[
International C. elegans Meeting,
1991]
Twitchin, the 753,570 Da polypeptide encoded by the
unc-22 gene, was the first member of a growing family of intracellular and mostly muscle proteins, found in diverse animals, composed of multiple copies of motif I (fibronectin type III domain-like) and motif II ( immunoglobulin C2 domain-like). To date, this family includes, smooth muscle and non-muscle myosin light chain kinases, titin, C-protein, 86 kDa protein, skelemin and probably insect projectin. The similarities to motifs found in extracellular and cell surface proteins engaged in recognition or adhesion suggests that motifs I and II of muscle proteins are also involved in binding--probably to myosin, other thick filament components, and also binding of these proteins to themselves and other family members. We hope to be able to demonstrate binding of small numbers of these motifs to thick filament components both in vitro and in vivo. Crystallographic structure determination will be done in collaboration with Pamela Bjorkman (Cal Tech). Using the pGEX- 2T vector, we have been able to express and purify large quantities of motif I, motif II and the predominant triplet I,I,II as glutathione-S- transferase (GST) fusion proteins in E. coli. We have succeeded with the thrombin cleavage of the motifs from GST and are trying ELISA-type binding assays with nematode and rabbit myosin. For in vivo 'binding' studies, we have cloned motif I, motif II, the trio I,I,II and the C- terminal 5 motif IIs into Andy Fire's pPD30.38 vector which directs body wall muscle expression. These constructs will be microinjected into
unc-22 nulls and transgenics examined by immunofluorescence for co-localization to myosin.
[
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 "filtering step" 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.
[
West Coast Worm Meeting,
2004]
Regulatory motifs are short sequences of DNA that regulate the level, timing, and location of gene expression. Identifying these motifs and their functions is crucial in our understanding of gene regulation and disease processes. We developed CompareProspector, a motif-finding program that takes advantage of cross-species sequence comparison to identify putative regulatory motifs from sets of co-regulated genes [1] . We applied CompareProspector to 30 sets of genes with very similar patterns of expression, identified from the C. elegans topomap [2] and individual DNA microarray experiments. The statistical significance of each candidate motif identified was evaluated using criteria such as motif enrichment-the ratio of prevalence of the motif in a given set of promoters to its prevalence elsewhere in the genome, and the expression coherence of genes with the motif. We identified twelve significant regulatory motifs, three of which have literature evidence confirming they are true regulatory motifs. Overall, these twelve motifs are found in the upstream regulatory regions of 2970 different genes, and may be involved in gene regulation in 24 clusters of co-expressed genes. The first known motif, with the consensus TGATAA, matches the consensus of known binding sites for GATA factors. As GATA factors are known to be involved in worm intestine development [3] and hyperdermis development, it is not surprising that the GATA motif is identified from a set intestine-specific genes (F. Pauli, unpublished), mount08 of the topomap, which is enriched in genes from the intestine, and several collagen-related datasets (mount14, 17, and 35 of the topomap). We correctly identified GATA sites in the promoters of genes known to be regulatory by GATA factors. Interestingly, the GATA motif is also identified from several data sets involved in the aging process. This result parallels that of Murphy and colleagues, who independently identified this motif from their data set of DAF-16 target genes [4] . Both our result and the result from Murphy suggest that GATA factors may be involved in worm aging. Motif 2, which is identified in the two heat shock-related data sets, matches the consensus of known binding sites for heat shock factors [5] . Motif 3 matches the consensus of heat shock associated sites (HSAS), a motif that was first predicted computationally to be involved in the heat shock process [6] and later experimentally validated to be involved in ethanol stress response (14 th International C. elegans Conference abstract 1113C). We are currently in the process of validating the rest of the motifs and their individual binding sites using mutagenesis studies of promoters with predicted motifs. 1. Liu, Y., Liu, X.S., Wei, L., Altman, R.B. and Batzoglou, S. (2004) Eukaryotic regulatory element conservation analysis and identification using comparative genomics . Genome Res. 14 , 451-8. 2. Kim, S.K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J.M., Eizinger, A., Wylie, B.N. and Davidson, G.S. (2001) A gene expression map for Caenorhabditis elegans . Science. 293 , 2087-92. 3. Maduro, M.F. and Rothman, J.H. (2002) Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm . Dev Biol. 246 , 68-85. 4. Murphy, C.T., McCarroll, S.A., Bargmann, C.I., Fraser, A., Kamath, R.S., Ahringer, J., Li, H. and Kenyon, C. (2003) Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans . Nature. 424 , 277-83. 5. Amin, J., Ananthan, J. and Voellmy, R. (1988) Key features of heat shock regulatory elements . Mol Cell Biol. 8 , 3761-9. 6. GuhaThakurta, D., Palomar, L., Stormo, G.D., Tedesco, P., Johnson, T.E., Walker, D.W., Lithgow, G., Kim, S. and Link, C.D. (2002) Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods . Genome Res. 12 , 701-12 .
Grill, Stephan W., Bois, Justin S., Kumar, K. Vijay, Hoege, Carsten, Gross, Peter, Julicher, Frank, Goehring, Nathan 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.