Tingting Zhang et al. (2005) International Worm Meeting "C. elegans PcG proteins form an RNA binding complex"

Hansong Deng, Yuxia Zhang, Yinyan Sun, Hong Zhang, E Tian, Tingting Zhang

[International Worm Meeting, 2005]

Epigenetic gene silencing involves the formation of an inherited repressive chromatin structure, which is established in a stepwise manner and involves the concerted action of proteins and RNA. For example, silencing of genes in the heterochromatic domains in S. pombe requires an RNAi effector complex termed RITS (RNA induced initiation of transcriptional gene silencing), which contains Argonaute homolog Ago1, chromodomain proteins Chp1, Tas3, and siRNAs derived from the RNAi machinery. However, the protein complex in other RNA mediated gene silencing processes, such as in the Xist RNA mediated X chromosome inactivation in mammals, has not been identified yet.RNA components have also been suggested to play a role in the Polycomb group genes mediated Hox gene repression, which is thought to involve the formation of localized repressive chromatin structures. Our lab uses C. elegans, which has a comparatively simple PcG complex, as a model to define essential features of the physiological role of PcG in gene regulation. Our previous studies demonstrated that sop-2 functions as a PcG gene in Hox gene regulation in C. elegans. SOP-2 contains a protein-protein interaction SAM domain and binds directly to RNA, properties that are conserved in PH and SCM, components of the PcG complex in other organisms. Binding of RNA appears to be essential for the role of sop-2 in Hox gene repression.We have identified a new PcG gene in C. elegans, sor-1, and demonstrated that SOR-1 and SOP-2 form an RNA binding complex, further underscoring the importance of RNA in PcG mediated gene silencing. Mutations in sor-1 cause widespread ectopic expression of Hox genes and homeotic transformations. Functions of sor-1 and sop-2 are partially redundant in Hox gene repression. sor-1 encodes a novel protein with a weak tenasin motif, found also in SOP-2 and PH. SOR-1 and SOP-2 are colocalized in the nuclear bodies, and they interact directly with each other in vitro and in vivo. Remarkably, SOR-1 possesses RNA binding activity through a previously uncharacterized RNA binding motif. Thus, SOR-1 and SOP-2 constitute an RNA binding complex in Hox gene repression. Surprisingly, homologs of SOR-1 and SOP-2 are not found in other organisms, even C. briggase. Thus, although the transcriptional regulatory mechanisms involved in the global repression of Hox genes are conserved, the protein components, however, are capable of undergoing major changes during the evolutionary divergence of animal phyla.

Ye Tian et al. (2008) "A genome-wide RNAi screen for genes that interact with the C. elegans PcG genes sop-2 and sor-3"

Dongjia Yang, Zhipeng Li, Hong Zhang, Ye Tian

[2008]

" Polycomb group (PcG) proteins play an important role in specifying the positional identities of cells by transcriptionally regulating the expression of Hox genes. PcG proteins also function in other processes such as in tumorigenesis and in the self-renewal of stem cells. However, the study of molecular mechanisms of PcG-mediated gene repression in fly and mammals is hindered by the complexity and redundancy of the PcG complexes. We previously showed that C. elegans contains relatively simple PcG complexes (1,2). sop-2 encodes a novel protein that is functionally and structurally related to PH and SCM of the PRC1 PcG complex. sor-3 encodes a novel protein containing an MBT domain, suggesting that it may constitute the PhoRC-like PcG complex in C. elegans. We also demonstrated that mutations in sop-2 cause synthetic lethal in combination of a mutation in sor-1 (encoding a component of the PRC1 complex), sor-3 or mes-6 (encoding a component of the ESC/E(Z) complex). Furthermore, the expression domains of the Hox gene egl-5 were dramatically expanded in the double mutants. The genetic interactions suggest that the distinct PcG complex could function together in gene regulation or, alternatively, that they act in parralle pathways. To further understand the underlying mechanisms of PcG-mediated gene repression, we performed a genome-wide RNAi screen to identify mutants that are synthetic lethal with sop-2 or sor-3 mutants. 70 RNAi clones were identified from 16,540 RNAi clones screened (targeting about 86% of the genome). We found that a large number of identified genes were previously implicated in chromatin remodeling or in regulating the germline tumor formation. We are further characterizing these putative PcG enhancers. Our study shows that PcG genes interact with multiple chromatin remodeling complexes in regulating the expression of Hox genes. Furthermore, PcG genes function in many biological processes, including vulva formation, specification of dopaminergic and serotonergic neurons and germline tumor formation.1. Zhang et al., Dev. Cell 20032. Zhang et al., Development 2006"

E Tian et al. (2007) International Worm Meeting "Identification and characterization of novel PcG-like genes in C.elegans."

E Tian, Hong Zhang

[International Worm Meeting, 2007]

Hox genes, which encode conserved homeodomain-containing transcription factors, control the positional identities of cells along the anterior-posterior axis. Inappropriate expression of Hox genes leads to homeotic transformations, whereby body structures are lost or duplicated. Thus, identification of factors required for the establishment of the spatially restricted expression domains of Hox genes is critical for understanding how cellular positional identities are specified during animal development. The C. elegans Hox cluster consists of lin-39, ceh-13, mab-5 and egl-5 (orthologs of Drosophila Scr, labial,ftz and Abd-B, respectively). Each C. elegans Hox gene is expressed in restricted regions of multiple diverse tissues and lineally unrelated cells and defines the region-specific differentiation characteristics. C. elegans Hox genes, as in mammals, are also globally repressed by Polycomb group (PcG)proteins. Two evolutionarily conserved PcG complexes, the ESC/E(Z) complex and the PRC1 complex, that are involved in modulating repressive chromatin structures, have been identified in fly and mammals. The C. elegans PcG-like complex, SOP-2/SOR-1, which shares many structural and functional properties with the PRC1 complex, is involved in the global repression of Hox gene expression. In sop-2 and sor-1 mutants, the C. elegans Hox genes are globally derepressed, resulting in gross homeotic transformations. What are other components that are involved in globally repressing Hox gene expression? We used mab-5::gfp as a marker to identify mutants with expanded expression domains of mab-5, as in sop-2 and sor-1 mutants. From 3,000 genomes screened, we identified several alleles of sop-2 and sor-1. We also identified a mutation, which defines a new genetic locus, with ectopic expression of mab-5::gfp in intestine, hypodermis and neuroblasts. The mutant homozygotes are arrested at the L2 larval stage. The mutation was mapped in a small genetic interval on chromosome III. Currently, we are attempting to clone this gene by transformation rescue experiments. 1.Zhang et al., 2003 2.Ross and Zarkower, 2003 3.Zhang et al., 2006.

Wei-meng Woo et al. (2004) West Coast Worm Meeting "The extracellular matrix protein F-spondin is essential for muscle attachment and epidermal elongation"

Chris Suh, Yishi Jin, Andrew D Chisholm, Mei Ding, Christie Emigh, Wei-Meng Woo, Jian Cao

[West Coast Worm Meeting, 2004]

In C. elegans epidermal intermediate filaments (IFs) and their associated structures, the trans-epidermal attachments, are essential for embryonic epidermal elongation (Woo et al 2004). The formation of muscle contractile units and trans-epidermal attachments are mutually dependent during epidermal elongation. To understand how the connection between epidermis and muscle is established and how the two tissues communicate during organogenesis, we performed a screen for epidermal elongation-defective mutants. One locus identified in this screen was defined by three lethal alleles and mapped to the cluster of LG II. Subsequent analysis showed that these mutations were allelic to vab-13 and ven-3 . By genetic mapping and allele sequencing we showed that all these mutations affect F10E7.4, which encodes the C. elegans member of the F-spondin family of secreted proteins. F-spondin has been shown to play roles in axon guidance, cell migration, and angiogenesis. Our genetic analysis shows that in C. elegans F-spondin is required for epidermal elongation and muscle attachment, as well as for proper positioning of neuronal processes. Using GFP reporters, we found that F-spondin is expressed in body muscle cells and is a secreted protein. Thus, F-spondin may function in embryogenesis in communication between muscle and epidermis. Immunostaining of F-spondin mutants suggest that F-spondin may indirectly affect the organization of epidermal actin microfilaments and trans-epidermal attachments . We are examining the expression patterns of muscle and basement membrane components in F-spondin mutants. To study the signaling pathways regulated by F-spondin, we are testing mutations in candidate receptor genes for genetic interactions with F-spondin mutations.

Bonnie Kaas et al. (2008) C.elegans Neuronal Development Meeting "Regulation of Levels of UNC-13 at Synapses"

Rebecca Lawson, Rebecca Kohn, Thomas Limouze, Ryan Wennell, Bonnie Kaas

[C.elegans Neuronal Development Meeting, 2008]

Release of neurotransmitters from neurons is highly regulated. Several proteins play roles in this process, including UNC-13, and decreased release of neurotransmitters in unc-13 mutants results in paralysis. We identified an F-box protein that interacts with UNC-13. F-box proteins participate in ubiquitin ligase complexes and in Drosophila, DUNC-13 is degraded via the ubiquitin proteasome pathway. This UNC-13/F-box interaction may therefore indicate that UNC-13 is tagged for proteasomal degradation with ubiquitin by the ligase complex in C. elegans. The C. elegans knockout consortium isolated a strain with a large deletion in the coding region of the gene that codes for the F-box protein. If the F-box protein is indeed involved in the degradation of UNC-13, this strain would be expected to have higher levels of UNC-13, which could result in changes in phenotypes. We characterized the F-box deletion mutant by assaying brood size, developmental rate, and body bends per minute. Aldicarb assays were used to determine whether a deletion in the gene coding for the F-box protein alters the response to inhibitors of acetylcholinesterase. We found that the deletion resulted in some changes in developmental rate and in aldicarb sensitivity. We are continuing to study strains with mutations in both the gene coding for the F-box protein and in unc-13.

Nathalie Velarde et al. (2005) International Worm Meeting "F-actin dynamics in early C. elegans development"

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2005]

Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.

Atsushi Yamanaka et al. (2001) International C. elegans Meeting "Biochemical and functional analysis of SKP1 family in Caenorhabditis elegans"

Yasumi Ohshima, Kei-ichi Nakayama, Masayoshi Yada, Atsushi Yamanaka, Hiroyuki Imaki

[International C. elegans Meeting, 2001]

The ubiuquitin-proteasome pathway is a key mechanism for substrate-specific degradation to control the abundance of a number of proteins. SCF complex, one of ubiquitin-protein ligases (E3s), regulates cell cycle progression, signal transduction, and many other biological systems. The SCF complex consists of invariable components, such as Skp1, Cul-1 and Rbx1, and variable components called F-box proteins that bind to Skp1 through the F-box motif. F-box proteins are substrate-specific adaptor subunits that recruit substrates to the SCF complex. Surprisingly, we found that the genome of Caenorhabditis elegans ( C. elegans ) contains at least 20 Skp1-like sequenses, whereas one or a few Skp1 is present in humans. Therefore, we studied C. elegans Skp1-like proteins (CeSkp1) that are likely to be variable components of SCF complex in addition to F-box proteins. At least, seven CeSkp1s were associated with C. elegans Cul-1 (CeCul-1) in yeast two-hybrid system as well as co-immunoprecipitation assay in mammalian cells, and these expression patterns were different in C. elegans . By RNA interference (RNAi), two of these CeSkp1s showed embyonic lethality and four showed the phenotype of slow growth. There were differences among CeSkp1s in ability to interact with F-box proteins. These results suggest that CeSkp1s, like F-box proteins, act as variable components of SCF complex in C. elegans .

Soto, Martha et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Arp2/3-based Regulation of Junctional Dynamics During Morphogenetic Cell Movements and Polarization."

Bernadskaya, Yelena, Patel, Falshruti, Soto, Martha

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Morphogenesis requires dynamic interaction between the adherens junctions and the actin cytoskeleton. This interaction is mediated by ?-catenin, which was proposed to be a stable bridge between the junctions and F-actin at the apical regions of polarized epithelial cells. However, ?-catenin cannot bind to F-actin and its adherens junction partner, ?-catenin, simultaneously, suggesting the bridge is dynamic and complex. ?-catenin was thought to inhibit Arp2/3-based branched actin at the junctions for normal maturation of adherens junctions. Our research suggests a new positive role for Arp2/3-dependent branched actin in junctional maturation and during embryonic cell movements of C. elegans . In this study we investigate how progressive WAVE/Scar and Arp2/3-depen dent accumulation of ?-catenin at the adherens junctions contributes to the accumulation of F-actin at apical regions of epithelial cells. We find that removing Arp2/3-dependent actin nucleation disrupts junctional maturation and results in altered levels of adherens junctional proteins in epithelial tissues. In particular, there is a drop in both ?-catenin and F-actin levels at the apical intestine, and altered organization of the apical intestine. Subcellular fractionation reveals that loss of Arp2/3-dependent actin nucleation reduces the amount of ?-catenin in the membrane-associated pool. These data demonstrate an essential role for Arp2/3 to dynamically remodel F-actin to support adherens junctions and polarized F-actin during cell migration and tissue morphogenesis.

Ge Gao et al. (2004) Mid-west Worm Meeting "Loss of pan-1 causes a Peter Pan-like phenotype in C. elegans"

Ge Gao, Karen Bennett

[Mid-west Worm Meeting, 2004]

P-granules are complexes of proteins and RNA found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the germline specific P-granules, which are necessary for fertility in C. elegans . PAN-1, a P-granule associated novel protein, was identified as a GLH-binding protein in yeast two hybrid assays. PAN-1 contains some conserved amino acids of N-terminal F-box motifs, as well as sixteen leucine-rich repeats and a weak FOG-2 homology (FTH) motif, each found in F-box proteins. F-box proteins, in the SCF (SKP-1, Cullin, F-box) complex, utilize ubiquitin-mediated substrate degradation. When pan-1 is eliminated by RNA interference (RNAi), the larvae arrest between the L1 and L2 stages and can survive eight days at 20 0 C. A pan-1(gk142) deletion strain exhibits the same 'forever-young' phenotype. mRNA analysis and protein expression show that PAN-1 is not germline specific but is germline enhanced. Experiments are ongoing to separate potential germline and somatic functions of PAN-1. If PAN-1 belongs to the family of F-box proteins, it may be implicated in regulating GLH protein levels, as are two other GLH binding proteins, CSN-5 and KGB-1.

De Fruyt, Nathan et al. (2021) International Worm Meeting "Neuropeptidergic modulation of C. elegans learning behavior"

Van Damme, Sara, Schoofs, Liliane, Watteyne, Jan, De Fruyt, Nathan, Beets, Isabel, Fadda, Melissa

[International Worm Meeting, 2021]

Neuropeptides are an evolutionarily conserved group of neuromodulators that regulate a wide range of adaptive behaviors, such as learning. Yet, unravelling the molecular and circuit mechanisms underlying this neuropeptidergic modulation is challenging due to the diversity of neuropeptide signaling pathways, and their 'wireless' extrasynaptic mode of action. Using reverse pharmacology, we have constructed a molecular map of the C. elegans neuropeptide-receptor network. Phylogenetic reconstruction of the evolutionary history of nematode neuropeptide systems across bilaterian animals revealed several nematode-specific diversifications of neuropeptide signaling in addition to evolutionarily ancient neuropeptide pathways. One of these ancient, conserved pathways is a neuropeptide Y/F (NPY/F)-like signaling system that is an important regulator of learning behavior both in Proto- and Deuterostomia. We found that NPY/F-like FLP-34 neuropeptides are required in serotonergic neurons for aversive olfactory associative learning, which is functionally similar to the role of NPY in vertebrate learning as well as to the role of NPF in invertebrate learning. NPY/F-like neuropeptides are released from serotonergic neurons and signal through the G protein-coupled receptor NPR-11 in the excitatory AIA interneurons to facilitate olfactory aversive learning. In addition, signaling through NPY/F-like receptor NPR-11 also affects learning in salt gustatory plasticity, a gustatory associative learning paradigm. NPY/F-like signaling is not the only neuropeptidergic signaling system affecting learning behavior; we discovered additional neuropeptides that appear to be important to the learning process as well, including peptides that are expressed in non-neuronal cells. Our current research focuses on unravelling the functions of such non-neuronal neuropeptide messengers in learning and other types of behavioral plasticity.