Epigenetic licensing of germline gene expression by maternal RNA in C. elegans.

RNA can act as a regulator of gene expression with roles in transposon silencing, antiviral defense, and cell fate determination. Here, we show that in Caenorhabditis elegans a maternal transcript of the sex-determining gene fem-1 is required to license expression of a wild-type fem-1 allele in the zygotic germ line. Females homozygous for fem-1 deletions produce heterozygous offspring exhibiting germline feminization, reduced fem-1 activity, and transcript accumulation. Injection of fem-1 RNA incapable of encoding a protein into the maternal germ line rescues this defect in the progeny. The defect in zygotic fem-1 expression is heritable, suggesting that the gene is subject to epigenetic silencing that is prevented by maternal fem-1 transcripts. This mechanism may contribute to protecting the identity and integrity of the germ line.

A CUL-2 ubiquitin ligase containing three FEM proteins degrades TRA-1 to regulate C. elegans sex determination.

In Caenorhabditis elegans, the Gli-family transcription factor TRA-1 is the terminal effector of the sex-determination pathway. TRA-1 activity inhibits male development and allows female fates. Genetic studies have indicated that TRA-1 is negatively regulated by the fem-1, fem-2, and fem-3 genes. However, the mechanism of this regulation has not been understood. Here, we present data that TRA-1 is regulated by degradation mediated by a CUL-2-based ubiquitin ligase complex that contains FEM-1 as the substrate-recognition subunit, and FEM-2 and FEM-3 as cofactors. CUL-2 physically associates with both FEM-1 and TRA-1 in vivo, and cul-2 mutant males share feminization phenotypes with fem mutants. CUL-2 and the FEM proteins negatively regulate TRA-1 protein levels in C. elegans. When expressed in human cells, the FEM proteins interact with human CUL2 and induce the proteasome-dependent degradation of TRA-1. This work demonstrates that the terminal step in C. elegans sex determination is controlled by ubiquitin-mediated proteolysis.

The C. elegans sex-determining GLI protein TRA-1A is regulated by sex-specific proteolysis.

TRA-1A is the sole representative in Caenorhabditis elegans of the Gli transcription factor family. Its activity is required to specify all somatic female cell fates in XX hermaphrodites. We have found that TRA-1 protein levels are much higher in hermaphrodites than in males, and that the difference is attributable to the predominance in hermaphrodites of C-terminally truncated isoforms that are nearly undetectable in males. Our results support a model in which TRA-1A is negatively regulated by male-specific proteolysis that depends upon specific TRA-1A protein sequences and upon the activity of the fem genes. C-terminally truncated TRA-1 isoforms are stable and can inappropriately feminize XO males, suggesting that they escape this negative regulation. Thus, although C. elegans appears to lack a Hedgehog-signaling pathway, our results indicate that proteolytic processing and degradation of Gli family transcription factors, commonly seen during Hedgehog signaling in other organisms, also control C. elegans sex determination.

Gene CATCHR--gene cloning and tagging for Caenorhabditis elegans using yeast homologous recombination: a novel approach for the analysis of gene expression.

Expression patterns of gene products provide important insights into gene function. Reporter constructs are frequently used to analyze gene expression in Caenorhabditis elegans, but the sequence context of a given gene is inevitably altered in such constructs. As a result, these transgenes may lack regulatory elements required for proper gene expression. We developed Gene Catchr, a novel method of generating reporter constructs that exploits yeast homologous recombination (YHR) to subclone and tag worm genes while preserving their local sequence context. YHR facilitates the cloning of large genomic regions, allowing the isolation of regulatory sequences in promoters, introns, untranslated regions and flanking DNA. The endogenous regulatory context of a given gene is thus preserved, producing expression patterns that are as accurate as possible. Gene Catchr is flexible: any tag can be inserted at any position without introducing extra sequence. Each step is simple and can be adapted to process multiple genes in parallel. We show that expression patterns derived from Gene Catchr transgenes are consistent with previous reports and also describe novel expression data. Mutant rescue assays demonstrate that Gene Catchr-generated transgenes are functional. Our results validate the use of Gene Catchr as a valuable tool to study spatiotemporal gene expression.

TRA-1/GLI controls development of somatic gonadal precursors in C. elegans.

TRA-1/GLI is best known as a master regulator of sex determination in the nematode C. elegans, but its fly and vertebrate homologs (e.g. Ci, GLI) regulate embryonic patterning and cell proliferation. In this paper, we show that TRA-1/GLI controls development of the two somatic gonadal precursors (SGPs) in both XX and XO animals, in addition to its role in sex determination. Normally, SGPs reside at the poles of the gonadal primordium and divide according to intrinsic gonadal axes. In tra-1-null mutants, however, SGPs assume non-polar positions and the polarity of one SGP is reversed. Consistent with its SGP function, TRA-1 protein is present in SGPs during embryogenesis and early larval development. Previous studies have shown that the ehn-3 gene also affects SGP positions, and we report here that tra-1 and ehn-3 interact genetically. Whereas SGPs in tra-1 and ehn-3 single mutants are largely normal and generate many descendants, those in tra-1; ehn-3 double mutants do not mature or divide. Furthermore, tra-1 is a dominant enhancer of the ehn-3 gonadal defect, which includes the enhancement of a weak sexual transformation in the gonad. We cloned ehn-3, and found that it encodes a C2H2 zinc-finger protein. A rescuing EHN-3::GFP reporter is predominantly nuclear and expressed specifically in SGPs. The EHN-3 protein is therefore likely to regulate gene expression. We propose that TRA-1/GLI and EHN-3 have overlapping roles in regulation of multiple steps of SGP development. We speculate that regulation of SGP development may be an evolutionarily ancient role of TRA-1/GLI in nematode development.

Caenorhabditis elegans triple null mutant lacking UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I.

We have previously reported, from the nematode worm Caenor-habditis elegans, three genes (gly-12, gly-13 and gly-14) encoding enzymically active UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid, paucimannose and complex N-glycan synthesis. We now describe a worm with null mutations in all three GnT I genes, gly-14 (III);gly-12 gly-13 (X) (III and X refer to the chromosome number). The triple-knock-out (TKO) worms have a normal phenotype, although they do not express GnT I activity and do not synthesize 31 paucimannose, complex and fucosylated oligomannose N-glycans present in the wild-type worm. The TKO worm has increased amounts of non-fucosylated oligomannose N-glycan structures, a finding consistent with the site of GnT I action. Five fucosylated oligomannose N-glycan structures were observed in TKO, but not wild-type, worms, indicating the presence of unusual GnT I-independent fucosyltransferases. It is concluded that wild-type C. elegans makes a large number of GnT I-dependent N-glycans that are not essential for normal worm development under laboratory conditions. The TKO worm may be more susceptible to mutations in other genes, thereby providing an approach for the identification of genes that interact with GnT I.

Isolation of null alleles of the Caenorhabditis elegans gly-12, gly-13 and gly-14 genes, all of which encode UDP-GlcNAc: alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I activity.

UDP-GlcNAc: alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (GnT I) is a Golgi-resident enzyme which transfers a GlcNAc residue in beta1,2 linkage to the Manalpha1,3Manbeta-terminus of (Manalpha1,6(Manalpha1,3)Manalpha1,6)(Manalpha1,3)Manbeta1,4GlcNAcbeta1,4GlcNAc-Asn-protein, thereby initiating the synthesis of hybrid N-glycans. Three Caenorhabditis elegans genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) have been cloned. All three cDNAs encode proteins with GnT I enzyme activity. We report in this paper the preparation by ultra-violet (UV) light irradiation in the presence of trimethylpsoralen, of mutants lacking either gly-12, gly-13 or gly-14. A double null mutation in the gly-12 and gly-14 genes (gly-14; gly-12) has also been prepared. These mutations are intragene deletions, removing large portions of the GnT I catalytic domain, and are therefore, all molecular nulls. The gly-12 and gly-14 mutants as well as the gly-14; gly-12 double mutant all displayed wild-type phenotypes, indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. In contrast, about 60% of the mutants lacking the gly-13 gene arrested as L1 larvae at 20 degrees C and the remaining 40% homozygous worms grew to adulthood but displayed severe morphological and behavioral defects despite the presence of the other two GnT I genes, gly-12 and gly-14. Attempts to rescue the gly-13 null phenotype with the wild type transgene were not successful. However, lethality co-segregated with the gly-13 deletion within 0.02 map units (mu) in genetic mapping experiments, suggesting that the gly-13 mutation is responsible for the phenotype.

Synthesis of paucimannose N-glycans by Caenorhabditis elegans requires prior actions of UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I, alpha3,6-mannosidase II and a specific membrane-bound beta-N-acetylglucosaminidase.

We have previously reported three Caenorhabditis elegans genes ( gly-12, gly-13 and gly-14 ) encoding UDP- N -acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2- N -acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid and complex N-glycan synthesis. GLY-13 was shown to be the major GnT I in worms and to be the only GnT I cloned to date which can act on [Manalpha1,6(Manalpha1,3)Manalpha1,6](Manalpha1,3)Manbeta1, 4GlcNAcbeta1,4GlcNAc-R, but not on Manalpha1,6(Manalpha1,3)Manbeta1- O -R substrates. We now report the kinetic constants, bivalent-metal-ion requirements, and optimal pH, temperature and Mn(2+) concentration for this unusual enzyme. C. elegans glycoproteins are rich in oligomannose (Man(6-9)GlcNAc(2)) and 'paucimannose' Man(3-5)GlcNAc(2)(+/-Fuc) N-glycans, but contain only small amounts of complex and hybrid N-glycans. We show that the synthesis of paucimannose Man(3)GlcNAc(2) requires the prior actions of GnT I, alpha3,6-mannosidase II and a membrane-bound beta- N -acetylglucosaminidase similar to an enzyme previously reported in insects. The beta- N -acetylglucosaminidase removes terminal N -acetyl-D-glucosamine from the GlcNAcbeta1, 2Manalpha1,3Manbeta- arm of Manalpha1,6(GlcNAcbeta1,2Manalpha1,3) Manbeta1,4GlcNAcbeta1,4GlcNAc-R to produce paucimannose Man(3)GlcNAc(2) N-glycan. N -acetyl-D-glucosamine removal was inhibited by two N -acetylglucosaminidase inhibitors. Terminal GlcNAc was not released from [Manalpha1,6(Manalpha1,3)Manalpha 1,6] (GlcNAcbeta1,2Manalpha1,3)Manbeta1,4GlcNAcbeta1,4GlcNAc-R nor from the GlcNAcbeta1,2Manalpha1,6Manbeta- arm. These findings indicate that GLY-13 plays an important role in the synthesis of N-glycans by C. elegans and that therefore the worm should prove to be a suitable model for the study of the role of GnT I in nematode development.

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II in Caenorhabditis elegans.

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II) are key enzymes in the synthesis of Asn-linked hybrid and complex glycans. We have cloned cDNAs from Caenorhabditis elegans for three genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) and one gene homologous to mammalian GnT II. All four cDNAs encode proteins which have the domain structure typical of previously cloned Golgi-type glycosyltransferases and show enzymatic activity (GnT I and GnT II, respectively) on expression in transgenic worms. We have isolated worm mutants lacking the three GnT I genes by the method of ultraviolet irradiation in the presence of trimethylpsoralen (TMP); null mutants for GnT II have not yet been obtained. The gly-12 and gly-14 mutants as well as the gly-14;gly-12 double mutant displayed wild-type phenotypes indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. This finding and other data indicate that the GLY-13 protein is the major functional GnT I in C. elegans. The mutation lacking the gly-13 gene is partially lethal and the few survivors display severe morphological and behavioral defects. We have shown that the observed phenotype co-segregates with the gly-13 deletion in genetic mapping experiments although a second mutation near the gly-13 gene cannot as yet be ruled out. Our data indicate that complex and hybrid N-glycans may play critical roles in the morphogenesis of C. elegans, as they have been shown to do in mice and men.

Functional post-translational proteomics approach to study the role of N-glycans in the development of Caenorhabditis elegans.

Glycosylation is one of the most common post-translational protein modifications. Carbohydrate-mediated interactions between cells and their environment are important in differentiation, embryogenesis, inflammation, cancer and metastasis and other processes. Humans and mice with mutations that prevent normal N-glycosylation show multi-systemic defects in embryogenesis, thereby proving that these molecules are essential for normal development; however, a large number of proteins undergo defective glycosylation in these human and mouse mutants, and it is therefore difficult to determine the precise molecular roles of specific N-glycans on individual proteins. We describe here a 'functional post-translational proteomics' approach that is designed to determine the role of N-glycans on individual glycoproteins in the development of Caenorhabditis elegans.