Hannah Nicholas et al. (2007) International Worm Meeting "The C. elegans CTBP-1 repressor protein contains a DNA-binding THAP domain."

Joel Mackay, Chu Kong Liew, Tina Wu, Merlin Crossley, Hannah Nicholas

[International Worm Meeting, 2007]

Transcriptional co-repressors of the C-terminal binding protein (CtBP) family interact with other proteins that contain amino acid motifs of the form PXDLS. These motifs occur in many transcription factors, and serve to recruit CtBP and its associated repression complex to DNA-regulatory elements. In mammals there are two CtBP genes, with overlapping functions. Disruption of both genes leads to early lethality. In C. elegans there is a single CtBP-related gene. We have shown that the worm CtBP (CTBP-1) binds PXDLS-containing transcription factors and can repress transcription, indicating that the nematode protein is functionally orthologous to other CtBPs. C. elegans therefore provides a tractable model system for investigating CtBP function. Interestingly, the CTBP-1 protein contains an additional domain not found in other characterised members of the CtBP family, which homology searches have identified as a THAP domain. We have shown that this domain can bind to double-stranded DNA and, through site-selection experiments, we have identified a consensus binding sequence. Using nuclear magnetic resonance spectroscopy we have determined the structure of the THAP domain of CTBP-1 and shown that it contains a positively charged face that may correspond to the DNA-contact surface. The C. elegans CtBP protein is the first CtBP protein with intrinsic DNA-binding capacity and may serve as a DNA-binding transcriptional repressor in vivo. In order to examine the in vivo roles of CTBP-1, we have obtained a ctbp-1 deletion allele from the C. elegans knockout consortium. Worms carrying this allele display a mild uncoordinated phenotype. Progress towards a molecular understanding of this phenotype will be presented.

Hannah Nicholas et al. (2007) First Australian C. elegans Meeting "Caenorhabditis elegans as a model in which to investigate the functions of a transcriptional co-repressor"

Joel Mackay, Hannah Nicholas, Tina Wu, Merlin Crossley, Chu Kong Liew

[First Australian C. elegans Meeting, 2007]

Transcriptional co-repressors of the C-terminal binding protein (CtBP) family interact with other proteins that contain amino acid motifs of the form PXDLS. These motifs occur in many transcription factors, and serve to recruit CtBP and its associated repression complex to DNA-regulatory elements. In mammals there are two CtBP genes, with overlapping functions. Disruption of both genes leads to early lethality. We have identified a single CtBP-related gene in C. elegans. This worm CtBP (CTBP-1) binds PXDLS-containing transcription factors and can repress transcription, indicating that the nematode protein is functionally orthologous to other CtBPs. C. elegans therefore provides a tractable model system for investigating CtBP function. We have begun investigating CTBP-1. This protein contains an additional domain not found in other characterised members of the CtBP family, which homology searches have identified as a THAP domain. We have shown that the domain can bind to double-stranded DNA. We have also determined the structure of this domain and shown that it contains a positively charged face that may correspond to the DNA-contact surface. The C. elegans CtBP protein is the first CtBP protein with an intrinsic DNA-binding function and may serve as a DNA-binding transcriptional repressor in vivo.. In order to examine the in vivo roles of CTBP-1, we have obtained a ctbp-1 deletion allele from the C. elegans knockout consortium. Worms carrying this allele display a mild uncoordinated phenotype. Progress towards a molecular and cellular explanation of this phenotype will be presented.

Jeffers, Tess E. et al. (2013) International Worm Meeting "Nucleosome organization in C. elegans gamete chromatin."

Lieb, Jason D., Jeffers, Tess E.

[International Worm Meeting, 2013]

We are interested in how the genome is packaged in gametes (sperm and oocytes), and how differences might contribute to the specification of zygotic gene expression programs. Using fem-1 and him-5 mutants, we separately isolated pure populations of C. elegans sperm and oocytes and performed RNA-seq and nucleosome mapping through MNase-seq. Preliminary transcriptome analysis reveals ~700 transcripts with higher relative levels in sperm, and ~2000 transcripts with higher relative levels in oocytes, in agreement with quantities reported by previous microarray and proteomics experiments (Reinke et al., Mol. Cell 2000 & Development 2004, Chu et al., Nature 2006). Sperm are enriched in transcripts encoding proteins that function in the cytoskeleton, as chaperones, and in the mitochondria while oocytes are enriched for transcripts that encode proteins functioning in mRNA processing, mitosis and meiosis. By integrating our nucleosome and transcriptome data, we find nucleosome depletion at the promoters of genes highly transcribed in the germline. Future experiments will investigate the use of differential histone isoforms and histone modifications in gamete genome packaging.

Chung Man Chan et al. (2006) East Asia C. elegans Meeting "Sex pheromone in dioecious nematode: its production and perception"

King L. Chow, Chung Man Chan, Jeffrey Chasnov, Wai Kar So

[East Asia C. elegans Meeting, 2006]

Two different mating systems are adopted by Caenorhabditis species, that is dioecy and androdioecy. For dioecious species, finding a mating partner is an obligatory process for survival. We have identified a sex-specific attractant produced by females of the dioecious Caenorhabditis remanei for attracting the mating partners. We have further characterized the nature and the constituents of this sex pheromone-like substance, the locale of its production, its perception requirement and related sex pheromones produced by other Caenorhabditis species. These attractant substances are produced by the somatic gonad as confirmed by laser ablation of gonadal progenitor cells in different combination. Their activity could be abolished by mating and can be recovered in the absence of further mating. While perception of these attractants could be observed in males of the androdioecious C. elegans, the perception pathway was delineated by repeated testing of various genetic mutants affecting either specific cell fate or molecular function. Our results show that two different sensory neurons and an interneuron are required. Moreover, a signaling pathway dependent of kinase activity and G-protein subunit are found to be crucial for the pheromone perception. A hypothesis of conservation of the pheromone perception pathway across different species will be discussed. (The research is funded by Research Grants Council, Hong Kong.)

Wan, Xuan et al. (2017) International Worm Meeting "Identification of nematodes volatile sex pheromone and the molecular basis of its perception."

WAN, Xuan, Chow, King L.

[International Worm Meeting, 2017]

Nematodes rely on their sensory modalities to locate mating partner. Volatile sex pheromone attracts potential mating partners from afar. Intriguingly, sexually matured females C. remanei and self-sperm exhausted hermaphrodites C. elegans produce a volatile attractant that is detectable by both con-specific and its relative adult males, qualifying this attractant as a long-range sex pheromone. We previously reported a class D GPCR serpentine receptor (SRD-1), a receptor in AWA neuron, is responsible for detecting this pheromone and several components in the olfactory pathway are involved in this pheromone signal transduction. The GC-MS results of chemo-attractants from female C. remanei verified several volatile chemicals. Two of them exhibited sex-, stage-, and species-specific attractiveness on wild-type males. Noticeably, these compounds could neither attract receptor-defective mutant nor pheromone-perception-defective mutant males. We also visualized AWA neuron excitation by employed a calcium indicator, GCaMP5. The excitation of the AWA neurons was observed only in wild-type males, but not in srd-1 mutants, upon induction by both candidates separately. We concluded that these two chemicals are the active components of this pheromone through the GC-MS analysis, signal transduction pathway mutant strain studies and also the demonstration of ligand-receptor relationships in calcium imaging and behavioural assays. (The study is supported by Research Grants Council, Hong Kong.)

FU, Lan et al. (2011) International Worm Meeting "The identification of the functional components of the male-specific CEM neurons."

Chow, King L., Fu, Lan, CHAN, Gus C. M.

[International Worm Meeting, 2011]

CEMs are the only four male-specific cephalic neurons located in the head region of Caenorhabditis elegans. They are required for sex pheromone perception possibly for sensory input (Chasnov et al., 2007). However, their specific role and molecular function in this process is largely unknown. In order to define the cellular identity of CEM and active elements conferring its function, we are analyzing the CEMs' transcriptome using Illumina sequencing technique. Although ced-4 mutant hermaphrodites have non-functional CEMs preserved, these animals are not able to respond to sex pheromone. Ectopic fem-3 expression in the nervous system could masculinize CEMs and significantly raised the sensitivity of ced-4 hermaphrodites to sex pheromone. Some cellular properties of CEM could also be partially restored with this fem-3 expression. PolyA RNA from the functional CEMs was obtained from such masculinized ced-4 mutant hermaphrodites and was compared with the polyA RNA from ced-4 mutant hermaphrodites with feminized CEMs. FLAG-tagged poly(A)-binding protein (PABP) was expressed under the CEM-specific pkd-2 promoter and the protein product crosslinked with the CEM-specific polyA RNA. The uniquely expressed or highly enriched RNA species in these functional CEMs are recovered. These unique components reflecting the molecular properties of CEM will be presented, where their potential involvement of the biological activity in the CEMs will be discussed. (The study is supported by Research Grants Council, Hong Kong.).

Li, Ching-Ki et al. (2015) International Worm Meeting "The male-specific CEM neurons sense sex pheromone via Go-mediated signaling in C. elegans."

CHOW, King-Lau, Li, Ching-Ki

[International Worm Meeting, 2015]

Finding a mating partner is an obligated process for the survival of sexual species. C. elegans males show prominent chemo-attractive response to the sex pheromone released by females of a dioecious species - C. remanei, implicating a mating cue. This chemotactic response towards sex pheromone is controlled by a pair of male-specific CEM neurons. Despite its role in chemo-sensation implicated by their ciliary endings, the biological function of these neurons remains elusive. We showed that C. elegans males lacking intact CEM cilia by depleting IFT (Intraflagellar transport) proteins displayed a notable weakened sex pheromone response, suggesting that CEMs are sensory neurons. We also sought to identify the molecular components acting in CEMs, including G proteins. The C. elegans genome encodes 21 G-alpha, 2 G-beta and 2 G-gamma subunits. We systematically examined the expression of these genes in CEM and evaluated their function in CEM by cell-specific knockdown. We found that goa-1 (gene encoding G-alpha), gpb-1 (gene encoding G-beta) and gpc-2 (gene encoding G-gamma) are expressed in CEMs, and knockdown of these genes impaired animal response towards sex pheromone. These results indicate Go-mediated signaling occurs in the CEMs to regulate sex pheromone chemotaxis. How these G proteins are coupled with the receptor for sex pheromone perception by CEM will be discussed. (This study is supported by Research Grants Council, Hong Kong.).

Ang, Y.J. et al. (2011) International Worm Meeting "Interaction of TGFb regulators, LON-1 and LON-2 with its downstream target, DBL-1."

Lee, J.C.C., Chow, K.L., Wong, C.K.H., Ang, Y.J., Mok, M.

[International Worm Meeting, 2011]

In C. elegans, TGFb pathway impacts on the body size of the organism in addition to a myriad of other developmental features. The body size of a worm is primarily controlled by endoreduplication that affects the cell size instead of the cell number. Mutants of Lon genes, e.g., lon-1, lon-2 and lon-3, produce worms 1.5 times longer than wild type. These Lon gene products act in the extracellular matrix regulating the body size, and they encode a PR-related protein, a glypican and a collagen, respectively. Their regulatory mechanism, however, remains unclear. Here, we are particularly interested in lon-1 and lon-2 genes which act as negative regulators of the BMP-ligand, DBL-1 in the TGFb pathway. In C, elegans, the effect of LON-1 exerts on DBL-1 was likely through the BMP ligand facilitator, CRM-1, possibly through physical interaction. We mapped the interaction domain between LON-1 and CRM-1 and demonstrated that the LON-1 SCP domain interacts with CR domains on CRM-1. In addition, lon-2 null mutant allele attributed to two large deletions was characterized. While glypican has the ability to bind BMP ligand, we would investigate this interaction between LON-2 glypican and DBL-1 ligand in vivo. Tagged version of both LON-2 and DBL-1 are expressed in transgenic animals for co-immunoprecipitation and domain mapping. The data from these experiments and the implication will be discussed in this poster. (This study is funded by the Research Grants Council, Hong Kong.).

XIE, Dongying et al. (2015) International Worm Meeting "The role of a claudin superfamily gene nsy-4 in male sensory organ development of C. elegans."

Chow, King L., Xie, Dongying

[International Worm Meeting, 2015]

Epithelial cell junctions are crucial for cell adhesion, cell-cell communication and morphogenesis. In C. elegans, how the components of adherens junctions are assembled in organogenesisis is largely unknown. We have previously reported that a claudin-like protein NSY-4, located at the apical junctions, is required for sensory ray assembly. nsy-4 knockdown in either ray hypodermis or structural cells alone is sufficient to generate ray-missing males. Like its claudin homologs in vertebrates, NSY-4 contains a functional PDZ binding motif and intracellular C-terminus, which are dispensable for correct localization, while mutation of the GLW motif leads to intracellular retention of the protein. These results suggest that NSY-4 functions similarly as its vertebrate counterparts do and may mediate adhesion by direct homophilic binding. NSY-4 also requires HMR-1, the classical cadherin, for correct localization and the two co-localize at the ray tips. hmr-1 and nys-4 double knockdown enhances the ray missing phenotype, implying functional interaction between the two proteins. In addition, homeobox transcription factor ceh-43 knockdown reduces nsy-4 expression level via a transcriptional reporter assay, pointing nsy-4 as a downstream target of ceh-43. Given these results, we propose NSY-4 is an additional building block in C. elegans male sensory ray. Further studies on NSY-4, such as homophilic interaction, mapping of membrane localization domain and interaction with other adherens junctional proteins, should reveal more information on the roles of claudin-like proteins in nematode organ formation.(This study is supported by Research Grants Council, Hong Kong).

W.K. So et al. (2006) European Worm Meeting "Sex pheromone production in dioecious nematodes and the perception pathway delineated by genetics"

W.S. Yeung, M.Y. Wu, C.M. Chan, W.K. So, K.L. Chow

[European Worm Meeting, 2006]

W.K. So, C.M. Chan, M.Y. Wu, W.S. Yeung and K.L. Chow. In dioecious nematodes, finding the mating partner and successful mating is instrumental to their reproductive success. To ensure these tasks are accomplished, sex pheromone production is a common strategy adopted in the nematode world. In Caenorhabditis remanei, a dioecious species, a sex-specific attractant was identified to be produced by females for attracting males. In this study, we focus on four areas of characterization, the properties of the pheromone, its regulatory production in female, male perception and species specification. In the characterization part, our GC-MS analysis result identifies two candidate chemicals in the samples, the exact identity of which will be confirmed by chemical synthesis coupled with bioassays. These attractant substances are produced under tight developmental control in the gonad as confirmed by laser ablation of gonadal progenitor cells in different combination. Their activity could be abolished by mating with males. Based on our pervious results, the males of the androdioecious can be attracted by this sex pheromone. So for the male perception part, we employed males from various Caenorhabditis elegans mutants with defects in cell lineage or molecular function to delineate the perception pathway. Our results show that two different sensory neurons and an interneuron are required. Moreover, a signaling process dependent of G protein coupled receptor is crucial for the pheromone perception, where modulation by other molecular components has been observed. Hypothesis suggesting a conservation of the pheromone perception pathway across different species will be tested using chemo-attraction assay on con-specific and hetero-specific combination of testing animals. (The project is funded by Research Grant Council, Hong Kong.)