Walker, Amy K. et al. (2011) International Worm Meeting "Control of one-carbon cycle genes by SBP-1 links SAMe production, PC biosynthesis and lipogenesis."

Watts, Jennifer L., Jacobs, Rene, Walker, Amy K., Rottiers, Veerle, Hart, Anne, Naar, Anders, Hansen, Malene

[International Worm Meeting, 2011]

To understand regulation of lipogenesis, we have examined the SBP-1/SREBP transcription factors necessary for fatty acid, phospholipid and cholesterol biosynthetic gene expression and lipid accumulation in vivo. Inactive SREBPs reside cytoplasmically in the ER and are activated by transit to the Golgi, where proteases release transcriptionally active forms to the nucleus. We have found a new set of SBP target genes in the 1-carbon cycle (1CC), including sams-1, an enzyme which produces s-adenosylmethionine (SAMe), the major methyl donor. We also found a feedback loop increasing SBP-1-dependent transcription and lipogenesis in C. elegans, murine liver and human cells when SAMe is limited. Deficiencies folate or other 1C metabolites predispose human fatty liver disease; thus, our studies linking SAMe production to SBP-1-dependent lipogenesis in C. elegans are likely relevant to metabolic disorders common in Western populations. sbp-1(RNAi) animals are lean and several SBP-1 targets, such as fat-7 are also required for lipogenesis. However, sams-1(RNAi) animals have increased lipid accumulation and lipogenic gene expression which depend on sbp-1, suggesting a feedback loop activating SBP-1 when SAMe is limiting. Metabolomic profiling after sams-1 RNAi revealed defects in phosphatidylcholine (PC) biosynthesis, which can require multiple methylation steps. Genetic ablation of PC biosynthesis coupled with dietary rescue experiments show that SBP-1 activity is sensitive to PC levels. We also found that murine liver or human cells with diminished SAMe or PC synthesis had increases in lipogenesis and SREBP-1 activity. Because PC is a major membrane component, we reasoned that SBP-1 activation was mechanistically linked to ER or Golgi function. We found that ER stress responses are strongly activated after sams-1 RNAi, but are not contributors to SBP-1 activation. However, Golgi markers were disrupted. Therefore we examined localization of Golgi-resident, SREBP-activating proteases in human cells and found that limiting SAMe or PC caused these proteases to relocalize to the ER, allowing processing of SREBP-1 and activation. Our observations show SBP-1/SREBP-1 may be over activated when SAMe and PC are low and may also reveal how methyl-deficient diets contribute to human fatty liver disease.

Khursheed A Wani et al. (2005) International Worm Meeting "C. elegans A, B and B3- type cyclins have both distinct and overlapping functions"

Khursheed A Wani, Sander Van Den Heuvel, Monique A Lorson, John S Satterlee, Jessica Koranda

[International Worm Meeting, 2005]

We are interested in understanding the roles of different Cyclin-dependent kinase (Cdk) complexes in the developmental regulation of cell-cycle progression. The C. elegans cell cycle uses multiple Cdks and various cyclins, possibly representing the entire range of Cyclin/Cdk complexes in mammals. C. elegans and other metazoans express distinct A- and B-type cyclins, as well as a cyclin B3 protein that shares specific motifs with both other mitotic cyclins. The evolutionary conservation of these distinct cyclin subfamilies may point to specificity in function. However, genetic studies in Drosophila have demonstrated that neither cyclin B nor cyclin B3 is essential for viability, and that cyclin A, B and B3 all cooperate during mitosis [Jacobs et al, Genes Dev. 12(23),1998].In previous unpublished studies, we observed that C. elegans B-type cyclins CYB-1 and CYB-3 show similar developmental expression patterns and largely overlapping subcellular localizations. However, inactivation of either gene by RNAi resulted in specific meiotic and mitotic defects. Inhibition of both cyclins simultaneously resulted in an earlier and more dramatic cell-cycle arrest and resembled cdk-1/ncc-1 RNAi. These results indicated that cyb-1 and cyb-3 have specific as well as overlapping M-phase functions. We wanted to expand these observations by including null mutations and characterization of cya-1 cyclin A. We generated a cya-1 deletion allele in a PCR-based approach. cya-1(he158) homozygous mutants and cya-1(RNAi) animals displayed a late larval arrest and protruding vulva phenotype. Stronger and more variable larval defects were seen in animals homozygous for either the cyb-1(gk35) or cyb-3(gk195) deletion allele (kindly provided by the C. elegans Knockout Consortium). Combination of cya-1 RNA and cyb-3(gk195) further increased the defects and resulted in a high percentage embryonic lethality. These results confirm and expand the RNAi-based observations, and indicate that cya-1, cyb-1 and cyb-3 each are individually required but also have overlapping functions.

Simon Tuck et al. (2003) International Worm Meeting "A positive role for lin-1 during VPC fate specification."

Simon Tuck, Teresa Tiensuu, Emma Vernersson

[International Worm Meeting, 2003]

During vulval development, P6.p is induced to adopt the 1o cell fate by a cell in the somatic gonad, the anchor cell. This signaling event is mediated by a receptor-type tyrosine kinase, LET-23 that functions by activating a conserved Ras/MAP kinase signal transduction pathway. Previous work has suggested that the pathway functions in part by phosphorylating and inactivating LIN-1, an ETS protein 1, 2. lin-1 loss of function mutations cause a Multivulva phenotype similar to that caused by constitutive activation of let-60 ras. We have obtained evidence that the role of lin-1 in the induction of the 1o fate may be more complicated than previously thought. Specifically we find that, contrary to expectation, lin-1 is required positively for the expression of several markers for the 1o fate. For example whereas in WT, Ras/MAP kinase signaling induces the expression of egl-17::gfp in P6.p, expression of the marker is abolished in lin-1(0). The requirement for lin-1 for expression of the marker is downstream of or in parallel to let-60 ras since lin-1(0) let-60(gf) and lin-1(0); lin-15(0) double mutants fail to express the marker in P6.p. Furthermore, the effect of lin-1 on egl-17::gfp expression appears not to be caused by inappropriate activation or expression of lin-12, lip-1 or mab-5, three genes that each can inhibit the 1o vulval cell fate. Analysis of egl-17::gfp expression in the background of lin-1 mutations that affect a MAP kinase docking site in the protein suggest that LIN-1 might require modification by MAPK-1 Map kinase in order to function positively. A positive role for lin-1 in the expression of markers for the 1o fate is consistent with the SynVul phenotype seen in lin-1 double mutants with eor-1 or eor-23. In the regulation of egl-17::gfp lin-1 appears to function independently of lin-31(0), another target of the pathway, since expression is not affected in lin-31(0) but is abolished in lin31(0); lin-1(0) double mutants. In order to investigate lin-1's positive role further we have identified conserved elements in the egl-17 gene promoter that mediate induction of expression in P6.p. We are presently testing for binding to these elements. We are also carrying out experiments to determine the focus of lin-1 with respect to the expression of markers for the 1o fate. (1) Beitel, G. J., S. Tuck, I. Greenwald, and H. R. Horvitz (1995). Genes & Dev. 9: 3149-3162; (2) Jacobs, D., G. J. Beitel, S. G. Clark, H. R. Horvitz, and K. Kornfeld (1998). Genetics 149: 1809-1822; (3) Horward, R. and M. V. Sundaram (2002). Genes & Dev. 16: 1815-1827.

van Luenen HGAM et al. (1996) European Worm Meeting "The use of Tc1 and Tc3 transposons in Caenorhabditis elegans and other species"

Vos JC, van der Linden KM, Hazendonk E, De Baere I, van Luenen HGAM, Plasterk RHA

[European Worm Meeting, 1996]

Tc1 and Tc3 transposable elements are the most active transposons in the nematode C. elegans. They are responsible for most of the spontaneous mutations in certain strains. Tc1 and Tc3 belong to a large family of transposons found in many different organisms, ranging from fungi to vertebrates, including man. We have investigated the mechanism of excision and integration of Tc1 and Tc3 (1). The requirements for this reaction are limited: the transposase protein encoded by the element (2), and the sequences at both ends of the transposon (see the abstract of Rene Rezsohazy). These limited requirements for transposition might explain the successful spread of the Tc1-like elements throughout the animal kingdom. The limited requirements for transposition might allow the use of Tc1 or Tc3 as a gene delivery system in C. elegans and other species. We are currently collaboration with several groups to investigate whether Tc1/Tc3 transposes in mice, zebrafish and insects. The C. elegans genome sequencing project will be making the complete genomic sequence available within two years. Efficient reverse genetics will be needed to study the functions of the newly identified genes. One method to determine the function of a gene is to disrupt the coding sequence by the insertion of a transposon. We have developed an efficient method for the isolation of transposon insertion mutants in a specific sequence (3). A large collection of different cultures of strain MT3126 has been generated and frozen away. The strain MT3126 contains many Tc1 transposons which are mobile in the germ line. DNA samples of all these cultures have been pooled in an organized way. With only a few PCR's we can identify a transposon insertion in a specific sequence and the frozen culture can be thawed. A transposon insertion in a gene is not a guaranteed null allele of a gene. Therefore, isolation of a transposon insertion is followed by the isolation of a transposon excision induced deletion of the genomic region. We screen our collection on a routine basis for laboratories all over the world and we have obtained thus far transposon insertions in over a 100 different genes. Transposons can also be used for forward genetic analysis. We have developed a quick method to display all transposon insertions in a genome in a single lane of a sequencing gel. Screening a panel of different cultures, it is possible to determine which transposon insertion co-segregates with a phenotype caused by a transposon integration. This band can be excised from gel and the transposon flank can be sequenced. This sequence is used to screen the data base and the mutated gene can identified. With this method it is possible to identify the gene that causes the phenotype within 2 or 3 days, whereas with the classical method of transposon tagging and cloning it would at least take a couple of months. Secondly, we have determined most Tc1 insertion sites in three strains with a high Tc1 copy number. These insertions can be visualized by PCR and they can be used for mapping mutant genes (see abstract of Rik Korswagen). 1. Van Luenen, H. G. A. M., Colloms, S. D. & Plasterk, R. H. A. (1994) Cell 79, 293-301. 2. Vos, J. C., de Baere, I. & Plasterk, R. H. A. (1996) Genes & Dev. 10, 755-761. 3. Zwaal, R. R., Broeks, A., Van Meurs, J., Groenen, J. T. M. & Plasterk, R. H. A. (1993) Proc. Natl. Acad. Sci. USA 90, 7431-7435.

Chris D Suh et al. (2004) West Coast Worm Meeting "Talin functions in neuronal axon guidance through integrin signaling in C. elegans"

Cori Bargmann, Alexandr Gonchrov, Yishi Jin, Chris D Suh, Scott Clark

[West Coast Worm Meeting, 2004]

Axons rely on both long-range and short-range guidance cues to reach their targets. Many of the known guidance cues are evolutionarily conserved from C. elegans to higher vertebrates. The best studied are netrins and its receptors, slit and Robo, and semaphorins and plexins. Integrins are heterodimeric cell surface receptors and involved in many different fundamental biological processes such as cell adhesion, signaling, differentiation, proliferation and cell migration. Recent studies have revealed specific roles of integrins in guiding axons to their targets (Nakamoto et al., 2004). For example, netrin-1 serves as an adhesive ligand that associates with integrins in epithelial cells in mammals (Yebra et al., 2003). In Drosophila , slit, a midline repellent molecule, has been shown to interact with integrin in dosage dependent manner (Stevens and Jacobs, 2002). How integrin signaling is transduced in the nervous system is not well understood. We report here the function of talin, a cytoplasmic signal tranducer for the integrin receptors, in motor neuron axon guidance. To identify novel genes involved in axon guidance, we carried out a genetic screen using a GFP marker that visualizes the GABAergic motor neurons, and searched for mutants with pleiotropic defects in fertility, larval viability, and motor neuron morphology. One mutation was an allele of unc-35 . By allele sequencing, we found that unc-35 encodes Talin. Talin binds directly to integrin receptors and actin cytoskeleton, thereby re-organizing the cytoskeleton in many different cell types in response to environmental stimuli. C. elegans talin/UNC-35 shows 36% identity to fly, mouse, and human talins. unc-35 mutations exhibit a broad range of defects in muscle, nervous system and gonad. We have focused on the role of unc-35 /talin and its interaction with integrins in the nervous system. The ventral cord motor neurons extend circumferential axons with stereotypical trajectories along the left or right side of the animal (Brown, 2000). For example, most GABAergic motor neurons (DD and VD) commissures exit the ventral cord on the right side, while most of the cholinergic DA neuron commissures extend to the left side. In unc-35 mutants this left/right motor axon asymmetry is altered. unc-35 mutants also show a striking axon guidance phenotype such that many commissures of the DA and DB neurons fail to reach the dorsal nerve cord, instead, turn laterally at a sub-dorsal position along the edge of the dorsal body muscle, as revealed by Nomarski DIC and electron microscopy (EM). C. elegans has two alpha-integrins and one beta-integrins. Mutations in the alpha integrin, ina-1 , and beta integrin, pat-3 , cause a similar, but weaker, axon guidance phenotype than unc-35 . Double mutant analysis between unc-35 and the integrin mutants are consistent with Talin functioning through the ina-1 and pat-3 integrin receptors. Using transgene RNAi, we found that unc-35 and integrins most likely functions cell autonomously in GABAergic motor neurons. Although many studies have shown how long range cues guide growth cones to their proper targets, we know very little about how short range or contact mediated signals affect growth cone guidance. Our analysis of integrins and talin mutants demonstrates that integrin and talin mediate specific short-range axon guidance at the left/right axon decision and at the dorsal muscle edge choice.