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BMC Evol Biol
2012 Jun 12;12:81. doi: 10.1186/1471-2148-12-81.
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Analysis of C. elegans NR2E nuclear receptors defines three conserved clades and ligand-independent functions.
Weber KP
,
Alvaro CG
,
Baer GM
,
Reinert K
,
Cheng G
,
Clever S
,
Wightman B
.
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BACKGROUND: The nuclear receptors (NRs) are an important class of transcription factors that are conserved across animal phyla. Canonical NRs consist of a DNA-binding domain (DBD) and ligand-binding domain (LBD). While most animals have 20-40 NRs, nematodes of the genus Caenorhabditis have experienced a spectacular proliferation and divergence of NR genes. The LBDs of evolutionarily-conserved Caenorhabditis NRs have diverged sharply from their Drosophila and vertebrate orthologs, while the DBDs have been strongly conserved. The NR2E family of NRs play critical roles in development, especially in the nervous system. In this study, we explore the phylogenetics and function of the NR2E family of Caenorhabditis elegans, using an in vivo assay to test LBD function.
RESULTS: Phylogenetic analysis reveals that the NR2E family of NRs consists of three broadly-conserved clades of orthologous NRs. In C. elegans, these clades are defined by nhr-67, fax-1 and nhr-239. The vertebrate orthologs of nhr-67 and fax-1 are Tlx and PNR, respectively. While the nhr-239 clade includes orthologs in insects (Hr83), an echinoderm, and a hemichordate, the gene appears to have been lost from vertebrate lineages. The C. elegans and C. briggsae nhr-239 genes have an apparently-truncated and highly-diverged LBD region. An additional C. elegans NR2E gene, nhr-111, appears to be a recently-evolved paralog of fax-1; it is present in C. elegans, but not C. briggsae or other animals with completely-sequenced genomes. Analysis of the relatively unstudied nhr-111 and nhr-239 genes demonstrates that they are both expressed--nhr-111 very broadly and nhr-239 in a small subset of neurons. Analysis of the FAX-1 LBD in an in vivo assay revealed that it is not required for at least some developmental functions.
CONCLUSIONS: Our analysis supports three conserved clades of NR2E receptors, only two of which are represented in vertebrates, indicating three ancestral NR2E genes in the urbilateria. The lack of a requirement for a FAX-1 LBD suggests that the relatively high level of sequence divergence for Caenorhabditis LBDs reflects relaxed selection on the primary sequence as opposed to divergent positive selection. This observation is consistent with a model in which divergence of some Caenorhabditis LBDs is allowed, at least in part, by the absence of a ligand requirement.
Figure 1. Phylogenetic analysis of NR2E NRs. Maximum Likelihood trees of ClustalW-aligned amino acid sequences were calculated using MEGA 5.0 [35,36]. Highly-divergent regions N-terminal to the DBD and C-terminal to the LBD were not included. A consensus tree generated from 500 bootstrap replicates and rooted to the Homo sapiens retinoic acid receptor gamma outgroup sequence is shown. Further details are provided in Methods. Species abbreviations (used throughout): Ce (or no annotation):Caenorhabditis elegans, Cb: Caenorhabditis briggsae, Bm: Brugia malayi, Pp: Pristionchus pacificus, Dm: Drosophila melanogaster, Ag: Anopheles gambiae, Tc: Tribolium castaneum, Sk: Saccoglossus kowalevskii, Sp: Strongylocentrotus purpuratus, Dr: Danio rerio, Xl: Xenopus laevis, Mm: Mus musculus, Gg: Gallus gallus, Hs: Homo sapiens, Nv: Nematostella vectensis, Aa: Aedes aegypti. Tree is proportionally scaled, with the scale bar indicating sequence distance in units of substitutions. Single diamonds identify branchpoints that were supported by >50 % of bootstrap replicates; double diamonds identify branchpoints that were supported by >95 % of bootstrap replicates. Numbers in red above selected branches show Ka/Ks ratios for the LBD portion only of NRs.
Figure 2. Alignments of NR2E DBDs. Annotated alignments of NR2E DBDs based on ClustalW multiple alignments as described in Methods. The reference sequence of the human retinoic acid receptor gamma subunit is shown at the top. The red box identifies the P box, which plays a critical role in DNA binding site specificity. The blue box identifies the D box, which functions in protein dimerization. Underlined amino acid positions identify points of contact between RAR/RXR heterodimers and DNA bases, and asterisks identify protein dimerization contacts in structural studies [46]. Color scheme distinguishes ecdysozoans (black), non-vertebrate deuterostomes (red), and vertebrates (blue). Vertical boxes identify amino acids common to a particular clade (TLL/TLX, PNR/FAX-1, Hr83/NHR239). Species abbreviations are given in Figure 1. The Brugia malayi sequence is derived from separate candidate coding sequences that have not been assembled into a gene model or confirmed as an expressed gene. The C-terminal portion of the predicted Pristionchus pacificus NHR-239 ortholog was not included since it aligned very poorly and it is unclear whether this reflects true divergence, incorrect gene assembly, or a pseudogene. It is also possible that the B. malayi ortholog shown here is actually a pseudogene, since it has not been confirmed by a cDNA.
Figure 3. C. elegans nhr-111 and nhr-239 are expressed genes. The nhr-111 and nhr-239 genes and their expression patterns are shown. A. Schematic of the nhr-111 and nhr-239 genes showing the basic gene structure and point of fusion to GFP in expression constructs. B. Expression of the nhr-111 and nhr-239 genes over time as measured in qRT-PCR experiments. Arbitrary expression units are 2-ΔCtx1010. Error bars represent the standard deviation among replicates. Data from embryos (EMB) and larval L1, L2/L3, and L4 preparations of wild-type cDNA are shown. C. Expression of the nhr-111::gfp transgene in L1, L2 and adult (Ad) animals. The Z1 and Z4 gonad precursors in the L1 are labelled. The L2 image shows expression throughout the developing somatic gonad. The PVD neurons and ventral nerve cord (vnc) are indicated in the adult panels. Arrowhead identifies the location of a prominent dorsal neuron that appears to be sensory. D. Expression of the nhr-239::gfp transgene in a two-fold embryo (EMB) and adult (Ad) hermaphrodites. Individual neuron pairs are identified by lower case letters: a, pair of pharyngeal neurons; b, pair of mid-lateral neurons or neuronal support cells; c, pair of dorsal neurons just posterior to the nerve ring with sensory dendritic processes (arrowhead). Due to the low level of nhr-239::gfp expression, brightness of the images in D was significantly increased relative to those in C.
Figure 4. Evaluation of LBD function for FAX-1. Design and results of the experiments with LBD swaps among different NR2E LBDs and a deletion of the FAX-1 LBD. A. Schematic showing design of the genomic constructs created to test LBD function, as described in Methods. Percentages above LBD boxes indicate the percent identity of the swapped LBD to the C. elegans FAX-1 LBD. B. Results of movement assays. Figure shows forward movement rates for control wild-type, fax-1(gm83)fax-1(gm83) lin-15(n765) and FAX-1::INV FAX-1 strains, as well as each swap construct transgene. C. Rescue of the AVKR pathfinding defect in fax-1(gm83) mutants by LBD swap transgenes. The wild-type hermaphrodite shows a prominent single AVKR axon located in its proper position in the left bundle of the ventral nerve cord [56]. In the FAX-1:: INV FAX-1 negative control, the AVKR axon is missing from the ventral nerve cord due to misrouting [24]. The FAX-1::FAX-1 and FAX-1:: Δ LBD transgenic animals show the rescued wild-type anatomy. Rescue by FAX-1::Cb FAX-1, FAX-1::NHR-111, and FAX-1::NHR-67 transgenes was equivalent to the examples shown. The circular circuitry of FMRFamide-positive axons around the vulva is at the right side of each figure. All views are ventral views. D. Immunofluorescence demonstrating expression of fusion and deletion transgenes in fax-1(gm83) lin-15(n765) embryos. Left panels show Cy3 fluorescence detecting the FAX-1 DBD, right panels show matching DAPI staining of nuclei. The FAX-1::FAX-1 and FAX-1::Δ LBD embryos are a somewhat earlier stage (“late comma”), as compared to the FAX-1::NHR-111 and FAX-1::NHR-67 embryos (“two-fold elongation”). The anterior side of each embryo is oriented toward the top. Because of movement of elongation-stage embryos within the egg, orientation of each embryos varies.
Antebi,
Nuclear hormone receptors in C. elegans.
2006, Pubmed
Antebi,
Nuclear hormone receptors in C. elegans.
2006,
Pubmed
Arda,
Functional modularity of nuclear hormone receptors in a Caenorhabditis elegans metabolic gene regulatory network.
2010,
Pubmed
Bates,
The unfulfilled gene is required for the development of mushroom body neuropil in Drosophila.
2010,
Pubmed
Bergsten,
A review of long-branch attraction.
2005,
Pubmed
Bertrand,
Evolutionary genomics of nuclear receptors: from twenty-five ancestral genes to derived endocrine systems.
2004,
Pubmed
Blaustein,
Minireview: Neuronal steroid hormone receptors: they're not just for hormones anymore.
2004,
Pubmed
Blumberg,
Orphan nuclear receptors--new ligands and new possibilities.
1998,
Pubmed
Bonneton,
Annotation of Tribolium nuclear receptors reveals an increase in evolutionary rate of a network controlling the ecdysone cascade.
2008,
Pubmed
Borchert,
Proteogenomics of Pristionchus pacificus reveals distinct proteome structure of nematode models.
2010,
Pubmed
Brenner,
The genetics of Caenorhabditis elegans.
1974,
Pubmed
Bridgham,
Protein evolution by molecular tinkering: diversification of the nuclear receptor superfamily from a ligand-dependent ancestor.
2010,
Pubmed
Bryson,
Protein structure prediction servers at University College London.
2005,
Pubmed
Chalfie,
The neural circuit for touch sensitivity in Caenorhabditis elegans.
1985,
Pubmed
Cheng,
Nuclear receptors in Bombyx mori: insights into genomic structure and developmental expression.
2008,
Pubmed
Daniel,
The control of cell fate in the embryonic visual system by atonal, tailless and EGFR signaling.
1999,
Pubmed
DeMeo,
Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of Caenorhabditis elegans is partially mediated via a subclass-specific P-box residue.
2008,
Pubmed
Dieterich,
The Pristionchus pacificus genome provides a unique perspective on nematode lifestyle and parasitism.
2008,
Pubmed
Enmark,
Comparing nuclear receptors in worms, flies and humans.
2001,
Pubmed
Freeman,
cDNA sequences for transcription factors and signaling proteins of the hemichordate Saccoglossus kowalevskii: efficacy of the expressed sequence tag (EST) approach for evolutionary and developmental studies of a new organism.
2008,
Pubmed
Garriga,
Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans.
1993,
Pubmed
Ghedin,
Draft genome of the filarial nematode parasite Brugia malayi.
2007,
Pubmed
Glass,
The coregulator exchange in transcriptional functions of nuclear receptors.
2000,
Pubmed
Grasso,
The evolution of nuclear receptors: evidence from the coral Acropora.
2001,
Pubmed
Gui,
A tale of tailless.
2011,
Pubmed
Haerty,
Comparative analysis of function and interaction of transcription factors in nematodes: extensive conservation of orthology coupled to rapid sequence evolution.
2008,
Pubmed
Heid,
Real time quantitative PCR.
1996,
Pubmed
Hollemann,
The Xenopus homologue of the Drosophila gene tailless has a function in early eye development.
1998,
Pubmed
Howard-Ashby,
Gene families encoding transcription factors expressed in early development of Strongylocentrotus purpuratus.
2006,
Pubmed
,
Echinobase
Jones,
GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences.
1999,
Pubmed
Jones,
Protein secondary structure prediction based on position-specific scoring matrices.
1999,
Pubmed
Kato,
The C. elegans tailless/Tlx homolog nhr-67 regulates a stage-specific program of linker cell migration in male gonadogenesis.
2009,
Pubmed
King-Jones,
Nuclear receptors--a perspective from Drosophila.
2005,
Pubmed
Kozlova,
Steroid regulation of postembryonic development and reproduction in Drosophila.
2000,
Pubmed
Li,
A map of the interactome network of the metazoan C. elegans.
2004,
Pubmed
Lin,
Nuclear receptor unfulfilled regulates axonal guidance and cell identity of Drosophila mushroom body neurons.
2009,
Pubmed
Lowe,
Anteroposterior patterning in hemichordates and the origins of the chordate nervous system.
2003,
Pubmed
Maglich,
Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes.
2001,
Pubmed
Magner,
Caenorhabditis elegans nuclear receptors: insights into life traits.
2008,
Pubmed
Mangelsdorf,
The nuclear receptor superfamily: the second decade.
1995,
Pubmed
Mello,
Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.
1991,
Pubmed
Milam,
The nuclear receptor NR2E3 plays a role in human retinal photoreceptor differentiation and degeneration.
2002,
Pubmed
Monaghan,
Defective limbic system in mice lacking the tailless gene.
1997,
Pubmed
Motola,
Identification of ligands for DAF-12 that govern dauer formation and reproduction in C. elegans.
2006,
Pubmed
Much,
The fax-1 nuclear hormone receptor regulates axon pathfinding and neurotransmitter expression.
2000,
Pubmed
Nuclear Receptors Nomenclature Committee,
A unified nomenclature system for the nuclear receptor superfamily.
1999,
Pubmed
Parihar,
The genome of the nematode Pristionchus pacificus encodes putative homologs of RXR/Usp and EcR.
2010,
Pubmed
Rastinejad,
Structure of the RXR-RAR DNA-binding complex on the retinoic acid response element DR1.
2000,
Pubmed
Raška,
SMED-TLX-1 (NR2E1) is critical for tissue and body plan maintenance in Schmidtea mediterranea in fasting/feeding cycles.
2011,
Pubmed
Reitzel,
Nuclear receptor complement of the cnidarian Nematostella vectensis: phylogenetic relationships and developmental expression patterns.
2009,
Pubmed
Saitou,
The neighbor-joining method: a new method for reconstructing phylogenetic trees.
1987,
Pubmed
Sarin,
The C. elegans Tailless/TLX transcription factor nhr-67 controls neuronal identity and left/right asymmetric fate diversification.
2009,
Pubmed
Schinkmann,
Localization of FMRFamide-like peptides in Caenorhabditis elegans.
1992,
Pubmed
Sengupta,
The C. elegans gene odr-7 encodes an olfactory-specific member of the nuclear receptor superfamily.
1994,
Pubmed
Sluder,
The nuclear receptor superfamily has undergone extensive proliferation and diversification in nematodes.
1999,
Pubmed
Stiernagle,
Maintenance of C. elegans.
2006,
Pubmed
Strecker,
Graded requirement for the zygotic terminal gene, tailless, in the brain and tail region of the Drosophila embryo.
1988,
Pubmed
Sung,
The unfulfilled/DHR51 gene of Drosophila melanogaster modulates wing expansion and fertility.
2009,
Pubmed
Tamura,
MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.
2011,
Pubmed
Tan,
Identification and characterization of nuclear receptors from the red flour beetle, Tribolium castaneum.
2008,
Pubmed
Taubert,
Nuclear hormone receptors in nematodes: evolution and function.
2011,
Pubmed
Thompson,
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
1994,
Pubmed
Van Gilst,
Diversity and function of orphan nuclear receptors in nematodes.
2002,
Pubmed
Velarde,
Nuclear receptors of the honey bee: annotation and expression in the adult brain.
2006,
Pubmed
Verghese,
The tailless ortholog nhr-67 functions in the development of the C. elegans ventral uterus.
2011,
Pubmed
Vermeirssen,
Transcription factor modularity in a gene-centered C. elegans core neuronal protein-DNA interaction network.
2007,
Pubmed
Wightman,
The C. elegans nuclear receptor gene fax-1 and homeobox gene unc-42 coordinate interneuron identity by regulating the expression of glutamate receptor subunits and other neuron-specific genes.
2005,
Pubmed
Wightman,
Genes that guide growth cones along the C. elegans ventral nerve cord.
1997,
Pubmed
Yu,
The orphan nuclear receptor Tlx regulates Pax2 and is essential for vision.
2000,
Pubmed
Yu,
Relationship between Drosophila gap gene tailless and a vertebrate nuclear receptor Tlx.
1994,
Pubmed
de Rosny,
DHR51, the Drosophila melanogaster homologue of the human photoreceptor cell-specific nuclear receptor, is a thiolate heme-binding protein.
2008,
Pubmed