Hryshkevich, Uladzislau et al. (2009) International Worm Meeting "Motif composition and evolution of the core promoter."

Yanai, Itai, Hryshkevich, Uladzislau

[International Worm Meeting, 2009]

Even before specific transcription factors can begin to modulate gene expression, the initiation of transcription is crucially dependent upon the properties of core promoter sequence. While many short motifs such as the initiator motif and the TATA-box are known to occupy this region, a surprising level of variation is present across a genome''s core promoters. We hypothesized the existence of a code of short motifs distinguishing the functional properties of core promoters. Here we characterize 12 regulatory elements located in C. elegans core promoters. We found that these cluster into two mutually exclusive groups of motifs (Core1 and Core2). For example the initiator motif and T-repeats (with a consensus of TTTTTCCTCCATTTTT) from the Core1 group do not co-occur with the TATA-box and purine-repeats ((R)12), of the second group. Invoking gene expression data across embryonic development, we asked whether different core promoter motifs correlate with different expression levels. We found that genes with elements from the first group show significantly elevated expression level. For example genes with the initiator motif exhibit strong expression levels (z-score = 35.7), while genes with motifs from the second group such as the TATA-box show low expression (z-score = -1.7). Our analysis enables us to examine the evolution of motif architecture in the core promoter across different species. Such an analysis of the dynamics of motif composition may lead to an understanding of the tuning of gene expression level by motif combinatorics of the core promoter.

Akihiro Mori et al. (2006) East Asia C. elegans Meeting "New computer algorithm for motif prediction"

Akihiro Mori, Yuji KOHARA

[East Asia C. elegans Meeting, 2006]

Multi-cellular organisms needs appropriate regulatory systems which control to activate/in-activate transcriptional process of multiple genes at proper stages and in proper cells for development. In many cases, this is regulated through the binding of proteins to a specific region of the gene. Such protein binding sites are known as cis-regulatory elements or motifs. However, despite their hypothetical importance, cell- and stage-specific regulatory motifs in multi-cellular organisms remain largely unrevealed. Moreover, the prediction of motifs by in silico methods and the verification of putative motifs by experimental methods are both challenging problems. To address these problems, we developed a new computer algorithm for extracting cell-/stage-specific regulatory motifs of C. elegans genes. Since the size and position of such motif are not known before analysis, we have to search short sequences (5 bases or so) in long target sequence area (more than 1000 bases), which cause too many pseudo positives and too long computation time. Thus, we developed an algorithm named &quotfiltering step&quot which reduces the search space (and therefore pseudo positives) dramatically, without losing the real positives. We performed multiple benchmark tests using various sets of genes that contain known motifs and artificial sequences in which some motifs are inserted. The results show that this filtering procedure effectively works to identify motifs in up to 2500 base region. We also found that our algorithm can identify combinations of short motifs very effectively. To identify novel motifs, we plan to apply the method to the gene sets that are known to be expressed stage/cell specifically by systematic in situ hybridization analysis in this laboratory.

Evgeni Efimenko et al. (2003) International Worm Meeting "The X-BOX - a major regulatory motif of ciliogenesis in C. elegans"

Peter Swoboda, Evgeni Efimenko

[International Worm Meeting, 2003]

Cilia are widespread eukaryotic subcellular organelles that function in cell motility, movement of extracellular fluids and sensory reception. While structure and function of cilia are well known, there are many questions with regard to cilia formation and development. The gene daf-19, encoding an RFX-family transcription factor, is crucial for the formation of cilia of sensory neurons. DAF-19 regulates the activity of its target genes by binding to the x-box promoter motif. The C. elegans genome has more than 200 candidate x-box genes. We have initiated expression analysis of these candidate genes. Our results suggest that only genes with x-boxes of a certain nucleotide composition will be expressed in most or all ciliated neurons. Using this consensus sequence we have identified new genes such as the dynein-complex gene xbx-1 that is involved in retrograde cilium transport, and genes D1009.5 and F20D12.3 that are also thought to be very important for cilium structure and function. The roles of new cilium genes are being determined by using RNAi, by generating knockouts and by interaction screens with already known cilium components. Based on the data obtained so far we conclude that DAF-19 typically regulates genes for cilium structure and transport machinery, whereas genes for signal reception and transduction do not require its function. Thus, the x-box search strategy represents a powerful tool for discovering new cilium genes in C. elegans. The knowledge of cilium genes and their function is very important for understanding human pathologies that are caused by defects in cilia.

Joh K et al. (1991) International C. elegans Meeting "CLONING OF GENE(S) CODING A PROTEIN CONTAINING WSX MOTIF."

Wadsworth WG, Hedgecock EM, Joh K

[International C. elegans Meeting, 1991]

unc-5 is a gene functioning in the guidance of migration of pioneer axons and mesodermal cells on the epidermis. Cloning and sequence analysis of the gene revealed that its putative product is a class I transmembrane protein, possibly a receptor protein for unc-6 laminin. The extracellular region of unc-5 consists of one immunoglobulin C2 domain and two WSX domains, and seems to be important for adhesiverecognition of extracellular ligands, such as unc-6. The WSX domain contains an unique sequence DGGWSXWSXWSXC. This WSX motif has been found in some of adhesive proteins, such as thrombospondin, properidin and complement components C6-C9. Considering these facts, it is possible that C. elegans has other proteins containing the WSX motif, which function in cell-cell interactions, recognition of molecules on extracellular matrix and so on. Thus we are attempting to clone genes coding a protein with the WSX motif. We will discuss our attempts to clone by two methods. One is amplification of sequences of such genes by PCR using degenerate primers for WSX sequences. Another is screening by hybridization with synthetic oligo-probe for WSX sequences.

Lum DH et al. (1995) International C. elegans Meeting "A kinesin light chain motif in the sex-determining protein FEM-1"

Elliott J, Lum DH, Spence AM, Chin-Sang ID

[International C. elegans Meeting, 1995]

The fem-1 gene is one of three genes required for male development in C. elegans. Its product contains six copies of the evolutionarily conserved cdc10/SWI6 or ANK motif which, in diverse proteins, mediates specific protein-protein interactions. Recently we noticed that a region of FEM-1 encoded almost entirely by exon 9, exhibits about 35% sequence identity and 48% similarity to a repeated motif found in kinesin light chains from several organisms. The kinesin light chain motif is 42 amino acids long and is of unknown function. The related sequence in FEM-1 corresponds to about 1.2 copies of the motif. Two pieces of evidence suggest that this region of FEM-1 is important to its activity. First, we have identified one partial loss-of-function mutation, fem- 1(e2177ts), which alters a residue within the region. Second, a fem-1 transgene with an in-frame deletion of exon 9 has no detectable masculinizing activity. We have isolated a number of suppresssors of fem- 1(e2177ts), hoping to identify genes which interact with fem-1, perhaps via its kinesin light chain-like motif.

Jessica Shinn et al. (2006) Development & Evolution Meeting "Regulation of the Cellular Fusogen EFF-1 via Motif-Based Protein Interactions"

Jessica Shinn, Jacob del Campo, William Mohler, Martin Schiller

[Development & Evolution Meeting, 2006]

Cell-cell fusion is an important developmental process occurring by a largely unknown mechanism. The cellular fusogen EFF-1 in C. elegans offers a valuable molecule and system to study the precise regulation of cell-cell fusion. EFF-1 is necessary and sufficient for most cell fusions in the worm, and it localizes specifically to the membrane border between fusion partner cells. While EFF-1 is a critical component of the mechanism, its sequence is unique (so far) to only nematode genomes. We and others are using several approaches to better understand the regulation of EFF-1 function and localization (Mohler et al., 2002; Shemer et al., 2004; del Campo et al., 2005). We utilized Minimotif Miner (http://mnm.engr.uconn.edu), a motif database that screens proteins for short motifs, to identify potential regulatory motifs in the cytoplasmic tail of EFF-1. Two of the highest scoring motifs were 14-3-3-binding motifs. In vivo analyses of site-directed mutations show that these motifs are required for timely, normal cell fusion events. In vitro binding assays demonstrate that the human isoform 14-3-3 (eta) binds to EFF-1, and that binding is reduced upon mutation of the 14-3-3-binding motifs (Balla et al., 2006). Our results suggest that 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate EFF-1 mediated cell fusion events by affecting either the localization or activation of EFF-1. We are now working to test this prediction in yeast 2-hybrid experiments and within embryonic cells in vivo. In addition, our initial studies of EFF-1 expression in cultured mammalian cells are allowing us to contrast its localization and function in endogenous and ectopic proteomic contexts.

Cheng, Yuzhu et al. (2021) International Worm Meeting "Significance of RNA Binding Motif Protein (RBM-39) in developmental processes in C. elegans"

Cheng, Yuzhu, Killian, Darrell, Killian, Eugenia Olesnicky

[International Worm Meeting, 2021]

RNA Binding Motif Protein 39 (or Caperalpha) is a conserved RBP that is known to act as an alternative splicing activator. Caper (RBM39 homolog in Drosophila) has been studied in the development and maintenance of the nervous system, and in cancers. Deficiency in Caper/RBM-39 compromises dendrite morphology, sensory neuron development, neuromuscular junction morphogenesis, and lifespan, mainly in Drosophila. However, the full phenotypic effect of RBM-39 remains obscured due to the absence of a knockout mutant. Using C. elegans as a model, we have successfully generated an rbm-39 knockout mutant (cnj4) via CRISPR. Homozygous deletion of rbm-39 leads to severe developmental defects, including sterility, early death, and larval arrest. We used a genetic balancer tmC25 to maintain the recessive sterile allele in heterozygotes. To investigate the potential causes of mutant sterility, a phenotypic analysis of germline development defects in rbm-39 mutants was conducted. Our results show that rbm-39(cnj4) animals have either delayed or failed in oogenesis. We found that the mitotic zone length of the germline was shorter in the homozygous rbm-39(cnj4) than N2 (wild type) and heterozygotes, suggesting disruptions in proliferation and/or maintenance of the germline stem cells. Gonad visualization also revealed abnormal oocytes that were seemingly endomitotic in rbm-39(cnj4) animals. Taken together, RBM-39 is essential for proper germline development and oogenesis. Since RBM-39 is predominantly an alternative splicing factor, genes that control aspects of germline development may be mis-spliced in the mutant. In general, our study highlights the importance of this conserved splicing factor, RBM-39, in reproductive and early development. We have established a novel mutant strain for future investigations while also expanding the knowledge of this protein's functions in C. elegans. Given that germline development in C. elegans is well studied and the processes are highly coordinated with defined signaling pathways, the mechanism of RBM-39 in germline development can be studied using the rbm-39(cnj4) strain. Since rbm-39(cnj4) mutants exhibited mild uncoordinated locomotion, which is strongly associated with neuronal or muscular defects, future studies should examine the RBM-39 knockout phenotype in neurons and dendritic branches.

E Efimenko et al. (2004) European Worm Meeting "THE X-BOX REGULATORY MOTIF DRIVES THE EXPRESSION OF A "CILIARY MODULE" IN C. ELEGANS"

E Efimenko, P Swoboda

[European Worm Meeting, 2004]

Cilia are widespread eukaryotic subcellular organelles that function in cell motility, movement of extracellular fluids and sensory reception. While structure and function of cilia are well known, there are many questions with regard to cilia formation and development. The RFX-transcription factor DAF-19 is crucial for the formation of cilia of sensory neurons. DAF-19 regulates the activity of its target genes by binding to the X-box promoter motif. We conducted a genome-wide search for X-boxes in promoters of C. elegans with subsequent expression and some mutant analyses of the group of positive matches in wild-type and daf-19 mutant backgrounds. Of the more than 30 transcriptional fusions tested so far in worms, we identified 18 X-box genes that function in the development of ciliated sensory neurons. We found that DAF-19 regulates not only genes for the cilium structure and transport machinery (che-2, che-13, osm-1, osm-5, osm-6, bbs-1, bbs-2, bbs-7, bbs-8, xbx-1) but also possible receptors (M04D8.6, T24A11.2) and other factors (odr-4, nhr-44). Most of them have homologous X-box sequences in promoters of C. briggsae, D. melanogaster and humans. We also observed that the actual X-box and it's position within the promoter can help us to predict expression patterns in ciliated neurons (in most or all / in some / in none). We propose a model where the gene daf-19 controls the expression of a "ciliary module" which leads to the formation of functional ciliated endings in sensory neurons in C. elegans. Different structural and functional parts of the "ciliary module" correspond to certain X-box compositions.

Gross, Peter et al. (2015) International Worm Meeting "PAR polarity in C. elegans zygotes is established by a mechanochemical feedback-patterning motif."

Goehring, Nathan W., Julicher, Frank, Gross, Peter, Hoege, Carsten, Bois, Justin S., Kumar, K. Vijay, Grill, Stephan W.

[International Worm Meeting, 2015]

Many conserved morphogenetic processes are orchestrated by a well-controlled interplay between mechanical forces and biochemical regulation. A key example is the early embryonic development of the Caenorhabditis elegans zygote, where large-scale flows of the actomyosin cortex occur simultaneously with the establishment of a polarity pattern in partitioning defective (PAR) proteins. However, how the PAR system interacts with and regulates cortical flow has remained elusive. By combining quantitative fluorescence microscopy, cell biology analysis and a physical theory, we here identify a novel mechanochemical pattern-generating motif, which represents the mechanism that drives the patterning of the PAR polarity proteins in the C. elegans zygote. Using Fluorescence Recovery After Photobleaching (FRAP) and RNA interference (RNAi), we demonstrate that the PAR domains feed back on the mechanics by establishing and maintaining a non-muscle myosin II (NMY-2) - based contractility gradient. To study the consequence of this PAR-mediated feedback on NMY-2, we first measured the dynamics of the PAR and NMY-2 system. Using calibrated, quantitative fluorescence microscopy, we measured the spatiotemporal evolution of the membrane-associated protein concentration of the posterior PAR-2, the anterior PAR-6 and NMY-2 as the mechanical force generator, as well as the cortical flow field. Next we show that these measured dynamics of PAR polarity establishment can be quantitatively recapitulated, using a reaction-diffusion-advection theory for the concentration fields of NMY-2, the posterior PAR-2 and the anterior PAR-6, in combination with a thin-film active-fluids theory for the flow field generated by NMY-2 gradients. Essential for this was the biochemical control of the PAR domains on the NMY-2 binding kinetics, which closes the mechanochemical feedback loop. Remarkably, our physical theory can, for the first time, fully recapitulate the spatiotemporal evolution of all the measured PAR-2, PAR-6 and NMY-2 membrane-concentration fields as well as the actomyosin flow field in the polarization process. We demonstrate that the function of this mechanochemical feedback is to amplify and stabilize cortical flows and thus to promote a rapid transition to the patterned state of the PAR system. We anticipate that this wok will open new avenues in our quantitative understanding of the emergence of patterns during the development of an organism.

Ilya Ruvinsky et al. (2005) International Worm Meeting "A cis-regulatory motif that allows identification of pan-neuronal genes in C. elegans"

Uwe Ohler, Christopher Burge, Ilya Ruvinsky, Gary Ruvkun

[International Worm Meeting, 2005]

Pan-neuronal genes are expressed in most or all types of neurons and thus define generic neuronal features and provide a window into the developmental origin of the nervous system. We computationally predicted a ten-nucleotide cis-regulatory motif shared by many pan-neuronal genes and demonstrated that it is required for broad neuronal expression. Our results suggest that global and cell-type specific controls act in concert to establish pan-neuronal gene expression. Using the newly discovered motif and genome-level gene expression data, we systematically identified a set of 234 candidate pan-neuronal genes, the largest collection of such genes in any organism. The known involvement of many of these genes in neurogenesis and physiology of the nervous system supports the utility of this set for future targeted analyses.