

CHR3 (nhr-23, NF1F4), the homologue of Drosophila DHR3 and mammalian ROR/RZR/RevErbA nuclear hormone receptors, is important for proper epidermal development and molting in the nematode Caenorhabditis elegans. Disruption of CHR3 (nhr-23) function leads to developmental changes, including incomplete molting and a short, fat (dumpy) phenotype. Here, we studied the role of CHR3 during larval development by using expression assays and RNA-mediated interference. We show that the levels of expression of CHR3 (nhr-23) cycle during larval development and reduction of CHR3 function during each intermolt period result in defects at all subsequent molts. Assaying candidate gene expression in populations of animals treated with CHR3 (nhr-23) RNA-mediated interference has identified dpy-7 as a potential gene acting downstream of CHR3. These results define CHR3 as a critical regulator of all C. elegans molts and begin to define the molecular pathway for its function.

SKIP is an indispensable factor for Caenorhabditis elegans development

SKI-binding protein (SKIP) is a transcription cofactor present in all eukaryotes. Here we show that SKIP is a unique protein that is required for Caenorhabditis elegans viability and development. Expression of CeSKIP (skp-1) assayed by RT-PCR and by GFP fluorescence in transgenic lines starts in embryos and continues to adulthood. Loss of CeSKIP activity by RNA-mediated inhibition results in early embryonic arrest similar to that seen following inhibition of RNA polymerase II. RNA polymerase II phosphorylation appears normal early in CeSKIP RNA-mediated inhibition treated embryos although the expression of several embryonic GFP reporter genes is severely restricted or absent. Our data suggest that CeSKIP is an essential component of many RNA polymerase II transcription complexes and is indispensable for C. elegans development.

NHR-23 dependent collagen and hedgehog-related genes required for molting.

NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in Caenorhabditis elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparative expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa.

BIR-1, a Caenorhabditis elegans homologue of Survivin, regulates transcription and development

bir-1, a Caenorhabditis elegans inhibitor-of-apoptosis gene homologous to Survivin is organized in an operon with the transcription cofactor C. elegans SKIP (skp-1). Because genes arranged in operons are frequently linked functionally, we have asked whether BIR-1 also functions in transcription. bir-1 inhibition resulted in multiple developmental defects that overlapped with C. elegans SKIP loss-of-function phenotypes: retention of eggs, dumpy, movement defects, and lethality. bir-1 RNA-mediated interference decreased expression of several gfp transgenes and the endogenous genes dpy-7 and hlh-1. Immunoblot analysis revealed decreased phosphoacetylated histones in bir-1 RNA-mediated interference-treated worms. In a heterologous transfection system, BIR-1 augments thyroid hormone-regulated transcription and has an additive effect with SKIP. These results show that BIR-1 functions in the regulation of transcription and development.

Perilipin-related protein regulates lipid metabolism in C. elegans

Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.

Nuclear receptors in nematode development: Natural experiments made by a phylum.

The development of complex multicellular organisms is dependent on regulatory decisions that are necessary for the establishment of specific differentiation and metabolic cellular states. Nuclear receptors (NRs) form a large family of transcription factors that play critical roles in the regulation of development and metabolism of Metazoa. Based on their DNA binding and ligand binding domains, NRs are divided into eight NR subfamilies from which representatives of six subfamilies are present in both deuterostomes and protostomes indicating their early evolutionary origin. In some nematode species, especially in Caenorhabditis, the family of NRs expanded to a large number of genes strikingly exceeding the number of NR genes in vertebrates or insects. Nematode NRs, including the multiplied Caenorhabditis genes, show clear relation to vertebrate and insect homologues belonging to six of the eight main NR subfamilies. This review summarizes advances in research of nematode NRs and their developmental functions. Nematode NRs can reveal evolutionarily conserved mechanisms that regulate specific developmental and metabolic processes as well as new regulatory adaptations. They represent the results of a large number of natural experiments with structural and functional potential of NRs for the evolution of the phylum. The conserved and divergent character of nematode NRs adds a new dimension to our understanding of the general biology of regulation by NRs. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Proteomic analysis uncovers a metabolic phenotype in C. elegans after nhr-40 reduction of function

Caenorhabditis elegans has an unexpectedly large number (284) of genes encoding nuclear hormone receptors, most of which are nematode-specific and are of unknown function. We have exploited comparative two-dimensional chromatography of synchronized cultures of wild type C. elegans larvae and a mutant in nhr-40 to determine if proteomic approaches will provide additional insight into gene function. Chromatofocusing, followed by reversed-phase chromatography and mass spectrometry, identified altered chromatographic patterns for a set of proteins, many of which function in muscle and metabolism. Prompted by the proteomic analysis, we find that the penetrance of the developmental phenotypes in the mutant is enhanced at low temperatures and by food restriction. The combination of our phenotypic and proteomic analysis strongly suggests that NHR-40 provides a link between metabolism and muscle development. Our results highlight the utility of comparative two-dimensional chromatography to provide a relatively rapid method to gain insight into gene function.

GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in Caenorhabditis elegans

NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although C. elegans has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in Drosophila or C. elegans. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in C. elegans and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the gei-8 coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA class. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous gei-8 mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.

Diversification of fasting regulated transcription in a cluster of duplicated nuclear hormone receptors in C. elegans

The genome of Caenorhabditis elegans encodes more than 280 nuclear hormone receptors (NHRs) in contrast to the 48 NHRs in humans and 18 NHRs in Drosophila. The majority of the C. elegans NHRs are categorized as supplementary nuclear receptors (supnrs) that evolved by successive duplications of a single ancestral gene. The evolutionary pressures that lead to the expansion of NHRs in nematodes, as well as the function of the majority of supnrs, are not known. Here, we have studied the expression of seven genes organized in a cluster on chromosome V: nhr-206, nhr-208, nhr-207, nhr-209, nhr-154, nhr-153 and nhr-136. Reverse transcription-quantitative PCR and analyses using transgenic lines carrying GFP fusion genes with their putative promoters revealed that all seven genes of this cluster are expressed and five have partially overlapping expression patterns including in the pharynx, intestine, certain neurons, the anal sphincter muscle, and male specific cells. Four genes in this cluster are conserved between C. elegans and Caenorhabditis briggsae whereas three genes are present only in C. elegans, the apparent result of a relatively recent expansion. Interestingly, we find that a subset of the conserved and non-conserved genes in this cluster respond transcriptionally to fasting in tissue-specific patterns. Our results reveal the diversification of the temporal, spatial, and metabolic gene expression patterns coupled with evolutionary drift within supnr family members.

Trichoplax adhaerens reveals a network of nuclear receptors sensitive to 9-cis-retinoic acid at the base of metazoan evolution

Trichoplax adhaerens, the only known species of Placozoa is likely to be closely related to an early metazoan that preceded branching of Cnidaria and Bilateria. This animal species is surprisingly well adapted to free life in the World Ocean inhabiting tidal costal zones of oceans and seas with warm to moderate temperatures and shallow waters. The genome of T. adhaerens (sp. Grell) includes four nuclear receptors, namely orthologue of RXR (NR2B), HNF4 (NR2A), COUP-TF (NR2F) and ERR (NR3B) that show a high degree of similarity with human orthologues. In the case of RXR, the sequence identity to human RXR alpha reaches 81% in the DNA binding domain and 70% in the ligand binding domain. We show that T. adhaerens RXR (TaRXR) binds 9-cis retinoic acid (9-cis-RA) with high affinity, as well as high specificity and that exposure of T. adhaerens to 9-cis-RA regulates the expression of the putative T. adhaerens orthologue of vertebrate L-malate-NADP+ oxidoreductase (EC 1.1.1.40) which in vertebrates is regulated by a heterodimer of RXR and thyroid hormone receptor. Treatment by 9-cis-RA alters the relative expression profile of T. adhaerens nuclear receptors, suggesting the existence of natural ligands. Keeping with this, algal food composition has a profound effect on T. adhaerens growth and appearance. We show that nanomolar concentrations of 9-cis-RA interfere with T. adhaerens growth response to specific algal food and causes growth arrest. Our results uncover an endocrine-like network of nuclear receptors sensitive to 9-cis-RA in T. adhaerens and support the existence of a ligand-sensitive network of nuclear receptors at the base of metazoan evolution.