3,5-T2 is an alternative ligand for the thyroid hormone receptor ß1.

Several liganded nuclear receptors have alternative ligands acting in a tissue-specific fashion and playing important biological roles. We present evidence that 3,5-diiodothyronine (T(2)), a naturally occurring iodothyronine that results from T(3) outer-ring deiodination, is an alternative ligand for thyroid hormone receptor ß1 (TRß1). In tilapia, 2 TRß isoforms differing by 9 amino acids in the ligand-binding domain were cloned. Binding and transactivation studies showed that T(2) activates the human and the long tilapia TRß1 isoform, but not the short one. A chimeric human TRß1 (hTRß1) that contained the 9-amino-acid insert showed no response to T(2), suggesting that the conformation of the hTRß1 naturally allows T(2) binding and that other regions of the receptor are implicated in TR activation by T(2). Indeed, further analysis showed that the N terminus is essential for T(2)-mediated transactivation but not for that by T(3) in the long and hTRß1, suggesting a functional interaction between the N-terminal domain and the insertion in the ligand-binding domain. To establish the functional relevance of T(2)-mediated TRß1 binding and activation, mRNA expression and its regulation by T(2) and T(3) was evaluated for both isoforms. Our data show that long TRß1expression is 10(6)-fold higher than that of the short isoform, and T(3) and T(2) differentially regulate the expression of these 2 TRß1 isoforms in vivo. Taken together, our results prompted a reevaluation of the role and mechanism of action of thyroid hormone metabolites previously believed to be inactive. More generally, we propose that classical liganded receptors are only partially locked to very specific ligands and that alternative ligands may play a role in the tissue-specific action of receptors.

The rapid divergence of the ecdysone receptor is a synapomorphy for Mecopterida that clarifies the Strepsiptera problem.

In arthropods, the regulation by ecdysteroids is mediated by the heterodimer between the ecdysone receptor (ECR; NR1H1) and ultraspiracle (USP/RXR; NR2B4) nuclear receptors. Both ECR and USP/RXR ligand-binding domains experienced a strong acceleration of evolutionary rate in Diptera and Lepidoptera, which belong to the superorder Mecopterida. We performed a phylogenetic analysis of 28 ECR and 30 USP/RXR protein sequences from 36 arthropod species, including representatives from Trichoptera, Mecoptera and Siphonaptera. Our data show that the acceleration of ECR and USP/RXR was a unique event in the ancestor of Mecopterida. Our analysis shows further that Strepsiptera ECR and USP/RXR sequences are unambiguously placed outside of the Mecopterida clade. Protein alignments reveal that eight of 11 synapomorphies support an affinity between Strepsiptera and Coleoptera sequences. The affiliation of Strepsiptera to Diptera should therefore be rejected.

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.

Dimerization is required for transactivation by estrogen-receptor-related (ERR) orphan receptors: evidence from amphioxus ERR.

The estrogen-receptor-related (ERR) receptors are orphan members of the nuclear receptor superfamily that bind to their specific DNA target sites as homodimers. However, it has not been shown whether this mode of binding is required for the transcriptional activation they drive. We here show that heterodimerization can also occur between these receptors. Furthermore, we demonstrate that the unique amphioxus ortholog of ERR genes (AmphiERR) is expressed as two isoforms differing by an in-frame insertion. While the short isoform behaves like its mammalian counterparts, the long isoform (AmphiERR(L)) displays divergent transcriptional properties according to the target site to which it binds. Indeed, AmphiERR(L) binds as a monomer but does not activate transcription through the SF1 response element (SFRE). On the contrary, this isoform binds as a homodimer and activates transcription through the classical estrogen-response element. Our results strongly suggest that dimerization is required for transactivation exerted by the ERR receptors.

The chordate amphioxus: an emerging model organism for developmental biology.

The cephalochordate amphioxus is the closest living invertebrate relative of the vertebrates. It is vertebrate-like in having a dorsal, hollow nerve cord, notochord, segmental muscles, pharyngeal gill slits and a post-anal tail that develops from a tail bud. However, amphioxus is less complex than vertebrates, lacking neural crest and having little or no mesenchyme. The genetic programs patterning the amphioxus embryo are also similar to those patterning vertebrate embryos, although the amphioxus genome lacks the extensive gene duplications characteristic of vertebrates. This relative structural and genomic simplicity in a vertebrate-like organism makes amphioxus ideal as a model organism for understanding mechanisms of vertebrate development.

Expression of the orphan nuclear receptor ERRalpha is under circadian regulation in estrogen-responsive tissues.

Circadian gene expression has been demonstrated in many tissues and involves both positive and negative regulatory loops. The potential interferences of circadian rhythmicity with other well-known biologic rhythms, such as the ovarian cycle, at least in part controlled by estrogens, has not been questioned. The estrogen receptor-related receptor (ERR)alpha is an orphan nuclear receptor that is widely expressed in estrogen-responsive tissues such as liver, uterus and bone. In addition, expression of the ERRalpha gene has been proposed to be transcriptionally controlled by estrogens in the uterus. Here we show that the expression of ERRalpha displays a circadian rhythmicity in liver, bone and uterus. This is in contrast to other uterine estrogen-regulated genes. Analysis of clock/clock mutant mice shows that ERRalpha is an output gene of the circadian clock oscillator. The expression of clock-control genes, such as Bmal1 and Rev-erbalpha, also displays diurnal oscillations in the uterus, but not in bone. In this tissue, however, Per2 displayed a rhythmic expression, altogether suggesting unconventional loops in the regulation of circadian rhythm in bone.

Evidence for two distinct functional glucocorticoid receptors in teleost fish.

Using RT-PCR with degenerated primers followed by screening of a rainbow trout (Oncorhynchus mykiss) intestinal cDNA library, we have isolated from the rainbow trout a new corticosteroid receptor which shows high sequence homology with other glucocorticoid receptors (GRs), but is clearly different from the previous trout GR (named rtGR1). Phylogenetic analysis of these two sequences and other GRs known in mammals, amphibians and fishes indicate that the GR duplication is probably common to most teleost fish. The open reading frame of this new trout GR (named rtGR2) encodes a protein of 669 amino acids and in vitro translation produces a protein of 80 kDa that appears clearly different from rtGR1 protein (88 kDa). Using rtGR2 cDNA as a probe, a 7.3 kb transcript was observed in various tIssues suggesting that this gene would lead to expression of a steroid receptor. In vitro studies were used to further characterize this new corticosteroid receptor. Binding studies with recombinant rtGR1 and rtGR2 proteins show that the two receptors have a similar affinity for dexamethasone (GR1 K(d)=5.05+/-0.45 nM; GR2 K(d)=3.04+/-0.79 nM). Co-transfection of an rtGR1 or rtGR2 expression vector into CHO-K1 or COS-7 cells, along with a reporter plasmid containing multiple consensus glucocorticoid response elements, shows that both clones are able to induce transcriptional activity in the presence of cortisol and dexamethasone. Moreover, at 10(-)(6 )M 11-deoxycortisol and corticosterone partially induced rtGR2 transactivation activity but were without effect on rtGR1. The other major teleost reproductive hormones, as well as a number of their precursors or breakdown products of these and corticosteroid hormones, were without major effects on either receptor. Interestingly, rtGR2 transactivational activity was induced at far lower concentrations of dexamethasone or cortisol (cortisol EC(50)=0.72+/-0.87 nM) compared with rtGR1 (cortisol EC(50)=46+/-12 nM). Similarly, even though RU486 inhibited transactivation activity in both rtGR1 and rtGR2, rtGR1 was more sensitive to this GR antagonist. Altogether, these results indicate that these two GR sequences encode for two functionally distinct GRs acting as ligand-inducible transcription factors in rainbow trout.

A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors.

The orphan receptors Rev-erbalpha and Rev-erbbeta are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbalpha is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbbeta. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbalpha and Rev-erbbeta proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbalpha is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbalpha deletion mutants showed that the NLS is located within a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbbeta fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located within the DBD. GFP/DBD. RORalpha or GFP/DBD.RORbeta remained cytoplasmic, in contrast to GFP/DBD. Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD. Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD. RORalpha towards the Rev-erbalpha residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.

Characterization of oestrogen receptors in zebrafish (Danio rerio).

We cloned the cDNAs corresponding to three oestrogen receptors (ERs) in zebrafish (Danio rerio). Sequence analysis and phylogenetic studies demonstrated that two of these genes, ER beta.1 and ER beta.2, arose from duplication of the original ER beta in many species of the fish phylum, whereas ER alpha is unique. Zebrafish ERs behaved as oestrogen-dependent transcription factors in transactivation assays. However, their reactivity to various oestrogen modulators was different compared with that of mouse ERs. ER mRNA expression during zebrafish development is restricted to distinct time periods, as observed by RNase protection assays. ER beta.2 is initially expressed as maternally transmitted RNA, until 6 h after fertilization, when expression disappears. Between 6 and 48 h after fertilization, no ER expression could be observed. After 48 h after fertilization, all ERs, but predominantly ER alpha, began to be expressed. We conclude that oestrogen signal transduction can operate during zebrafish development only within discrete time windows.

How many nuclear hormone receptors are there in the human genome?

The sequence of the human genome now allows the definition of the complete set of genes for specific protein families in humans. Because of their involvement in many physiological and pathological processes, the nuclear hormone receptors are a superfamily of crucial medical significance. Although 48 human nuclear receptor genes were identified previously, their total number is unclear from early human genome reports. Here, we report the identification and classification of all nuclear receptor genes in the human genome, and we discuss corresponding transcriptome and proteome diversity.

Circadian regulation of diverse gene products revealed by mRNA expression profiling of synchronized fibroblasts.

Genes under a 24-h regulation period may represent drug targets relevant to diseases involving circadian dysfunctions. As a testing model of the circadian clock system, we have used synchronized rat fibroblasts that are known to express at least six genes in a circadian fashion. We have determined the expression patterns of 9957 transcripts every 4 h over a total period of 76 h using high density oligonucleotide microarrays. The spectral analysis of our mRNA profiling data indicated that approximately 2% (85 genes) of all expressed genes followed a robust circadian pattern. We have confirmed the circadian expression of previously known clock or clock-driven genes, and we identified 81 novel circadian genes. The majority of the circadian-regulated gene products are known and are involved in diverse cellular functions. We have classified these circadian genes in seven clusters according to their phase of cycling. Our pathway analysis of the mRNA profiling data strongly suggests a direct link between circadian rhythm and cell cycle.

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.

Modulation of the far-upstream enhancer of the rat alpha-fetoprotein gene by members of the ROR alpha, Rev-erb alpha, and Rev-erb beta groups of monomeric orphan nuclear receptors.

Expression of the oncodevelopmental alpha-fetoprotein (AFP) gene is tightly regulated and occurs in the yolk sac, fetal liver and intestine, and cancerous liver cells. Transcription of the AFP gene is under the control of three enhancers that are very tissue specific. We have shown that the most upstream of these enhancers, located at -6 kb, works through the combined action of liver-enriched factors and nuclear receptors that bind to three regions of this DNA regulatory element. This study showed that orphan nuclear receptors of the ROR alpha, Re-verb alpha, and Rev-erb beta groups can bind as monomers with high affinity and specificity to an evolutionarily conserved AGGTCA motif in the functionally important region 1 of this AFP enhancer. Transient transfection experiments performed with human HepG2 hepatoma cells showed that overproduction of ROR alpha 4 stimulated the activity of the AFP enhancer in a dose-dependent manner, while that of Rev-erb alpha and Rev-erb beta had the opposite effect. These effects were highly specific and required the integrity of the AGGTCA motif. The action of these nuclear receptors also occurred in the context of the entire 7-kb regulatory region of the rat AFP gene. These results suggest that altering the amounts or activities of these orphan receptors in cells of hepatic or endodermal origin could modulate AFP gene expression in response to a variety of developmental or carcinogenic stimuli.

Circadian and glucocorticoid regulation of Rev-erbalpha expression in liver.

Rev-erbalpha [NR1D1], a member of the nuclear receptor superfamily, is an orphan receptor that constitutively represses gene transcription. Rev-erbalpha has been shown to play a role in myocyte differentiation and to be induced during adipogenesis. Furthermore, Rev-erbalpha is a regulator of lipoprotein metabolism. It was recently shown that Rev-erbalpha messenger RNA (mRNA) levels oscillate diurnally in rat liver. Here, we report that the circadian rhythm of Rev-erbalpha in liver is maintained in primary cultures of rat hepatocytes. Because glucocorticoids have been shown to regulate other transcription factors with circadian expression, it was furthermore examined whether hepatic Rev-erbalpha expression is also regulated by glucocorticoids. Treatment of rats with dexamethasone resulted in a decrease of Rev-erbalpha mRNA levels by 70% after 6 h. Furthermore, dexamethasone decreased Rev-erbalpha expression in rat primary hepatocytes in a dose-dependent fashion. This effect was mediated by the glucocorticoid receptor because simultaneous addition of the glucocorticoid antagonist RU486 prevented the decrease in Rev-erbalpha mRNA levels by dexamethasone. Protein synthesis inhibition with cycloheximide markedly induced Rev-erbalpha mRNA levels; however, this induction was reduced by dexamethasone supplementation in both rat and human primary hepatocytes. Treatment with actinomycin D blocked the repression of Rev-erbalpha expression by dexamethasone in rat hepatocytes, suggesting that glucocorticoids regulate Rev-erbalpha expression at the transcriptional level. Transient transfection experiments further indicated that Rev-erbalpha promoter activity is repressed by dexamethasone in the presence of cotransfected glucocorticoid receptor. Taken together, these data demonstrate that Rev-erbalpha expression is under the control of both the circadian clock and glucocorticoids in the liver.

Fast evolution of interleukin-2 in mammals and positive selection in ruminants.

Interleukin-2 (IL-2) is a cytokine involved in induction and regulation of the immune response in mammals. There have been numerous reports about the search for IL-2 in species other than mammals, and recently an IL-2-like gene has been isolated in chicken. Using PCR, we searched for IL-2 gene sequences in a wide variety of mammals, including marsupials and monotremes, as well as in birds. Although we can readily amplify IL-2 gene fragments in placental mammals, no amplification was obtained in other species. This is best explained by very high substitution rates. This suggest that strategies to isolate IL-2 homologous genes outside mammals should involve functional assays, as for the chicken gene, and not hybridization-based techniques. Nonsynonymous substitution rates are especially high in ruminants, due to positive selection acting on regions important in term of structure-function. We suggest that, although globally similar, the immune response of various mammals is not identical, mainly at the level of cytokine-mediated regulations.

Candidate gene analysis of thyroid hormone receptors in metamorphosing vs. nonmetamorphosing salamanders.

We used two different experimental approaches to test the hypothesis that thyroid hormone receptor (TR) variation is associated with alternate life cycles modes in ambystomatid salamanders. In the first experiment, the inheritance of TRalpha and TRbeta genotypes was determined for metamorphic and non metamorphic offspring from backcrosses between Ambystoma mexicanum (an obligate metamorphic-failure species) and metamorphic F1 hybrids (A. mexicanum x A. tigrinum tigrinum). The segregation of TR genotype was independent of the expression of life cycle mode phenotype, and neither TR locus was linked to DNA markers that flank a major-effect locus for life cycle mode. In the second experiment, a portion of the ligand-binding domain of TRalpha and TRbeta was cloned and sequenced for DNA samples from 14 different ambystomatid salamander populations, including obligate metamorphic, facultative metamorphic, and obligate metamorphic-failure taxa. Nucleotide sequence variation was found for both TRalpha and TRbeta, with several nonsynonomous substitutions that presumably code for nonconservative amino acid replacements. However, no general relationship was found between TR allelic variation and life cycle mode among populations or species. These data do not implicate TRs as candidate loci involved in the current maintenance or past evolution of alternate life cycle modes in members of the tiger salamander complex.

DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss.

DMRT1 has been suggested to be the first conserved gene involved in sex differentiation found from invertebrates to human. To gain insight on its implication for fish gonadal differentiation, we cloned a DMRT1 homologue in the rainbow trout, Oncorhynchus mykiss (rtDMRT1), and showed that this gene is expressed during testicular differentiation, but not during ovarian differentiation. After 10 days of steroid treatment, expression was shown to be decreased in estrogen-treated male differentiating gonads but not to be restored in androgen-treated differentiating female gonads. This clearly reinforces the hypothesis of an important implication for DMRT1 in testicular differentiation in all vertebrates. In the adults a single 1.5 kb transcript was detected by Northern blot analysis in the testis, and its expression was found to be sustained throughout spermatogenesis and declined at the end of spermatogenesis (stage VI). Along with this expression in the testis we also detected by reverse transcriptase-polymerase chain reaction a slight expression in the ovary. We also obtained new DM-domain homologous sequences in fish, and their analysis suggest that at least four different genes bearing 'DM-domain' (DMRT genes) exist in fish just as in all vertebrate genomes.