Batchelder C et al. (1997) International C. elegans Meeting "TRANSCRIPTIONAL REPRESSION BY THE PIE-1 PROTEIN"

Mello CC, Batchelder C, Choy B, Cassie C, Suh Y, Blackwell TK

[International C. elegans Meeting, 1997]

PIE-1 is a maternally-expressed protein which is required for germline specification in the early C. elegans embryo. PIE-1 appears to maintain the germ cell precursors in a transcriptionally inactive state, which may be a mechanism to ensure that their fate is not restricted and remains pluripotent. We are interested in establishing if PIE-1 acts directly as a transcriptional repressor, and in elucidating how it functions. PIE-1 is composed of 335 amino acids and contains 2 putative zinc fingers (C3H) in its central region. In order to examine the transcriptional activity of PIE-1, we have set up a transient transfection assay system in mammalian cells. We have observed that PIE-1 can repress transcription from many promoters transcribed by RNA polymerase II, and inhibit a variety of transcriptional activators, including SKN-1. To determine which sequences have repression activity, we have divided PIE-1 into 3 regions (N-terminal, central zinc finger and C-terminal) which were fused to the GAL4 DNA-binding domain. Both the zinc finger and C-terminal regions repressed transcription, the C-terminal region being the stronger repressor by several fold. The C-terminal region was dissected into several sub-regions in order to more precisely define the sequences that mediate the repression. A smaller region with repression activity has been identified and we are currently evaluating the significance of sequence motifs within the region, and whether they might be involved in an interaction with the transcriptional machinery. To further study the function of the PIE-1 zinc finger region, we are presently making several zinc finger mutations, and are also comparing the transcriptional activity of PIE-1 with that of other C3H zinc finger proteins.

Batchelder C et al. (1995) International C. elegans Meeting "REGULATION OF TRANSCRIPTION BY THE SKN-1 AND PIE-1 PROTEINS"

Mello CC, Suh Y, Batchelder C, Blackwell TK

[International C. elegans Meeting, 1995]

Maternally expressed Skn-1 protein is involved in committing blastomeres in the early C. elegans embryo to produce pharyngeal and intestinal cells. However, in germ line precursor cells, Skn-1 must be prevented from functioning by a pathway requiring the maternally expressed Pie-1 protein. At its C-terminal end, Skn-1 protein has a unique DNA binding domain, composed of elements from two distinct families of DNA-binding proteins, suggesting that it may act as a transcription factor and function by regulating transcription of downstream genes. Lacking established Skn-1 targets, we used a mammalian cell transfection assay to determine that Skn-1 is able to bind through its own site, and strongly activate transcription of a reporter gene. When co-expressed in the Skn-1 transfection assay, Pie-1 appears to inhibit the ability of Skn-1 to activate transcription. However, Pie-1 does not act specifically on Skn-1; Pie-1 does not disrupt Skn-1 binding and can repress transcription mediated through other DNA binding domains and by different types of activators. Although our findings are preliminary, they are consistent with Pie-1 functioning as a broadly- acting repressor of transcription.

Batchelder C et al. (1998) East Coast Worm Meeting "Transcriptional repression by C. elegans PIE-1"

Cassie C, Batchelder C, Choy B, Suh Y, Mello CC, Blackwell TK

[East Coast Worm Meeting, 1998]

In the developing C. elegans embryo, maternally-expressed PIE-1 protein is required for specification of germ cell precursors. In pie-1 (pharyngeal and intestinal excess) mutants, these cells instead form pharyngeal and endodermal cells by a pathway that is dependent on the skn-1 gene. Germ cell precursors normally appear to lack both embryonic mRNA transcription, and a particular phosphoepitope on the C-terminal repeat of RNA Polymerase II. PIE-1 is expressed specifically in these cells and is required for their transcriptional silencing, and thus for preventing them from being directed to differentate by SKN-1 or other transcription factors. The sequence of PIE-1 does not suggest an obvious model for how it might function. PIE-1 contains two predicted Cys(3)-His zinc finger motifs and an intervening RS-rich region. Other Cys(3)-His zinc finger proteins have been implicated in RNA processing, post-transcriptional gene regulation, and growth factor responses, but the roles of their zinc fingers remain undefined. An effect of PIE-1 on transcription could be mediated at any of a variety of levels, including signaling, an effect on expression or modification of a general transcription factor, or a direct repressive effect on the transcriptional machinery. We have used a mammalian cell transfection assay to investigate the last of these possibilities. Our experiments indicate that PIE-1 can repress transcription driven by various types of RNA Polymerase II promoters in mammalian cell transfection assays. By attaching portions of PIE-1 to the yeast GAL4 DNA binding domain, we have identified a region that can function as a powerful repressor domain when it is thus tethered to a promoter. Critical residues within this repressor domain are strikingly similar to the Pol II C-terminal repeat sequence, suggesting that PIE-1 might act on proteins that bind to this repeat structure. Experiments are in progress to test this model. Our findings demonstrate that PIE-1 contains sequences that can act directly at promoters to repress transcription. We hypothesize that PIE-1 acts on an evolutionarily conserved component of the transcriptional regulatory machinery, and brings to it residues that are capable of effecting repression.

Ellen Batchelder et al. (2001) International C. elegans Meeting "Investigating the possibe role of calmodulin in cytokinesis"

Jeff Walker, John White, Ellen Batchelder

[International C. elegans Meeting, 2001]

Cytokinesis, the physical separation of a cell during cell division, is fundamentally important to all cell types and must be tightly regulated. Both spatial and temporal regulation are required to ensure proper segregation of chromosomes and molecules involved in processes such as development and differentiation. Calmodulin is a good candidate for a regulator of cytokinesis. It has been localized to the cleavage furrow and/or mitotic apparatus in different organisms and is a known regulator in other calcium dependent pathways, through its calcium sensing properties. Our work has focused on determining the role of calmodulin in regulating cytokinesis in C. elegans using a combination of standard and novel techniques. There are several candidate calmodulins in the worm database, the most conserved of which is CMD-1. Surprisingly, RNAi experiments using cmd-1 did not show cytokinetic defects, although a dramatic embryonic arrest at morphogensis was observed. Other candidate calmodulins are currently being examined with RNAi. Localization studies are also underway. Additionally, we are attempting to phenocopy the RNAi results and to study calmodulin in a more temporally defined manner using a caged calmodulin inhibitor.

Mello CC et al. (1995) International C. elegans Meeting "PIE-1 PROTEIN LOCALIZATION CORRELATES WITH THE GERM CELL FATE DURING EARLY C. ELEGANS EMBRYOGENESIS"

Zhang W, Lobel R, Priess JR, Schubert CM, Mello CC, Draper BW

[International C. elegans Meeting, 1995]

The maternally expressed gene pie-1 is required for germline specification in the early C. elegans embryo. In pie-1 single mutants and in all double mutant combinations analyzed, the germ cell precursors adopt somatic cell fates, in addition, pie-1 mutations exhibit a synthetic dominant maternal effect sterile phenotype in specific genetic backgrounds (see abstracts by Flanagan and Rocheleau). Recent findings suggest that pie-1 may suppress somatic development in the germ lineage by acting as a general transcriptional repressor (see abstracts by Batchelder and Seydoux).

El Batchelder et al. (2005) International Worm Meeting "Post fertilization events in C. elegans embryos are not acutely sensitive to disruption of calcium signaling pathways"

JD Hardin, JG White, Cl Thomas-Virning, El Batchelder

[International Worm Meeting, 2005]

Calcium is an intracellular signal shown in many cell types to regulate cellular events such as fertilization and progression through mitosis. Little is known about calcium signaling in post fertilization events in Caenorhabditis elegans embryos. We have focused on calmodulin, a secondary messenger in calcium signaling pathways. Calmodulin has many downstream effectors, including Myosin Light Chain Kinase. MLCK phospharylates myosin Regulatory Light Chains resulting in myosin activation. A role for MLCK in cytokinesis has been suggested in part because of its function in regulating myosin II based contractility, and because active MLCK localizes to the cleavage furrow in some cells (Chew et al., J Cell Biol 2002; 156:3). We have shown that RNA-mediated interference directed at C.elegans calmodulin (cmd-1) does not lead to early embryonic defects, although there is a penetrant developmental arrest during morphogenesis (see also Karabinos et al., Eur J Cell Biol, 2003; 82). In cmd-1(RNAi) embryos the level of calmodulin is significantly reduced as determined by western blot and by the elimination of signal in a CMD-1::GFP strain. GFP::CMD-1 localizes diffusely to the cytoplasm, to centrosomes, mitotic spindles, nuclear membranes and the cortices of two adjacent cells. We hypothesized that Ca2+/ calmodulin activated MLCK might regulate myosin at the cleavage furrow. let-502(sb106), a C. elegans Rho Kinase mutant, gives rise to cleavage furrows that contain less phosphorylated mRLC than wild type and occasionally fail to complete (Piekny and Mains, J Cell Sci 2002; 115). To ask if a Ca2+/calmodulin activated kinase acts along with RhoK to activate myosin, we looked at let-502(sb106);cmd-1(RNAi) embryos. These embryos do not have more frequent or severe furrow defects than let-502(sb106) embryos. Attempting to more severely disrupt calcium signaling pathways in the embryo, we looked at itr-1(jc5);cmd-1(RNAi) embryos. InsP3 Receptors control release of internal calcium stores and itr-1 encodes a single C. elegans IP3R. itr-1(jc5) embryos have few early defects but later in development the epithelial sheet that normally encloses the embryo is disrupted (Thomas-Virnig, et al., Curr Biol 2004;14). itr-1(jc5);cmd-1(RNAi) embryos have a more severe enclosure defect (Thomas-Virnig, pers. comm.) but no early embryonic defects. Contrary to our expectations, Ca2+/calmodulin does not appear to be involved in signaling cleavage furrow activation in C. elegans early embryos and early C. elegans early embryos do not appear to be acutely sensitive to disruption of calcium signaling pathways.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.