Katherine Weber et al. (2006) Development & Evolution Meeting "Domain Functions of NR2E Nuclear Receptors"

Danielle Snowflack, Stephen DeMeo, Melissa Cronin, Katherine Weber, Bruce Wightman, Rebecca Lombel, Sheila Clever

[Development & Evolution Meeting, 2006]

The nuclear receptors of the NR2E class play important roles in pattern formation and nervous system development. We are studying the function of NR2E DNA-binding domains (DBD) and ligand-binding domains (LBD) in C. elegans. We define two conserved groups of NR2E genes: the NR2E1 subclass, which includes C. elegans nhr-67, Drosophila tailless and dissatisfaction, and vertebrate Tlx (NR2E2, NR2E4, NR2E1), and the NR2E3 subclass, which includes C. elegans fax-1 and vertebrate PNR (NR2E5, NR2E3). PNR and Tll nuclear receptors have been shown to bind the hexamer half-site AAGTCA, instead of the hexamer AGGTCA recognized by most other nuclear receptors, suggesting unique DNA-binding properties for NR2E class members. FAX-1 (NR2E5), unlike NHR-67 and other NR2E1 subclass members, binds to hexamers with relaxed specificity: it will bind hexamers with the sequence ANGTCA, although it prefers a purine to a pyrimidine at the second position. We used site-directed mutagenesis to demonstrate that the difference between FAX-1 and NHR-67 binding preference is partially mediated by a conserved Asn residue at position 19 of the DNA-binding domain. This amino acid position is part of the “P box” that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. We are currently examining the non-NR2E receptors NHR-4 and NHR-34 (provided by M. Van Gilst), which also have an Asn-19, to test whether Asn-19 is sufficient to confer the same relaxed specificity of DNA binding. The relaxed specificity allows FAX-1 to bind a large repertoire of potential binding sites. These findings define subclass-specific DNA-binding specificities for the NR2E1 and NR2E3 subclasses, and suggest that the relaxed specificity of binding observed for the NR2E3 subclass is partially mediated by a conserved Asn-19 in the P box. We are evaluating the specificity of LBD function by swapping the LBD"s from various NR2E receptors, including C. briggsae FAX-1, NHR-67, NHR-111, and human PNR, for the FAX-1 LBD, and testing the fusion genes for function in C. elegans by rescue of fax-1 mutants.

Weber M et al. (2000) European Worm Meeting "Regulation of programmed cell death in the Germ line of C. elegans Hermaphrodites"

Weber M, Conradt B

[European Worm Meeting, 2000]

Four genes, egl-1 (for egg laying defective), ced-9 (for cell death defective), ced-4, and ced-3, form the general PCD (Programmed Cell Death) pathway in somatic tissues of C. elegans (METZSTEIN et al. 1998). Although ced-9, ced-4, and ced-3, are also involved in PCD in the germ line of hermaphrodites, the regulatory, proapoptotic egl-1 gene is not required for PCD in this tissue (CONRADT & HORVITZ 1998, GUMIENNY et al. Q999). This indicates that different pathways regulate the process of PCD in the soma and in the germ line. To identify loss-of-function mutations in proapoptotic genes acting specifically in the germ line, we are screening for mutants with reduced PCD in the germ line. To that end, the numbers of cell corpses in the germ line of the F2 progeny of EMS-treated animals are analyzed using Nomarski optics. The screen is performed in a ced-1 mutant background, in which the engulfment of germ cell corpses is partially blocked and in which cell corpses therefore accumulate in the germ line. The ced-1 mutant background hence increases the sensitivity of the screen and allows the identification of mutants with weak defects. The extend of PCD in the germ line of hermaphrodites increases with age (SULSTON 1988, GUMIENNY et al. 1999). In addition, it has been reported that the death of germ cells may be affected by environmental factors (SULSTON 1988). To determine whether PCD in the germ line varies with environmental conditions, and therefore might be regulated cell non-autonomously, we are in the process of examining the effect of stress and food-deprivation on PCD in the germ line. Conradt, B. and Horvitz, H. R. (1998). Cell 93, 9-529. Gumienny, T. L., Lambie, E., Hartwieg, E., Horvitz, H. R. and Hengartner, M. O. (1999). Development 126, 1011-1022. Metzstein, M. M., Stanfield, G. H. and Horvitz, H. R. (1998). Trends Genet., 14: 408-414. Sulston, J. (1988) in The Nematode Caenorhabditis elegans, (ed. W. B. Wood), pp. 123-155. Cold Spring Harbor.

Katherine P Weber et al. (2007) International Worm Meeting "Conservation of Ligand Binding Domain Function Among NR2E Nuclear Receptors."

Katherine P Weber, Bruce Wightman

[International Worm Meeting, 2007]

Nuclear receptors (NHRs) are transcriptional regulators that consist of a highly conserved DNA-binding domain (DBD) and a more diverged ligand binding domain (LBD). Some LBDs bind hormonal ligands, and they may also regulate transcription through roles in dimerization and association with transcription regulatory complexes. Nematodes are unusual in that they have a particularly large number and diversity of nuclear hormone receptors (there are 21 known genes encoding NHRs in the Drosophila melanogaster</I> genome, 48 in the human genome, and 284 in that of C. elegans</I>). The evolutionary reason for this diversity is unknown. Considerable divergence in nuclear receptor gene sequences is contained in the LBD, so general questions about evolutionary pressure on nhr genes and nhr function can also be asked in regard to LBDs. fax-1</I> is a nuclear receptor of the NR2E class that plays an important role in neuron identity and axon pathfinding. We are evaluating the specificity of LBD function by swapping the LBDs of nuclear receptors with varying degrees of sequence identity to FAX-1, including C. briggsae</I> FAX-1, NHR-111, NHR-67, and human PNR, for the FAX-1 LBD. We are testing the fusion genes for function in C. elegans</I> by rescue of fax-1</I> mutants and have found that the FAX-1 DBD::NHR-111 LBD fusion protein weakly rescues the fax-1</I> phenotype, suggesting that an LBD with only 25% identity can partially substitute for the function of the FAX-1 LBD. Results from the remaining fusion constructs will provide insight about the evolutionary pressure on C. elegans</I> nuclear receptor LBDs.

Katherine Weber et al. (2009) European Worm Neurobiology Meeting "DETERMINING THE GENETIC BASIS OF NATURALLY VARYING C. ELEGANS OXYGEN RESPONSE AND SOCIAL FEEDING BEHAVIOURS"

Katherine Weber, Antoine Verslype, Mario De Bono

[European Worm Neurobiology Meeting, 2009]

We are studying the genetic basis of behavioural variation using C. elegans as a model system. Even as whole-genome association studies become faster and cheaper, the genetic underpinnings of natural variation in behaviour have proven difficult to elucidate in most organisms. This difficulty arises because complex behaviours are usually controlled by a combination of minor effects of variation in many genes. C. elegans provides a powerful system for studying the genetics of behaviour because of its genetic tractability, the availability of strains collected from around the world, and the opportunity to move from genetic association studies to determination of the molecular mechanisms behind phenotypic variation. We have constructed Recombinant Inbred Advanced Intercross Lines (RIAILs) between N2 and CB4856 (Hawaiian) worms. We have genotyped these lines using PCR to detect polymorphisms between N2 and CB4856 with the assistance of a high-throughput PCR robot. A number of oxygen-sensing phenotypes vary between N2 and CB4856, and this variation is independent of npr-1 and glb-5, genes in which polymorphisms between N2 and CB4856 have previously been shown to affect behaviour. We have assayed a subset of RIAILs for these oxygen response behaviours, and we are using this genotyping and phenotyping data to perform QTL analysis and identify the loci responsible for the natural phenotypic variation.

Weber, Katherine P. et al. (2011) International Worm Meeting "High throughput identification of mutations affecting aggregation using a novel Illumina sequencing analysis pipeline."

Baier, Felix, Weber, Katherine P., de Bono, Mario, De, Subhajyoti

[International Worm Meeting, 2011]

The standard C. elegans reference strain, N2, feeds in isolation, but wild-caught C. elegans aggregate on food or after starvation. Failure of N2 to aggregate is due to a gain-of-function mutation in the neuropeptide receptor npr-1: when this gene is knocked out N2 strongly aggregate. To identify molecules that promote or inihibt aggregation we mutagenized, in a series of independent screens, npr-1(null) or N2 animals and isolated mutants that suppressed or enhanced aggregation. This approach has been very fruitful: we have identified a number of genes involved in aggregation behavior. However, traditional genetic mapping and gene cloning is time-consuming, and recent work from other groups has shown that Illumina sequencing can be used to more rapidly identify mutations (Sarin et al. (2008); Doitsidou et al. (2010); Zuryn et al. (2010)). To increase the throughput of aggregation gene cloning, we have established a pipeline to sequence more than 200 uncloned modifiers of aggregation using Illumina technology. Many of these mutants have been mapped roughly (usually to ~1 Mb) so examining their genome sequences allows us to rapidly identify the lesion responsible for their phenotype. We have adapted a protocol from Dan Turner at the Sanger Institute to make amplificaton-free libraries and sequenced these on both the Illumina Genome Analyzer II (GAIIX) and on the Illumina HiSeq machine. The HiSeq has increased our output approximately six-fold. We have also assembled a bioinformatics pipeline to analyse the gigabases of data generated. Our downstream analysis can be completed on an entire flow cell (7 lanes of sequence) in approximately 2 days: it uses the BWA (Burrows Wheeler Algorithm) program to align filtered sequence reads to the reference genome, further filters the alignment output for high confidence polymorphisms, and then implements a custom pipeline to highlight which of these introduce exonic and regulatory changes, indicate genes affected and predict specific coding changes.

Mongan NP et al. (1997) International C. elegans Meeting "AN EXTENSIVE NICOTINIC ACETYLCHOLINE RECEPTOR GENE FAMILY IN CAENORHABDITIS ELEGANS."

Baylis HA, Mongan NP, Sattelle DB

[International C. elegans Meeting, 1997]

Although the nicotinic acetylcholine receptors (nAChRs) are the best characterized ionotropic neurotransmitter receptors, the extent of the molecular and functional diversity of the nAChR gene family is not known for a single organism. A number of C. elegans nAChR subunit genes have been identified previously using genetic approaches [lev-1(non-a),unc-38(a),unc-29(non-a),deg-3(a)] and cross-species probing of C. elegans cDNA libraries [acr-2(non-a),acr-3(non-a),and Ce-21(a)]. nAChR a-subunits may be distinguished from all other ionotropic receptor subunits by the presence of adjacent cysteines at positions equivalent to 192,193 in the Torpedo a-subunit. We have applied an RT-PCR strategy to confirm the expression of 8 novel putative nAChR a-subunits predicted by GENEFINDER. Analysis of the regions of the a-subunit considered to (a) contribute to the acetylcholine binding site and (b) the channel lining, has revealed a diversity which may underlie distinct functional roles for members of this multi-gene family.

C Huang et al. (1999) International C. elegans Meeting "Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis"

WG Wadsworth, C Huang, PD Yurchenco

[International C. elegans Meeting, 1999]

Laminin is a heterotrimeric glycoprotein composed of a , b , and g subunits. The genome sequencing project reveals two a , one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin a A and laminin a B, are most similar to the vertebrate a 1/ a 2 and a 5 chains respectively, while the b and g resemble the b 1/ b 2 and g 1 chains. Thus, there are two predicted laminin trimers, a A bg and a B bg . Mutations in epi-1 , which encodes laminin a B, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin a A and a B. By immunostaining, both first appear at gastrulation between germ layers. However, laminin a A is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin a B is more posteriorly localized. Laminin a B becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin a A accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin a A is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3 , which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin a B has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin a A is restricted to specialized membranes and is required for the morphogenesis of specific tissues.

Yanjue Wu et al. (2005) International Worm Meeting "EGb 761 INHIBITS AMYLOID BETA OLIGOMERIZATION AND TOXICITY IN TRANSGENIC CAENORHABDITIS ELEGANS"

Christopher D Link, Yves Christen, Astrid G Zepeda, Yanjue Wu, Zhixin Wu, Yuan Luo

[International Worm Meeting, 2005]

Amyloid peptide aggregation has been postulated to link oxidative stress and neurodegeneration seen in Alzheimer's diesease (AD). We have previously demonstrated that the standardized Ginkgo Biloba extract, EGb 761, inhibits A aggregation in vitro and attenuates the expression of a stress response gene small heat shock protein (hsp 16-2) in Caenorhabditis elegans. In this study, we use EGb 761 as a pharmacological modulator to associate A species with the levels of oxidative free radicals and with A-induced toxicity in an A-expressing transgenic C.elegans model, which can express A in muscle cells after temperature up-shift. We observed that EGb 761 alleviated A-induced toxicity which correlated with its ability to specifically inhibit A oligomerization. Furthermore, EGb 761 reduces intracellular levels of hydrogen peroxide in the transgenic C.elegans. These findings suggest that A oligomers and A-induced oxidative strss are crucial for A toxicity, and EGb 761 has a clear therapeutic potential for prevention and treatment of AD.

Lyashenko, Alexey et al. (2015) International Worm Meeting "Line scanning fluorescent confocal microscope for a 3D multi-neuron imaging of C. elegans."

Polanco, Edward, Lyashenko, Alexey, Arisaka, Katsushi

[International Worm Meeting, 2015]

Conventional confocal microscope uses a physical aperture to reduce the amount of out of focus light to the image sensor. We developed a line scanning confocal microscope that utilizes a software controlled rolling shutter on a CMOS camera for a high-speed 3D volume imaging of dozens of active neurons. The microscope setup allows for a real time worm tracking for freely navigating C. elegans under a localized external stimulation for phototaxis and thermotaxis. An external photo stimulation for optogenetics was also realized.

Paul Maddox et al. (2007) International Worm Meeting "Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin."

Joost Monen, Arshad Desai, Francie Hyndman, Karen Oegema, Paul Maddox

[International Worm Meeting, 2007]

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.