Jan Burghoorn et al. (2007) International Worm Meeting "Transcriptional regulation of DAF-19 dependent genes."

Peter Swoboda, Jan Burghoorn, Kristian Jeppsson, Filip Ystrom, Sofie Sahlin

[International Worm Meeting, 2007]

The C. elegans protein DAF-19, a Regulatory Factor X (RFX) transcription factor (TF), is a key regulator for cilia development (Swoboda et al. 2000). Via its recognition site in promoters of target genes, the X-box, DAF-19 directly controls the expression of genes that are important for ciliary structure and function. Cilia are specialized sub-cellular organelles that in C. elegans are present only on the endings of sensory neuron dendrites. In genome-wide searches for candidate genes with X-box promoter motifs, hundreds of genes were predicted to be DAF-19 dependent, and as a consequence many new ciliary genes could be identified (Blacque et al. 2005; Efimenko et al. 2005; Chen et al. 2006). Despite its successful application, the accuracy of the X-box prediction method for ciliary genes is still worth improving, especially since most candidate genes chosen for experimental verification, encode protein domains frequently found in other, already described ciliary genes. Moreover, a number of genes found in those studies were not expressed at all in ciliated neurons (so called false positives). To significantly improve the X-box prediction method that can distinguish true direct DAF-19 targets from false positives, we selected all TFs that have a promoter X-box motif. We consider this a more unbiased group of genes, since TFs are not directly involved in ciliary structure and function. Of 50 selected TFs we analyzed more than 30 TF::GFP fusions in transgenic worms. One third turned out to be expressed in ciliated neurons in a DAF-19 dependent manner. These results, together with information from previously experimentally proven direct DAF-19 targets and cross species comparisons with C. briggsae and C. remanei, allowed us to establish a high-accuracy X-box prediction method. In addition, we could clearly split X-box promoter motifs into two groups. Group I X-boxes have near-palindromic sequences, locate close to gene starts, are conserved across species, and direct gene expression in most or all ciliated neurons. Group II X-boxes have less palindromic sequences, are less conserved across species and direct gene expression in only one or a few ciliated neurons. We are currently creating a system in which we can not only predict a candidate genes DAF-19 dependence with high precision, but also whether its expression pattern will be in all or only a subset of the ciliated neuron class. Moreover, prelimenary results show that other cis-regulatory motifs, together with the X-box, are essential for the proper expression of DAF-19 dependent genes. Our work will help to find all the genes necessary for structurally and functionally correct sensory neuron cilia.

Jan CH et al. (2011) Nature "Formation, regulation and evolution of Caenorhabditis elegans 3'UTRs."

Ruby JG, Bartel DP, Friedman RC, Jan CH

[Nature, 2011]

Post-transcriptional gene regulation frequently occurs through elements in mRNA 3' untranslated regions (UTRs). Although crucial roles for 3'UTR-mediated gene regulation have been found in Caenorhabditis elegans, most C. elegans genes have lacked annotated 3'UTRs. Here we describe a high-throughput method for reliable identification of polyadenylated RNA termini, and we apply this method, called poly(A)-position profiling by sequencing (3P-Seq), to determine C. elegans 3'UTRs. Compared to standard methods also recently applied to C. elegans UTRs, 3P-Seq identified 8,580 additional UTRs while excluding thousands of shorter UTR isoforms that do not seem to be authentic. Analysis of this expanded and corrected data set suggested that the high A/U content of C. elegans 3'UTRs facilitated genome compaction, because the elements specifying cleavage and polyadenylation, which are A/U rich, can more readily emerge in A/U-rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,480 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes use palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3'UTRs have median length only one-sixth that of mammalian 3'UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying post-transcriptional gene regulation.

Jan Burghoorn et al. (2002) European Worm Meeting "Taste perception by the nematode Caenorhabditis elegans"

Gert Jansen, Jan Burghoorn

[European Worm Meeting, 2002]

We use Caenorhabditis elegans to study heterotrimeric G-protein mediated signal transduction. Only seven pairs of chemosensory cells are involved in the detection of tastants. Five pairs of cells were found to be important for attraction, ADF, ASE, ASG, ASI and ASK and two pairs of cells for aversion, ADL and ASH. Despite this limited number of chemosensory cells, the nematode is able to detect several different compounds and distinguish between them. This is most likely accomplished by the expression of multiple chemosensory receptors per cell. Furthermore, the observation that these cells express multiple different G protein alpha subunits implies that multiple signal transduction pathways might exist for perception per cell. In this project we will determine which G alpha subunits are involved in taste, and whether these signals are modulated. We use a water soluble compound chemotaxis assay (Wicks et al 2000, Dev Biol 221, 295-307) to determine the effect of loss- or gain-of-function of the various alpha subunits. We searched for suitable compounds of the taste modalities salt, sweet and bitter. NaCl (salt) was clearly recognized by C. elegans. It is thought that NaCl perception relies on ion channels and that G proteins are not involved. However the loss-of-function mutant of gpa-3 (a G alpha subunit) gives a weaker response to salt compared to the wild type, suggesting that G- protein signaling is involved. Trehalose was a suitable sweet compound. In Drosophila trehalose acts via G-protein coupled receptors (GPCR). C. elegans avoids trehalose, however, mutations in the G-alpha subunit ODR- 3 and GPA-10 affect trehalose aversion. This clearly shows that G proteins are involved. An appropriate bitter compound is denatonium. In mammals the denatonium signaling pathway acts via GPCR. For C. elegans denatonium is a strong atractant, yet in time it becomes very repulsive to C. elegans. Mutations in the G-alpha subunits GPA-1, 2, 3, 10, 11, 14 and 15 affect chemotaxis to denatonium. We use genetic cell specific rescue (Massimo et al 2001, Int Worm Meeting, abstract 206) to determine which cells are involved in the perception of denatonium in our assay. Subsequently, we will determine which G alpha subunits are involved, and whether these signals are modulated.

Jan A et al. (2016) Acta Neuropathol "eEF2K inhibition blocks A42 neurotoxicity by promoting an NRF2 antioxidant response."

Taubert S, Sorensen PH, Bhanshali F, Jan A, Vandal M, Hayden MR, Delaidelli A, MacKenzie I, Jansonius B, Moerman D, Somasekharan SP, Calon F, Negri GL

[Acta Neuropathol, 2016]

Soluble oligomers of amyloid- (A) impair synaptic plasticity, perturb neuronal energy homeostasis, and are implicated in Alzheimer's disease (AD) pathogenesis. Therefore, significant efforts in AD drug discovery research aim to prevent the formation of A oligomers or block their neurotoxicity. The eukaryotic elongation factor-2 kinase (eEF2K) plays a critical role in synaptic plasticity, and couples neurotransmission to local dendritic mRNA translation. Recent evidence indicates that A oligomers activate neuronal eEF2K, suggesting a potential link to A induced synaptic dysfunction. However, a detailed understanding of the role of eEF2K in AD pathogenesis, and therapeutic potential of eEF2K inhibition in AD, remain to be determined. Here, we show that eEF2K activity is increased in postmortem AD patient cortex and hippocampus, and in the hippocampus of aged transgenic AD mice. Furthermore, eEF2K inhibition using pharmacological or genetic approaches prevented the toxic effects of A42 oligomers on neuronal viability and dendrite formation in vitro. We also report that eEF2K inhibition promotes the nuclear factor erythroid 2-related factor (NRF2) antioxidant response in neuronal cells, which was crucial for the beneficial effects of eEF2K inhibition in neurons exposed to A42 oligomers. Accordingly, NRF2 knockdown or overexpression of the NRF2 inhibitor, Kelch-Like ECH-Associated Protein-1 (Keap1), significantly attenuated the neuroprotection associated with eEF2K inhibition. Finally, genetic deletion of the eEF2K ortholog efk-1 reduced oxidative stress, and improved chemotaxis and serotonin sensitivity in C. elegans expressing human A42 in neurons. Taken together, these findings highlight the potential utility of eEF2K inhibition to reduce A-mediated oxidative stress in AD.

Jan Burghoorn et al. (2003) International Worm Meeting "G-protein signaling in sensory neuron development"

Jan Burghoorn, Gert Jansen

[International Worm Meeting, 2003]

The nematode Caenorhabitis elegans has nonmotile cilia, which are situated at the dendritic endings of sensory neurons. Most of the ciliated nerve endings are exposed directly to the external environment. Fluorescent dye filling of the phasmid and most of the amphid neurons typically indicates normal cilia ultrastructure. Mutants like osm-6 on the other hand, which have a developmental defect of the cilia, show no dye filling. OSM-6 is a component of the transport system known as intraflagellar transport (IFT), that is important for the formation and maintenance of many cilia, such as sensory cilia. IFT is driven from the base of the cilium (transition zone) to its distal tip. The G-alpha subunit GPA-3 is specifically expressed in many sensory neurons and over expression of a dominant active mutant of this G-alpha subunit leads to a non-dye filling phenotype (Zwaal et al., (1997), Genetics. 145:715-27.). To investigate this defect we used a GPA-13::GFP (Jansen et al., (1999) Nat. Genet. 21:414-9.) fusion to look at the AWC fan-like cilia structure. This fusion protein is highly enriched in the AWC ciliated endings in the N2 Bristol. In contrast, in the gpa-3QL and osm-6 mutants GPA-13::GFP is uniformly expressed throughout the AWC neuron with accumulation in the transition zone. In addition, similar results were obtained using anti-GPA-3 immunostaining of wild type, gpa-3QL and osm-6, which visualize the sensory neurons with rod-like cilia structures. This strongly suggests a role for GPA-3 in the development of the sensory cilia. To determine the signaling pathways in which GPA-3 participates we screened for suppressors of the gpa-3 non-dye filling phenotype. Nine independent suppressors mutants where isolated and preliminary mapping revealed mutations in at least at three independent loci. Two mutants had a dye filling pattern comparable to wild type animals. Seven other mutants showed dye-filling of only a subset of the sensory neurons or morphological defects of the sensory neurons. GPA-13::GFP expression was determined in one mutant thus far. This mutant revealed an almost full recovery of the AWC fan-like cilia structure.

Jan Burghoorn et al. (2006) European Worm Meeting "Involvement of DAF-19 in the General Development and Subtype Specification of Cilia"

Jan Burghoorn, Peter Swoboda, Sofie Sahlin

[European Worm Meeting, 2006]

The highly conserved C. elegans DAF-19 protein, a regulatory factor X (RFX) transcription factor was originally identified as a key regulator for cilia development (Swoboda et al. 2000). Cilia are specialized subcellular organelles that in C. elegans are present on the endings of sensory neuron dendrites. They are functional compartments that mediate signal reception and transduction from the environment. In order to be able to receive many different signals from the environment, cell type specific expression of signal transduction and cilia structural genes have evolved. This resulted in, for example, rod-like cilia, which are involved in the perception of soluble chemotaxis compounds. By contrast, the wing-like cilia are involved in smell perception. However, how different cilia subtypes develop is hardly known. Recent data suggests the involvement of developmental cascades in the promotion of cell specific neuron cilia subtypes (Lanjuin and Sengupta 2004). In this cascade well-conserved transcription factors through possibly multiple transcription events are thought to specify the identity of sensory neuron cilia subtypes. This is similar to the neuron cell (sub)type specification cascade. We are interested whether DAF-19 is involved in this cilia subtype specification cascade. This would imply that DAF-19 does not only play a role in general ciliogenesis, but also plays a role in cilia subtype specification. In a genome-wide search for candidate genes with a DAF-19 recognition site in the promoter region, the X-box, many transcription factors were identified (Blacque et al. 2005; Efimenko et al. 2005). We are currently establishing the expression patterns of more than 20 selected transcription factors, for three of which we could already demonstrate to be expressed in ciliated neurons. In addition we will establish if those transcription factors, expressed in ciliated sensory neurons, are transcriptionally dependent on DAF-19 and determine if they play a role in cilia subtype specification.

Jan E et al. (1997) EMBO J "Conservation of the C.elegans tra-2 3'UTR translational control."

Goodwin EB, Yoon JW, Jan E, Walterhouse D, Iannaccone P

[EMBO J, 1997]

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.

Jan Burghoorn et al. (2008) European Worm Meeting "Environmental cues and G protein signalling regulate intraflagellar transport."

Jan Burghoorn, Martijn Dekkers, Evgeni Efimenko, Rob Willemsen, Ton de Jong, Suzanne Rademakers, Gert Jansen, Peter Swoboda

[European Worm Meeting, 2008]

Cilia are important for various functions, including sensory signaling.. Recent studies suggest that the structure of cilia and the sensory. signaling machinery in the cilia is regulated by intraflagellar transport. (IFT). In C. elegans this regulation may be achieved by a structural. compartmentalization of the cilia in middle and distal segments and the. coordination of two kinesin-2 motor complexes used for anterograde IFT,. kinesin II and OSM-3. The kinesins are loaded with complex A and B particle. proteins and cargo molecules. Both kinesins enter the cilia middle segments. and move together, but only OSM-3 enters the distal segments.. We found that in gain-of-function (gpa-3QL) mutants of the heterotrimeric. Ga gpa-3 cilia length is changed. Using EM and cilium specific GFP markers. we show that the length of a subset of amphid cilia is reduced and that. they are sometimes posteriorly displaced. The morphology is affected in a. dose dependent and reversible way. gpa-3 loss-of-function (lf) animals do. not display any morphological abnormalities. Interestingly, IFT proteins. are located normally in the cilia, but IFT is perturbed in both gpa-3(lf). and gpa-3QL mutants: kinesin II speed is reduced and OSM-3 kinesin speed is. increased in the middle segments of the cilia. However, complex A/B speed. is approximately wild type. These results suggest that kinesin II and OSM-3. kinesin are partially uncoupled in gpa-3 mutants, but both motors are still. able to transport complex A/B proteins. Previous studies have shown that. GPA-3 functions in the developmental switch that regulates dauer formation.. Interestingly, exposure to dauer pheromone affected ASI dye filling and OSM-. 3 speed, similar to mutation of gpa-3. We suggest a model where. environmental cues regulate cilia length by regulating the constitution of. IFT particles.. To identify additional genes that function in the regulation of IFT by G-. protein signaling, we performed a genetic screen for suppressors of gpa-3QL. (sql). We identified sql-1, which encodes the homologue of the mammalian. Golgi protein GMAP-210, and uev-3, which encodes a ubiquitin-conjugating. enzyme variant that lacks the cysteine necessary for catalytic activity.. sql-1(lf) suppresses the dye filling defect of gpa-3QL animals and restores. the length of the ASI cilia. Moreover sql-1(lf) has a similar effect on the. speeds of OSM-3 and kinesin II as mutation of gpa-3.. POSTER

Jan Burghoorn et al. (2006) European Worm Meeting "G Proteins and Environmental Cues Regulate the Localization of Ciliary Signalling Molecules"

Suzanne Rademakers, Evgeni Efimenko, Gert Jansen, Jan Burghoorn, Peter Swoboda

[European Worm Meeting, 2006]

Jan Burghoorn1, Suzanne Rademakers1, Evgeni Efimenko2, Peter Swoboda2 & Gert Jansen1 . Sensory perception requires the proper localization of signalling molecules in sensory cilia. Intraflagellar transport (IFT), a system essential for the assembly and maintenance of cilia, plays a critical role in trafficking cargo. Although many IFT proteins have been identified, not much is known about the regulation and specificity of IFT. We were intrigued by the finding that gain-of-function mutation of the G? protein GPA-3 in C. elegans (gpa-3QL) causes a dye filling defect (Zwaal et al., 1997, Genetics). Since many dye filling defects are caused by mutations in genes important for IFT, we hypothesized that gpa-3QL might affect IFT. Defects in the IFT machinery often strongly affect the microtubular axoneme of the cilia. However, we found no defects in the microtubular axoneme of gpa-3QL animals. Next, we looked at more specific IFT defects in the cilia of gpa-3QL animals. To this end we determined the localisation of several sensory sensory G? subunits using GFP fusion constructs, since they are possible cargo molecules. Surprisingly, gpa-3QL interfered with entry into the distal segments of the cilia of three sensory G? proteins, GPA-4, GPA-9 and GPA-15, but did not affect other G? proteins. In wild type animals GPA-4 and GPA-15 can be transported in the cilia middle segments by both kinesin II and OSM-3 kinesin motor complexes, and in the distal segments by OSM-3. In gpa-3QL animals the two G? proteins are predominantly transported by kinesin II; OSM-3 mediated transport can only be seen in the absence of kinesin II. However, we found no evidence that gpa-3QL affects the two kinesin complexes, the retrograde motor complex or several other IFT proteins. In contrast, gpa-3QL strongly affects the localization of OSM-5, the C. elegans homologue of the mouse Polaris/Tg737 IFT protein. We propose a model in which G protein mediated signals regulate the localization of specific signalling molecules in the cilia by modulating, via OSM-5/Polaris, the assembly of motor protein - cargo complexes. Finally, we show that a similar effect on the localization of GPA-4 and GPA-15 in the cilia can be accomplished by exposing C. elegans larvae to dauer pheromone. We hypothesize that our findings reveal a novel mechanism that allows structural plasticity in response to environmental cues.

Jan Karbowski et al. (2004) West Coast Worm Meeting "Nematode locomotion: Integrating theory with genetic perturbations and comparative analysis."

Jane E Mendel, Christopher J Cronin, Adeline Seah, Jan Karbowski, Paul W Sternberg

[West Coast Worm Meeting, 2004]

We are interested in understanding the mechanism controlling the undulatory locomotion of nematodes. We recorded movement of worms with our automated movement analysis system, focusing on genetic perturbations to potential controlling parameters, including neurons, muscle, and cuticle. We also examined the undulatory locomotion of different Caenorhabditis species. We constructed a theoretical model that combines neural dynamics, mechanics, and biophysics, and that can be directly related to parameters measured experimentally. These parameters include velocity of movement, frequency, amplitude, and wavelength of undulations. The model fits the data well. For the cases we studied, the undulatory locomotion is robust. We suggest that the control mechanisms, implemented by mechano-sensory feedback, are robust as well, and optimize the velocity.