Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> is partially mediated via a subclass-specific P-box residue

Background  

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 C. elegans nhr-67, Drosophila tailless and dissatisfaction, and vertebrate Tlx (NR2E2, NR2E4, NR2E1), and the NR2E3 subclass, which includes C. elegans fax-1 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.     

A comparative genomic analysis of the small heat shock proteins in Caenorhabditis elegans and briggsae

The small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones. We have identified 18 sHSPs in the Caenorhabditis elegans genome and 20 sHSPs in the Caenorhabditis briggsae genome. Analysis of phylogenetic relationships and evolutionary dynamics of the sHSPs in these two genomes reveals a very complex pattern of evolution. The sHSPs in C. elegans and C. briggsae do not display clear orthologous relationships with other invertebrate sHSPs. But many sHSPs in C. elegans have orthologs in C. briggsae. One group of sHSPs, the HSP16s, has a very unusual evolutionary history. Although there are a number of HSP16s in both the C. elegans and C. briggsae genomes, none of the HSP16s display orthologous relationships across these two species. The HSP16s have an unusual gene pair structure and a complex evolutionary history shaped by gene duplication, gene conversion, and purifying selection. We found no evidence of recent positive selection acting on any of the sHSPs in C. elegans or in C. briggsae. There is also no evidence of functional divergence within the pairs of orthologous C. elegans and C. briggsae sHSPs. However, the evolutionary patterns do suggest that functional divergence has occurred between the sHSPs in C. elegans and C. briggsae and the sHSPs in more distantly related invertebrates.     

Comparative genetics of sex determination: masculinizing mutations in Caenorhabditis briggsae

The nematodes Caenorhabditis elegans and C. briggsae independently evolved self-fertile hermaphroditism from gonochoristic ancestors. C. briggsae has variably divergent orthologs of nearly all genes in the C. elegans sex determination pathway. Their functional characterization has generally relied on reverse genetic approaches, such as RNA interference and cross-species transgene rescue and more recently on deletion mutations. We have taken an unbiased forward mutagenesis approach to isolating zygotic mutations that masculinize all tissues of C. briggsae hermaphrodites. The screens identified loss-of-function mutations in the C. briggsae orthologs of tra-1, tra-2, and tra-3. The somatic and germline phenotypes of these mutations are largely identical to those of their C. elegans homologs, including the poorly understood germline feminization of tra-1(lf) males. This overall conservation of Cb-tra phenotypes is in contrast to the fem genes, with which they directly interact and which are significantly divergent in germline function. In addition, we show that in both C. briggsae and C. elegans large C-terminal truncations of TRA-1 that retain the DNA-binding domain affect sex determination more strongly than somatic gonad development. Beyond these immediate results, this collection of mutations provides an essential foundation for further comparative genetic analysis of the Caenorhabditis sex determination pathway.     

Functional elements and domains inferred from sequence comparisons of a heat shock gene in two nematodes

Caenorhabditis elegans and Caenorhabditis briggsae are two closely related nematode species that are nearly identical morphologically. Interspecific cross-hybridizing DNA appears to be restricted primarily to coding regions. We compared portions of the hsp-3 homologs, two grp 78-like genes, from C. elegans and C. briggsae and detected regions of DNA identity in the coding region, the 5' flanking DNAs, and the introns. The hsp-3 homologs share approximately 98% and 93% identity at the amino acid and nucleotide levels, respectively. Using the nucleotide substitution rate at the silent third position of the codons, we have estimated a lower limit for the date of divergence between C. elegans and C. briggsae to be approximately 23-32 million years ago. The 5' flanking DNAs and one of the introns contain elements that are highly conserved between C. elegans and C. briggsae. Some of the regions of nucleotide identity in the 5' flanking DNAs correspond to previously detected identities including viral enhancer sequences, a heat shock element, and an element present in the regulatory regions of mammalian grp78 and grp94 genes. We propose that a comparison of C. elegans and C. briggsae sequences will be useful in the detection of potential regulatory and structural elements.     

Genetic analysis of dauer formation in Caenorhabditis briggsae

Molecular changes that underlie evolutionary changes in behavior and physiology are not well understood. Dauer formation in Caenorhabditis elegans is a temperature-sensitive process controlled through a network of signaling pathways associated with sensory neurons and is potentially an excellent system in which to investigate molecular changes in neuronal function during evolution. To begin to investigate the evolution of dauer formation in the genus Caenorhabditis at the molecular level, we isolated dauer-formation mutations in C. briggsae, a species closely related to the model organism C. elegans. We identified mutations in orthologs of C. elegans genes daf-2 (insulin receptor), daf-3 (Smad), and daf-4 (TGF-beta type 2 receptor), as well as genes required for formation of sensory cilia. Phenotypic analyses revealed that functions of these genes are conserved between C. elegans and C. briggsae. Analysis of C. briggsae mutations also revealed a significant difference between the two species in their responses to high temperatures (>26 degrees). C. elegans is strongly induced to form dauers at temperatures above 26 degrees, near the upper limit for growth of C. elegans. In contrast, C. briggsae, which is capable of growth at higher temperatures than C. elegans, lacks this response.     

Evolutionary selection across the nuclear hormone receptor superfamily with a focus on the NR1I subfamily (vitamin D,pregnane X,and constitutive androstane receptors)

Background

The nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with diverse roles in metabolic homeostasis, development, and detoxification. In general, NRs are strongly conserved between vertebrate species, and few examples of molecular adaptation (positive selection) within this superfamily have been demonstrated. Previous studies utilizing two-species comparisons reveal strong purifying (negative) selection of most NR genes, with two possible exceptions being the ligand-binding domains (LBDs) of the pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3), two proteins involved in the regulation of toxic compound metabolism and elimination. The aim of this study was to apply detailed phylogenetic analysis using maximum likelihood methods to the entire complement of genes in the vertebrate NR superfamily. Analyses were carried out both across all vertebrates and limited to mammals and also separately for the two major domains of NRs, the DNA-binding domain (DBD) and LBD, in addition to the full-length sequences. Additional functional data is also reported for activation of PXR and the vitamin D receptor (VDR; NR1I1) to gain further insight into the evolution of the NR1I subfamily.

Results

The NR genes appear to be subject to strong purifying selection, particularly in the DBDs. Estimates of the ratio of the non-synonymous to synonymous nucleotide substitution rates (the ω ratio) revealed that only the PXR LBD had a sub-population of codons with an estimated ω ratio greater than 1. CAR was also unusual in showing high relative ω ratios in both the DBD and LBD, a finding that may relate to the recent appearance of the CAR gene (presumably by duplication of a pre-mammalian PXR gene) just prior to the evolution of mammals. Functional analyses of the NR1I subfamily show that human and zebrafish PXRs show similar activation by steroid hormones and early bile salts, properties not shared by sea lamprey, mouse, or human VDRs, or by Xenopus laevis PXRs.

Conclusion

NR genes generally show strong sequence conservation and little evidence for positive selection. The main exceptions are PXR and CAR, genes that may have adapted to cross-species differences in toxic compound exposure.

     

Smoke without fire: most reported cases of intron gain in nematodes instead reflect intron losses

Identification of recently gained spliceosomal introns would provide crucial evidence in the continuing debate concerning the age and evolutionary significance of introns. A previously published genomic analysis reported to have identified 122 introns that had been gained since the divergence of the nematodes Caenorhabidits elegans and Caenorhabditis briggsae approximately 100 MYA. However, using newly available genomic sequence from additional Caenorhabditis species, we show that 74% (60/81) of the reported gains in C. elegans are present in a C. briggsae relative. This pattern indicates that these introns represent losses in C. briggsae, not gains in C. elegans. In addition, 61% (25/41) of the reported gains in C. briggsae are present in the more distant C. briggsae relative, in a pattern suggesting that additional reported gains in C. elegans and/or C. briggsae may in fact represent unrecognized losses. These results underscore the dominance of intron loss over intron gain in recent eukaryotic evolution, the pitfalls associated with parsimony in inferring intron gains, and the importance of genomic sequencing of clusters of closely related species for drawing accurate inferences about genome evolution.     

Conformational adaptation of nuclear receptor ligand binding domains to agonists: potential for novel approaches to ligand design

Ligands occupy the core of nuclear receptor (NR) ligand binding domains (LBDs) and modulate NR function. X-ray structures of NR LBDs reveal most NR agonists fill the enclosed pocket and promote packing of C-terminal helix 12 (H12), whereas the pockets of unliganded NR LBDs differ. Here, we review evidence that NR pockets rearrange to accommodate different agonists. Some thyroid hormone receptor (TR) ligands with 5′ extensions designed to perturb H12 act as antagonists, but many are agonists. One mode of adaptation is seen in a TR/thyroxine complex; the pocket expands to accommodate a 5′ iodine extension. Crystals of other NR LBDs reveal that the pocket can expand or contract and some agonists do not fill the pocket. A TRβ structure in complex with an isoform selective drug (GC-24) reveals another mode of adaptation; the LBD hydrophobic interior opens to accommodate a bulky 3′ benzyl extension. We suggest that placement of extensions on NR agonists will highlight unexpected areas of flexibility within LBDs that could accommodate extensions; thereby enhancing the selectivity of agonist binding to particular NRs. Finally, agonists that induce similar LBD structures differ in their activities and we discuss strategies to reveal subtle structural differences responsible for these effects.     

Clustered organization of reproductive genes in the C. elegans genome

Defining the forces that sculpt genome organization is fundamental for understanding the origin, persistence, and diversification of species. The genomic sequences of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae provide an excellent opportunity to explore the dynamics of chromosome evolution. Extensive chromosomal rearrangement has accompanied divergence from their common ancestor, an event occurring roughly 100 million years ago (Mya); yet, morphologically, these species are nearly indistinguishable and both reproduce primarily by self-fertilization. Here, we show that genes expressed during spermatogenesis (sperm genes) are nonrandomly distributed across the C. elegans genome into three large clusters located on two autosomes. In addition to sperm genes, these chromosomal regions are enriched for genes involved in the hermaphrodite sperm/oocyte switch and in the reception of sperm signals that control fertilization. Most loci are present in single copy, suggesting that cluster formation is largely due to gene aggregation and not to tandem duplication. Comparative mapping indicates that the C. briggsae genome differs dramatically from the C. elegans genome in clustering. Because clustered genes have a direct role in reproduction and thus fitness, their aggregated pattern might have been shaped by natural selection, perhaps as hermaphroditism evolved.     

Conservation of gene organization and trans-splicing in the glyceraldehyde-3-phosphate dehydrogenase-encoding genes of Caenorhabditis briggsae.

The genes encoding body-wall-specific glyceraldehyde-3-phosphate dehydrogenase from Caenorhabditis briggsae were sequenced and compared to the homologous genes from Caenorhabditis elegans. The direct tandem organization of these genes, gpd-2 and gpd-3, and the size and location of the two introns in each gene are the same in C. elegans and C. briggsae. Primer-extension studies demonstrated that the two genes in C. briggsae are trans-splice differentially with the same splice leader (SL) RNAs as are observed in C. elegans. The gdp-2 gene is trans-spliced with SL1 while gdp-3 is trans-spliced with SL2. Significant sequence conservation was observed within the promoter regions of each species and may indicate those regions responsible for body-wall-muscle-specific gene expression and/or differential trans-splicing. Comparisons of the sequences suggest that the tandem repeat of the genes has been subjected to concerted evolution and that C. briggsae and C. elegans diverged much earlier than would be anticipated based on morphological similarities alone. Finally, an open reading frame found several hundred nucleotides upstream from gpd-2, in both species, appears to be homologous to the ATP synthase subunit, ATPase inhibitor protein, from bovine mitochondria.     

Changes in Nuclear Receptor and Vitellogenin Gene Expression in Response to Steroids and Heavy Metal in Caenorhabditis elegans

To gain basic understanding of the reproductive and developmental effects of endocrine disrupting chemicals in invertebrates, we have used C. elegans as an animal model. The completion of the C. elegans genome sequence brings to bear microarray analysis as a tool for these studies. We previously showed that the C. elegans genome was responsive to vertebrate steroid hormones, and changes in gene expression of traditional biomarkers used in environmental studies were detected; i.e., vitellogenin (vtg), cytochrome P450 (cyp450), glutathione-S-transferase (gst) and heat shock proteins (hsp). The data were interpreted to suggest that exogenous lipophilic compounds can be metabolized via cytochrome P450 proteins, and that the resulting metabolites can bind to members of the Nuclear Receptor (NR) class of proteins and regulate gene expression. In the present study, using DNA microarrays, we examined the pattern of gene expression after progesterone (10(-5), 10(-7) M), estradiol (10(-5) M), cholesterol (10(-9) M) and cadmium (0.1, 1 and 10 μM) exposure, with special attention to the members of NRs. Of approximately 284 NRs in C. elegans, expression of 25 NR genes (representing 9% of the total NRs in C. elegans) was altered after exposure to steroids. Of note, each steroid activated or inhibited different subsets of NR genes, and only estradiol regulated NR genes implicated in neurogenesis. These results suggest that NRs respond to a variety of exogenous steroids, which regulate important metabolic and developmental pathways. The response of the C. elegans genome to cholesterol and cadmium was analyzed in more detail. Cholesterol is a probable precursor to signaling molecules that may interact with NRs and we focused on expression of genes related to lipid metabolism (cyp450), transport and storage (i.e., vitellogenin). Worms exposed to cadmium respond principally by activating the expression of genes encoding stress-responsive proteins, such as mtl-2 and cdr-1, and no significant changes in expression of NRs or vtg genes were observed. The possible implications of these results with regard to the evolution of steroid receptors, endocrine disruption and the role of vitellogenin as a lipid transporter are discussed.     

Hakuna Nematoda: genetic and phenotypic diversity in African isolates of Caenorhabditis elegans and C. briggsae

Caenorhabditis elegans and C. briggsae have many parallels in terms of morphology, life history and breeding system. Both species also share similar low levels of molecular diversity, although the global sampling of natural populations has been limited and geographically biased. In this study, we describe the first cultured isolates of C. elegans and C. briggsae from sub-Saharan Africa. We characterize these samples for patterns of nucleotide polymorphism and vulva precursor cell lineage, and conduct a series of hybrid crosses in C. briggsae to test for genetic incompatibilities. The distribution of genetic diversity confirms a lack of geographic structure to C. elegans sequences but shows genetic differentiation of C. briggsae into three distinct clades that may correspond to three latitudinal ranges. Despite low levels of molecular diversity, we find considerable variation in cell division frequency in African C. elegans for the P3.p vulva precursor cell, and in African C. briggsae for P4.p, a variation that was not previously observed in this species. Hybrid crosses did not reveal major incompatibilities between C. briggsae strains from Africa and elsewhere, and there was some evidence of inbreeding depression. These new African isolates suggest that important ecological factors may be shaping the patterns of diversity in C. briggsae, and that despite many similarities between C. elegans and C. briggsae, there may be more subtle differences in their natural histories than previously appreciated.     

Conservation of the C.elegans tra-2 3'UTR translational control.

The Caenorhabditis elegans sex-determination gene, tra-2, is translationally regulated by two 28 nt elements (DREs) located in the 3'UTR that bind a factor called DRF. This regulation requires the laf-1 gene activity. We demonstrate that the nematode Caenorhabditis briggsae tra-2 gene and the human oncogene GLI are translationally regulated by elements that are functionally equivalent to DREs. Here, we rename the DREs to TGEs (tra-2 and GLI elements). Similarly to the C.elegans tra-2 TGEs, the C.briggsae tra-2 and GLI TGEs repress translation of a reporter transgene in a laf-1 dependent manner. Furthermore, they regulate poly(A) tail length and bind DRF. We also find that the C.elegans TGEs control translation and poly(A) tail length in C.briggsae and rodent cells. Moreover, these same organisms contain a factor that specifically associates with the C.elegans TGEs. These findings are consistent with the TGE control being present in C.briggsae and rodent cells. Three lines of evidence indicate that C.briggsae tra-2 and GLI are translationally controlled in vivo by TGEs. First, like C.elegans tra-2 TGEs, the C.briggsae tra-2 and GLI TGEs control translation and poly(A) tail lengths in C.briggsae and rodent cells, respectively. Second, the same factor in C.briggsae and mammalian cells that binds to the C.elegans tra-2 TGEs binds the C.briggsae tra-2 and GLI TGEs. Third, deletion of the GLI TGE increases GLI's ability to transform cells. These findings suggest that TGE control is conserved and regulates the expression of other mRNAs.