Analysis of C. elegans NR2E nuclear receptors defines three conserved clades and ligand-independent functions.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.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

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

<p>Abstract</p> <p>Background</p> <p>The nuclear receptors of the NR2E class play important roles in pattern formation and nervous system development. Based on a phylogenetic analysis of DNA-binding domains, we define two conserved groups of orthologous NR2E genes: the NR2E1 subclass, which includes <it>C. elegans nhr-67, Drosophila tailless </it>and <it>dissatisfaction</it>, and vertebrate Tlx (NR2E2, NR2E4, NR2E1), and the NR2E3 subclass, which includes <it>C. elegans fax-1 </it>and vertebrate PNR (NR2E5, NR2E3). PNR and Tll nuclear receptors have been shown to bind the hexamer half-site AAGTCA, instead of the hexamer AGGTCA recognized by most other nuclear receptors, suggesting unique DNA-binding properties for NR2E class members.</p> <p>Results</p> <p>We show that NR2E3 subclass member FAX-1, unlike NHR-67 and other NR2E1 subclass members, binds to hexamer half-sites with relaxed specificity: it will bind hexamers with the sequence ANGTCA, although it prefers a purine to a pyrimidine at the second position. We use site-directed mutagenesis to demonstrate that the difference between FAX-1 and NHR-67 binding preference is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position 19 of the DNA-binding domain. This amino acid position is part of the "P box" that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. The relaxed specificity allows FAX-1 to bind a much larger repertoire of half-sites than NHR-67. While NR2E1 class proteins bind both monomeric and dimeric sites, the NR2E3 class proteins bind only dimeric sites. The presence of a single strong site adjacent to a very weak site allows dimeric FAX-1 binding, further increasing the number of dimeric binding sites to which FAX-1 may bind <it>in vivo</it>.</p> <p>Conclusion</p> <p>These findings identify subclass-specific DNA-binding specificities and dimerization properties for the NR2E1 and NR2E3 subclasses. For the NR2E1 protein NHR-67, Asp-19 permits binding to AAGTCA half-sites, while Asn-19 permits binding to AGGTCA half-sites. The apparent conservation of DNA-binding properties between vertebrate and nematode NR2E receptors allows for the possibility of evolutionarily-conserved regulatory patterns.</p

A biomechanical model of the effect of subtalar arthroereisis on the adult flexible flat foot.

OBJECTIVE: The hypothesis tested was that the increased load on the medial arch in the adult flat foot can be reduced through a 6 mm subtalar arthroereisis. DESIGN: A three-dimensional multisegment biomechanical model was used in conjunction with experimental data and data from the literature. BACKGROUND: Biomechanical models have been used to study the plantar fascia, medial arch height, subtalar motion, medial displacement calcaneal osteotomy and distribution of forces in the foot. METHODS: Responses of a normal foot, a flat foot, and a flat foot with a subtalar arthroereisis to an applied load of 683 N were analyzed and the distribution of support among the metatarsal heads and the moment about various joints were computed. RESULTS: The flattened foot results in an increase in the load on the head of the first metatarsal from 10% to 24% of the body weight, and an increase in the moment about the talo-navicular joint from 3.4 to 11.9 Nm. Insertion of a 6 mm cylinder into the sinus tarsi, subtalar arthroereisis, results in a shift of the load back toward the lateral column, decreasing the load on the first metatarsal to 6% of the body weight and decreasing the moment about the talo-navicular joint to 6.0 Nm. CONCLUSIONS: Our analysis indicates that a 6 mm subtalar arthroereisis in an adult flat foot model decreases the load on the medial arch

The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3′UTR

Caenorhabditis elegans let-7, a founding member of the microRNA family, is predicted to bind to six sites in the 3′UTR of the mRNA of its target gene, lin-41, to down-regulate LIN-41. Here, we demonstrate that wild-type let-7 microRNA binds in vitro to RNA from the lin-41 3′UTR. This interaction is dependent on two conserved let-7 complementary sites (LCSs). A 27-nucleotide sequence between the LCSs is also necessary for down-regulation in vivo. LCS mutations compensatory to the lesion in let-7(n2853) can partially restore lin-41 3′UTR function in a let-7(n2853) background, providing the first experimental evidence for an animal miRNA binding directly to its validated target in vivo

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue-2

<p><b>Copyright information:</b></p><p>Taken from "Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue"</p><p>http://www.biomedcentral.com/1471-2199/9/2</p><p>BMC Molecular Biology 2008;9():2-2.</p><p>Published online 7 Jan 2008</p><p>PMCID:PMC2225407.</p><p></p>irs, respectively). Competition experiments with DR2A, DR3A, and DR1A are shown at 10-fold and 100-fold molar excess (wedges) or 10-fold molar excess only (DR3A). We observed additional shifted species in this experiment, however these bands could not be competed with equivalent unlabeled oligonucleotides

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue-3

<p><b>Copyright information:</b></p><p>Taken from "Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue"</p><p>http://www.biomedcentral.com/1471-2199/9/2</p><p>BMC Molecular Biology 2008;9():2-2.</p><p>Published online 7 Jan 2008</p><p>PMCID:PMC2225407.</p><p></p>smid derived from pLacZi that contained a single DR1A binding site upstream of the gene. Each strain included either no activator or a fusion construct containing a nematode nuclear receptor DBD fused to the yeast GAL4 activation domain. B. β-galactosidase activity for yeast containing DR1G binding sites. Error bars show standard deviations. Asterisks indicate results that are significantly different than no activator control by student's t-test (p < 0.05). The difference between the FAX-1 and FAX-1 N19D mutant activators on DR1A sites is also statistically significant. We performed equivalent experiments using strains containing negative control DRNC sites and activator constructs. These strains did not show β-gal activity relative to controls that had no activator (data not shown). We performed equivalent experiments using strains containing MON1 sites and HRWS sites (both AAGTCA monomers) and a FAX-1 DBD activator, which also did not show β-gal activity relative to controls (data not shown)

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue-0

<p><b>Copyright information:</b></p><p>Taken from "Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue"</p><p>http://www.biomedcentral.com/1471-2199/9/2</p><p>BMC Molecular Biology 2008;9():2-2.</p><p>Published online 7 Jan 2008</p><p>PMCID:PMC2225407.</p><p></p>-1 antiserum [31]. B. EMSA of FAX-1 protein binding to DR1A sequences (AAGTCA DR1) and failing to bind to DRNC sequences (AATTCA repeats). Binding could be competed with 10-fold and 100-fold molar excess of unlabeled competitor DR1G and DR1A DNA, but not DRNC DNA (wedges). Similar results (not shown) were obtained using radioactively-labeled DR1G DNA (AGGTCA DR1). C. and D. EMSA of FAX-1 protein binding to DR1T sequences (ATGTCA DR1) and DR1C sequences (ACGTCA DR1), respectively