A conserved retinoid X receptor (RXR) from the mollusk Biomphalaria glabrata transactivates transcription in the presence of retinoids.

Retinoid X receptors (RXR) are members of the nuclear receptor superfamily of ligand-activated transcription factors that have been characterized in a wide variety of metazoan phyla. They act as heterodimer partners of other nuclear receptors, and in vertebrates also activate transcription as homodimers in the presence of a ligand, 9-cis retinoic acid. In order to test the hypothesis that retinoic acid signaling pathways involving RXRs are present in the Lophotrochozoa, we have sought to isolate conserved members of this family from the platyhelminth parasite Schistosoma mansoni and its intermediate host, the mollusk Biomphalaria glabrata. Here we report that an RXR ortholog from B. glabrata (BgRXR) is better conserved, compared with mouse RXRalpha, both in the DNA-binding domain (89% identity) and in the ligand-binding domain (LBD) (81% identity), than are arthropod homologs. In EMSA, BgRXR binds to the direct repeat response element DR1 as a homodimer or as a heterodimer with mammalian RARalpha, LXR, FXR or PPARalpha. When transfected alone into mammalian cell lines, BgRXR transactivated transcription of a reporter gene from the Apo-A1 promoter in the presence of 9-cis retinoic acid or DHA. Constructs with the Gal4 DNA binding domain fused to the hinge and LBDs of BgRXR were used to show that ligand-dependent activation of transcription by BgRXR required its intact AF-2 activation domain, and that the LBD can form homodimers. Finally, the binding of 9-cis retinoic acid preferentially protected the LBD of BgRXR from degradation by trypsin in a proteolysis protection assay. Our results show that BgRXR binds and is activated by retinoids and suggest that retinoid signaling pathways are conserved in the Lophotrochozoa. The nucleotide sequence reported in this paper has been submitted to the GenBank/EBI Data Bank with accession no. AY048663.

Endogenous beta-galactosidase activity in amphioxus: a useful histochemical marker for the digestive system.

Endogenous beta-galactosidase activity has been shown in the digestive tract of amphioxus from the larval to the adult stage and it can be easily followed as a histochemical marker. Enzymatic activity first appeared in 30-h larvae, became evident in 36-h larvae and remained in adults. In situ detection of beta-galactosidase activity was used to monitor morphological and functional differentiation of the digestive system and the posteriorization of the endodermal structures in retinoic-acid treated embryos. The endogenous beta-galactosidase activity was distinguished from the bacterial lacZ reporter by incubation at low pH.

Euteleost fish genomes are characterized by expansion of gene families.

The presence of additional hox clusters in the zebrafish has led to the hypothesis that there was a whole genome duplication at the origin of modern fish. To investigate the generality of this assumption, we analyzed all available actinopterygian fish gene families, and sequenced nuclear receptors from diverse teleost fish. The origin and timing of duplications was systematically determined by phylogenetic analysis. More genes are indeed found in zebrafish than in mouse. This abundance is shared by all major groups of euteleost fish, but not by eels. Phylogenetic analysis shows that it may result from frequent independent duplications, rather than from an ancestral genome duplication. We predict two zebrafish paralogs for most mouse or human genes, thus expressing a note of caution in functional comparison of fish and mammalian genomes. Redundancy appears to be the rule in fish developmental genetics. Finally, our results imply that the outcome of genome projects cannot be extrapolated easily between fish species.

Molecular cloning and characterization of thyroid hormone receptors in teleost fish.

Thyroid hormones are pleiotropic factors important for many developmental and physiological functions in vertebrates. Their effects are mediated by two specific receptors (TRalpha and TRbeta) which are members of the nuclear hormone receptor superfamily. To clarify the function of these receptors, our laboratory has started a comparative study of their role in teleost fish. This type of approach has been hampered by the isolation of specific clones for each fish species studied. In this report, we describe an efficient reverse transcription/PCR procedure that allows the isolation of large fragments corresponding to TRalpha and TRbeta of a wide range of teleost fish. Phylogenetic analysis of these receptors revealed a placement consistent with their origin, sequences from teleost fish being clearly monophyletic for both TRalpha and TRbeta. Interestingly, this approach allowed us to isolate (from tilapia and salmon) several new TRalpha or TRbeta isoforms resulting from alternative splicing. These isoforms correspond to expressed transcripts and thus may have an important physiological function. In addition, we isolated a cDNA encoding TRbeta in the Atlantic salmon (Salmo salar) encoding a functional thyroid hormone receptor which binds specific thyroid hormone response elements and regulates transcription in response to thyroid hormones.

Evidence of tissue-specific, post-transcriptional regulation of NRF-2 expression.

Mitochondrial respiratory function requires the expression of genes both from the mitochondrial and nuclear genomes. Nuclear respiratory factor 2 (NRF-2) is a transcription factor required for the expression of several nuclear-encoded mitochondrial proteins, including the specific mitochondrial transcription factor Tfam. This makes NRF-2 a likely candidate to coordinate expression of mitochondrial components. NRF-2 is a multisubunit complex of which the alpha subunit binds DNA and the beta subunit enhances this binding, respectively. We have analysed in vivo the expression patterns of NRF-2 subunits both at the mRNA and protein level, in three rat tissues, liver, testis and brain. In contrast with Tfam or the 'housekeeping' beta-actin expressions in which a parallel gradient was observed, no correlation was found between NRF-2 mRNAs and proteins levels, thus suggesting post-transcriptional regulation.

Ligand binding and nuclear receptor evolution.

Nuclear receptors form a superfamily of ligand-activated transcription factors that regulate various physiological functions, from development to homeostasis, in metazoans. The superfamily contains not only receptors for known ligands but also a large number of so-called orphan receptors for which ligands do not exist or have not been identified. The evolution of ligand-binding capacity of nuclear receptors may involve either secondary loss in orphan receptors, or evolutionary acquisition of ligand-binding capacity in liganded receptors. In this review, we present arguments from phylogenetic, functional and structural studies that support the hypothesis that there have been several independent gains of ligand-binding ability of nuclear receptors during metazoan evolution.

Hormones and nuclear receptors in schistosome development.

A substantial but disparate body of evidence suggests that hormones affect the development of schistosomes within their definitive hosts. Here, Raymond Pierce and colleagues review such evidence for host steroid and thyroid hormones, and for ecdysteroids, and link this to the expanding knowledge of the nuclear receptors for these hormones. Phylogenetic analysis of the nuclear receptor superfamily and the characterization of the first schistosome nuclear receptors suggest that steroids and thyroid hormone probably act indirectly, or by pathways not involving the control of gene transcription. However, the probability that schistosome nuclear receptors exist for a variety of unique ligands opens up exciting possibilities for targeted drug development.

Structural and functional divergence of a nuclear receptor of the RXR family from the trematode parasite Schistosoma mansoni.

We describe the cloning and functional characterization of Schistosoma mansoni retinoid-X-receptor (SmRXR; NR2B4-B), a novel member of the nuclear receptor superfamily from S. mansoni, a homologue of vertebrate retinoid-X-receptor. The DNA-binding C domain of SmRXR shows 80% sequence identity to both human RXRalpha and Drosophila ultraspiracle (USP), but a much lower level of conservation of the ligand-binding E domain (22-25% identity). Phylogenetic analysis places SmRXR within the RXR group as an early offshoot of this clade. SmRXR mRNA is expressed at all life-cycle stages but at higher levels in the free-living larval stages. However, the SmRXR protein is expressed at markedly different levels, being almost absent from eggs while present at the highest concentration in schistosomula. Recombinant SmRXR fails to bind to the consensus direct repeat response elements, either alone, or as a heterodimer with mouse retinoic acid receptor alpha or the Drosophila ecdysone receptor. However, the use of chimaeric constructions shows that the C domain of SmRXR will bind to conventional response elements as a heterodimer, and that its specificity is modified by the presence of the D and E domains. In accordance with these results, native SmRXR failed to transactivate the transcription of a reporter gene after cotransfection of mammalian cell lines.

The complete nucleotide sequence of the mitochondrial DNA of the agnathan Lampetra fluviatilis: bearings on the phylogeny of cyclostomes.

There are two competing theories about the interrelationships of craniates: the cyclostome theory assumes that lampreys and hagfishes are a clade, the cyclostomes, whose sister group is the jawed vertebrates (gnathostomes); the vertebrate theory assumes that lampreys and gnathostomes are a clade, the vertebrates, whose sister group is hagfishes. The vertebrate theory is best supported by a number of unique anatomical and physiological characters. Molecular sequence data from 18S and 28S rRNA genes rather support the cyclostome theory, but mtDNA sequence of Myxine glutinosa rather supports the vertebrate theory. Additional molecular data are thus needed to elucidate this three-taxon problem. We determined the complete nucleotide sequence of the mtDNA of the lamprey Lampetra fluviatilis. The mtDNA of L. fluviatilis possesses the same genomic organization as Petromyzon marinus, which validates this gene order as a synapomorphy of lampreys. The mtDNA sequence of L. fluviatilis was used in combination with relevant mtDNA sequences for an approach to the hagfish/lamprey relationships using the maximum-parsimony, neighbor-joining, and maximum-likelihood methods. Although trees compatible with our present knowledge of the phylogeny of craniates can be reconstructed by using the three methods, the data collected do not support the vertebrate or the cyclostome hypothesis. The present data set does not allow the resolution of this three-taxon problem, and new kinds of data, such as nuclear DNA sequences, need to be collected.

Amphicoup-TF, a nuclear orphan receptor of the lancelet Branchiostoma floridae, is implicated in retinoic acid signalling pathways.

In vertebrates, the orphan nuclear receptors of the COUP-TF group function as negative transcriptional regulators that inhibit the hormonal induction of target genes mediated by classical members of the nuclear hormone superfamily, such as the retinoic acid receptors (RARs) or the thyroid hormone receptors (TRs). To investigate the evolutionary conservation of the roles of COUP-TF receptors as negative regulators in the retinoid and thyroid hormone pathways, we have characterized AmphiCOUP-TF, the homologue of COUP-TFI and COUP-TFII, in the chordate amphioxus (Branchiostoma floridae), the closest living invertebrate relative of the vertebrates. Electrophoretic mobility shift assays (EMSA) showed that AmphiCOUP-TF binds to a wide variety of response elements, as do its vertebrate homologues. Furthermore, AmphiCOUP-TF is a transcriptional repressor that strongly inhibits retinoic acid-mediated transactivation. In situ hybridizations revealed expression of AmphiCOUP-TF in the nerve cord of late larvae, in a region corresponding to hindbrain and probably anterior spinal cord. Although the amphioxus nerve cord appears unsegmented at the gross anatomical level, this pattern reflects segmentation at the cellular level with stripes of expressing cells occurring adjacent to the ends and the centers of each myotomal segment, which may include visceral motor neurons and somatic motor neurons respectively, among other cells. A comparison of the expression pattern of AmphiCOUP-TF with those of its vertebrate homologues, suggests that the roles of COUP-TF in patterning of the nerve cord evolved prior to the split between the amphioxus and vertebrate lineages. Furthermore, in vitro data also suggest that Amphi-COUP-TF acts as a negative regulator of signalling by other nuclear receptors such as RAR, TR or ER.

Expression of mitochondrial genes and of the transcription factors involved in the biogenesis of mitochondria Tfam, NRF-1 and NRF-2, in rat liver, testis and brain.

Mitochondrial function requires genes encoded in both mitochondrial and nuclear genomes. Tfam, the activator of mammalian mitochondrial transcription, is encoded in the nucleus and its expression has been shown in in vitro studies to be controlled by nuclear respiratory factors NRF-1 and NRF-2. In order to understand the physiological dependence of mitochondrial gene expression, we have analyzed in rat liver, testis and brain the expression level of mitochondrial genes in parallel with those of the three transcription factors. We found that: a) Tfam expression is down-regulated in rat testis, both at the protein and transcript level. The three-fold reduction in the abundance of Tfam protein in rat testis does not result in low steady-state levels of mitochondrial gene transcripts, suggesting that Tfam is in excess and does not limit transcription in vivo; and b) NRF-1 and NRF-2 (alpha, beta and gamma subunits) mRNAs were analyzed by Northern blotting; for each mRNA, several transcripts were observed as well as tissue-specific patterns of expression. The mRNA steady-state levels of NRF-1 and NRF-2 were higher in testis than in liver or brain. These data suggest that the low expression level of Tfam found in testis is not due to decreased NRF-1 and/or NRF-2 expression and further suggest the existence of tissue-specific post-transcriptional regulatory mechanisms for the expression of NRF-1/NRF-2 genes.

Ligand binding was acquired during evolution of nuclear receptors.

The nuclear receptor (NR) superfamily comprises, in addition to ligand-activated transcription factors, members for which no ligand has been identified to date. We demonstrate that orphan receptors are randomly distributed in the evolutionary tree and that there is no relationship between the position of a given liganded receptor in the tree and the chemical nature of its ligand. NRs are specific to metazoans, as revealed by a screen of NR-related sequences in early- and non-metazoan organisms. The analysis of the NR gene duplication pattern during the evolution of metazoans shows that the present NR diversity arose from two waves of gene duplications. Strikingly, our results suggest that the ancestral NR was an orphan receptor that acquired ligand-binding ability during subsequent evolution.

Rat mammary-gland transferrin: nucleotide sequence, phylogenetic analysis and glycan structure.

The complete cDNA for rat mammary-gland transferrin (Tf) has been sequenced and also the native protein isolated from milk in order to analyse the structure of the main glycan variants present. A lactating-rat mammary-gland cDNA library in lambda gt10 was screened with a partial cDNA copy of rat liver Tf and subsequently rescreened with 5' fragments of the longest clones. This produced a 2275 bp insert coding for an open reading frame of 695 amino acid residues. This includes a 19-amino acid signal sequence and the mature protein containing 676 amino acids and one N-glycosylation site in the C-terminal domain at residue 490. Phylogenetic analysis was carried out using 14 translated Tf nucleotide sequences, and the derived evolutionary tree shows that at least three gene duplication events have occurred during Tf evolution, one of which generated the N- and C-terminal domains and occurred before separation of arthropods and chordates. The two halves of human melanotransferrin are more similar to each other than to any other sequence, which contrasts with the pattern shown by the remaining sequences. Native rat milk Tf is separated into four bands on native PAGE that differ only in their sialic acid content: one biantennary glycan is present containing either no sialic acid residues or up to three. The complete structures of the two major variants were determined by methylation, m.s. and 400 MHz 1H-n.m.r. spectroscopy. They contain either one or two neuraminic acid residues (alpha 2-->6)-linked to galactose in conventional biantennary N-acetyl-lactosamine-type glycans. Most contain fucose (alpha 1-->6)-linked to the terminal non-reducing N-acetylglucosamine.