Chan-Hsien Yeh et al. (2004) East Asia Worm Meeting "Screen for TLK-1-interacting proteins"

Chan-Hsien Yeh, Yi-Chun Wu

[East Asia Worm Meeting, 2004]

Tousled-like kinases (TLKs) constitute a family of serine/threonine kinases conserved in multi-cellular organisms and have important functions during cell-cycle progression. Altering TLK activity in loss or gain-of-function experiments caused aberrant chromosome morphology and arrested nuclear divisions. Human and fly TLKs appear to function in chromosome assembly and nuclear division, at least in part, through phosphorylation of histone H3 and histone chaperone protein Asf-1 (anti-silencing function protein 1) (Li Y. et al, 2001; Carrera P. et al, 2003). Our studies of the tlk-1 gene by RNAi showed that tlk-1 is important for DNA condensation and segregation during mitosis. In tlk-1(RNAi) embryos, mitotic DNA was not condensed normally and DNA segregation defects were observed. The phosphorylation of histone H3 has been implicated in mitotic DNA condensation. However, histone H3 was still phosphorylated in tlk(RNAi) mutants as detected by immuno-staining using the antibody against phospho-histone H3 (Han Z. et al, 2003).To understand how TLK-1 functions, we performed a yeast two-hybrid screen using TLK-1 as bait. In a pilot screen, we obtained TLK-1-interacting proteins with previously identified roles in translation, ubiquitination, signal transduction, cell-cycle regulation or cytoplasmic cargo translocation. We are currently testing the physiological significance of the interactions and performing a large-scale yeast two-hybrid screen for more TLK-1 interacting candidates. In parallel, we are establishing a TLK-1 kinase assay using the TLK-1 protein expressed in the baculovirus system. References 1.Carrera, P., Moshkin, Y.M., Gronke, S., Sillje, H.H.W., Nigg, E.A., Jackle, H., and Karch, F. (2003) Genes & Dev., 17, 2578-2590. 2.Han, Z., Saam J.R., Adams, H.P., Mango S.E., and Schumacher, J.M. (2003) Curr. Biol., 13, 1921-1929. 3.Li, Y., DeFatta, R., Anthony, C., Sunavala, G., and DeBenedetti, A. (2001) Oncogene, 20, 726-738.

Ju-Ling Liu et al. (2005) International Worm Meeting "TLK-1 functions in chromosome condensation and segregation during mitosis"

Ju-Ling Liu, Chan-Hsien Yeh, Yi-Chun Wu

[International Worm Meeting, 2005]

TLKs (tousled-like kinases) belong to a conserved serine/ threonine kinase family in multicellular organisms. Studies in flies and humans showed that TLKs function in chromatin assembly at S phase and chromosome condensation at M phase during cell cycle (1, 2). Recent studies showed that C. elegans tlk-1 plays an essential role in transcription (3). We found that tlk-1 is also important for chromosome condensation and segregation during mitosis. tlk-1(RNAi) mutants showed aneuploidy in DNA content and arrested at an approximately 55-cell stage with uneven distribution of small and large nuclei. To further characterize the tlk-1(RNAi) phenotype, we used the XA3505 strain carrying GFP-tagged histone H2B and TBB-2 to monitor DNA and tubulin dynamics during mitosis in living embryos. Prometaphase chromosomes of tlk-1(RNAi) embryos did not condense to a rod-like shape as those of wild-type embryos did. Although metaphase chromosomes of tlk-1(RNAi) mutants aligned in the metaphase plate but did not condense properly. Chromosome bridges and unequal (or even no) chromosome segregation were frequently observed during anaphase. These results show that tlk-1 is important for DNA condensation and segregation during mitosis. We are currently performing a yeast two-hybrid screen and tlk-1 suppressor screen to identify molecules that may directly or indirectly interact with tlk-1 during mitosis. References: 1.Carrera, P., Moshkin, Y.M., Gronke, S., Sillje, H.H.W., Nigg, E.A., Jackle, H., and Karch, F. (2003) Genes & Dev., 17, 2578-2590. 2.Li, Y., DeFatta, R., Anthony, C., Sunavala, G., and DeBenedetti, A. (2001) Oncogene, 20, 726-738. 3.Han, Z., Saam J.R., Adams, H.P., Mango S.E., and Schumacher, J.M. (2003) Curr. Biol., 13, 1921-1929.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Ju-Ling Liu et al. (2004) East Asia Worm Meeting "TLK-1 functions in chromosome condensation and segregation during mitosis."

Yi-Chun Wu, Ju-Ling Liu

[East Asia Worm Meeting, 2004]

TLKs, tousled-like kinases, is a conserved serine/ threonine kinases family in multicellular organism. Previous studies in flies and humans showed that TLKs function in chromatin assembly at S phase and chromosome condensation at M phase during cell cycle (1,2). Recent studies showed that C.elegans TLK-1 plays an essential role in transcription (3). We found that C.elegans tlk-1 is also important for chromosome condensation and segregation during mitosis.. The tlk-1(RNAi) mutants arrested at about 55-cell stages with uneven distributions of small and large nuclei. When stained with DAPI, signals with various intensities and sizes were observed in tlk-1(RNAi) mutants. To quantify chromosomes in tlk-1(RNAi) mutants we utilized the PS2442 (constructed by Paul Sternberg lab) that carries integrated lacI ::GFP and lacO transgenes. LacI ::GFP binds lacO, thereby allowing for detection of lacO. In wild-type embryos we normally saw two GFP dots; however, cells with less than one dots or cells with six or eight dots were observed in tlk-1(RNAi) mutants, indicating the phenotype of aneuploidy. To further characterize the tlk-1(RNAi) phenotype, we used the XA3505 strain carrying both the GFP-tagged histone H2B and beta-tubulin to monitor the DNA and tubulin dynamics during mitosis. Prometaphase chromosomes of tlk-1(RNAi) embryos did not condense to a rod-like shape as did wild-type embryos. During metaphase chromosomes aligned in the metaphase plate but did not condense properly. Chromosome bridges and unequal (or even no) chromosome segregation were observed during anaphase. These results show that tlk-1 is important for DNA condensation and segregation during mitosis. Defects in chromosome condensation and segregation result in aneuploidy in tlk-1(RNAi) mutants. To understand how TLK function we are currently investigating the relationship of tlk-1 with various previously identified proteins involved in cell cycle progression.References:1. Carrera, P., Moshkin, Y.M., Gronke, S., Sillje, H.H.W., Nigg, E.A., Jackle, H., and Karch, F. (2003) Genes & Dev., 17, 2578-2590.2. Li, Y., DeFatta, R., Anthony, C., Sunavala, G., and DeBenedetti, A. (2001) Oncogene, 20, 726-738.3. Han, Z., Saam J.R., Adams, H.P., Mango S.E., and Schumacher, J.M. (2003) Curr. Biol., 13, 1921-1929.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Wendy Hanna-Rose, Rasika Kalamegham

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Schwartz, Hillel et al. (2013) International Worm Meeting "Developing Heterorhabditis nematodes as an experimental system for the study of mutualistic symbiosis."

Schwartz, Hillel, Sternberg, Paul

[International Worm Meeting, 2013]

Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora. Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility. In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.

Schwartz H et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Towards a SNP map for Heterorhabditis bacteriophora"

Sternberg P, Schwartz H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Heterorhabditis bacteriophora is a species of insect-parasitic nematode that lives in mutually beneficial symbiosis with pathogenic Photorhabdus luminescens bacteria. P. luminescens bacteria are lethal to insects and to other nematodes, including the soil nematode Caenorhabditis elegans, but are required for H. bacteriophora growth. The symbiosis between H. bacteriophora and P. luminescens therefore offers the potential to study the molecular genetic basis of their cooperative relationship. We are interested in developing tools to make such studies more feasible; in particular, we propose to generate tools for genetic mapping in H. bacteriophora. We will test independent isolates of H. bacteriophora to ensure that the isolates are cross-fertile. We will then examine the abilities of these isolates to grow on and to become infected with different wild-type and mutant Photorhabdus bacteria. From these tests we will select an isolate on which we will use next-generation high-throughput sequencing technology for the purpose of refining the existing draft genome sequence and for the creation of a SNP map. We anticipate that this SNP map will enable us and the wider insect-parasitic nematode community to identify induced mutations and natural variations affecting the interactions between H. bacteriophora nematodes and pathogenic Photorhabdus bacteria.

Chu, Yu-De et al. (2013) International Worm Meeting "An RNAi-based screen for the DExD/H-box RNA helicases involved in Caenorhabditis elegans microRNA function."

Chan, Shih-Peng, Chen, Shin-Kai, Chu, Yu-De, Chen, Guan-Rong, Huang, Tao

[International Worm Meeting, 2013]

MicroRNAs (miRNAs) are small non-coding regulatory RNAs that regulate gene expression at the post-transcriptional level and are involved in a broad spectrum of biological processes. Rearrangements of inter- or intra-molecular RNA structures and conformational changes of ribonucleoprotein complexes in the multiple processes of the miRNA pathway have led to the involvement of RNA helicase activities but little is known so far. In eukaryotes, RNA helicases generally belong to the superfamily 2 (SF2) in helicase classification, especially the DExD/H-box helicase family. The DExD/H-box proteins have been shown in association with many cellular processes involving RNA. To better understand the possible roles of DExD/H-box RNA helicases in miRNA function, we employed RNAi screen to identify genetic interaction between C. elegans DExD/H-box RNA helicases and the let-7 miRNA, which controls the timing of cell cycle exit and terminal differentiation. In addition to the RNA helicase p72, a component of Drosha Microprocessor complex, and CGH-1 that has been reported to facilitate the function of miRNA-induced silencing complex (miRISC), we found several DExD/H-box RNA helicases, which are involved in ribosomal RNA processing, pre-mRNA splicing and mRNA surveillance, may also take part in miRNA biogenesis and/or function. (Support: National Science Council, Taiwan. NSC 100-2311-B-002-006-MY3).

Alexandre Neves et al. (2006) Development & Evolution Meeting "A Notch-GATA Transcription Code for Endoderm-Specific Gene Activation in C.elegans"

James Priess, Alexandre Neves

[Development & Evolution Meeting, 2006]

Several Notch interactions occur in rapid succession during early C.elegans embryogenesis, each resulting in a distinct fate. We have previously shown that ref-1, a gene distantly related to Drosophila E(spl), is a direct Notch target in all these interactions. We show here that ref-1 expression is controlled by multiple, Notch-dependent enhancers that are specific for each interaction. We have identified a 150bp enhancer that is specific to Notch interactions in the endoderm, and found similar enhancers with the same activity in C.briggsae and C.remanei. The endoderm enhancer contains multiple, conserved binding sites for LAG-1/Su(H) and GATA transcription factors. We demonstrate that all LAG-1/Su(H) and GATA sites are required for full activity of this enhancer. We provide evidence that a GATA transcription factor called ELT-2 is a cooperative factor for Notch in the endoderm. Ectopic expression of ELT-2 in non-endodermal lineages caused activation of the endoderm enhancer only in Notch-signaled cells, suggesting that the presence of the cooperative factor dictates in which Notch interaction a Notch-dependent enhancer becomes responsive in vivo. Previous studies in Drosophila showed that synergy between Su(H) and the bHLH transcription factor Daughterless requires a specific configuration of Su(H) sites, known as a Su(H) paired site, SPS. The endoderm enhancer contains a putative SPS. However, we find that Notch-GATA synergy does not require a SPS, and instead requires a non-SPS configuration of oriented LAG-1/Su(H) sites. Thus, it appears that that each configuration couples Notch signaling with a specific class of transcription factor.