MacNeil, Lesley (2021) International Worm Meeting "ELT-3 regulates cuticle collagen expression in response to environmental stimuli"

MacNeil, Lesley

[International Worm Meeting, 2021]

The C. elegans cuticle is a collagen-rich structure that acts as a physical barrier to protect the animal from pathogens, desiccation, and other stresses. Over 170 cuticular collagens are predicted in the C. elegans genome, but the role of each individual collagen is unclear, as is the need for so many collagens. Stage-specific and dauer-specific specialization of the cuticle, achieved by modulating collagen gene expression, has been reported, suggesting that the C. elegans cuticle may be tailored to the specific needs of the animal during development. We find that there is likely also specialization of the cuticle in response to environmental factors. Taking advantage of missense mutations in collagen genes that result in a rolling phenotype, we were able to identify environmental factors that result in changes in the cuticle, either enhancing or suppressing the effects of these mutations. We find that diet, early developmental arrest, and population density can differentially influence the penetrance of rolling in these mutants. These effects are in part due to changes in collagen gene expression that are mediated by the GATA family transcription factor ELT-3. We propose a model by which ELT-3 regulates collagen gene expression in response to environmental stimuli to promote the assembly of a cuticle specialized to a given environment.

MacNeil, Lesley T. et al. (2011) International Worm Meeting "A transcription factor specific RNAi screen to identify regulators of intestinal gene expression."

Walhout, A.J. Marian, Arda, H. Efsun, MacNeil, Lesley T.

[International Worm Meeting, 2011]

The assembly of gene regulatory networks has been accomplished through a number of different, complementary approaches. Techniques that identify protein-DNA binding events, including chromatin immunoprecipitations and yeast one-hybrid assays, have identified a number of physical interactions between genes and transcription factors (TFs). However, the regulatory consequences of these interactions remain largely unknown. We sought to develop a complementary in vivo approach to identify regulatory rather than physical interactions between genes and their regulators. We generated a TF-specific RNAi feeding library. Using both ORFeome clones, clones from the Ahringer library and additional clones made in house, we were able to generate constructs for a total of 913 TFs, which is 97% of all 940 predicted C. elegans TFs. In order to identify gene-specific regulators of gene expression, we used this library to screen transgenic animals carrying intestine specific transcriptional GFP fusion constructs. RNAi-treated animals were visually examined for changes in GFP expression; either lower expression, higher expression or a spatial change in expression. Screening was accomplished in a 96-well format with each transgenic line screened in three independent replicates. TFs scoring positive in at least two experiments were considered true positives. As expected, we found that elt-2 regulates the expression of all intestinal transgenes assayed. This is consistent with the previous identification of elt-2 as a global regulator of intestinal expression (McGhee et al., 2009. Dev.Biol. 327) and demonstrates that we are able to identify functionally relevant interactions using this approach. In addition, we have identified specific regulators for a number of intestinal transgenes. Together, our data indicate that our RNAi screening platform will be a valuable addition to the toolkit of regulatory network studies.

MacNeil, Lesley T. et al. (2013) International Worm Meeting "An in vivo C. elegans gene regulatory network unveils post-developmental role of intestinal transcription factors."

Arda, H. Efsun, D'Elia, Lauren, Walhout, A.J. Marian, MacNeil, Lesley T.

[International Worm Meeting, 2013]

The C. elegans intestine must integrate dietary, stress and physiological information and respond with an appropriate gene expression program. To understand how these inputs are coordinated, we examined physical and regulatory interactions between TFs and the promoters of 19 genes expressed in the adult intestine. We selected a set of genes that include stress and diet-responsive genes, as well as genes that are stably expressed through a variety of conditions. The same promoter fragments were used in two different assays to compare physical and regulatory interactions. Physical interactions were identified by enhanced yeast one-hybrid assays. To identify regulatory interactions, we generated an RNAi-feeding library covering 95% of all C. elegans TFs and used this library to perform RNAi knockdown using fluorescent transcriptional reporters strains. In addition to screening transgenic animals under standard laboratory conditions, we screened these transgenes under specific stress and dietary conditions. In total, we identified approximately 200 physical interactions and 300 regulatory interactions. We find little overlap between physical and regulatory interactions suggesting that redundancy may mask the effects of physical interactions. Conversely, regulatory interactions that are not associated with physical binding events may occur indirectly as a result of one TF regulating another, or as a result of compensation. By combining our data for all 19 genes, we have identified TFs that are broad regulators of intestinal gene expression, including elt-2 and sbp-1, as well as more gene-specific or stress-specific regulators. In addition, we can begin to predict relationships between TFs based on shared regulatory targets and knowledge of physical interaction data. Our data suggest that the intestinal gene regulatory network is complex and that compensation and redundancy exist to modulate and/or buffer the appropriate levels of gene expression in response to genetic perturbations.

Lesley MacNeil et al. (2001) International C. elegans Meeting "A Screen for Suppressors of Distal Tip Cell Migration Defects Induced by Ectopic unc-129 Expression"

Joe Culotti, Lesley MacNeil, Antonio Colavita, Jeffrey Wrana

[International C. elegans Meeting, 2001]

unc-129 was first identified as a gene influencing axon guidance in C. elegans . The uncoordinated phenotype observed in unc-129 mutant animals results from defects in axon guidance of the DA, DB, VD and DD commissural classes of motorneurons. Cloning of the unc-129 gene showed that it encodes a TGF-beta ligand that functions in dorsal muscles to mediate axon guidance. Biochemical characterization confirmed that unc-129 is processed and dimerized, consistent with its identification as a TGF-beta ligand. TGF-beta ligands act through heterodimers of type I and type II serine threonine kinase receptors. In C. elegans there are two characterized TGF-beta pathways, one affecting body size and male tail formation, the other regulating dauer formation. These pathways use distinct type I receptors, SMA-6 and DAF-1, but share a common type II receptor (DAF-4). No other TGF-beta receptors have been identified in the genome sequence. Mutations in these receptors as well as their downstream mediators, the SMADs, have not been reported to result in uncoordinated phenotypes. In order to explore the possibility that unc-129 acts through known TGF-beta receptors, we examined receptor mutants for the presence of axon guidance defects. Mutations in the TGF-beta receptors did not cause axon guidance defects. Furthermore, a double mutant of the type I receptors, sma-6 and daf-1, did not display defects in axon guidance. These data suggest that UNC-129 does not act through the traditional type I and II receptors. In order to identify downstream mediators of unc-129 , we began a mutagenesis screen for suppressors of ectopic unc-129 . Ectopic expression of unc-129 in both dorsal and ventral muscles, which occurs in an unc-130 mutant, results in distal tip cell (DTC) migration defects. These defects are suppressed by mutations in unc-129 , which, on their own do not cause DTC defects. This suggests that defects in components of the unc-129 signaling pathway should suppress the DTC defects induced by ectopic unc-129 expression. Expression of unc-129 in both dorsal and ventral muscles using the myo3 promoter to drive expression also results in defects in distal tip cell (DTC) migration, mimicking the unc-130 mutant phenotype. In principle, defects in components of the unc-129 signaling pathway should suppress the DTC defects of myo-3::unc-129 transgenic animals. Using a small scale F2 screen, we have isolated 7 mutations that suppress the DTC migration defects of myo-3::unc-129 transgenic animals. These mutants do not display defects in body size, male tail formation or dauer formation.

Lesley MacNeil et al. (2008) C.elegans Neuronal Development Meeting "UNC-129 Alters the Response to UNC-6/Netrin by Regulating the Decision Between UNC-40 Dependent and Independent UNC-5 Signaling"

Jeffrey Wrana, Lesley MacNeil, William Hardy, Joseph Culotti

[C.elegans Neuronal Development Meeting, 2008]

UNC-129 is a TGF-beta family ligand that regulates ventral to dorsal migration of commissural motor axons in C. elegans. Mutations in unc-129 result in motoraxons that do not accomplish normal ventral to dorsal migration, but instead deviate from their proper course. Genetic evidence demonstrates that UNC-129 does not function as other TGF-beta family ligands, through the classical type I and type II serine/threonine kinase receptors, but instead regulates axon guidance through a novel signaling pathway. Genetic interactions between null alleles of unc-129 and unc-5, unc-40 and unc-6 revealed that unc-129 acts within the UNC-5 signaling pathway to regulate axon guidance, cell migration and patterning of the male tail. Analysis of unc-40 and unc-129 compound null mutants demonstrates that UNC-129 acts specifically to promote UNC-5+UNC-40 signaling. In contrast, overexpression studies using ectopically expressed UNC-129 suggest that UNC-129 acts to inhibit UNC-40 independent UNC-5 signaling. Together, these data suggest a model where UNC-129 regulates the decision between UNC-40 dependent and independent UNC-5 signaling. The result of this, for cells and axons migrating along the ventral-dorsal axis, is to increase the sensitivity of cells and axons to decreasing levels of UNC-6/Netrin by switching from UNC-5 signaling to UNC-5+UNC-40 signaling.

Lesley T MacNeil et al. (2005) International Worm Meeting "Identification and Characterization of a Novel Family of GPI-linked Proteins with Homology to the TGF-? Receptor Family"

Lesley T MacNeil, Jeffrey L Wrana, Joseph G Culotti

[International Worm Meeting, 2005]

TGF-? family ligands signal through heteromeric complexes of type I and type II serine threonine transmembrane receptors. In C. elegans, there exist two well-characterized TGF-? signaling cascades, one regulating body size and male tail formation and one regulating dauer formation. These pathways share a common type II receptor, DAF-4, and use unique type I receptors, SMA-6 and DAF-1. Sequence analysis of the genome has determined that there are no additional type I or type II receptors in C. elegans. In addition to the ligands that stimulate the two characterized pathways, there exist two additional ligands, UNC-129 and TIG-2. unc-129 mutations cause defects in axon guidance, which do not appear to result from de-regulation of the canonical TGF-? signaling pathways. In mammalian systems as well as in Drosophila, novel mechanisms of TGF-? signaling exist that do not use traditional serine/threonine receptors. For example, GDNF, a divergent member of the TGF-? family, functions through the receptor tyrosine kinase RET and a GPI-linked co-receptor, GNDFR?. GDNF has also been shown to modulate the effects of NCAM, using GDNFR-? to traffic NCAM into lipid rafts. In order to uncover novel methods of TGF-? signaling, we attempted to identify new TGF-? receptors based on a pattern of cysteine spacing that is conserved in the extracellular domains of TGF-? and BMP receptors. We have identified five genes with this pattern of cysteine spacing that are predicted to encode GPI-linked proteins. We have made GFP reporter constructs for these genes and show that they have distinct, but overlapping, expression patterns and that they are expressed in a number of compartments where traditional TGF-? receptors are known to function as well as in compartments where UNC-129 receptors are believed to function. These patterns of expression are consistent with a potential role as unc-129 co-receptors and also as modulators of the canonical TGF-? signaling pathways. We will present preliminary characterization of these genes.

DiBernardo, Mercedes et al. (2021) International Worm Meeting "Exposure to human microbiota isolates during development impacts Caenorhabditis elegans susceptibility to Pseudomonas aeruginosa infection"

MacNeil, Lesley, Surette, Michael, Burrows, Lori, DiBernardo, Mercedes

[International Worm Meeting, 2021]

Commensal microorganisms that colonize host surfaces can modulate susceptibility to infection. This can occur through direct inhibition of pathogen growth via competitive exclusion, or by modulation of the host environment to prevent pathogen dissemination and infection-mediated damage. However, understanding the mechanisms underlying these interactions is challenging due to the complexity of the microbiome. Here, we investigate the influence of human respiratory tract microbiota isolates on C. elegans susceptibility to Pseudomonas aeruginosa infection. Identifying isolates that differentially regulate resistance to infection will provide insight into how particular strains isolated from the human microbiome may improve or worsen infection outcomes. In order to identify microbiotal isolates that can protect C. elegans from P. aeruginosa infection, a screen of bacterial isolates derived from the human respiratory tract was carried out using a liquid-based infection assay. Animals were first exposed to lawns of individual microbiotal isolates during development, prior to infection with a pathogenic strain of P. aeruginosa, PA14. We find that pre-exposure to two non-pathogenic isolates of P. aeruginosa is able to increase survival during PA14 liquid killing, compared to animals pre-exposed to E. coli OP50. This protective phenotype can also be induced by other mildly pathogenic strains of P. aeruginosa, including a laboratory strain with genetically attenuated virulence, PAO1deltavfr. Current work focuses on identifying host pathways underlying this protective phenotype, as well as how previous exposure to other Gram-negative pathogens can regulate P. aeruginosa infection outcomes in C. elegans.

Mesbahi, Hiva et al. (2021) International Worm Meeting "Impact of microbiota on neurodegeneration in C. elegans models of tauopathy"

Surette, Michael, Mesbahi, Hiva, MacNeil, Lesley, Pho, Kim

[International Worm Meeting, 2021]

Alterations in the microbiota have been observed in many human diseases including diseases of neurodegeneration. However, specific microbiome factors that either promote or protect against neurodegeneration are largely unknown. We are examining the effects of human microbiota in tauopathies, a class of age-associated neurodegenerative diseases that are characterized by the accumulation and aggregation of the microtubule-associated protein Tau. Mutations in the gene encoding Tau (MAPT) result in Frontotemporal Lobal Degenerations (FTLDs). Using a Caenorhabditis elegans model of this disease, we examined the impact of exposure to specific bacteria present in the human microbiome on neurodegeneration. We identified strains of bacteria that are neuroprotective in this model of tauopathy, and one that promotes neurodegeneration. Rothia species that suppress neurodegeneration also influence fat metabolism in C. elegans. We determined that the ability of Rothia species to promote neuroprotection in the PLM neurons requires the fatty acid desaturate fat-3. fat-3 mutants lack D6 fatty acids and are depleted in C20 fatty acids. We are currently investigating the mechanism of fat-3 dependent neuroprotection and investigating how Rothia exposure modifies lipid metabolism.

Rafikova, Adilya et al. (2015) International Worm Meeting "Role of SEM-4 in BRAP-2/SKN-1/ROS detoxification pathway."

Rafikova, Adilya, Walhout, Marian, MacNeil, Lesley, Kubiseski, Terrance J.

[International Worm Meeting, 2015]

Oxidative stress causes damage to cells by creating reactive oxygen species (ROS), which are harmful molecules that destroy cellular structure and impair protein function. The overproduction of ROS is detrimental, having been linked to the onset of premature ageing and age-related diseases, such as Alzheimer's and Parkinson's. Our lab has previously found that a partial deletion of brap2 (BRCA-1 associated protein 2) significantly enhances nuclear localization of SKN-1 and increased the expression of gst-4, a phase II detoxification enzyme. An RNAi screen for 940 transcription factors on a brap-2;gst-4::gfp strain resulted in more than 20 candidates that are proposed to alter expression of gst-4 in BRAP-2/SKN-1/ROS detoxification pathway. One of those genes, sem-4, was chosen for the further studies. SEM-4 is a zinc-finger transcription factor and is important for development of neuronal, mesodermal and vulval lineages in C.elegans. C. elegans with sem-4 mutation, or overexpressing sem-4 are viable and readily available, which makes it possible to test for the importance of dosage effect of SEM-4 in BRAP-2/SKN-1/ROS detoxification pathway. While brap-2 mutant worms demonstrate higher levels of gst-4 expression, a significant reduction in gst-4 mRNA levels is observed in sem-4;brap-2 double mutants before and after paraquat stress. Although no physical interaction between SEM-4 and SKN-1 was observed, a luciferase construct containing the promoter of gst-4 was activated by co-expression of both SEM-4 and SKN-1 in 293T cells. We also found that higher levels of ROS were generated in sem-4 and sem4;brap-2 mutants in comparison to N2 worms, indicating that SEM-4 is required to prevent overproduction of ROS in vivo. Furthermore, the lifespan of skn-1 overexpressing worms was significantly prolonged by overexpression of sem-4 and shortened by deletion of sem-4. Together, these results indicate a newly identified role of SEM-4 in regulating expression of phase II detoxification enzymes and preventing the harmful effects caused by overproduction of ROS. .

Hu, Queenie et al. (2013) International Worm Meeting "The role of C. elegans BRAP-2 in the SKN-1 mediated oxygen radical detoxification response."

Kubiseski, Terrance J., Walhout, Marian, MacNeil, Lesley, Hu, Queenie

[International Worm Meeting, 2013]

To prevent cellular damage caused by reactive oxygen species (ROS), eukaryotes have developed conserved defense mechanisms to protect themselves from these stressors. The C. elegans BRCA1 associated protein 2, BRAP-2, has been identified to have a role in responding to ROS. A deletion mutant of brap-2 (EEED8.16) is sensitive to oxidizing conditions, resulting in developmental L1 larval arrest or lethality. However, the molecular mechanism of how BRAP-2 relieves stress is poorly understood. Thus, it is important to understand the precise signaling pathway in which BRAP-2 regulates stress. We have found that brap-2 mutants display an enhanced gst-4 expression in the intestine and hypodermis, and this increase is dependent on the transcription factor SKN-1/Nrf2. Although the exact mechanism of BRAP-2/SKN-1 regulation requires further investigation, our results indicate that BRAP-2 physically interacts with both LET-60/Ras and KSR-1/KSR-2, suggesting that MAPK is the pathway involved in the regulation of SKN-1 by BRAP-2. We have used a genetic approach in order to identify novel genes that function in the BRAP-2/SKN-1 detoxification pathway. Using a transcription factor specific library, an RNAi screen was used to identify factors required for the enhanced gst-4::gfp expression in the brap-2 strain. We have identified twenty suppressors that potentially represent either a novel transcription factor or co-activator of SKN-1 to promote its biological effect. Our results indicate that elt-3 is a potential candidate involved in the SKN-1/Nrf2 signaling pathway. We found that ELT-3/GATA is required for enhanced gst-4 expression in the brap-2 mutant, through binding to SKN-1. Furthermore, no lifespan extension was observed in overexpressing SKN-1 worms when we knock down elt-3, indicating ELT-3 is crucial to co-activate SKN-1 in promoting longevity. Taken together, these results suggest a model where BRAP-2 acts as negative regulator of KSR and MAPK activity on SKN-1/ELT-3 dependent gst-4 expression.