Cheng Z et al. (2013) Nat Biotechnol "Conditional targeted genome editing using somatically expressed TALENs in C. elegans."

Liang X, Feng G, Yi P, Li W, Yang Y, Chai Y, Ou G, Cheng Z, Wang X, Zhu Z

[Nat Biotechnol, 2013]

We have developed a method for the generation of conditional knockouts in Caenorhabditis elegans by expressing transcription activator-like effector nucleases (TALENs) in somatic cells. Using germline transformation with plasmids encoding TALENs under the control of an inducible or tissue-specific promoter, we observed effective gene modifications and resulting phenotypes in specific developmental stages and tissues. We further used this method to bypass the embryonic requirement of cor-1, which encodes the homolog of human severe combined immunodeficiency (SCID) protein coronin, and we determined its essential role in cell migration in larval Q-cell lineages. Our results show that TALENs expressed in the somatic cells of model organisms provide a versatile tool for functional genomics.

Ketel, CS et al. (2005) Mol Cell Biol "Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes."

Vargas, ML, Andersen, EF, Strome, S, Ketel, CS, Suh, J, Simon, JA

[Mol Cell Biol, 2005]

The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The four-subunit complex is > 1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. We describe missense mutations in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. We also find that a recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set.

Wan G et al. (2021) EMBO J "ZSP-1 is a Z granule surface protein required for Z granule fluidity and germline immortality in Caenorhabditis elegans."

Pagano D, Dodson AE, Bajaj L, Kennedy S, Fei Y, Wan G, Fields B

[EMBO J, 2021]

Germ granules are biomolecular condensates that form in germ cells of all/most animals, where they regulate mRNA expression to promote germ cell function and totipotency. In the adult Caenorhabditis elegans germ cell, these granules are composed of at least four distinct sub-compartments, one of which is the Z granule. To better understand the role of the Z granule in germ cell biology, we conducted a genetic screen for genes specifically required for Z granule assembly or morphology. Here, we show that zsp-1, which encodes a low-complexity/polyampholyte-domain protein, is required for Z granule homeostasis. ZSP-1 localizes to the outer surface of Z granules. In the absence of ZSP-1, Z granules swell to an abnormal size, fail to segregate with germline blastomeres during development, and lose their liquid-like character. Finally, ZSP-1 promotes piRNA- and siRNA-directed gene regulation and germline immortality. Our data suggest that Z granules coordinate small RNA-based gene regulation to promote germ cell function and that ZSP-1 helps/is need to maintain Z granule morphology and liquidity.

Khan, Munzareen et al. (2021) International Worm Meeting "

Khan, Munzareen, Sengupta, Piali, Dwyer, Noelle, Hartmann, Anna, Bargmann, Cori, Piccione, Madeline

[International Worm Meeting, 2021]

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Tonks NK (2009) Cell "Pseudophosphatases: grab and hold on."

Tonks NK

[Cell, 2009]

Catalytically inactive pseudophosphatases are able to signal in the absence of enzymatic activity. Analyzing the oocyte-to-zygote transition in the worm, Cheng et al. (2009) and Parry et al. (2009) now show how two pseudophosphatases, EGG-4 and EGG-5, can regulate signaling by the DYRK family kinase MBK-2.

Krishna P et al. (1988) J Biol Chem "Yolk proteins from nematodes, chickens, and frogs bind strongly and preferentially to left-handed Z-DNA."

Krishna P, Kennedy BP, van de Sande JH, McGhee JD

[J Biol Chem, 1988]

Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left-handed Z-DNA probe, brominated poly(dG-dC). We estimate that the nematode proteins bind to Z-DNA with an association constant of at least 10(4) (M-1) and that this association constant is at least 40-50-fold higher than the association constant to B-DNA. Thus, yolk proteins have a higher Z-DNA specificity than most of the Z-DNA binding proteins previously isolated from other sources. Although yolk protein binding to Z-DNA is poorly competed by a wide variety of nucleic acids, the interaction is strongly competed by the phospholipids cardiolipin and phosphatidic acid (500-1000-fold better than by the same mass of B-DNA). We suggest that Z-DNA interacts with the yolk protein phospholipid binding site. In general, our results emphasize the danger of using physical properties to infer biological function. In particular, our results should raise serious questions about the biological relevance of previously isolated Z-DNA binding proteins.

Riga, Amalia et al. (2019) International Worm Meeting "The role of basolateral polarity regulators in epithelial tissue homeostasis."

Boxem, Mike, Pires, Helena, Riga, Amalia

[International Worm Meeting, 2019]

Polarization of epithelial cells into apical and basolateral domains is essential for the functioning of epithelia as selectively permeable barriers. The basolateral Scribble group proteins (Scrib, Lgl, Dlg) were originally identified as tumor suppressors in D. melanogaster and were later shown to play key roles in the establishment and maintenance of polarity in a broad range of cell types. In addition to promoting basolateral identity, Scribble group proteins regulate diverse processes including tissue growth, differentiation and directed cell migration, and therefore are major regulators of tissue development and homeostasis. In C. elegans, loss of LET-413/Scrib and DLG-1/Dlg results in defects in epithelial polarization and junction formation during embryonic development, resulting in embryonic lethality. However, their importance in larval epithelia is less well understood. To study the role of LET-413 in larval epithelia, we generated an allele that allows inducible protein degradation using the auxin-inducible system recently developed for C. elegans (1). We find that ubiquitous degradation of LET-413 in larval tissues results in a developmental arrest. Tissue-specific degradation of LET-413 in the larval intestine does not cause defects in development. In contrast, degradation of LET-413 in the seam cells and hypodermis mimics the developmental arrest seen upon ubiquitous LET-413 degradation. Live-cell imaging of seam cells shows a severe effect on outgrowth and reattachment following the L1 asymmetric division. In addition, we observed that loss of LET-413 results in punctate and discontinuous localization pattern of DLG-1, similar to previous observations in C. elegans embryos. We are currently following the effects of LET-413 inactivation on cell recognition and migration pathways to understand the outgrowth defects of the seam cells. (1) Zhang L, Ward JD, Cheng Z, Dernburg AF. Development. 2015 Dec 15;142(24):4374-84.

Wu S-L et al. (1996) East Coast Worm Meeting "CHARACTERIZATION OF AN ATYPICAL PROTEIN KINASE C IN C. Elegans."

Rubin CS, Wu S-L

[East Coast Worm Meeting, 1996]

Protein kinase Cz (PKCz) is an atypical member of the PKC superfamily. PKCz contains Cys-rich zinc binding and catalytic domains that are homologous with corresponding regions in other PKCs. Unlike other PKCs, the zeta isoform in not activated by diacyl glycerol, TPA or calcium. However, PKCz appears to be a target for activation by alternative lipid second messengers (3,4,5-phosphoinositol, ceramide) generated by PI-3-kinase and/or sphingomyelinase. Activated PKCz has been implicated in the transmission of regulatory signals from the cytoplasm to the nucleus, but its precise physiological roles remain to be rigorously determined. We have initiated studies on the structure, function and regulation of an atypical C. elegans PKC (designated PKC-Z) that is related to mammalian PKCz. A cDNA that encodes full-length PKC-Z was cloned and sequenced. The predicted PKC-Z protein (598 amino acid residues, molecular weight = 68,000) is only ~55% identical with mammalian PKCz. N-terminal and central portions of the two proteins are highly divergent. However, the catalytic, psuedosubstrate and Cys-rich domains are highly conserved. Comparison of the cDNA sequence with data from the C. elegans genome project indicates that the PKC-Z gene (LGII) encompasses 3.6 kbp of DNA and contains 9 exons. A C terminal fragment (120 amino acids) of PKC-Z was expressed as a His-tagged fusion protein in E. coli, purified and injected into rabbits to produce antiserum. Western immunoblot analysis revealed that PKC-Z is most abundantly expressed in the soluble fraction of embryos. The kinase is also evident in L1-L4 larvae and adult nematodes, but the bulk of the enzyme is tightly associated with the particulate fractions of post-embryonic animals. Consistent with the latter observation, immunostaining and confocal microscopy of permeabilized C. elegans revealed that PKC-Z is localized and concentrated in discrete intracellular clusters. The nature of the intracellular structure at which PKC-Z accumulates is not yet known. PKC-Z is being expressed in baculovirus/Sf9 cells and in mammalian cells for enzymic characterization. In addition, injected anti-sense oligonucleotides are being used to probe the function(s) of PKC-Z in embryogenesis.

Ding Z et al. (2008) Sheng Wu Gong Cheng Xue Bao "[Molecular cloning and characterization of an acetylcholinesterase gene Dd-ace-2 from sweet potato stem nematode Ditylenchus destructor]"

He W, Peng D, Gao B, Ding Z, Huang W

[Sheng Wu Gong Cheng Xue Bao, 2008]

A cDNA, named Dd-ace-2, encoding an acetylcholinesterase (AChE, EC3.1.1.7), was isolated from sweet-potato-stem nematode, Ditylenchus destructor. The nucleotide and amino acid sequences among different nematode species were compared and analyzed with DNAMAN5.0, MEGA3.0 softwares. The results showed that the complete nucleotide sequence of Dd-ace-2 gene of Ditylenchus destructor contains 2425 base pairs from which deduced 734 amino acids (GenBank accession No. EF583058). The homology rates of amino acid sequences of Dd-ace-2 gene between Ditylenchus destructor and Meloidogyne incognita, Caenorhabditis elegans, Dictyocaulus viviparous were 48.0%, 42.7%, 42.1% respectively. The mature acetylcholinesterase sequences of Ditylenchus destructor may encode by the first 701 residues of deduced 734 amino acids.The conserved motifs involved in the catalytic triad, the choline binding site and 10 aromatic residues lining the catalytic gorge were present in the Dd-ace-2 deduced protein. Phylogenetic analysis based on AChEs of other nematodes and species showed that the deduced AChE formed the same cluster with ACE-2s.

Gorgon S et al. (2024) Cancer Res Commun "A drug discovery pipeline for MAPK/ERK pathway inhibitors in C. elegans."

Gorgon S, Hemmingsson O, Billing O, Eriksson AU

[Cancer Res Commun, 2024]

Oncogenic signaling through the MAPK/ERK pathway drives tumor progression in many cancers. While targeted MAPK/ERK pathway inhibitors improve survival in selected patients, most tumors are resistant. New drugs could be identified in small animal models that, unlike in vitro models, can address oral uptake, compound bioavailability and toxicity. This requires pharmacological conformity between human and model MAPK/ERK pathways, and available phenotypic assays. Here, we test if the conserved MAPK/ERK pathway in C. elegans could serve as a model for pharmacological inhibition and we develop in vivo pipelines for high throughput compound screens. Using fluorescence-based image analysis of vulva development as a readout for MAPK/ERK activity, we obtained excellent assay Z-scores for the MEK inhibitors trametinib (Z = 0.95), mirdametinib (Z = 0.93), and AZD8330 (Z = 0.87), as well as the ERK inhibitor temuterkib (Z = 0.86). The throughput was 800 wells per hour, with an average seed density of 25.5 animals per well. Readouts included drug efficacy, toxicity, and pathway specificity, which was tested against pathway activating upstream (lin-15)- and downstream (lin-1) mutants. To validate the model in a high throughput setting, we screened a blinded library of 433 anti-cancer compounds and identified four MEK inhibitors among seven positive hits. Our results highlight a high degree of pharmacological conformity between C. elegans and human MAPK/ERK pathways and the presented high-throughput pipeline may discover and characterize novel inhibitors in vivo.