Joshua M Stuart et al. (2003) International Worm Meeting "Discovering conserved pathways and core molecular machinery on a global scale."

Stuart Kim, Joshua M Stuart, Daphne Koller, Eran Segal

[International Worm Meeting, 2003]

The genome projects have paved the way for new genome-wide functional studies on several organisms. We can now use the rich supply of DNA microarray data to find expression patterns that reflect physiologically meaningful relationships. We have developed a computational technique for identifying conserved co-expression patterns in multiple organisms. Co-expression that represents a functionally-relevant coupling between two genes will confer a selective advantage to the organism and hence be selected over the course of evolution while irrelevant co-expression will be lost. We constructed a multi-species expression network combining microarray data from over 3000 microarray experiment from four evolutionarily distant organisms H. sapiens, S. cerevisiae, C. elegans, and D. melanogaster. We found over 10,000 examples of pairs of genes that are strongly co-expressed in microarray data from at least two organisms. These gene correlations form a network revealing many relationships between genes involved in ancient pathways and complexes, spanning several cellular and multicellular core processes. The network includes over 300 genes that encode novel proteins, and we can assign potential functions to these genes based on their co-expression with genes of known function. We are in the process of making the network available as a resource for the academic community.

Haining Zhang et al. (2006) Development & Evolution Meeting "The Small GTPase RAB-11 is Required for Normal Spindle Alignment in C. elegans Early Embryos"

Haining Zhang, John White

[Development & Evolution Meeting, 2006]

Communication between intracellular membrane trafficking and the cytoskeleton for regulating biological processes is important yet poorly understood. For example, spindle alignment is crucial for generating cell diversity in many organisms (Betschinger and Knoblich, 2004; Segal and Bloom, 2001), yet little is known about the role of membrane trafficking in this process. RAB-11, a small GTPase enriched in recycling endosomes, regulates protein transport and recycling back to the plasma membrane. When RAB-11 was depleted by RNAi in C. elegans, the P0 spindle failed to rotate and the anaphase spindle underwent violent movement. The length of the astral microtubules, specifically at metaphase, was significantly reduced in rab-11(RNAi) embryos, which may give rise to the observed spindle alignment defects. RAB-11 localization resembles ER distribution around the spindle, and proper ER morphology requires functional RAB-11, in particular during metaphase. This suggests that proteins necessary for normal spindle alignment may be transported to ER by RAB-11 to regulate microtubule length during metaphase and ensure proper spindle alignment in C. elegans early embryos.

Albritton, Sarah et al. (2011) International Worm Meeting "Regulation of X chromosome transcription in Caenorhabditis species."

Albritton, Sarah, Ercan, Sevinc

[International Worm Meeting, 2011]

Animals with different numbers of X chromosomes in males and females possess mechanisms to compensate for the difference in the X-linked gene dose between the two sexes. In addition to the X chromosome dose difference between the sexes, the presence of a single-copy X chromosome per two-copy diploid autosomes creates an important problem for males, because all X-linked genes are haploinsufficient compared to the autosomal genes. In C. elegans, hermaphrodites (XX) contain two Xes, whereas males (XO) contain a single X, therefore facing X haploinsufficiency. By performing microarray analysis of RNA abundance in XX and XO worms, we observed that the overall transcript levels from the X chromosome in both XX and XO animals is similar to that of overall expression from autosomes. This suggests that transcription from the single X in XO L3 hermaphrodites (TY2205, her-1(e1520) sdc-3(y126) V; xol-1(y9) X) is increased approximately two-fold. The mechanism of this upregulation is unclear. We had shown that the X chromosome promoters have higher GC content compared to the autosomes (Ercan et al 2010), suggesting a DNA-encoded mechanism of transcriptional regulation. We will study X upregulation by comparing transcription of orthologus genes that are on the X versus autosomes in four Caenorhabditis species. Ercan S, Lubling Y, Segal E, Lieb JD. High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans. Genome Res. 2011 Feb;21(2):237-44. PMID:21177966.

Don Fox et al. (2001) International C. elegans Meeting "Securin and"

Don Fox, Barry Chestnut, Andy Golden

[International C. elegans Meeting, 2001]

Cell division results in the precise halving of genetic material. In yeast, such partitioning requires the protease separin 1 . This protease acts at the metaphase to anaphase transition to degrade sister chromatid cohesion, which in turn permits chromosomes to segregate to opposite poles of a dividing cell . In addition to facilitating chromosome segregation, separin promotes spindle elongation in S. cerevisiae through the activity of a calcium-binding domain 2 . Separin activity also depends heavily on the anaphase inhibitor securin, which localizes separin to both the nucleus and spindle mid-zone, and inhibits its protease activity until anaphase. Here, we report a requirement for the C. elegans homologue of yeast separin during oocyte meiosis. RNAi of this homologue, which contains both the protease and calcium binding domains of S. cerevisiae separin, results in multi-nucleated one-cell embryos with multiple spindles. In these embryos, the chromosomes are disorganized and the embryos fail to produce polar bodies. Taken together, these results suggest a role for C.elegans separin in chromosome segregation and cytokinesis in the early embryo. Currently, we are investigating if C. elegans separin also plays a role in exit from M-phase of the cell cycle. Additionally, we hope to identify protein partners of C.elegans separin by using the protein as bait in a yeast 2-hybrid screen. This screen may prove the most effective way of identifying a C. elegans securin homologue, as all of the known securins from other organisms demonstrate no amino acid conservation. Also, to date, no separin interacting proteins other than securin have been identified. Results of the screen will be discussed at the meeting. 1.Uhlman, F., Wernic, D., Poupart, M., Koonin, E., and K. Nasmyth. 2000. Cleavage of Cohesin by the CD Clan Protease Separin Triggers Anaphase in Yeast. Cell 103:375-386. 2.Jensen, S., Segal, M., Clarke, D., and S. Reed. 2001. A Novel Role of the Budding Yeast Separin Esp1 in Anaphase Spindle Elongation: Evidence that Proper Spindle Associatioin of Esp1 is Regulated by Pds1. J.Cell Biol. 152:27-40.

Mara Schvarzstein et al. (2003) International Worm Meeting "Regulation of tra-1 by sex-specific phosphorylation, processing, and nuclear localization."

Andrew M Spence, Mara Schvarzstein

[International Worm Meeting, 2003]

The tra-1 gene is the terminal global regulator of sex determination in C. elegans. Its activity is required for all female somatic cell fates and is sufficient for oogenesis, though it also has a role in sustaining spermatogenesis. Its major product, TRA-1A, is a zinc finger transcription factor of the Gli family, which includes the targets of Hedgehog signaling in Drosophila and mammals.Western analysis of nematode lysates with an antibody against a bacterially expressed GST-TRA-1 fusion protein reveals striking differences between hermaphrodites and males. Lysates of both sexes contain a band of apparent molecular weight 140 kD, approximately the predicted size of TRA-1A (125 kDa), but hermaphrodites accumulate several bands of apparent molecular weights 90-100 kDa that are absent from males. All of these bands are absent from lysates of tra-1 null mutant pseudomales, and preincubation of the antibody with a bacterial lysate expressing a MBP-TRA-1 fusion protein blocks their detection, suggesting that all are tra-1 products. The hermaphrodite-specific bands are much too large to be encoded by the small tra-1 transcript tra-1b, and we suggest that they derive from TRA-1A by sex-specific proteolysis. The more slowly migrating bands in the 90-100 kDa size range are eliminated by phosphatase treatment of the lysates, suggesting that they are phosphorylated forms of the smaller bands.Males and hermaphrodites also differ with respect to the subcellular localization of TRA-1 as assayed by immunofluorescence with our antibody. Whereas adult males exhibit diffuse TRA-1 immunoreactivity throughout the cell in most tissues, adult hermaphrodites show predominantly nuclear TRA-1 immunofluorescence in somatic nuclei and distal germline nuclei. Segal et al. also reported increased nuclear accumulation of TRA-1 in hermaphrodites (Dev.Cell 1, 539-51. 2001). Nuclear accumulation of TRA-1 first becomes apparent in most somatic tissues of hermaphrodites during the L3 larval stage.We suggest that TRA-1A activity is regulated by sex-specific phosphorylation and proteolysis as well as by differential subcellular localization. Truncated, hermaphrodite-specific isoforms of TRA-1 may be responsible for specifying female cell fate by repressing target genes such as egl-1, mab-3, and fog-3. The regulation of TRA-1A may be analogous to that of Drosophila Ci and mammalian Gli2, both of which undergo ligand-independent proteolysis to yield transcriptional repressors.

Morris F Maduro et al. (2003) International Worm Meeting "Towards the Use of Zinc Finger Proteins to Generate Artificial Transcription Factors and Rapid Gene Knockouts in C. elegans"

Morris F Maduro, Joel H Rothman

[International Worm Meeting, 2003]

The Cys2-His2 class of zinc finger (Zif) proteins is among the largest class of proteins in nature. A single Cys2-His2 domain is ~30aa long, has a structure, and typically binds a three-base sequence of DNA. Owing to their modularity, such fingers can be joined together to generate polydactyl DNA-binding proteins of high specificity: a single six-fingered protein can recognize a unique sequence among 70 billion unrelated sequences, which is unique in any genome. Work by Carlos Barbas, David Segal and others has focused on identifying specific amino acid residue changes in the -helix to expand the repertoire of available 3-bp target modules. To date, their work has identified zinc fingers that permit access to target sites of the form 5'-GNN-3' and 5'-ANN-3'. We are interested in two broad applications of this technology as additions to the rich repertoire of tools available with the C. elegans system. First is the generation of artificial transcription factors. Fusions of six-fingered proteins with effector (activator or repressor) domains have been shown to function in plants and tissue culture (e.g. Beerli et al., 1998). We are currently constructing 6xZif-effector proteins for testing on the ges-1 gene. Potential downstream applications of this technology include lineage-specific transcriptional repression of endogenous genes, and the generation of expression systems similar to the GAL4 system used in Drosophila. The second application is the generation of gene knockouts by gene conversion. Pairs of triplet zinc fingers can be joined to endonuclease domains to create a bipartite endonuclease that can be targeted to a single chromosomal locus. In Drosophila, such zinc finger nucleases were shown to mediate generation of small-deletion mutants in the y gene following activation of a nuclease and recovery of germline non-homologous end joining (NHEJ) events (Bibikova et al., 2002). In C. elegans, double-stranded (ds) breaks created by Tc1 excision have been shown to promote gene conversion from multicopy extrachromosomal transgenes (Plasterk and Groenen, 1992). Our goal is to fuse these approaches to generate a system for gene conversion: i.e., we propose to use zinc finger nucleases to generate ds breaks in the presence of homologous repair transgenes. We are currently constructing zinc finger nucleases that will target the unc-22 locus for gene conversion from a mutated transgene. Progress with these experiments will be described.

Natalia G Starostina et al. (2005) International Worm Meeting "The global sex determination factor TRA-1 is negatively regulated by a ubiquitin ligase complex that contains FEM-1 and CUL-2"

Mara Schvarzstein, Natalia G Starostina, Edward T Kipreos, Andrew Spence

[International Worm Meeting, 2005]

TRA-1 is a transcription factor that functions as the most downstream component in the somatic sex determination pathway and also has an important role in germline sex determination. TRA-1 transcriptional activity promotes female development and represses male development. In males, TRA-1 is negatively regulated by FEM-1. FEM-1 activity is associated with a decrease in the levels of nuclear TRA-1 (Segal et al., 2001 Dev Cell 1:539) and total TRA-1 accumulation (our unpublished results). However, the mechanism through which FEM-1 regulates TRA-1 accumulation has remained elusive. We present evidence that FEM-1 functions as part of a CUL-2 ubiquitin ligase. CUL-2 is the scaffold for the assembly of a multisubunit complex that includes core components and differentially-associated substrate recognition subunits (SRSs). In an effort to identify SRSs, we employed affinity purification of an epitope-tagged CUL-2 expressed in transgenic animals coupled with mass spectrometry. Among the CUL-2-associated proteins, we identified all core components of the CUL-2 complex, as well as novel interactors, including FEM-1. SRSs bind to the CUL-2 complex through a BC-box motif that mediates interaction with the core complex component ELC-1. We have found that FEM-1 has a BC-box motif which exactly matches the consensus that we find in the BC-box of other C. elegans SRSs. Further, in vitro binding studies have demonstrated that FEM-1 can bind directly to ELC-1. These data strongly suggest that FEM-1 functions as an SRS in CUL-2 ubiquitin ligase complex. The recent finding that the human FEM-1 homolog is also a component of a CUL-2 complex suggests that interaction of FEM-1 with CUL-2 is evolutionarily conserved. To determine if CUL-2 has a role in sex determination, we observed cul-2 mutant males that were the progeny of heterozygous parents (i.e., the males had maternal product). We found that these males have an intersex phenotype that is indistinguishable from that of fem-1 mutant males that are the progeny of heterozygous parents, namely the presence of oocyte-like cells and defective male tail structure. Immunofluorescence reveals that the level of nuclear TRA-1 in the germline and intestine is significantly higher in cul-2 males than in wild type, suggesting that the FEM-1-CUL-2 complex regulates TRA-1 protein stability in both the soma and the germline. We propose that TRA-1 is a substrate of a ubiquitin ligase complex containing CUL-2 and FEM-1, and that the final step of the global sex determination pathway is controlled by the ubiquitin-mediated degradation of TRA-1.