Conserved elements associated with ribosomal genes and their trans-splice acceptor sites in Caenorhabditis elegans.

The recent publication of the Caenorhabditis elegans cisRED database has provided an extensive catalog of upstream elements that are conserved between nematode genomes. We have performed a secondary analysis to determine which subsequences of the cisRED motifs are found in multiple locations throughout the C. elegans genome. We used the word-counting motif discovery algorithm DME to form the motifs into groups based on sequence similarity. We then examined the genes associated with each motif group using DAVID and Ontologizer to determine which groups are associated with genes that also have significant functional associations in the Gene Ontology and other gene annotation sources. Of the 3265 motif groups formed, 612 (19%) had significant functional associations with respect to GO terms. Eight of the first 20 motif groups based on frequent dodecamers among the cisRED motif sequences were specifically associated with ribosomal protein genes; two of these were similar to mouse EBP-45, rat HNF3-family and Drosophila Zeste transcription factor binding sites. Additionally, seven motif groups were extensions of the canonical C. elegans trans-splice acceptor site. One motif group was tested for regulatory function in a series of green fluorescent protein expression experiments and was shown to be involved in pharyngeal expression.

Characterization of the octamer, a cis-regulatory element that modulates excretory cell gene-expression in Caenorhabditis elegans.

BACKGROUND: We have previously demonstrated that the POU transcription factor CEH-6 is required for driving aqp-8 expression in the C. elegans excretory (canal) cell, an osmotic regulatory organ that is functionally analogous to the kidney. This transcriptional regulation occurs through a CEH-6 binding to a cis-regulatory element called the octamer (ATTTGCAT), which is located in the aqp-8 promoter. RESULTS: Here, we further characterize octamer driven transcription in C. elegans. First, we analyzed the positional requirements of the octamer. To do so, we assayed the effects on excretory cell expression by placing the octamer within the well-characterized promoter of vit-2. Second, using phylogenetic footprinting between three Caenorhabditis species, we identified a set of 165 genes that contain conserved upstream octamers in their promoters. Third, we used promoter::GFP fusions to examine the expression patterns of 107 of the 165 genes. This analysis demonstrated that conservation of octamers in promoters increases the likelihood that the gene is expressed in the excretory cell. Furthermore, we found that the sequences flanking the octamers may have functional importance. Finally, we altered the octamer using site-directed mutagenesis. Thus, we demonstrated that some nucleotide substitutions within the octamer do not affect the expression pattern of nearby genes, but change their overall expression was changed. Therefore, we have expanded the core octamer to include flanking regions and variants of the motif. CONCLUSIONS: Taken together, we have demonstrated that octamer-containing regions are associated with excretory cell expression of several genes that have putative roles in osmoregulation. Moreover, our analysis of the octamer sequence and its sequence variants could aid in the identification of additional genes that are expressed in the excretory cell and that may also be regulated by CEH-6.

High-throughput in vivo analysis of gene expression in Caenorhabditis elegans.

Using DNA sequences 5' to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5' DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index) and through WormBase (http://www.wormbase.org).

Basal body dysfunction is a likely cause of pleiotropic Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized primarily by retinal dystrophy, obesity, polydactyly, renal malformations and learning disabilities. Although five BBS genes have been cloned, the molecular basis of this syndrome remains elusive. Here we show that BBS is probably caused by a defect at the basal body of ciliated cells. We have cloned a new BBS gene, BBS8, which encodes a protein with a prokaryotic domain, pilF, involved in pilus formation and twitching mobility. In one family, a homozygous null BBS8 mutation leads to BBS with randomization of left-right body axis symmetry, a known defect of the nodal cilium. We have also found that BBS8 localizes specifically to ciliated structures, such as the connecting cilium of the retina and columnar epithelial cells in the lung. In cells, BBS8 localizes to centrosomes and basal bodies and interacts with PCM1, a protein probably involved in ciliogenesis. Finally, we demonstrate that all available Caenorhabditis elegans BBS homologues are expressed exclusively in ciliated neurons, and contain regulatory elements for RFX, a transcription factor that modulates the expression of genes associated with ciliogenesis and intraflagellar transport.

Polymorphic segmental duplication in the nematode Caenorhabditis elegans.

BACKGROUND: The nematode Caenorhabditis elegans was the first multicellular organism to have its genome fully sequenced. Over the last 10 years since the original publication in 1998, the C. elegans genome has been scrutinized and the last gaps were filled in November 2002, which present a unique opportunity for examining genome-wide segmental duplications. RESULTS: Here, we performed analysis of the C. elegans genome in search for segmental duplications using a new tool -- OrthoCluster -- we have recently developed. We detected 3,484 duplicated segments -- duplicons -- ranging in size from 234 bp to 108 Kb. The largest pair of duplicons, 108 kb in length located on the left arm of Chromosome V, was further characterized. They are nearly identical at the DNA level (99.7% identity) and each duplicon contains 26 putative protein coding genes. Genotyping of 76 wild-type strains obtained from different labs in the C. elegans community revealed that not all strains contain this duplication. In fact, only 29 strains carry this large segmental duplication, suggesting a very recent duplication event in the C. elegans genome. CONCLUSION: This report represents the first demonstration that the C. elegans laboratory wild-type N2 strains has acquired large-scale differences.

Characterizing the transcriptional regulation of let-721, a Caenorhabditis elegans homolog of human electron flavoprotein dehydrogenase.

LET-721 is the Caenorhabditis elegans ortholog of electron-transferring flavoprotein dehydrogenase (ETFDH). We are studying this protein in C. elegans in order to establish a tractable model system for further exploration of ETFDH structure and function. ETFDH is an inner mitochondrial membrane localized enzyme that plays a key role in the beta-oxidation of fatty acids and catabolism of amino acids and choline. ETFDH accepts electrons from at least twelve mitochondrial matrix flavoprotein dehydrogenases via an intermediate dimer protein and transfers the electrons to ubiquinone. In humans, ETFDH mutations result in the autosomal recessive metabolic disorder, multiple acyl-CoA dehydrogenase deficiency. Mutants of let-721 in C. elegans are either maternal effect lethals or semi-sterile. let-721 is transcribed in the pharynx, body wall muscle, hypoderm, intestine and somatic gonad. In addition, the subcellular localization of LET-721 agrees with predictions that it is localized to mitochondria. We identified and confirmed three cis-regulatory sequences (pha-site, rep-site, and act-site). Phylogenetic footprinting of each site indicates that they are conserved between four Caenorhabditis species. The pha-site mapped roughly 1,300 bp upstream of let-721's translational start site and is necessary for expression in pharyngeal tissues. The rep-site mapped roughly 830 bp upstream of the translational start site and represses expression of LET-721 within pharyngeal tissues. The act-site mapped roughly 800 bp upstream of the translational start site and is required for expression within spermatheca, body wall muscle, pharynx, and intestine. Taken together, we find that LET-721 is a mitochondrially expressed protein that is under complex transcriptional controls.

The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport.

The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet-Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.

Functional genomics of the cilium, a sensory organelle.

Cilia and flagella play important roles in many physiological processes, including cell and fluid movement, sensory perception, and development. The biogenesis and maintenance of cilia depend on intraflagellar transport (IFT), a motility process that operates bidirectionally along the ciliary axoneme. Disruption in IFT and cilia function causes several human disorders, including polycystic kidneys, retinal dystrophy, neurosensory impairment, and Bardet-Biedl syndrome (BBS). To uncover new ciliary components, including IFT proteins, we compared C. elegans ciliated neuronal and nonciliated cells through serial analysis of gene expression (SAGE) and screened for genes potentially regulated by the ciliogenic transcription factor, DAF-19. Using these complementary approaches, we identified numerous candidate ciliary genes and confirmed the ciliated-cell-specific expression of 14 novel genes. One of these, C27H5.7a, encodes a ciliary protein that undergoes IFT. As with other IFT proteins, its ciliary localization and transport is disrupted by mutations in IFT and bbs genes. Furthermore, we demonstrate that the ciliary structural defect of C. elegans dyf-13(mn396) mutants is caused by a mutation in C27H5.7a. Together, our findings help define a ciliary transcriptome and suggest that DYF-13, an evolutionarily conserved protein, is a novel core IFT component required for cilia function.

The conserved Mediator subunit MDT-15 is required for oxidative stress responses in Caenorhabditis elegans.

Reactive oxygen species (ROS) play important signaling roles in metazoans, but also cause significant molecular damage. Animals tightly control ROS levels using sophisticated defense mechanisms, yet the transcriptional pathways that induce ROS defense remain incompletely understood. In the nematode Caenorhabditis elegans, the transcription factor SKN-1 is considered a master regulator for detoxification and oxidative stress responses. Here, we show that MDT-15, a subunit of the conserved Mediator complex, is also required for oxidative stress responses in nematodes. Specifically, mdt-15 is required to express SKN-1 targets upon chemical and genetic increase in SKN-1 activity. mdt-15 is also required to express genes in SKN-1-dependent and SKN-1-independent fashions downstream of insulin/IGF-1 signaling and for the longevity of daf-2/insulin receptor mutants. At the molecular level, MDT-15 binds SKN-1 through a region distinct from the classical transcription-factor-binding KIX-domain. Moreover, mdt-15 is essential for the transcriptional response to and survival on the organic peroxide tert-butyl-hydroperoxide (tBOOH), a largely SKN-1-independent response. The MDT-15 interacting nuclear hormone receptor, NHR-64, is specifically required for tBOOH but not arsenite resistance, but NHR-64 is dispensable for the transcriptional response to tBOOH. Hence, NHR-64 and MDT-15's mode of action remain elusive. Lastly, the role of MDT-15 in oxidative stress defense is functionally separable from its function in fatty acid metabolism, as exogenous polyunsaturated fatty acid complementation rescues developmental, but not stress sensitivity phenotypes of mdt-15 worms. Our findings reveal novel conserved players in the oxidative stress response and suggest a broad cytoprotective role for MDT-15.

Identification and analysis of internal promoters in Caenorhabditis elegans operons.

The current Caenorhabditis elegans genomic annotation has many genes organized in operons. Using directionally stitched promoterGFP methodology, we have conducted the largest survey to date on the regulatory regions of annotated C. elegans operons and identified 65, over 25% of those studied, with internal promoters. We have termed these operons "hybrid operons." GFP expression patterns driven from internal promoters differ in tissue specificity from expression of operon promoters, and serial analysis of gene expression data reveals that there is a lack of expression correlation between genes in many hybrid operons. The average length of intergenic regions with putative promoter activity in hybrid operons is larger than previous estimates for operons as a whole. Genes with internal promoters are more commonly involved in gene duplications and have a significantly lower incidence of alternative splicing than genes without internal promoters, although we have observed almost all trans-splicing patterns in these two distinct groups. Finally, internal promoter constructs are able to rescue lethal knockout phenotypes, demonstrating their necessity in gene regulation and survival. Our work suggests that hybrid operons are common in the C. elegans genome and that internal promoters influence not only gene organization and expression but also operon evolution.

Transcriptional regulation of AQP-8, a Caenorhabditis elegans aquaporin exclusively expressed in the excretory system, by the POU homeobox transcription factor CEH-6.

Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5' deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 bp upstream of the translational start site of aqp-8. Analysis of this region revealed a sequence corresponding to a known DNA functional element (octamer motif), which interacts with POU homeobox transcription factors. Phylogenetic footprinting showed that this site is perfectly conserved in four nematode species. The octamer site's function was further confirmed by deletion analyses, mutagenesis, functional studies, and electrophoretic mobility shift assays. Of the three POU homeobox proteins encoded in the C. elegans genome, CEH-6 is the only member that is expressed in the excretory cell. We show that expression of AQP-8 is regulated by CEH-6 by performing RNA interference experiments. CEH-6's mammalian ortholog, Brn1, is expressed both in the kidney and the central nervous system and binds to the same octamer consensus binding site to drive gene expression. These parallels in transcriptional control between Brn1 and CEH-6 suggest that C. elegans may well be an appropriate model for determining gene-regulatory networks in the developing vertebrate kidney.

Caenorhabditis elegans dpy-14: an essential collagen gene with unique expression profile and physiological roles in early development.

We describe the molecular characterisation of Caenorhabditis elegans dpy-14, a gene encoding an essential cuticular collagen annotated as col-59. Expression of dpy-14 starts at the 16 E cell stage, making it the earliest-expressing collagen reported to date. SAGE data and dpy-14 promoter::GFP reporter constructs indicate that the gene is transcribed mainly during embryogenesis, specifically in ciliated neurons and hypoderm. Water permeability assays and lectin staining showed that a mutation in the DPY-14 collagen results in defects in the channels of the amphids, which are a class of ciliated neuron, while the amphids appear morphologically normal by dye filling methods. Behavioural assays showed that the ciliated neurons expressing the gene are functional in dpy-14 mutants. All together, our data suggest that ciliated neurons and their hypodermal support cells collaborate in the transcription and synthesis of DPY-14, which then becomes a component of the amphid channels but not of the amphids proper. Interestingly, seam cells of dpy-14 mutants do not properly fuse to form a syncytium. This novel phenotype due to collagen mutations further stresses that dpy-14 plays a fundamental role in C. elegans physiology, since it is required for the proper development of the hypoderm.

Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics.

BACKGROUND: The recent availability of genome sequences of multiple related Caenorhabditis species has made it possible to identify, using comparative genomics, similarly transcribed genes in Caenorhabditis elegans and its sister species. Taking this approach, we have identified numerous novel ciliary genes in C. elegans, some of which may be orthologs of unidentified human ciliopathy genes. RESULTS: By screening for genes possessing canonical X-box sequences in promoters of three Caenorhabditis species, namely C. elegans, C. briggsae and C. remanei, we identified 93 genes (including known X-box regulated genes) that encode putative components of ciliated neurons in C. elegans and are subject to the same regulatory control. For many of these genes, restricted anatomical expression in ciliated cells was confirmed, and control of transcription by the ciliogenic DAF-19 RFX transcription factor was demonstrated by comparative transcriptional profiling of different tissue types and of daf-19(+) and daf-19(-) animals. Finally, we demonstrate that the dye-filling defect of dyf-5(mn400) animals, which is indicative of compromised exposure of cilia to the environment, is caused by a nonsense mutation in the serine/threonine protein kinase gene M04C9.5. CONCLUSION: Our comparative genomics-based predictions may be useful for identifying genes involved in human ciliopathies, including Bardet-Biedl Syndrome (BBS), since the C. elegans orthologs of known human BBS genes contain X-box motifs and are required for normal dye filling in C. elegans ciliated neurons.

Identification of a nematode chemosensory gene family.

Taking advantage of the recent availability of the whole genome sequence of Caenorhabditis briggsae, a closely related nematode to Caenorhabditis elegans, we have examined the chemosensory gene superfamily by using comparative genomic methods. We have identified a chemosensory gene family, serpentine receptor class ab (srab), which exists in both species with 25 members in C. elegans and 14 members in C. briggsae. More than 20% of these gene models are reannotated. The srab family is similar to, but distinct from, the previously described serpentine receptor class a (sra) family and shows a differential expansion in C. elegans similar to that previously described for sra. The cellular expression patterns for multiple members of the srab family in both phasmid neurons in the tail and amphid neurons in the head supports the conclusion that they are chemosensory genes and suggests that they may play a role in integrating chemosensory inputs from both ends of the organism. The expansion of both the srab and sra gene families in C. elegans relative to C. briggsae is due to multiple rounds of tandem duplication and translocation of individual genes.

Loss of C. elegans BBS-7 and BBS-8 protein function results in cilia defects and compromised intraflagellar transport.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous developmental disorder whose molecular basis is largely unknown. Here, we show that mutations in the Caenorhabditis elegans bbs-7 and bbs-8 genes cause structural and functional defects in cilia. C. elegans BBS proteins localize predominantly at the base of cilia, and like proteins involved in intraflagellar transport (IFT), a process necessary for cilia biogenesis and maintenance, move bidirectionally along the ciliary axoneme. Importantly, we demonstrate that BBS-7 and BBS-8 are required for the normal localization/motility of the IFT proteins OSM-5/Polaris and CHE-11, and to a notably lesser extent, CHE-2. We propose that BBS proteins play important, selective roles in the assembly and/or function of IFT particle components. Our findings also suggest that some of the cardinal and secondary symptoms of BBS, such as obesity, diabetes, cardiomyopathy, and learning defects may result from cilia dysfunction.