Cunningham KA et al. (2009) Cell Metab "Fat rationing in dauer times."

Ashrafi K, Cunningham KA

[Cell Metab, 2009]

The fundamental task of maintaining energy balance is complex when nutrient levels are plentiful, but it becomes even more challenging when nutrients are dynamic or scarce. A recent Nature report delineates a role of the AMP kinase pathway in rationing energy stores for the long-term survival of Caenorhabditis elegans dauers (Narbonne and Roy, 2009).

Xie M et al. (2016) PLoS Genet "Correction: The Causative Gene in Chanarian Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans."

Xie M, Roy R

[PLoS Genet, 2016]

[This corrects the article DOI: 10.1371/journal.pgen.1005284.]. In the title of this article, the words Chanarian Dorfman should read Chanarin-Dorfman. The correct title is: The Causative Gene in Chanarin-Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans. The correct citation is: Xie M, Roy R (2015) The Causative Gene in Chanarin-Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans. PLoS Genet 11(6): e1005284. doi:10.1371/journal.pgen.1005284

Das, Moushumi et al. (2019) International Worm Meeting "3D Genomic Architecture and Transcriptional Regulation."

Semple, Jennifer, Ridder, Jeroen de, Meister, Peter, Allahyar, Amin, Das, Moushumi, Straver, Roy

[International Worm Meeting, 2019]

Understanding how our genes are regulated is a central question in biology. Studies have highlighted multiple levels of regulation for genomic expression, ranging from transcription factors binding to nuclear organization of the genome. Recent technical optimization of chromosome conformation capture (3C) analysis has revealed new levels of chromosome compartmentalization. Interphase chromosomes are arranged into specific kilo- to mega-base sized domains, known as Topologically Associated Domains (TADs). Functionally, most enhancer-promoter interactions occur within the same TAD. Moreover, during differentiation, genes clustered within the same TAD display a similar expression pattern, and alteration of these domains result in aberrant expression of the genes, suggesting their involvement in transcriptional regulation. TADs appear remarkably stable between cell types, but can differ in the level of compaction inside individual TADs. TADs encompassing inactive genes are more compact than TADs comprising active ones. This raises the question whether TAD compaction directly regulates or somehow limits gene expression. This project aims at answering this question using a model system, dosage compensation (DC) in the nematode, Caenorhabditis elegans. It has been previously shown that on the X chromosome, more compact TADs correlate with the down-regulation of X-linked genes in hermaphrodites compared to males. This provides an ideal system to understand the mechanism of transcriptional regulation by TAD formation. I have setup a chromatin conformation capture technique coupled with long molecule sequencing (Oxford Nanopore Technology) to capture gene structures at high resolution, in DC and non-DC environment. In contrast to standard HiC that captures pair-wise contacts, this approach can directly identify multi-way chromosomal contacts from within single cells. My aim is to understand how chromatin structure is modified at the gene level by TAD formation and whether and how this regulates gene expression, thereby deciphering the importance of genome folding in transcriptional regulation.

Labbe JC et al. (2006) Clin Genet "New developmental insights from high-throughput biological analysis in Caenorhabditis elegans."

Labbe JC, Roy R

[Clin Genet, 2006]

Labbe J-C, Roy R. New developmental insights from high-throughput biological analysis in Caenorhabditis elegans.The use of Caenorhabditis elegans as a model system for understanding animal development and human disease has long been recognized as an efficient tool of discovery. Recent developments, particularly in our understanding of RNA-mediated interference and its ability to modify gene activity, have facilitated the use of C. elegans in determining gene function via high-throughput analysis. These new strategies have provided a framework that allows investigators to analyse gene function globally at the genomic level and will likely become a prototypic model for biological analysis in the post-genome era.

Dana Byrd et al. (2006) Development & Evolution Meeting "Understanding the Molecular Properties of a Stem Cell Niche"

Judith Kimble, Dana Byrd, Daniel Hesselson

[Development & Evolution Meeting, 2006]

Distal tip cells (DTCs) serve at least two critical roles during development of the C. elegans hermaphrodite gonad. They function as migratory leader cells to regulate morphogenesis of the U-shaped gonad arms and as niche cells to regulate germline proliferation. We have taken a microarray approach to determine the gene expression profiles of the DTC during development to further explore the molecular basis of its leader and niche cell functions. We are enriching for DTC transcripts by immunoprecipitation of Flag-tagged PAB-1 (poly-A binding protein) with bound RNAs (Roy et al. 2002). DTC expression will be validated with promoter-GFP constructs, and function will be assessed by cell-specific RNAi. Our progress will be described.

Sharma, Sunanda et al. (2021) International Worm Meeting "Forward Genetic screening to identify novel regulators of neuronal Microtubule cytoskeleton"

Sharma, Sunanda, Thyagarajan, Pankajam, Puri, Dharmendra, Ponniah, Keerthana, Ghosh-Roy, Anindya

[International Worm Meeting, 2021]

A neuron has a polarized structure with a long axon and several dendrites which is established and maintained by microtubule cytoskeleton. Microtubules are heteropolymers of alpha and beta-tubulin dimers and have intrinsic polarity with a fast growing plus end and a slow growing minus end. We are using Touch receptor neurons (TRN) of C. elegans, to understand the basis of this organization and regulation of neuronal microtubules. One of the major factors that depolymerize microtubules is the kinesin-13 family protein KLP-7. The loss of klp-7 causes excess stabilization of microtubule cytoskeleton leading to ectopic neurite extension in touch neurons and other neurons as well (Ghosh-Roy et al 2012; Puri et al 2019). We found that this phenotype in klp-7(0) can be reversed by a microtubule-destabilizing drug colchicine or in backgrounds lacking either alpha or beta tubulin. We hypothesized that the suppressors of klp-7(0) neuronal phenotype might encode for novel regulators of the neuronal microtubule cytoskeleton. With this idea, we did a forward genetic screen in klp-7(0) mutant using Ethyl Methane Sulfonate(EMS). We isolated 26 mutants that suppress the ectopic posterior extension phenotype of ALM neuron in klp-7(0), out of 12,422 F1's from a clonal screening. To identify the exact causal mutant, the suppressors were crossed four times with (N2) Bristol to collect 10 individual recombinants. We sequenced the pooled lysates of these recombinants and the FASTQ sequences of Whole Genome Sequencing (WGS) data were analyzed on the Galaxy user interface using MimodD tools. The list of SNP variants was used to generate mapping plots, which gave putative homozygous EMS SNPs for a given suppressor. These SNPs affect genes that encode for tubulin subunits, RNA-binding protein, Ca2+ dependent secretion activators, kinases and ECM components. We will present a comprehensive analysis of the characterization of a few mutants to unravel their roles in regulating microtubule cytoskeleton. References: Ghosh-Roy, A., Goncharov, A., Jin, Y., Chisholm A.D. (2012). Kinesin-13 and tubulin posttranslational modifications regulate microtubule growth in axon regeneration. Developmental Cell 23(4), 716-728 Puri D, Ponniah K, Biswas K, Basu A, Lundquist EA, Ghosh-Roy A. (2019) WNT Signaling Establishes Microtubule Polarity in Neuron Through the Regulation of Kinesin 13 Family Microtubule Depolymerizing Factor. (Sneak peek in Cell Press). http://dx.doi.org/10.2139/ssrn.3456296

Mariam Alexander et al. (2005) International Worm Meeting "A Screen For Genes Required for Muscle Arm Development."

Mariam Alexander, Peter J Roy

[International Worm Meeting, 2005]

In C. elegans, body wall muscles (BWMs) extend membrane projections called muscle arms to the nerve cord to form the neuromuscular junction. Our lab and others have provided evidence that there is likely two distinct phases of muscle arm development; embryonic muscle arms arise by a passive mechanism (CR Norris, IA Bazykina, DH Hall, EM Hedgecock, Worm Breeder's Gazette 14(5): 37), while larval arms actively seek out motor axons (Dixon and Roy, submitted). Although the mechanism by which larval muscle arms reach motor axons is not clear, one model is that a chemotropic cue secreted by motor axons guide muscle arm migration to the nerve cord. To better understand muscle arm development, I performed a forward genetic screen to isolate mutants with muscle arm extension defects. I have screened 36,000 haploid genomes and isolated five mutants with highly penetrant and expressive muscle arm defects. Complementation tests show that tr50 is allelic to unc-60, a gene encoding the C. elegans ADF/cofilin orthologue, while tr51 is allelic to unc-22, which encodes twitchin. The remaining mutants do not map near other genes known to be required for larval muscle arm extension, including lev-11 and unc-54 (Dixon and Roy, submitted), suggesting that these genes may provide novel insight into the development of muscle arms. Surprisingly, one of mutants that has a dramatic reduction in the number of muscle arms (tr64) is not uncoordinated. This suggests that proper muscle arm development is not necessary for wild type movement. I am currently refining the map positions of the genes corresponding to the remaining mutations.

Florencia Pauli et al. (2005) International Worm Meeting "Global profile of intestine-specific gene expression in C. elegans"

Yueyi Liu, Stuart Kim, Florencia Pauli

[International Worm Meeting, 2005]

The simple body plan, many tissues, and complete genome sequence of C. elegans allow for a systems biology approach to the question of which genes specify a tissue. A global profile of tissue-specific gene expression of this organism would expand our knowledge of tissue development and maintenance, regulation of gene expression, and higher order chromosome structure. We used mRNA tagging (Roy et al.) to identify genes expressed in the intestine of the worm. Animals expressing an epitope-tagged protein that binds the poly-A tail of mRNAs (FLAG::PAB-1) from an intestine-specific promoter (ges-1) were used to immunoprecipitate (IP) FLAG::PAB-1/mRNA complexes from the intestine. Genes enriched by intestinal IP were identified with DNA microarrays. The average ranks of enrichment from 8 repeats of this experiment identified 1938 intestine-expressed genes (p<0.001). First, we compared the intestine-expressed genes to those expressed in the muscle (Roy et al.) and germline (Reinke et al.), and identified 510 genes enriched in all 3 tissues and 246 intestine-, 115 muscle-, and 219 germline-enriched genes. Second, we showed that the 1938 intestine genes were physically clustered on the chromosomes, suggesting that the order of genes in the genome is influenced by the effect of chromatin domains on gene expression. Furthermore, the commonly-expressed genes showed more chromosomal clustering than the tissue-enriched genes, suggesting that chromatin domains may influence housekeeping genes more than tissue-specific genes. Third, in order to gain further insight into the regulation of intestinal gene expression, we searched for regulatory motifs. We used a modified Gibbs sampling method called CompareProspector (Liu et al.) to identify motifs that were conserved with C. briggsae and over-represented in the 1 kb upstream region of the 1938 intestine-expressed genes. This analysis found that the promoters of the intestine genes were enriched for the consensus sequence for GATA transcription factors. We experimentally verified these results by showing that GATA motifs are required in cis and that GATA transcription factors are required in trans for expression of these intestinal genes. Previous work had shown that GATA transcription factors (med-1, end-1, etc.) are important for intestinal differentiation, and our work has identified about 800 intestinal genes that may be the direct targets of the GATA transcription factors. Liu, Y. et al. (2004). Genome Research, 14(3), 451-458. Reinke, V. et al. (2004). Development, 131(2), 311-323. Roy, P. J. et al. (2002). Nature, 418(6901), 975-979.

Florencia Pauli et al. (2004) West Coast Worm Meeting "Global profile of intestine-specific gene expression in C. elegans"

Yueyi Liu, Florencia Pauli, Stuart K Kim

[West Coast Worm Meeting, 2004]

Although many advances have been made by studying individual genes, the global picture of tissue-specific gene expression in an organism remains dim. The relatively simple body plan, yet representative of many specific tissues, and complete genome sequence of the nematode C. elegans allows for a systems biology approach to the question of which genes specify a tissue. A global profile of the tissue-specific gene expression of this organism would expand our knowledge of tissue development and maintenance, regulation of gene expression, higher order chromosome structure, and the housekeeping genes that characterize a cell. In order to identify the genes expressed in the intestine of the worm we have taken the mRNA-tagging approach described by Roy et al (2002) to identify genes expressed in muscle cells. Animals expressing FLAG::PAB-1 from the intestine-specific promoter ges-1 were used to immunoprecipitate FLAG::PAB-1/mRNA complexes from the intestine and the number of enriched genes relative to whole worm lysate was determined by microarrays. The average ranks from eight repeats of this experiment identified 1938 intestine-enriched genes. First, we compared the genes expressed in the intestine to those expressed in muscle (Roy et al. 2002), and thereby identified 807 genes expressed in both tissues and 645 genes enriched in the intestine versus muscle. Second, we showed that the 1938 intestine-enriched genes were also positionally clustered on the chromosomes, suggesting that the order of genes in the genome is influenced by the effect of chromatin domains on gene expression. Furthermore, the tissue-specific lists showed less chromosomal clustering than the list of genes expressed in both the intestine and muscle. This observation suggests that chromatin domains may influence housekeeping genes more than tissue-specific genes. Third, in order to gain further insight into the regulation of expression of intestine-enriched genes, we searched for regulatory motifs in the set of intestine-enriched genes. We used a modified Gibbs sampling method called CompareProspector (Liu, 2004) to identify motifs that were conserved with C. briggsae and over-represented in the 1kb upstream region of the 645 intestine-specific genes. This analysis found that the promoter regions of the intestine genes were enriched for the consensus sequence for GATA transcription factors at a rate two-fold over random. In order to test the functionality of the GATA motif, we are using transcriptional GFP fusions of several intestinal markers with wild type and mutated GATA sites. Liu, Y., Liu, X. S., Wei, L., Altman, R. B., & Batzoglou, S. (2004). Eukaryotic regulatory element conservation analysis and identification using comparative genomics. Genome Research, 14(3), 451-458. Roy, P. J., Stuart, J. M., Lund, J., & Kim, S. K. (2002). Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans. Nature, 418(6901), 975-979.

Roy, Vincent et al. MicroPubl Biol

Gagne, Olivier, Hamiche, Karim, Narbonne, Patrick, Labbe, Jean-Claude, Roy, Vincent

[MicroPubl Biol, 2018]

PAR-4/LKB1 is maternally expressed and required for the asymmetrical distribution of early embryonic determinants and viability in C. elegans (Morton et al. 1992; Watts et al. 2000; Tenlen et al. 2008). It is also implicated in a variety of postembryonic processes, including germline stem cell quiescence (Narbonne and Roy 2006; Narbonne et al. 2017), neuronal growth and polarity (Kim et al. 2010; Teichmann and Shen 2011), cytoskeletal rearrangements (Narbonne et al. 2010; Chartier et al. 2011), and metabolism (Narbonne and Roy 2009). Its expression pattern and subcellular localization has been determined in fixed animals by antibody staining (Watts et al. 2000). Here, we used CRISPR/Cas9-mediated genome editing to fluorescently label the two longest endogenous PAR-4 isoforms, PAR-4A and PAR-4C, with monomeric Neon Green (mNG) (Shaner et al. 2013), using the self-excising cassette (SEC) system (Dickinson et al. 2015) (Fig. 1A). We found ubiquitous mNG::par-4a &amp; c expression and cytoplasmic and cortical enrichment of mNG::PAR-4A &amp; C proteins in the germ line and early embryos (Fig. 1B-H), as previously described (Watts et al. 2000). Interestingly, we find that mNG::PAR-4A &amp; C cortical enrichment is transiently lost in the pachytene area of the germ line (Fig. 1F), although it remains unclear whether this is functionally relevant. The intermediate strain that still contains the SEC (Dickinson et al. 2015) is predicted to be null for par-4a &amp; c, potentially leaving the shorter par-4b isoform functional. To evaluate the requirement for par-4a &amp; c we examined the embryonic lethality (at 25C) of the generated SEC-containing and SEC-excised strains. We found that the self-progeny of homozygous SEC-containing (e.g. par-4a &amp; c null) animals is viable (Fig. 1H). This suggests that par-4b alone is sufficient to establish embryonic polarity and sustain the essential function of par-4. Consistent with this, to our knowledge, all existing par-4 alleles that impair embryonic development disrupt par-4b (Morton et al. 1992; Watts et al. 2000).