Yucel, Duygu et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Insights into C-terminal Binding Protein (CtBP) via a mutagenesis screen in C. elegans"

Yucel, Duygu, Crossley, Merlin, Nicholas, Hannah

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

CtBP is a transcriptional co-repressor which plays roles in development and apoptosis. There are two highly related CtBP genes in vertebrates. Knock-out of both CtBP1 and CtBP2 results in lethality early in embryogenesis. Recently, we cloned a single CtBP in C. elegans. Like its mammalian counterparts, the C. elegans CtBP, called CTBP-1, functions as a transcriptional co-repressor and docks onto transcription factors containing an amino acid motif of the form PXDLS. We have analysed the expression pattern of CTBP-1 using a translational reporter construct (CTBP-1::GFP) which has shown that CTBP-1 is expressed in numerous cells, some of which have been identified as neurons. We are using various reporter markers to assess whether CTBP-1 is involved in the developmen t or specification of these neurons. One such marker showed that ctbp-1 loss of function mutant worms have fewer of one particular motor neuron cell type. In order to identify novel mutations that interfere with correct CTBP-1 activity, we carried out a mutagenesis screen. We treated the CTBP-1::GFP transgenic line with EMS and isolated worms which showed changes in the expression of the fusion protein. We have isolated six mutant lines, two of which show particularly interesting phenotypes. In one of the mutants named aus5, the expression of the CTBP-1::GFP fusion protein is dramatically decreased. In the other mutant line, aus3, the fusion protein is ectopically expressed. The aus5 mutant line may have a mutation in an activator of CTBP-1 such that CTBP-1 is switched off. In the aus3 mutant line, the mutation could be in a repressor of CTBP-1 allowing CTBP-1 to be switched on ectopically. Whole genome sequencing is in progress to identify the genes affected by these mutati ons which alter CTBP-1 activity. Taken together, our results hold the potential to unravel the roles of CTBP-1 in neuronal regulation and to reveal novel components of the gene regulatory networks in which CTBP-1 is involved.

Nicholas, Hannah R. et al. (2011) International Worm Meeting "Defining target genes of the transcriptional repressor protein CTBP-1."

Crossley, Merlin, Liu, Chu-Kong, Lun, Aaron, Tan, Melinda S-Y, Setiyaputra, Surya, Mackay, Joel, Kant, Sashi, Nicholas, Hannah R.

[International Worm Meeting, 2011]

Mammalian members of the C-terminal binding protein family of transcriptional repressors are recruited to promoters through interactions with DNA-bound transcription factors that contain amino acid motifs of the form PXDLS. Although similarly able to interact with PXDLS-containing transcription factors, we have found that the sole C. elegans member of this protein family, CTBP-1, also contains intrinsic DNA binding capacity in the form of a THAP domain. We have identified additional THAP domain-containing CtBPs in the nematode, echinoderm and cephalochordate lineages. The distribution of these lineages within the animal kingdom suggests that the ancestral form of the animal CtBPs may have contained a THAP domain that was subsequently lost in the vertebrate lineage. Since determining the structure of the THAP domain of CTBP-1 by nuclear magnetic resonance spectroscopy, we have used a variety of biophysical approaches to define the DNA contact surface of this domain and to assess the affinity of binding to an 11 bp consensus binding site derived from site selection experiments. Using the CisOrtho program1 we have identified promoters that contain putative CTBP-1-THAP binding sites, representing candidate CTBP-1 target genes. With reference to both our own and published2 microarray datasets comparing transcripts from wild type animals with those from ctbp-1 mutants, and to expression pattern data, we have defined a sub-set of these as likely in vivo targets of CTBP-1-mediated repression. Given the reported role of CTBP-1 in the regulation of lifespan and stress resistance2, and other investigations implicating CTBP-1 in aspects of neuronal development (D. Yucel, unpublished), our identification of CTBP-1 target genes will make an important contribution to understanding the function of this transcriptional regulator in a range of contexts. 1.Bigelow HR, Wenick AS, Wong A, Hobert O. 2004. BMC Bioinformatics 5: 27 2.Chen S, Whetstine JR, Ghosh S, Hanover JA, Gali RR, et al. 2009. Proc Natl Acad Sci U S A 106: 1496-501.

Lord , A. et al. (2019) International Worm Meeting "TXBP-3 regulates C. elegans cell adhesion and spermathecal contractility."

Cram, Erin, Zaidel-Bar, Ronen, Lord , A.

[International Worm Meeting, 2019]

Proteins containing PDZ domains function primarily as molecular scaffolds that recruit target proteins in order to promote cell signaling, cell adhesion, and protein scaffolding. TAX1BP3 is a highly conserved protein with a single atypical PDZ domain. Little is known about the C. elegans homolog, TXBP-3, although it has been shown to interact with the FERM family protein NFM-1/merlin. TXBP-3 is expressed in the pharynx, vulval precursor cells, spermatheca, and rectal epithelial cells of C. elegans. The current study investigates the role of TXBP-3 in the spermatheca as a model for understanding the coordination of contractility in a tube and to elucidate the function of TXBP-3 in the regulation of anchoring proteins, such as actin, to the cytoskeleton. TXBP-3 relocalizes from the apical to the basal surface of the spermatheca upon ovulation of oocytes into the spermatheca. Overexpression of TXBP-3 results in more rapid embryo transit times during the first three ovulations, which may be caused by a downstream effect on RHO-1, a major contributor to spermathecal contractility. Previous research indicates that TAX1BP3 is necessary for the redistribution of Rhotekin, a Rho GTPase effector that is conserved in C. elegans, which suggests a potential role of TXBP-3 in the regulation of this important pathway. We also demonstrate that CRISPR deletion of a coding region of TXBP-3 results in slower transit times and causes a "lace nose" phenotype, where the worm appears to have large vacuoles along both sides of the nose, possibly due to mis-localization of anchoring proteins. We aim to characterize the TXBP-3 gene and identify its interactors in order to better understand how it affects cytoskeletal organization and cell signaling.

Verena Goebel et al. (2001) International C. elegans Meeting "The cytoskeletal linker ERM is required for the integrity of epithelial luminal surfaces of internal organs in C. elegans"

John Fleming, Olaf Bossinger, Verena Goebel, Peter Barrett, David H Hall

[International C. elegans Meeting, 2001]

We are interested in the role of submembraneous cytoskeletal linker proteins of the protein 4.1 family in growth regulation and morphogenesis and have previously reported the identification of two C. elegans members of the ezrin-radixin-moesin-merlin family, erm and nfm (previously called nf2 ). The highly conserved C. elegans homolog of ezrin-radixin-moesin, erm , is predominantly expressed in apical epithelial membranes of the digestive tract, excretory system and somatic gonad. Its loss during embryonic development induces early larval lethality with extensive morphogenesis defects in the digestive and excretory organs. Death occurs at L1 subsequent to the appearance of cysts that were found to be diverticula of the lumina of the intestine and excretory system. The intestinal cysts can run the full length of the gut, and the excretory cysts can fill the head, pressing the pharynx or anterior intestine against the bodywall. Confocal analysis of the intestine using antibodies against proteins localized to adherens junctions, to the apical and baso-lateral membranes and to actin, indicate that the polarity of the epithelium is preserved, but that cell shapes can be grossly distorted by the cysts or by defects of the epithelial tube formation. Ultrastructural analysis of L1s by TEM demonstrates loss of microvilli in the intestinal lumen in the cystic region, but adherens junctions are still intact. Judging by TEM, posterior intestinal cells appear to be less well organized into an epithelial tube. By TEM, the excretory canals are found to be missing, shortened and/or to contain large cysts, and connections between the canal, duct and pore cells are often missing. ERM is also expressed within the somatic gonad in the spermatheca, the uterus and the vulval opening. Loss of erm during larval development induces severe defects in gonad morphogenesis. The uterine wall (and perhaps the spermatheca) may fail to form a proper epithelium or may fail during use, so that developing embryos and adult sperm leak into the body cavity. Thus all three organ systems show defects in luminal epithelia. We propose that erm is required for the integrity of luminal surfaces of internal organs in C. elegans .