Van de Walle, P. et al. (2019) International Worm Meeting "Interacting TALE Hox cofactors regulate vitellogenesis."

Geens, E., Van De Walle, P., Temmerman, L., Schoofs, L.

[International Worm Meeting, 2019]

The onset of sexual maturity is accompanied by dramatic changes in physiology and gene expression in many organisms. In Caenorhabditis elegans, the production of yolk proteins destined for the nascent oocytes, termed vitellogenesis, must be switched on only in adult hermaphrodites. Previous work has identified the rapidly evolving TALE-class homeodomain-containing transcription factor CEH-60 as necessary for adequate transcription of the vit genes that code for yolk proteins. However, the mechanism by which CEH-60 is able to regulate vitellogenesis remained poorly understood. Fluorescent reporters show that CEH-60 is expressed in the intestine, but only from adulthood on, coinciding with both the time and place of yolk protein production in C. elegans. Using bimolecular fluorescence complementation, we show that CEH-60 interacts in vivo with another TALE transcription factor, UNC-62, which has been shown to bind directly to the promoter of the yolk protein coding vit genes. We additionally show that this interaction depends on the PBC-domain of CEH-60, which is predicted to be its UNC-62 interacting domain. Using differential proteomics, we show that, in addition to all yolk proteins, several proteins involved in the synthesis of the cuticle are differentially expressed in ceh-60 mutants. Correspondingly, the cuticle of ceh-60 mutants appears to be more permeable to small molecules, including fluorescent dyes and oxidative stressors. Taken together, these findings suggest that CEH-60 regulates yolk protein production through interaction with UNC-62 and cuticle permeability through an as yet unknown mechanism.

Van de Walle P et al. (2020) PLoS One "DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans."

Temmerman L, Schoofs L, Van de Walle P, Munoz-Jimenez C, Askjaer P

[PLoS One, 2020]

Transcription factors govern many of the time- and tissue-specific gene expression events in living organisms. CEH-60, a homolog of the TALE transcription factor PBX in vertebrates, was recently characterized as a new regulator of intestinal lipid mobilization in Caenorhabditis elegans. Because CEH-60's orthologs and paralogs exhibit several other functions, notably in neuron and muscle development, and because ceh-60 expression is not limited to the C. elegans intestine, we sought to identify additional functions of CEH-60 through DNA adenine methyltransferase identification (DamID). DamID identifies protein-genome interaction sites through GATC-specific methylation. We here report 872 putative CEH-60 gene targets in young adult animals, and 587 in L2 larvae, many of which are associated with neuron development or muscle structure. In light of this, we investigate morphology and function of ceh-60 expressing AWC neurons, and contraction of pharyngeal muscles. We find no clear functional consequences of loss of ceh-60 in these assays, suggesting that in AWC neurons and pharyngeal muscle, CEH-60 function is likely more subtle or redundant with other factors.

Van de Walle P et al. (2019) PLoS Biol "CEH-60/PBX regulates vitellogenesis and cuticle permeability through intestinal interaction with UNC-62/MEIS in Caenorhabditis elegans."

Van de Walle P, Temmerman L, Baggerman G, Askjaer P, Schoofs L, Geens E, Jose Naranjo-Galindo F

[PLoS Biol, 2019]

The onset of sexual maturity involves dramatic changes in physiology and gene expression in many animals. These include abundant yolk protein production in egg-laying species, an energetically costly process under extensive transcriptional control. Here, we used the model organism Caenorhabditis elegans to provide evidence for the spatiotemporally defined interaction of two evolutionarily conserved transcription factors, CEH-60/PBX and UNC-62/MEIS, acting as a gateway to yolk protein production. Via proteomics, bimolecular fluorescence complementation (BiFC), and biochemical and functional readouts, we show that this interaction occurs in the intestine of animals at the onset of sexual maturity and suffices to support the reproductive program. Our electron micrographs and functional assays provide evidence that intestinal PBX/MEIS cooperation drives another process that depends on lipid mobilization: the formation of an impermeable epicuticle. Without this lipid-rich protective layer, mutant animals are hypersensitive to exogenous oxidative stress and are poor partners for mating. Dedicated communication between the hypodermis and intestine in C. elegans likely supports these physiological outcomes, and we propose a fundamental role for the conserved PBX/MEIS interaction in multicellular signaling networks that rely on lipid homeostasis.

Detienne G et al. (2016) Worm "SKN-1-independent transcriptional activation of glutathione S-transferase 4 (GST-4) by EGF signaling."

Temmerman L, Detienne G, Van de Walle P, De Haes W, Schoofs L

[Worm, 2016]

In C. elegans research, transcriptional activation of glutathione S-transferase 4 (gst-4) is often used as a read-out for SKN-1 activity. While many heed an assumed non-exclusivity of the GFP reporter signal driven by the gst-4 promoter to SKN-1, this is also often ignored. We here show that gst-4 can also be transcriptionally activated by EOR-1, a transcription factor mediating effects of the epidermal growth factor (EGF) pathway. Along with enhancing exogenous oxidative stress tolerance, EOR-1 inde-pendently of SKN-1 increases gst-4 transcription in response to augmented EGF signaling. Our findings caution researchers within the C. elegans community to always rely on sufficient experimental controls when assaying SKN-1 transcriptional activity with a gst-4p::gfp reporter, such as SKN-1 loss-of-function mutants and/or additional target genes next to gst-4.

Zarkower D et al. (1994) Worm Breeder's Gazette "mab-3 YAC Rescue"

De Bono M, Hodgkin JA, Zarkower D

[Worm Breeder's Gazette, 1994]

mab-3 YAC rescue David Zarkower, Mario de Bono, and Jonathan Hodgkin MRC Laboratory of Molecular Biology, Cambridge, England

Minor, Paul et al. (2011) International Worm Meeting "FGF signaling cooperates with WNT signaling in C. elegans vulval cell lineage polarity."

Minor, Paul, Sternberg, Paul, Asthagiri, Anand

[International Worm Meeting, 2011]

The C. elegans vulva is formed from divisions of three vulval precursor cells (VPCs): P5.p, P6.p, and P7.p. P5.p and P7.p are induced and divide to form a 2 deg lineage pattern in which the daughter cells of P5.p and P7.p display mirror symmetry about the vulval center. Previous data shows the orientation of these cells is determined by the interaction of multiple Wnt signals. Two separate signal cascades beginning with Wnts from the anchor cell promote the wild-type, anterior-facing P7.p, vulval lineage, whereas the Wnt/EGL-20 signal from the tail promotes the daughter cells of P7.p to face the posterior. Here we show that EGL-20 acts through a member of the LDL receptor superfamily, LRP-2, in addtion to Ror/CAM-1, Van Gogh/VANG-1. All three proteins act together to control vulval polarity through the localization of the beta-catenin-like transcriptional coactivator SYS-1. We also show that the FGF/EGL-17 pathway acting through Raf/LIN-45 is also required for the P7.p wild-type lineage orientation. The role of FGF in the P7.p orientation decision presents the first evidence that the 1 deg daughter cells of P6.p influence the polarity of the neighboring 2 deg cells. Finally by observing SYS-1 localization we show that the FGF pathway converges with Wnt in determining P7.p orientation.

Sharma S et al. (2019) Int Microbiol "Lipidomic insights to understand membrane dynamics in response to vanillin in Mycobacterium smegmatis."

Sharma S, Fatima Z, Hameed S

[Int Microbiol, 2019]

Considering the emergence of multidrug resistance (MDR) in prevalent human pathogen, Mycobacterium tuberculosis (MTB), there is parallel spurt in development of novel strategies aimed to disrupt MDR. The cell envelope of MTB comprises a wealth of lipid moieties contributing towards long-term survival of pathogen that could be exploited as efficient antitubercular target owing to advancements made in mass spectrometry-based lipidomics technology. This study aimed to utilize the lipidomics approach to unveil several lipid associated changes in response to natural antimycobacterial compound vanillin (Van) in Mycobacterium smegmatis, a surrogate for MTB. Lipidomic analyses revealed that that Van alters the composition of fatty acid (FA), glycerolipid (GL), glycerophospholipid (GP), and saccharolipids (SL). Furthermore, Van leads to potentiation of ampicillin and displayed additive effect. The differential expressions of various lipid biosynthetic pathway genes by RT-PCR corroborated with the lipidomics data. Lastly, we demonstrated enhanced survival of Mycobacterium-infected Caenorhabditis elegans model in presence of Van. Thus, lipidomics approach provided detailed insight into mechanisms of membrane disruption by Van in Mycobacterium smegmatis. Our work offers the basis of further understanding the regulation of lipid homeostasis in MTB so that better therapeutic targets could be identified to combat MDR.

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[International Worm Meeting, 2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .

Aoki ST et al. (2021) Nat Commun "C. elegans germ granules require both assembly and localized regulators for mRNA repression."

Aoki ST, Crittenden SL, Bingman CA, Wickens M, Kimble J, Lynch TR

[Nat Commun, 2021]

Cytoplasmic RNA-protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein-RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5A, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.

Braeckman BP et al. (2002) Aging Cell "Rebuttal to Van Voorhies: 'the influence of metabolic rate on longevity in the nematode Caenorhabditis elegans'."

Braeckman BP, Houthoofd K, Vanfleteren JR

[Aging Cell, 2002]

The papers by Van Voorhies in Free Radical Biology & Medicine (33, 587-596, 2002) and in this journal claim that the major longevity-extending mutations in C. elegans essentially act by reducing metabolic rate as predicted by the rate-of-living theory, and do not alter any metabolically independent mechanism specific to aging. In contrast, we found no evidence of a reduction in metabolic rate in these mutants using different experimental approaches. Now, Van Voorhies challenges the accuracy of our experimental results.