Vertebrate Nuclear Receptors Research Articles

Methods for assessing the interaction of apocarotenoids with vertebrate nuclear receptors.

Apocarotenoids occur in the diets of human and other vertebrates and form from the metabolism of intact carotenoids. The metabolism of the provitamin A carotenoid, β-carotene, produces β-apo-15-carotenoic acid or retinoic acid. Retinoic acid functions as a hormone by binding to and activating nuclear retinoic acid receptors to modulate gene transcription. It is likely that other apocarotenoids may similarly modulate other ligand-activated nuclear receptors. This chapter describes in vitro biochemical and biophysical methods to characterize the direct binding of apocarotenoids to nuclear receptors and to assess their effect on binding of transcriptional coactivators to the receptor. It also provides a few widely used methods to study the consequences of nuclear receptor activation in intact cells. The effects of β-apocarotenoids on retinoid receptors provide examples.

Hormone-dependent activation and repression of microRNAs by the ecdysone receptor in the dengue vector mosquito Aedes aegypti

Female mosquitoes transmit numerous devastating human diseases because they require vertebrate blood meal for egg development. MicroRNAs (miRNAs) play critical roles across multiple reproductive processes in female Aedes aegypti mosquitoes. However, how miRNAs are controlled to coordinate their activity with the demands of mosquito reproduction remains largely unknown. We report that the ecdysone receptor (EcR)-mediated 20-hydroxyecdysone (20E) signaling regulates miRNA expression in female mosquitoes. EcR RNA-interference silencing linked to small RNA-sequencing analysis reveals that EcR not only activates but also represses miRNA expression in the female mosquito fat body, a functional analog of the vertebrate liver. EcR directly represses the expression of clustered miR-275 and miR-305 before blood feeding when the 20E titer is low, whereas it activates their expression in response to the increased 20E titer after a blood meal. Furthermore, we find that SMRTER, an insect analog of the vertebrate nuclear receptor corepressors SMRT and N-CoR, interacts with EcR in a 20E-sensitive manner and is required for EcR-mediated repression of miRNA expression in Ae. aegypti mosquitoes. In addition, we demonstrate that miR-275 and miR-305 directly target glutamate semialdehyde dehydrogenase and AAEL009899, respectively, to facilitate egg development. This study reveals a mechanism for how miRNAs are controlled by the 20E signaling pathway to coordinate their activity with the demands of mosquito reproduction.

The nuclear receptors of Biomphalaria glabrata and Lottia gigantea: implications for developing new model organisms.

Nuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.

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.

Expression and retinoic acid regulation of the zebrafish nr2f orphan nuclear receptor genes

The vertebrate nuclear receptor subfamily 2, group f (nr2f) genes encode orphan receptors that have the capacity to act as negative regulators of retinoic acid (RA) signaling. We describe embryonic and larval expression of four of the six zebrafish nr2f genes, nr2f1a, nr2f1b, nr2f2, and nr2f5. These genes show highly regulated patterns of expression within the central nervous system, including in the developing hindbrain, as well as in the mesoderm and endoderm. We also investigated the role of RA and fibroblast growth factor (Fgf) signaling in regulating early nr2f gene expression. RA is not required for nr2f expression in the hindbrain; however, exogenous RA can repress this expression. Conversely, we find that RA positively regulates nr2f1a expression in trunk endoderm and mesoderm. Fgf signaling is not required for nr2f expression onset in the hindbrain; however, it may play a role in maintaining rhombomere-specific expression. We report detailed expression analysis of four nr2f genes in all three germ layers. The onset of nr2f expression in the hindbrain does not require RA or Fgf signals. Our finding that RA positively regulates nr2f1a expression in the trunk supports the possibility that Nr2fs function in a negative feedback loop to modulate RA signaling in this region.

Physical association of the WC-1 photoreceptor and the histone acetyltransferase NGF-1 is required for blue light signal transduction in Neurospora crassa

In Neurospora crassa and other filamentous fungi, light-dependent-specific phenomena are regulated by transcription factors WC-1 and WC-2. In addition to its transcriptional activity, WC-1 is able to directly sense light stimuli through a LOV sensor domain. Its location in the nucleus and heterodimerization with WC-2, together with the presence of a zinc-finger DNA-binding domain and an environmental sensor domain, all resemble the functional evolutionary architecture adopted by vertebrate nuclear receptors (NRs). Here we describe a scenario in which WC-1 represents a functional orthologue of NRs and acts through association with the chromatin-modifying coactivator NGF-1, which encodes a homologue of the yeast Gcn5p acetyltransferase. To support this view, we show a direct association between WC-1 and NGF-1 that depends on a WC-1 region containing a conserved functional LXXLL motif, a signature previously described as being an exclusive feature of NR/coactivator interaction. Our data suggest that a WC-1/NGF-1 complex is preassembled in the dark on light-inducible promoters and that, after exposure to light stimulation, NGF-1-associated HAT activity leads to histone H3 acetylation and transcriptional activation. Finally, we provide evidence for a NGF-1-independent acetylated form of WC-1. Overall our data indicate that Neurospora and higher eukaryotes share a common mechanism for the signal transduction of environmental stimuli.

Drosophila DHR38 nuclear receptor is required for adult cuticle integrity at eclosion

DHR38 is the only Drosophila member of the NR4A subclass of vertebrate nuclear receptors, which have been implicated in multiple biological pathways, including neuronal function, apoptosis, and metabolism. Although an earlier study identified three point mutations in DHR38, none of these were shown to be a null allele for the locus, leaving it unclear whether a complete loss of DHR38 function might uncover novel roles for the receptor. Here we show that a specific DHR38 null allele, DHR38(Y214), leads to fully penetrant pharate adult lethality, similar to the most severe phenotype associated with the EMS-induced mutations. DHR38(Y214) mutants display minor effects on ecdysone-regulated transcription at the onset of metamorphosis. In contrast, cuticle gene expression is significantly reduced in DHR38(Y214) mutant pupae. These studies define the essential functions of DHR38 and provide a genetic context for further characterization of its roles during development.

Mediator Subunit Gal11p/MED15 Is Required for Fatty Acid-dependent Gene Activation by Yeast Transcription Factor Oaf1p

The yeast zinc cluster transcription factor Oaf1p activates transcription of target genes in response to direct binding of fatty acids in a manner analogous to the vertebrate nuclear receptor peroxisome proliferator-activated receptoralpha (PPARalpha). PPARs and other metazoan nuclear receptors productively engage several distinct LXXLL motif-containing co-activators, including p160 family members and the TRAP220/MED1 subunit of the Mediator co-activator, to promote ligand-dependent gene activation. Yeast, however, does not appear to harbor LXXLL motif co-activators, and the mechanism of fatty acid-dependent gene activation by the yeast PPARalpha analog Oaf1p is unknown. Here we show that the yeast Mediator subunit Gal11p/MED15 and its activator-targeted KIX domain plays a critical role in fatty acid-dependent transcriptional regulation of fatty acid beta-oxidation and peroxisomal genes by Oaf1p and for the ability of yeast to utilize fatty acids as a sole carbon source. Moreover, structural studies by NMR spectroscopy reveal that the Oaf1p activation domain interacts with the Gal11p/MED15 KIX domain in a manner similar to the yeast zinc cluster family member and xenobiotic receptor Pdr1p, revealing that the Gal11p/MED15 KIX domain is a key target of several ligand-dependent transcription factors in yeast. Together with previous work showing that the Caenorhabditis elegans Gal11p/MED15 homolog MDT-15 plays a critical role in regulation of fatty acid metabolism by the nematode PPAR-like nuclear receptor NHR-49, the findings presented here provide evidence for an ancient and essential role of a Mediator co-activator subunit in regulation of fatty acid metabolism by nuclear receptor-like transcription factors in eukaryotes.

Gene duplication, gene loss and evolution of expression domains in the vertebrate nuclear receptor NR5A (Ftz-F1) family

Fushi tarazu factor 1 (Ftz-F1, NR5A) is a zinc-finger transcription factor that belongs to the nuclear receptor superfamily and regulates genes that are involved in sterol and steroid metabolism in gonads, adrenals, liver and other tissues. To understand the evolutionary origins and developmental genetic relationships of the Ftz-F1 genes, we have cloned four homologous Ftz-f1 genes in zebrafish, called ff1a, ff1b, ff1c and ff1d. These four genes have different temporal and spatial expression patterns during development, indicating that they have distinct mechanisms of genetic regulation. Among them, the ff1a expression pattern is similar to mammalian Nr5a2, while the ff1b pattern is similar to that of mammalian Nr5a1. Genetic mapping experiments show that these four ff1 genes are located on chromosome segments conserved between the zebrafish and human genomes, indicating a common ancestral origin. Phylogenetic and conserved synteny analysis show that ff1a is the orthologue of NR5A2, and that ff1b and ff1d genes are co-orthologues of NR5A1 that arose by a gene-duplication event, probably a whole-genome duplication, in the ray-fin lineage, and each gene is located next to an NR6A1 co-orthologue as in humans, showing that the tandem duplication occurred before the divergence of human and zebrafish lineages. ff1c does not have a mammalian counterpart. Thus we have characterized the phylogenetic relationships, expression patterns and chromosomal locations of these Ftz-F1 genes, and have demonstrated their identities as NR5A genes in relation to the orthologous genes in other species.

Flies Reveal New Nuclear Receptor Signaling Mechanism

Ecdysteroids are hormones with important roles in regulating development and metamorphosis in insects. Like mammalian steroid hormones, they act to regulate transcription through a nuclear receptor, the ecdysone receptor (EcR). The EcR forms heterodimers with Ultraspiracle (USP), which is related to another nuclear receptor family member, the vertebrate retinoid X receptor (RXR). However, the EcR appears not to mediate all effects of ecdysone and related ecdysteroids found in insect hemolymph. Baker et al. therefore examined the function of DHR38, an insect nuclear receptor that heterodimerizes with USP. The authors constructed a reporter system that used the better-characterized RXR as a partner in place of USP and showed that transcriptional activity of the complex was activated in response to various ecdysteroids. Activation of complexes of DHR38 with RXR required ligand-induced activation of both receptors, and the same appeared to be true of complexes with USP. Particularly surprising were experiments showing that DHR38, although responsive to ecdysteroids, appears not to bind the ligands directly. An X-ray crystal structure of the protein confirmed that the ligand-binding pocket and the coactivator binding sites normally conserved in nuclear receptors are unlikely to function in DHR38. The authors speculate that a cofactor acting with the receptors at sites of transcriptional activation might confer sensitivity to ecdysteroids. Because DHR38 is related to a subfamily of mammalian orphan nuclear receptors (the NGFI-B subfamily), the new paradigm for activation revealed by DHR38 in flies may be relevant to signaling by vertebrate nuclear receptors as well.K. D. Baker, L. M. Shewchuk, T. Kozlova, M. Makishima, A. Hassell, B. Wisely, J. A. Caravella, M. H. Lambert, J. L. Reinking, H. Krause, C. S. Thummel, T. M. Willson, D. J. Mangelsdorf, The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway. Cell 113, 731-742 (2003). [Online Journal]

Flies Reveal New Nuclear Receptor Signaling Mechanism

Ecdysteroids are hormones with important roles in regulating development and metamorphosis in insects. Like mammalian steroid hormones, they act to regulate transcription through a nuclear receptor, the ecdysone receptor (EcR). The EcR forms heterodimers with Ultraspiracle (USP), which is related to another nuclear receptor family member, the vertebrate retinoid X receptor (RXR). However, the EcR appears not to mediate all effects of ecdysone and related ecdysteroids found in insect hemolymph. Baker et al . therefore examined the function of DHR38, an insect nuclear receptor that heterodimerizes with USP. The authors constructed a reporter system that used the better-characterized RXR as a partner in place of USP and showed that transcriptional activity of the complex was activated in response to various ecdysteroids. Activation of complexes of DHR38 with RXR required ligand-induced activation of both receptors, and the same appeared to be true of complexes with USP. Particularly surprising were experiments showing that DHR38, although responsive to ecdysteroids, appears not to bind the ligands directly. An X-ray crystal structure of the protein confirmed that the ligand-binding pocket and the coactivator binding sites normally conserved in nuclear receptors are unlikely to function in DHR38. The authors speculate that a cofactor acting with the receptors at sites of transcriptional activation might confer sensitivity to ecdysteroids. Because DHR38 is related to a subfamily of mammalian orphan nuclear receptors (the NGFI-B subfamily), the new paradigm for activation revealed by DHR38 in flies may be relevant to signaling by vertebrate nuclear receptors as well. K. D. Baker, L. M. Shewchuk, T. Kozlova, M. Makishima, A. Hassell, B. Wisely, J. A. Caravella, M. H. Lambert, J. L. Reinking, H. Krause, C. S. Thummel, T. M. Willson, D. J. Mangelsdorf, The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway. Cell 113 , 731-742 (2003). [Online Journal]

Direct repeats bind the EcR/USP receptor and mediate ecdysteroid responses in Drosophila melanogaster.

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional through binding to palindromic ecdysteroid mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with the receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transformation assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.

Relationship between Drosophila gap gene tailless and a vertebrate nuclear receptor Tlx.

We report here the identification of a unique vertebrate nuclear receptor, Tlx, which is expressed exclusively in the neuroepithelium of the embryonic brain. Sequence comparison reveals striking similarity to the product of the Drosophila terminal/gap gene tailless (tll), which is expressed in the embryonic brain and is required for brain development in flies. In vitro DNA-binding assays demonstrated that Tlx and Tll proteins share a target gene specificity that is unique among the nuclear receptor superfamily. Ectopic expression of Tlx in fly embryos caused a repression of segmentation comparable to that elicited by Tll. The similarities in structure, expression pattern, target gene specificity and phenotypes in transgenic flies suggest conservation of genetic programs upstream and downstream of this Tlx/Tll class of nuclear receptors during embryogenesis.

Homology of the ligand-binding regions of Rhizobium symbiotic regulatory protein NodD and vertebrate nuclear receptors

The signal specificity and structure of sensor-activator proteins from different species (NodD of Rhizobium bacteria and vertebrate nuclear receptors) were compared. Several compounds (including flavonoids, coumestrol and estradiol) that bind to mammalian receptors also interact with NodD proteins. NodD-dependent synergism of the signal compounds luteolin and catechin was observed suggesting that these compounds bind directly to NodD. Two regions comprising 63 and 37 amino acids in NodD showed 45% and 36% homology, respectively, with the estrogen receptor. These regions, designated as modules M1 and M2, coincide with conserved parts of the ligand-binding domains of the nuclear receptors. A part of NodD overlapping with the M1 module was predicted to be membrane associated and was 46% homologous to a membrane-spanning sensory segment of the Agrobacterium VirA protein. We suggest that the homologous polypeptide modules detected in NodD and the nuclear receptors originate from a common ancestor protein and may be directly involved in ligand binding.