Tauffenberger, Arnaud et al. (2009) International Worm Meeting "Age-dependent modification of expanded CAG toxicity in simple C. elegans models."

Parker, Alex, Tauffenberger, Arnaud, Bel-Hadj, Samar

[International Worm Meeting, 2009]

Arnaud Tauffenberger1,2,3, Samar Bel-Hadj1, Alex Parker1,2, 3 1CRCHUM, 2Centre of Excellence in Neuromics, 3Department de pathologie et biologie cellulaire, Universite da Montreal, Montreal, Quebec, Canada The expansion of CAG trinucleotides coding for glutamine is the cause of at least nine late-onset neurodegenerative diseases. The expanded CAG (expCAG) diseases may have some pathogenic mechanisms in common as the disease threshold for all of these disorders is around 40 CAG repeats and they display misfolded intracellular protein aggregation phenotypes. We have turned to the model organism C. elegans to explore two areas of interest: 1. Age-dependent toxicity: Although the expCAG diseases show anticipation, the size of CAG repeat only partially correlates with the variation in age-of-onset. Evidence suggests that even for these single trait neurodegenerative diseases, disease onset and progression may have additional environmental and genetic components. Using C. elegans transgenic worms expressing expCAG in neurons we are exploring the links between aging and late-onset neurodegeneration. We hypothesize that genes regulating longevity, stress response, their associated signaling pathways and downstream effectors may encode new therapeutic targets for age-dependent proteotoxicity in humans. 2. Mechanisms of expCAG toxicity: Long homopolymeric CAG nucleotide stretches are unstable and may be prone to translation errors. Mistranslation of expCAG to alternate reading frames may produce hybrid proteins with additional toxicity. We have created transgenic strains to investigate this phenomenon. A summary of our data and recent findings will be presented.

Frame IG et al. (2001) Gene "New BEL-like LTR-retrotransposons in Fugu rubripes, Caenorhabditis elegans, and Drosophila melanogaster."

Poulter RTM, Frame IG, Cutfield JF

[Gene, 2001]

The BEL group of retroelements is present in greater numbers, variety and taxonomic range than may have been thought previously. In addition to the insects, nematodes and schistosomes, BEL-like elements are present in echinoderms, urochordates, and at least two highly diverged species of fish. We describe one new full-length BEL-like element in Fugu that we call Suzu, another in Drosophila that we call Tinker, and seven new families in C. elegans. Many of the C. elegans elements have an unusual insertion at the 5' end. The previously known Roo, TRAM and Telemac are also BEL-like retrotransposons. Some BEL-like elements have captured an envelope gene, probably from other retroelements in some cases but from a phlebovirus in one case.

Demers-Lamarche, Julie et al. (2009) International Worm Meeting "Development of a C. elegans model for a hereditary laye-onset cerebellar ataxia (LOCA)."

Parker, Alex J., Brais, Bernard, Bel Hadj, Samar, Demers-Lamarche, Julie, Thiffault, Isabelle

[International Worm Meeting, 2009]

Recessive ataxias are a heterogeneous group of neurodegenerative diseases. Late-onset ataxias have largely been considered as either milder forms of dominant ataxias or sporadic diseases seemingly not caused by genetic factors. Genotyping of our present cohort, 58 affected cases belonging to 34 families, allowed us to link a form of late-onset cerebellar ataxia (LOCA) with a region on the chromosome 2. Sequencing of genes in the candidate region uncovered mutations in a gene coding for ubiquitin-like protein not previously known to be mutated in a disease. We investigated the effect of the inhibition of the C. elegans homologues on the phenotype. We showed that RNAi silencing in both wild-type N2 and neurosensitive rrf-3 strains reduced lifespan. The RNAi experience on N2 strain did not activate a number of in vivo stress reporters including sod-3::GFP, hsp-4::GFP, hsp-6::GFP and hsp-60::GFP. The identification of recessive mutations in a ubiquitin-like gene in LOCA cases suggested that abnormal protein folding also plays a role in this new form of late-onset neurodegenerative disorder so we reasoned that the RNAi gene homologue should affect proteotoxicity. We tested this hypothesis with transgenic worms expressing a polyglutamine-tract of 40 residues fused to YFP. The RNAi accelerated the mean onset of paralysis of these animals compare to control and the appearance of Q40::YFP aggregation puncta. We are now generating a transgenic C. elegans strain for this late-onset ataxia.

Lyczak R et al. (2000) West Coast Worm Meeting "Actin-dependent processes in the early C. elegans embryo require the profilin gene pfn-1, the FH gene cyk-1, and bel-1"

Severson AF, Lyczak R, Bowerman BA, Baillie DL

[West Coast Worm Meeting, 2000]

The actin cytoskeleton is essential for many processes in the early C. elegans embryo, including ruffling of the anterior cortex and pseudocleavage following fertilization, the asymmetric migration of pronuclei and position of the first mitotic spindle, and cytokinesis. A worm profilin gene, called pfn-1, is required for all of these processes. In pfn-1(RNAi) embryos, cortical ruffling and pseudocleavage are absent, the pronuclei meet in the center of the embryo, the first mitotic spindle is centrally positioned, and a cleavage furrow fails to form during cytokinesis. Profilin regulates the actin cytoskeleton by sequestering actin monomers and preventing their polymerization into microfilaments. In addition, profilin can stimulate actin assembly by converting actin into an assembly-competent GTP-bound state. In other systems, profilin can bind to FH proteins, which may recruit profilin/actin complexes to sites of actin assembly. The C. elegans FH protein CYK-1 is required for embryonic cytokinesis (Swan et al, 1998), and embryos produced by worms mutant for a strong combination of cyk-1 alleles fail early in furrow ingression. We are currently characterizing anti-PFN-1 antisera to examine PFN-1 localization in wild-type and CYK-1 mutant embryos. In addition, we are examining actin, myosin, and CYK-1 localization in pfn-1(RNAi) embryos to determine the effects of profilin inactivation on the actin cytoskeleton. While PFN-1 may be required for organized contraction of the actin cytoskeleton, a second gene may function to temporally and spatially restrict contractility in the embryo. In embryos mutant for a temperature-sensitive, embryonic lethal allele of this gene, waves of contraction sweep across the early embryo, resembling mobile cleavage furrows. We have named this gene bellydancer, or bel-1. To determine if the mechanism that underlies these waves is similar to the contractile ring, we are staining bel-1 mutant embryos with antibodies to actin, myosin, and CYK-1. In addition, we are mapping bel-1, which we hope to clone by a combination of RNAi phenocopy and transgenic rescue.

Burglin TR (1997) Nucleic Acids Res "Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals."

Burglin TR

[Nucleic Acids Res, 1997]

A new Caenorhabditis elegans homeobox gene, ceh-25, is described that belongs to the TALE superclass of atypical homeodomains, which are characterized by three extra residues between helix 1 and helix 2. ORF and PCR analysis revealed a novel type of alternative splicing within the homeobox. The alternative splicing occurs such that two different homeodomains can be generated, which differ in their first 25 amino acids. ceh-25 is an orthologue of the vertebrate Meis genes and it shares a new conserved domain of 130 amino acids with them. A thorough analysis of all TALE homeobox genes was performed and a new classification is presented. Four TALE classes are identified in animals: PBC, MEIS, TGIF and IRO (Iroquois); two types in fungi: the mating type genes (M-ATYP) and the CUP genes; and two types in plants: KNOX and BEL. The IRO class has a new conserved motif downstream of the homeodomain. For the KNOX class, a conserved domain, the KNOX domain, was defined upstream of the homeodomain. Comparison of the KNOX domain and the MEIS domain shows significant sequence similarity revealing the existence of an archetypal group of homeobox genes that encode two associated conserved domains. Thus TALE homeobox genes were already present in the common ancestor of plants, fungi and animals and represent a branch distinct from the typical homeobox genes.

Burglin TR et al. (2016) Chromosoma "Homeodomain proteins: an update."

Affolter M, Burglin TR

[Chromosoma, 2016]

Here, we provide an update of our review on homeobox genes that we wrote together with Walter Gehring in 1994. Since then, comprehensive surveys of homeobox genes have become possible due to genome sequencing projects. Using the 103 Drosophila homeobox genes as example, we present an updated classification. In animals, there are 16 major classes, ANTP, PRD, PRD-LIKE, POU, HNF, CUT (with four subclasses: ONECUT, CUX, SATB, and CMP), LIM, ZF, CERS, PROS, SIX/SO, plus the TALE superclass with the classes IRO, MKX, TGIF, PBC, and MEIS. In plants, there are 11 major classes, i.e., HD-ZIP (with four subclasses: I to IV), WOX, NDX, PHD, PLINC, LD, DDT, SAWADEE, PINTOX, and the two TALE classes KNOX and BEL. Most of these classes encode additional domains apart from the homeodomain. Numerous insights have been obtained in the last two decades into how homeodomain proteins bind to DNA and increase their specificity by interacting with other proteins to regulate cell- and tissue-specific gene expression. Not only protein-DNA base pair contacts are important for proper target selection; recent experiments also reveal that the shape of the DNA plays a role in specificity. Using selected examples, we highlight different mechanisms of homeodomain protein-DNA interaction. The PRD class of homeobox genes was of special interest to Walter Gehring in the last two decades. The PRD class comprises six families in Bilateria, and tinkers with four different motifs, i.e., the PAIRED domain, the Groucho-interacting motif EH1 (aka Octapeptide or TN), the homeodomain, and the OAR motif. Homologs of the co-repressor protein Groucho are also present in plants (TOPLESS), where they have been shown to interact with small amphipathic motives (EAR), and in yeast (TUP1), where we find an EH1-like motif in MAT2.

Rosa E Navarro et al. (2003) International Worm Meeting "vbh-1, the C. elegans homolog of Drosophila vasa and belle, is present in P granules and is essential for germline function."

Keith Blackwell, Rosa E Navarro, Sergio Zonszein

[International Worm Meeting, 2003]

DEAD box RNA helicases are involved in diverse cellular functions such as RNA splicing, ribosome assembly, initiation of translation, gametogenesis, embryogenesis, and cell growth. Several C. elegans RNA helicases have been identified to be components of P granules (mRNA-protein complexes that are present in the germline and that are also essential for its function). VASA is the first RNA helicase described as a Drosophila polar granule component, and is essential in the female germline. Based on interactions between VASA and translation factors it has been suggested that this protein might be involved in translational regulation; however, its function remains to be elucidated. In the C. elegans genome, the ORF most closely related to Drosophila vasa is Y54E10A.9. This gene is comparably similar to another RNA helicase from Drosophila known as belle. Belle is expressed in female and male somatic and germline tissues, and is required for oogenesis (1). Based on this homology, we have named Y54E10A.9 vasa belle like helicase, vbh-1. Using RNAi, we have found that vbh-1 is essential for germline function in C. elegans. Nearly half of the vbh-1(RNAi) F1 hermaphrodites are sterile, and the remainder show dramatically reduced brood size and produce a high percentage of sterile progeny. vbh-1(RNAi) F1 hermaphrodite gonads are characterized by normal germ cell numbers and size, but produce a reduced number of sperm. In many of these animals, the most proximal oocytes contain endomitotic nuclei. vbh-1(RNAi) males are also sterile. Antibody staining experiments have shown that the VBH-1 protein is present in both the male and hermaphrodite germline. VBH-1 is found in the germ cell cytoplasm, and is abundant in P granules in both hermaphodites and males. We have been unable to detected VBH-1 staining in the somatic gonad or other tissues, suggesting that this protein might be germline specific. We are continuing to analyze the vbh-1(RNAi) phenotype, and investigate VBH-1 function.Reference1. Johnstone, O., Deuring, R., Fuller, M.T. and Lasko, P. Bel is an essential DEAD-box protein with a requirement in the germline. A. Dros. Res. Conf. 2001:63A

Dennis, Shannon M et al. (2009) International Worm Meeting "Analysis of Viruslike Particles in the C. elegans germline."

Priess, James R, Dennis, Shannon M

[International Worm Meeting, 2009]

Retroviral DNA is reverse-transcribed in the cytoplasm and forms a large nucleoprotein pre-integration complex (PIC) that is transported to the nucleus. In non-dividing cells, the PIC must be imported through nuclear pores. P granules cover most nuclear pores in C. elegans germ cells except during cell division and in transcriptionally quiescent oocytes or early embryos. We are interested in how retroelements confront, or circumvent, P granules. The C. elegans genome contains an unusually small number of retroviral and retrotransposon sequences compared to other animal genomes, suggesting that nematodes have efficient methods of combating these elements. No natural virus infections or viruslike particles (VLPs) have been reported previously in nematodes. We used electron microscopy to search for VLPs in the adult gonads of 17 wild strains of C. elegans and in 4 other Caenorhabditis species. In a typical experiment, a total of from 800 to 1200 germ cells were scored from about 40 gonads, and each gonad was analyzed from a single plane of section to provide a minimum estimate. We found that about 4% of germ cells in N2 gonads contain VLPs; these were seen in multiple laboratory strains of N2 as well as in a fresh sample of the "ancestral" N2 strain from the CGC. Most of the wild C. elegans strains lacked VLPs, and none had more than N2; VLPs were abundant in C. japonica gonads, but absent in the other Caenorhabiditis species tested. The VLPs appear to form on P granules in a narrow zone of late pachytene germ cells, then exit the germ cells in tight association with long microtubules that extend from the germ nuclei into the gonad core. This indicates a possible role of P granules in the formation of VLPs, analogous to recent results for yeast Ty3 assembly in P bodies. The VLPs are also present in early oogonia, presumably through cytoplasmic flow within the gonad, but generally are not detected in the most proximal oocytes or in early embryos. We did not observe an increase in VLP frequency in a strain lacking the cell death and RNAi pathways [ced-1(e1735);ced-3(n717); rde-1(ne300)], however the number of VLPs was affected markedly (both increasing and decreasing) in mutants lacking various Argonaute family members. We are testing by RNAi which retroelements contribute to the VLPs. Thus far, RNAi for either CER1 or CER2 (Gypsy/Ty3 clade) elements decrease VLP number, while RNAi against CER13 (Bel clade) does not. We are currently making GFP reporters and immunological reagents to examine CER1 and CER2, and hope to test whether strains with increased numbers of VLPs are active for retrotransposition.