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 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.

Hu, Queenie et al. (2015) International Worm Meeting "BRAP-2 regulates SKN-1/ELT-3 in response to oxidative stress through the ERK/MAPK pathway."

Hu, Queenie, D'Amora, Dayana R., Walhout, Marian, MacNeil, Lesley T., Kubiseski, Terrance J.

[International Worm Meeting, 2015]

Cellular damage caused by reactive oxygen species (ROS) is believed to be a major contributor to the onset of detrimental diseases associated with aging. Among eukaryotes, the Nrf2 family transcription factors are conserved and act as the master regulators of the antioxidant response for protection against stress. SKN-1, a C. elegans ortholog of Nrf2, plays a crucial role in various aspects of organismal development, longevity and stress resistance. However, the molecular mechanisms of SKN-1 and its upstream regulation are poorly understood. Our lab previously introduced the C. elegans BRCA1 associated binding protein 2, BRAP-2, and discovered that brap-2 mutant worms arrest at L1 larval stage when exposed to oxidative stress. We have used both genetics and biochemical approaches to determine how BRAP-2 regulates SKN-1 and its target stress genes. Here we show that the brap-2(ok1492) mutation enhances the expression of skn-1 dependent gene gst-4 in the intestine and hypodermis, indicating that the presence of BRAP-2 may act as a potential inhibitor of SKN-1. Furthermore, the loss of BRAP-2 increases p-MAPK protein levels, and BRAP-2 physically interacts with LET-60/Ras and KSR-1/2. These results suggest that the ERK/MAPK pathway is involved in BRAP-2/SKN-1 regulation.To better define the BRAP-2/SKN-1 signaling cascade, we used reverse genetics to identify twenty novel transcription factors or co-activators of SKN-1 that elevate gst-4 expression in the brap-2(ok1492) mutant. The screen identified elt-3 as a potential candidate, and our results show that ELT-3/GATA is required for enhanced gst-4 expression in the brap-2(ok1492) mutant, through binding to SKN-1. Further analysis revealed that the activation of gst-4 by SKN-1 requires ELT-3, indicating that they may dimerize to increase gst-4 transcriptional activity. Lastly, we have observed that extended lifespan in overexpressing SKN-1 is dependent on functional ELT-3. Together, this suggests a new role for BRAP-2 in the regulation of the SKN-1/ELT-3 complex in response to oxidative stress through the ERK/MAPK pathway.

Leon-Guerrero, Victoria et al. (2021) International Worm Meeting "Characterization of a third SHC adaptor protein in Caenorhabditis elegans"

Hardy, W. Rod, Di Bernardo, Mercedes, Leon-Guerrero, Victoria, MacNeil, Lesley T.

[International Worm Meeting, 2021]

SHC proteins are a family of adaptor proteins that play an important role in signal transduction, they are characterized by three crucial domains: the phosphotyrosine binding (PTB) domain, a Src2 homology (SH2) domain and a less conserved collagen homolog (CH1) domain. Two Caenorhabditis elegans SHC proteins have been described: SHC-1 and SHC-2. We have identified a third SHC protein, K11E4.2, that is intestinally expressed. Our analysis revealed that K11E4.2 null mutant animals suffer from a diet-dependent change in fat accumulation and increased sensitivity to starvation and oxidative stress. C. elegans shc-1 plays a role in stress response and lifespan regulation through the insulin signaling pathway. Our data suggest that shc-1 and K11E4.2 do not act redundantly to regulate stress or starvation response, but rather each plays a distinct role in these processes.

Pho, Kim et al. (2019) International Worm Meeting "Investigating the impact of the human microbiota on Alzheimer's Disease using Caenorhabditis elegans as a model."

Surette, Michael G., Pho, Kim, Tench, Andrea, Lau, Tammy, MacNeil, Lesley T., McArthur, Andrew G.

[International Worm Meeting, 2019]

The risk of developing Alzheimer's Disease (AD) is influenced, not only by genetic factors, but also by environment and lifestyle. The multi-factored pathogenesis that leads to AD development poses a challenge for identifying causal factors that promote neurodegeneration. Two hallmarks of AD are amyloid-? (A?) plaques and neurofibrillary tangles composed of the protein Tau. We use Caenorhabditis elegans as a model of AD to measure the impact of human microbiota species on AD-related phenotypes. Using an A?-overexpressing C. elegans model, we identified several bacterial species that differentially impact paralysis and are characterizing a group of Enterobacteriaceae species that significantly reduce paralysis. Most of these bacteria also decreased neurodegeneration in animals that pan-neuronally express aggregate-prone Tau, providing additional evidence of microbiota-promoted neuroprotection. To explore these effects, we examined global changes in gene expression in animals exposed to neuroprotective bacteria. We identified several biological processes that are differentially regulated in response to the neuroprotective microbiota species, including innate immunity and defense response. Overall, by studying the impact of the human microbiota on models overexpressing A? or aggregate-prone Tau, we gain a greater understanding of conserved pathways involved in gene-environment interactions that influence the development of AD.

Sara Maxwell et al. (2002) European Worm Meeting "Functional analysis of the C. elegans T-box gene family"

Alison Woollard, Roger Pocock, Lizzie Clarke, Sara Maxwell

[European Worm Meeting, 2002]

T-box genes are a group of developmentally important transcription factors united by a common DNA binding domain. T-box genes are present in all metazoan species so far analysed but are absent from yeast. There are 20 T-box genes in C. elegans, more than twice the number found in Drosophila. Many of the C. elegans T-box genes are highly diverged from those found in other species while others have clear orthologues present throughout the metazoan kingdom. One highly conserved T-box gene is mab-9, a member of the tbx20 sub-family1. This was the first C. elegans T-box gene to be identified by mutation and is required for cell fate specification during hindgut and male tail development, and aspects of nervous system function. One other conserved T-box gene has recently been reported to be important for a particular muscle cell fate specification2. We have inactivated the remaining C. elegans T-box genes by RNAi and have found obvious phenotypes only in very few cases. These phenotypes include embryonic lethality, L1 lethality, and a Dpy phenotype with weakly penetrant male tail defects, and will be described in detail. The remaining T-box genes give no obvious phenotype by RNAi. Phylogenetic analysis reveals that several pairs of T-box genes are very similar to eachother and are therefore likely to be the result of recent duplications. This might suggest functional redundancy. Double RNAi experiments have revealed this to be the case with at least two of the T-box gene pairs (see also poster by Pocock et al). Study of the expression patterns of the whole T-box family may suggest other potential redundancy relationships which can be explored by RNAi. Comparison of the C. elegans T-box genes with the set of T-box genes now defined for C. briggsae is being used as a tool for defining potentially important regulatory regions present in orthologous genes.

Hisako Takeshita et al. (2005) International Worm Meeting "Direction of cell polarity is determined by Wnt in C.elegans"

Hisako Takeshita, Hitoshi Sawa, Kota Mizumoto

[International Worm Meeting, 2005]

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, asymmetric divisions of many cells are regulated by Wnt signaling. In particular, asymmetry of the T cell division is regulated by LIN-44/Wnt and the LIN-17/Frizzled receptor. LIN-44/Wnt is expressed in hypodermal cells at the tip of the tail, posterior to the T cell. In lin-44 mutants, the polarity of the T cell division is frequently reversed, while in lin-17 mutants, the division is symmetric. It has been proposed that LIN-44 activates the LIN-17 receptor on the posterior side of the T cell to polarize it. To determine whether LIN-44/Wnt acts as an asymmetric cue that polarizes the T cell, we ectopically expressed LIN-44 in cells anterior to the T cell using the egl-5 promoter (egl-5::LIN-44) to find that polarity reversal phenotype of lin-44 was strongly enhanced. Moreover lin-17 was required for egl-5::LIN-44 to reverse the T-cell polarity. These results suggest that LIN-44 polarize the T cell through the LIN-17 receptor. To further confirm that LIN-44 directly acts on LIN-17 in the T cell, we analyzed the localization of LIN-17::GFP fusion proteins expressed by the seam cell specific promoter (SCM::LIN-17::GFP). This construct rescued defects of lin-17 mutants in the T cell division, indicating that lin-17 functions cell-autonomously in the T cell. We found that LIN-17 localization was localized in the posterior cortex of the T cell in wild type. In lin-44 mutants, the LIN-17 localization was uniform on the cortex. Furthermore, egl-5::LIN-44 reversed the LIN-17 localization to the anterior cortex. These results strongly indicate that LIN-44 directly acts on LIN-17 in the T cell to orient the polarity.

Hryshkevich, Uladzislau et al. (2011) International Worm Meeting "Core promoter T-blocks correlate with gene expression levels in C. elegans."

Yanai, Itai, Hryshkevich, Uladzislau, Hashimshony, Tamar

[International Worm Meeting, 2011]

Core promoters mediate transcription initiation by the integration of diverse regulatory signals encoded in the proximal promoter and enhancers. It has been suggested that genes under simple regulation may have low-complexity permissive promoters. For these genes, the core promoter may serve as the principal regulatory element; however the mechanism by which this occurs is unclear. We report here a periodic poly-thymine motif, we term T-blocks, enriched in occurrences within core promoter forward strands in C. elegans. An increasing number of Tblocks on either strand is associated with increasing nucleosome eviction. Strikingly, only forward strand T-blocks are correlated with expression levels, whereby genes with ³6 T-blocks have five fold higher expression levels than genes with 3 T-blocks. We further demonstrate that differences in T-block numbers between strains predictably affect expression levels of orthologs. Highly expressed genes and genes in operons tend to have a large number of T-blocks, as well as the previously characterized SL1 motif involved in trans-splicing. The presence of T-blocks thus correlates with low nucleosome occupancy and the precision of a trans-splicing motif, suggesting its role at both the DNA and RNA levels. Collectively our results suggest that core promoters may tune gene expression levels through the occurrences of T-blocks, independently of the spatio-temporal regulation mediated by the proximal promoter.

TM Morse et al. (1999) International C. elegans Meeting "Time-series analysis of pirouettes in C. elegans chemotaxis."

TM Morse, JT Pierce, SR Lockery, ML Moravec, TC Ferree, SE Owens, AB Bates

[International C. elegans Meeting, 1999]

Chemotaxis in C. elegans involves a series of abrupt turns (pirouettes) triggered by movement down a gradient of chemical attractant * . Analysis of the time series of concentration change experienced by a worm, together with its pirouette record, suggests a three-stage chemotaxis mechanism in which chemical concentration (C(t)) is differentiated (dC(t)/dt), smoothed (q(t)), and converted into pirouette probability by a nonlinear function (P(q[t])). To test the plausibility of this mechanism, we constructed a computer model in which the smoothing filter and the nonlinearity were estimated from the time series of dC(t)/dt and the pirouette records of real worms. Pirouettes were modeled by sampling randomly, via a Poisson process with probability P(q[t]), from the distribution of direction changes associated with pirouettes in real worms. The average chemotaxis index (time-average of normalized C(t)) of model worms (0.47 +/- 0.05 SD, n = 2000) closely matched the average chemotaxis index of real worms (0.45 +/- 0.04 SD, n = 45), indicating that the three-stage mechanism is quantitatively sufficient to account for C. elegans chemotaxis. To determine the form of the smoothing filter and nonlinearity directly, we have devised a new behavioral assay that subjects unrestrained worms to negative-going impulses in dC/dt as they swim across a sharp border between high and low concentrations of attractant. Preliminary results show that immediately after a border crossing, large impulses make P(t)~= 1, while small impulses make 0 t) >< C. elegans . * Pierce, J.T., and Lockery, S.R., J. Neurosci. (Submitted)