An inherited functional circadian clock in zebrafish embryos.

Circadian clocks are time-keeping systems found in most organisms. In zebrafish, expression of the clock gene Period3 (Per3) oscillates throughout embryogenesis in the central nervous system and the retina. Per3 rhythmic expression was free-running and was reset by light but not by the developmental delays caused by low temperature. The time of fertilization had no effect on Per3 expression. Per3 messenger RNA accumulates rhythmically in oocytes and persists in embryos. Our results establish that the circadian clock functions during early embryogenesis in zebrafish. Inheritance of maternal clock gene products suggests a mechanism of phase inheritance through ovogenesis.

Nuclear receptors. Lonesome orphans.

Nuclear receptors typically bind to DNA as homodimers or heterodimers, but increasing numbers of orphan receptors have been identified that bind DNA as monomers.

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.

FTZ-F1-related orphan receptors in Xenopus laevis: transcriptional regulators differentially expressed during early embryogenesis.

Orphan receptors of the FTZ-F1-related group of nuclear receptors (xFF1r) were identified in Xenopus laevis by isolation of cDNAs from a neurula stage library. Two cDNAs were found, which encode full length, highly related receptor proteins, xFF1rA and B, whose closet relative known so far is the murine LRH-1 orphan receptor. xFF1rA protein expressed by a recombinant vaccinia virus system specifically binds to FTZ-F1 response elements (FRE; PyCAAGGPyCPu). In cotransfection studies, xFF1rA constitutively activates transcription, in a manner dependent on the number of FREs. The amounts of at least four mRNAs encoding full-length receptors greatly increase between gastrula and early tailbud stages and decrease at later stages. At early tailbud stages, xFTZ-F1-related antigens are found in all nuclei of the embryo.

Rel/NF-kappa B transcription factors and I kappa B inhibitors: evolution from a unique common ancestor.

From the sequences of Rel/NF-kappa B and I kappa B proteins, we constructed an alignment of their Rel Homology Domain (RHD) and ankyrin repeat domain. Using this alignment, we performed tree reconstruction with both distance matrix and parsimony analysis and estimated the branching robustness using bootstrap resampling methods. We defined four subfamilies of Rel/NF-kappa B transcription factors: (i) cRel, RelA, RelB, Dorsal and Dif; (ii) NF-kappa B1 and NF-kappa B2; (iii) Relish and (iv) NF-AT factors, the most divergent members. Subfamilies I and II are clustered together whereas Relish diverged earlier than other Rel/NF-kappa B proteins. Three subfamilies of I kappa B inhibitors were also defined: (i) NF-kappa B1 and NF-kappa B2; (ii) close to subfamily I, the short I kappa B proteins I kappa B alpha, I kappa B beta and Bcl-3; (iii) Relish that diverged earlier than other I kappa B inhibitors. Our definition of groups and subfamilies fits to structural and functional features of the Rel/NF-kappa B and I kappa B proteins. We also showed that ankyrin repeats of NF-kappa B1, NF-kappa B2 and Relish are short I kappa B-specific ankyrin motifs. These proteins defining a link between Rel/NF-kappa B and I kappa B families, we propose that all these factors evolved from a common ancestral RHD-ankyrin structure within a unique superfamily, explaining the specificities of interaction between the different Rel/NF-kappa B dimers and the various I kappa B inhibitors.

TATA-less promoters of some Ets-family genes are efficiently repressed by wild-type p53.

p53 has been reported to repress a number of TATA-containing promoters in transient transfection assays. TATA-less promoters are generally believed to be refractive to p53 repression. We report here that the TATA-less promoters of Ets-family genes (Ets-1 and Ets-2) are efficiently repressed by wild-type but not mutant p53 in transient co-transfection assays. Moreover, p53 was immunologically detected in protein complexes formed on oligonucleotides from both the TATA-containing and TATA-less promoters. Our data suggest that p53 is involved in the regulation of the expression of both promoter types, most probably by protein-protein interaction. A model for p53 function in promoter repression is proposed.

Activation of the thyroid hormone receptor alpha gene promoter by the orphan nuclear receptor ERR alpha.

The superfamily of nuclear receptors comprises transcription factors that depend on a ligand for their activity. In addition, the superfamily includes a number of orphan receptors, for which no ligand is known. We report here that the orphan receptor estrogen receptor related alpha receptor (ERR alpha) stimulates the expression of the thyroid hormone receptor alpha (TR alpha) gene promoter. We characterized a responsive site that is both necessary and sufficient for ERR alpha-induced transactivation. In addition, we show that both TR alpha and ERR alpha are coexpressed in embryonic intestine, brown fat and heart as well as in the adult gonads. In the testis, expression of both receptors can be found in the seminiferous tubes where it is totally restricted to spermatocytes I. Altogether this suggests that TR alpha is an in vivo target of ERR alpha.

Characterization of a functional promoter for the human thyroid hormone receptor alpha (c-erbA-1) gene.

The thyroid hormone receptor alpha (THRA or c-erbA-1) gene belongs to a family of genes that encode nuclear receptors for various hydrophobic ligands such as steroids, retinoic acid and thyroid hormones. We have previously described the genomic organization of the human THRA gene, which comprises 10 exons distributed along 27 kbp of genomic DNA. We describe here a promoter that initiates THRA transcription. This promoter contains no obvious TATA-like element but is very GC rich and harbors numerous Sp1 sites. It also contains several sites similar to previously described cis-acting sequences including hormone-responsive elements (HREs). When transfected into cultured HeLa cells, it drives the expression of a CAT reporter gene. The activity of this human THRA promoter is enhanced by the synthetic glucocorticoid dexamethasone but seems unaffected by thyroid hormones.

Molecular phylogeny of the ETS gene family.

We have constructed a molecular phylogeny of the ETS gene family. By distance and parsimony analysis of the ETS conserved domains we show that the family containing so far 29 different genes in vertebrates can be divided into 13 groups of genes namely ETS, ER71, GABP, PEA3, ERG, ERF, ELK, DETS4, ELF, ESE, TEL, YAN, SPI. Since the three dimensional structure of the ETS domain has revealed a similarity with the winged-helix-turn-helix proteins, we used two of them (CAP and HSF) to root the tree. This allowed us to show that the family can be divided into five subfamilies: ETS, DETS4, ELF, TEL and SPI. The ETS subfamily comprises the ETS, ER71, GABP, PEA3, ERG, ERF and the ELK groups which appear more related to each other than to any other ETS family members. The fact that some members of these subfamilies were identified in early metazoans such as diploblasts and sponges suggests that the diversification of ETS family genes predates the diversification of metazoans. By the combined analysis of both the ETS and the PNT domains, which are conserved in some members of the family, we showed that the GABP group, and not the ERG group, is the one most closely related to the ETS group. We also observed that the speed of accumulation of mutations in the various genes of the family is highly variable. Noticeably, paralogous members of the ELK group exhibit strikingly different evolutionary speed suggesting that the evolutionary pressure they support is very different.

Cooperative effect of v-myc and v-erbA in the chick embryo.

We show that a construct designated as MAHEVA, which encodes oncogenes v-myc from MH2 virus and v-erbA from AEV under the control of the LTR of MH2, induces rapidly growing heart rhabdomyosarcomas, when it is injected in E3 but not E5 chick embryos. A similar pathology has previously been observed with MC29, within the same limited time frame. The tumors, which expressed P61-63myc, P75gag-erbA and Pr76gag proteins were detectable from E14 onwards. Compared with MC29, MAHEVA induced a secondary anomaly, not detectable prior to E17. This is the appearance of cartilage nodules within the heart rhabdomyosarcomas. The constant location of these nodules inside the rhabdomyosarcomas and their delayed appearance suggests that the chondrocytes originate from myoblasts prevented from differentiating by the expression of the v-myc product. This interpretation is supported by the appearance of chondrocytes in E3 heart muscle cells infected in vitro with MAHEVA.

Overexpression of c-erbA proto-oncogene enhances myogenic differentiation.

Triiodothyronine (T3) positively regulates both the expression of the MyoD gene, a key myogenic regulator, and C2 muscle cell differentiation. To directly examine the role of its nuclear receptors in the control of myogenesis, we introduced a c-erbA expression vector into C2 muscle cells by transient or stable transfection. Our results show that c-erbA can play a potent role in the triggering of muscle terminal differentiation since its overexpression leads to: (1) a complete abrogation of the activity of the myogenesis inhibitor AP-1 (fos/jun) transcription factor; (2) an enhanced induction of MyoD expression upon T3 treatment; (3) the acquisition by T3 of the ability to trigger both growth arrest and terminal differentiation in the presence of large amounts of serum mitogens, a property that is otherwise specific to retinoic acid (RA). Thus, c-erbA is one of the two protooncogenes (with c-ski) that acts as positive regulator of muscle differentiation. Furthermore, the fact that c-erbA overexpression allows T3 to largely mimic the RA effects indicates that their biological differences in the modulation of myogenic program primarily rely on the differential expression of their receptors in C2 muscle cells rather than on an intrinsic specificity of their target genes.

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.

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.

The complete nucleotide sequence of the mitochondrial DNA of the dogfish, Scyliorhinus canicula.

We have determined the complete nucleotide sequence of the mitochondrial DNA (mtDNA) of the dogfish, Scyliorhinus canicula. The 16,697-bp-long mtDNA possesses a gene organization identical to that of the Osteichthyes, but different from that of the sea lamprey Petromyzon marinus. The main features of the mtDNA of osteichthyans were thus established in the common ancestor to chondrichthyans and osteichthyans. The phylogenetic analysis confirms that the Chondrichthyes are the sister group of the Osteichthyes.

Expression of the estrogen-related receptor 1 (ERR-1) orphan receptor during mouse development.

We studied the expression of the estrogen-related receptor 1 (ERR-1) during mouse embryonic development. ERR-1 is expressed at very early stages in ES cells and at E8.5 in the mesodermal cells of the visceral yolk sac. ERR-1 continues to be expressed later in mesodermal tissues and particularly in heart and in skeletal muscles. This expression persists during all the embryonic development and in adult stage. ERR-1 transcripts level increases during muscle differentiation. Accordingly, we show that ERR-1 expression increases during the myoblast to myotube transition in differentiating C2 myoblastic cells. ERR-1 has also been detected in the nervous system during embryonic development. At E10.5, a high level of ERR-1 transcripts can be observed in differentiated cells of the intermediate zone of the spinal cord which also suggests a role of ERR-1 in the differentiation of the nervous system. The same is observed in the telencephalon vesicules at E13.5. Later, at E15.5 and E17.5, expression persists in the spinal cord but decreases dramatically in the central nervous system. Moreover, ERR-1 expression increases during skin formation and is detected in the stratum spinosum which contains differentiated Malpighian cells. Finally, we also observed ERR-1 in endodermal derivatives such as the epithelium of intestine and urogenital system. The DNA target of ERR-1 has been identified to be the SF-1/FTZ-F1 responsive element (SFRE) and we show in this paper that SF-1/FTZ-F1 and ERR-1 bind to and activate transcription independently through the SFRE element. Our study suggests that ERR-1 may be implicated in numerous physiological or developmental functions, particularly in the muscle, the central and peripheral nervous system and the epidermis. Interestingly, in these various systems ERR-1 expression is correlated with post-mitotic cells stage, suggesting that ERR-1 may play a role in the differentiation process.

Identification and characterization of a novel corepressor interaction region in RVR and Rev-erbA alpha.

Rev-erbA alpha and RVR are orphan nuclear receptors that function as dominant transcriptional silencers. Ligand-independent repression of transcription by Rev-erbA alpha and RVR is mediated by the nuclear receptor corepressors, N-CoR and its variants RIP (RXR interacting protein) 13a and RIP13 delta 1. The physical association between the corepressors and Rev-erbA alpha and RVR is dependent on the presence of a receptor interaction domain (RID) in the N-CoR family. Our previous study demonstrated that the E region of RVR and Rev-erbA alpha is necessary and sufficient for the in vivo interaction with the nuclear receptor corepressor, RIP13 delta 1. The present investigation demonstrates that two corepressor interaction regions, CIR-1 and CIR-2, separated by approximately 150 amino acids in the E region of RVR, are required for the interaction with N-CoR, RIP13a, and RIP13 delta A. The D region is not required for the physical interaction. In contrast, the D and E regions of Rev-erbA alpha were necessary for the interaction with the N-CoR and RIP13a-RIDs in vivo, suggesting that RIP13 delta 1 and N-CoR/RIP13a differentially interact with Rev-erbA alpha. Mutagenesis of CIR-1, a novel domain that is highly conserved between RVR and Rev-erbA alpha, demonstrated that the N-terminal portion of helix 3 plays a key role and is absolutely necessary for the interaction with RIP13 delta 1, RIP13a, and N-CoR. The phenylalanine residues, F402 and F441, in RVR and Rev-erbA alpha, respectively, were critical residues in supporting corepressor interaction. Cotransfection studies demonstrated that repression of a physiological target, the human Rev-erbA alpha promoter, by RVR was significantly impaired by mutation of CIR-1 or deletion of CIR-2. Furthermore, overexpression of either the N-CoR/RIP13a or RIP13 delta 1-RIDs alleviated RVR-mediated repression of the Rev-erbA alpha promoter, demonstrating that corepressor binding mediates the repression of a native target gene by RVR. A minimal region containing juxtapositioned CIR-1 and CIR-2 was sufficient for corepressor binding and transcriptional repression. In conclusion, our study has identified a new corepressor interaction region, CIR-1, in the N terminus of helix 3 in the E region of RVR and Rev-erbA alpha, that is required for transcriptional silencing. Furthermore, we provide evidence that CIR-1 and CIR-2 may form a single corepressor interaction interface.

3,5,3'-Triiodothyronine positively regulates both MyoD1 gene transcription and terminal differentiation in C2 myoblasts.

Thyroid hormones are among the positive regulators of muscle development in vivo, but little is known about the way they work. We demonstrate here that MyoD1, one of the master genes controlling myogenesis, is a target of T3. After proliferating C2 myoblasts have been treated with T3 for 15 h, we observed a rise in MyoD1 expression at both the mRNA and protein levels. This is the first positive hormonal control of MyoD1 gene expression reported so far. We also provide data which suggest that T3 nuclear receptor(s) have a direct role on MyoD1 gene transcription: 1) C2 cells express the alpha 1 form of T3 nuclear receptors; 2) T3 up-regulates MyoD1 gene transcription and does not affect MyoD1 mRNA stability, as demonstrated by run-on and actinomycin D chase experiments, respectively; and 3) this transcriptional activation does not need the synthesis of intermediate protein(s) since it is not abolished by simultaneous treatment with cycloheximide. Moreover, in presence of T3, the increase of MyoD1 transcripts is associated with a faster terminal differentiation. Indeed we observed an earlier expression of various markers of myogenesis including myogenin (a regulatory gene of the MyoD1 family mainly involved in the triggering of terminal differentiation), myosin light chain 1A, and troponin T in T3-treated cells vs. untreated cells. We suggest that the regulation of a pivotal myogenic gene could be an important step in the control exerted by T3 on muscle development in vivo.

Identification of transcripts initiated from an internal promoter in the c-erbA alpha locus that encode inhibitors of retinoic acid receptor-alpha and triiodothyronine receptor activities.

The thyroid hormone receptor-coding locus, c-erbA alpha, generates several mRNAs originating from a single primary transcript that undergoes alternative splicing. We have identified for the first time two new transcripts, called TRdelta alpha1 and TRdelta alpha2 [mRNA for isoform alpha1 and alpha2 of the T3 receptor (TR), respectively], whose transcription is initiated from an internal promoter located within intron 7 of the c-erbA alpha gene. These two new transcripts exhibit tissue-specific patterns of expression in the mouse. These two patterns are in sharp contrast with the expression patterns of the full-length transcripts generated from the c-erbA alpha locus. TR alpha1 and TRdelta alpha2 mRNAs encode N-terminally truncated isoforms of T3R alpha1 and T3R alpha2, respectively. The protein product of TRdelta alpha1 antagonizes the transcriptional activation elicited by T3 and retinoic acid. This protein inhibits the ligand-induced activating functions of T3R alpha1 and 9-cis-retinoic acid receptor-alpha but does not affect the retinoic acid-dependent activating function of retinoic acid receptor-alpha. We predict that these truncated proteins may work as down-regulators of transcriptional activity of nuclear hormone receptors in vivo.

Transcriptional activities of the orphan nuclear receptor ERR alpha (estrogen receptor-related receptor-alpha).

Estrogen receptor-related receptor alpha (ERR alpha) is an orphan nuclear receptor closely related to the estrogen receptor (ER), whose expression covers various stages of embryonic development and persists in certain adult tissues. We show that ERR alpha binds as a homodimer on a specific target sequence, the SFRE (SF-1 response element), already known to respond to the orphan nuclear receptor SF-1. Target sequences that are related to the SFRE and that discriminate between ERR alpha and SF-1 were identified. We have also analyzed the transcriptional properties of the ERR alpha originating from various species. All ERR alpha orthologs act as potent transactivators through the consensus SFRE. ERR alpha activity depends on the putative AF2AD domain, as well as on a serum compound that is withdrawn by charcoal treatment, suggesting the existence of a critical regulating factor brought by serum.

The ERR-1 orphan receptor is a transcriptional activator expressed during bone development.

We studied the expression of estrogen-related receptor ERR-1 during mouse embryonic development. ERR-1 mRNA is present in bones formed by both the endochondral and intramembranous routes, and the onset of its expression coincides with bone formation. By RT-PCR experiments, we found that ERR-1, but not the related receptor ERR-2, is expressed in osteoblastic osteosarcoma cell lines as well as in primary osteoblastic cell populations derived from normal human bone. By gel shift analysis we found that ERR-1 binds as a monomer specifically to the SFRE sequence (SF-1-responsive-element; TCAAGGTCA). Mutation analysis revealed that both the core AGGTCA motif and the TCA 5'-extension are required for efficient ERR-1 binding. In transient transfection assays, ERR-1 acts as a potent transactivator through the SFRE sequence. This effect is cell-specific since ERR-1 activates transcription in the rat osteosarcoma cell line ROS 17.2/8 as well as in HeLa, NB-E, and FREJ4 cells but not in COS1 and HepG2 cells. Notably, the osteopontin (a protein expressed by osteoblasts and released in the bone matrix) gene promoter is a target for ERR-1 transcriptional regulation. Our findings suggest a role for ERR-1 in bone development and metabolism.