CHR3 (nhr-23, NF1F4), the homologue of Drosophila DHR3 and mammalian ROR͞RZR͞RevErbA 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) RNAmediated 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.molting ͉ development N uclear hormone receptors (NHRs) form a large superfamily of transcription factors that are important for the regulation of cell metabolism, development, and reproduction (1-3). The typical receptor consists of six domains. The most conserved domain is the DNA-binding domain (DBD) and is characteristic for the NHR superfamily. It is formed by two zinc finger motifs that are conserved through evolution (4-6). A second, less conserved domain is the ligand-binding domain, located in the C terminus of the molecule and involved in binding of small hydrophobic molecules, hormones of the steroid class, thyroid hormone, retinoic acid stereoisomers, farnesoids, prostaglandins, and terpenoids (2). The generally accepted classification of NHRs is based on homology in both the DBD and the ligandbinding domain and the specific ligands (if known), which activate the receptors. The majority of NHRs have no known ligands and usually are referred to as orphan receptors.The NHRs are present in a majority of Metazoan species higher than diploblasts. NHRs apparently evolved from a single gene present in a Metazoan ancestor before the divergence of diploblastic species (4-6). There are about 70 NHRs in vertebrates, 22 in Drosophila We previously have cloned the C. elegans orphan NHR CHR3 (19), which is a homologue of Drosophila DHR3 (20), Manduca sexta MHR3 (21), and mammalian ROR͞RZR͞Rev ErbA (22, 23). For clarity, we refer to it in this paper by using both its common and gene names, CHR3 (nhr-23). The gene is classified as NR1F4 in a unified nomenclature system for the NHR superfamily (24). We previously showed that CHR3 (nhr-23) is expressed in the epidermis of C. elegans throughout development and that it was required for proper molting (16).Molting is a complex of developmental processes characteristic for a clad Ecdysozoa, which includes arthropods, tardigrades, onychophorans, nematodes, nematomorphs, kinorhynchs, and priapulids (25). At each molt, there is production of the new outer-body cover, the exoskeleton, and the old part is shed. Molting represents...

Nuclear hormone receptors comprise a characteristic family of transcription factors found in vertebrates, insects and nematodes. Here we show by cDNA and gene cloning that a Cnidarian, Tripedalia cystophora, possesses a retinoid receptor (jRXR) with remarkable homology to vertebrate retinoic acid X receptors (RXRs). Like vertebrate RXRs, jRXR binds 9-cis retinoic acid (K d ‫؍‬ 4 ؋ 10 ؊10 M) and binds to the DNA sequence, PuGGTCA as a monomer in vitro. jRXR also heterodimerizes with Xenopus TR beta on a thyroid responsive element of a direct repeat separated by 4 bp. A jRXR binding half-site capable of interacting with (His 6 )jRXR fusion protein was identified in the promoters of three T. cystophora crystallin genes that are expressed highly in the eye lens of this jellyfish. Because crystallin gene expression is regulated by retionoid signaling in vertebrates, the jellyfish crystallin genes are candidate in vivo targets for jRXR. Finally, an antibody prepared against (His 6 )jRXR showed that fulllength jRXR is expressed at all developmental stages of T. cystophora except the ephydra, where a smaller form replaces is. These data show that Cnidaria, a diploblastic phylum ancestral to the triploblastic invertebrate and subsequent vertebrate lineages, already have an RXR suggesting that RXR is an early component of the regulatory mechanisms of metazoa.

NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in C. 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.

CHR3 is a Caenorhabditis elegans orphan nuclear hormone receptor highly homologous to Drosophila DHR3, an ecdysone-inducible gene product involved in metamorphosis. Related vertebrate factors include RORalpha/RZRalpha, RZRbeta and RevErb. Gel-shift studies show that CHR3 can bind the DR5-type hormone response sequence. CHR3 is a nuclear protein present in all blastomeres during early embryogenesis. During morphogenesis, both CHR3 protein and zygotically active reporter genes are detectable in epidermal cells and their precursors. Inhibition of the gene encoding CHR3 results in several larval defects associated with abnormal epidermal cell function, including molting and body size regulation, suggesting that CHR3 is an essential epidermal factor required for proper postembryonic development.

The large family of steroid/thyroid hormone receptor (STR) genes has been extensively studied in vertebrates and insects but little information is available on it in more primitive organisms. All members possess a DNA binding domain of zinc fingers of the C2, C2 type. We have used the polymerase chain reaction with degenerate oligonucleotide primers covering this region to clone three distinct members of this family from the nematode Caenorhabditis elegans. All three belong to the retinoic acid receptor (RAR), thyroid hormone receptor subfamily of genes. The cDNA of one of these clones shows such a high homology to DHR3, an early ecdysone response gene found in Drosophila, and MHR3, identified in Manduca sexta, that we have termed it CHR3. Furthermore, the C-terminal portion of the deduced protein sequence shows a box containing eight identical amino acids among CHR3, DHR3, and MHR3 suggesting an identical specific ligand for these proteins. CNR8 shows homology to NAK1, and CNR14 has homology to both the RAR-yl gene and to another ecdysone response gene, E78A. Neither of the latter two cDNAs is a clear homologue of any known gene and each is distinctive. All of these genes are expressed varyingly in both larval and adult stages of nematode development as shown by Northern blot analyses. These data demonstrate that the STR family of genes is represented in a nematode whose ancestor appeared well before the branching that gave rise to the Arthropoda and Chordata.More than 200 different cDNAs of the steroid/thyroid hormone receptor (STR) gene family have been cloned and sequenced from a variety of organisms. Many of these sequences are clear homologues-i.e., the thyroid hormone receptor (TR) genes from rat, chicken, mouse, human, and frog code for a highly similar protein that binds thyroid hormone. However, the term homologue can be ambiguous; in addition to the above, it can also mean evolutionarily related, as are all of these STR genes whether they bind steroids, retinoids, or thyroid hormone or have no known ligands. Our interest has been to discern the evolutionary history of this gene family. The family has been variously divided into 3-10 different groups (1-3). Sequences are available mostly from vertebrates and arthropods, although one sequence is known from an echinoderm (4). All of these genes can be divided into five or six domains denoted A-F (2, 3) (see Fig. 4 A). The A/B domain is most N terminal and is poorly conserved. The C domain is the zinc finger DNA binding domain and is so highly conserved as to identify the superfamily (5). The sequence of the distal part of the first zinc finger, termed the P box, distinguishes the three main subfamilies of the STR (2). The D, E, and F domains contain the activating domain, a dimerization domain, and the ligand binding domain (2, 3, 6, 7). We have searched for related genes in several invertebrate phyla and report here the identification and cDNA sequences of three genes from Caenorhabditis elegans. t The sequences of the P box of the cDNAs we have...

IntroductionIn the last few years, various studies have underlined a correlation between thyroid function and male sexual function, hypothesizing a direct action of thyroid hormones on the penis.AimTo study the spatiotemporal distribution of mRNA for the thyroid hormone nuclear receptors (TR) α1, α2 and β in the penis and smooth muscle cells (SMCs) of the corpora cavernosa of rats and humans during development.MethodsWe used several molecular biology techniques to study the TR expression in whole tissues or primary cultures from human and rodent penile tissues of different ages.Main Outcome MeasureWe measured our data by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) amplification, Northern blot and immunohistochemistry.ResultsWe found that TRα1 and TRα2 are both expressed in the penis and in SMCs during ontogenesis without development-dependent changes. However, in the rodent model, TRβ shows an increase from 3 to 6 days post natum (dpn) to 20 dpn, remaining high in adulthood. The same expression profile was observed in humans. While the expression of TRβ is strictly regulated by development, TRα1 is the principal isoform present in corpora cavernosa, suggesting its importance in SMC function. These results have been confirmed by immunohistochemistry localization in SMCs and endothelial cells of the corpora cavernosa.ConclusionsThe presence of TRs in the penis provides the biological basis for the direct action of thyroid hormones on this organ. Given this evidence, physicians would be advised to investigate sexual function in men with thyroid disorders. Carosa E, Di Sante S, Rossi S, Castri A, D'Adamo F, Gravina GL, Ronchi P, Kostrouch Z, Dolci S, Lenzi A, and Jannini EA. Ontogenetic profile of the expression of thyroid hormone receptors in rat and human corpora cavernosa of the penis. J Sex Med 2010;7:1381–1390.

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. transcription ͉ nuclear hormone receptors ͉ cofactors ͉ molting T he RNA polymerase II (RNA Pol II) transcription complex consists of the polymerase holoenzyme, general transcription factors, and gene-specific transcription factors. General factors include components such as TATA box-binding proteinassociated factors (TAFs) and thyroid hormone receptorassociated protein (TRAP)͞SRB-and MED-containing cofactor complex (SMCC)͞mediator comprised of several proteins (1). The RNA Pol II complex is also regulated by gene-specific transcription factors that recognize cis-acting regulatory sequences in promoters resulting in activation or repression of the target gene.One cofactor that has been identified in both stimulatory and inhibitory Pol II complexes is the SKI-binding protein (SKIP). SKIP was first identified in Drosophila melanogaster (2), and was subsequently identified in many other species (3-7). SKIP is an essential protein in yeast (3). Several papers (4-10) report interactions between SKIP and other proteins involved in the repression and͞or activation of transcription. In addition, SKIP is known to be a cofactor in Notch, transforming growth factor (TGF)-␤ and vitamin D receptor (VDR) pathways where it acts as a coactivator. The ubiquitous presence of SKIP, from yeast to plants and animals, and its possible involvement in the regulation of transcription by diverse regulatory systems raises the possibility that this protein may be functioning as a fundamental cofactor in the RNA Pol II complex.We have been studying the role of SKIP during Caenorhabditis elegans development. In this study, we demonstrate that C. elegans SKIP (CeSKIP) is essential for viability and is a critical component of multiple regulatory pathways during C. elegans development. Our results also show that despite phosphorylation of the RNA Pol II C-terminal domain (CTD), CeSKIP RNAmediated inhibition (RNAi) embryos arrest with a phenotype similar to the loss of RNA Pol II activity and fail to express multiple embryonic green fluorescent protein (GFP) reporter genes. In C. elegans, CeSKIP is cotranscribed with bir-1, an inhibitor of apoptosis that is homologous to mammalian Survivin. We confirm earlier studies demons...

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.