Choi, S. et al. (2017) International Worm Meeting "Real time observation of DNA unwinding mode of Caenorhabditis elegans HIM-6 helicase."

Cho, H., Kim, H., Ahn, B., Choi, S.

[International Worm Meeting, 2017]

Bloom's syndrome(BS) is caused by mutations in the BLM gene encoding BLM helicase. BLM helicase contains conserved RecQ helicase domain which includes 3'-5' helicase activity. BLM unwinds various DNA helicase substrates including replication forks. Cells from BS patients, that lack the BLM, show defects in the response to replicative stress and contain a multitude of chromosomal aberrations. A BLM homolog, him-6, has been identified in C. elegans based on its close homology with human BLM. Here, we used single-molecule FRET to measure DNA unwinding in real time. Our results showed that the HIM-6 repetitively unwound a long forkd DNA depending on ATP concentrations and resolved flap and D-loop DNA, all of which are intermediates in DNA repair. Also, we found that HIM-6 unwound at most 25 base pairs of duplex DNA in a speed of 28 nucleotides per second before moving back. Besides, repetitive unwinding mode of HIM-6 was changed to unidirectional unwinding in the presence of RPA, indicating that its unwinding mode can be modulated by proteins in vivo. This study can be used to search proteins which would modulated HIM-6 unwinding mode on different DNA structures, such as Holliday junction and G4-quadruplex.

Melissa M Grabowski et al. (2005) International Worm Meeting "Mutation in the C. elegans him-6 Blooms Ortholog Causes a Mutator Phenotype"

Nenad Svrzikapa, Melissa M Grabowski, Heidi A Tissenbaum

[International Worm Meeting, 2005]

Individuals with Bloom's Syndrome display a high incidence of cancer caused by genomic instability. Bloom Syndrome (BS) is caused by mutation in a member of the RecQ helicase family, the Bloom helicase (BLM). We use C. elegans to elucidate the in vivo functions of BLM in maintaining genomic stability. We find him-6 mutants show a mutator phenotype that specifically results in an increased amount of insertions and deletions. This leads to the appearance of random visible phenotypes in a mutant him-6 population. We also observe additional germ line phenotypes in him-6 mutants including an increased level of germ line apoptosis and a mortal germ line. Finally, the increased genomic instability in the him-6 mutants leads to a shortened life span. Our data suggests a model where loss of HIM-6 causes genomic instability either due to an increased number of DNA lesions, an inability to repair these lesions, or low fidelity recombination events.

Glenwinkel, Lori et al. (2013) International Worm Meeting "CisOrtho V 2.0: a comparative genomic approach to genome wide identification of transcription factor target genes."

Glenwinkel, Lori, Hobert, Oliver

[International Worm Meeting, 2013]

CisOrtho V 2.0 is able to predict novel regulatory target genes using an experimentally derived PSSM and species conservation among five nematode genomes. CisOrtho ranks transcription factor (TF) binding sites (BS) by the PSSM log-odds score, BS enrichment, and inter-species preservation of BSs in orthologous regions. I have validated CisOrtho's predictive power using a bioinformatics approach and in vivo validation of novel regulatory targets. Three sets of regulatory target genes for the cholinergic, AIY, and ASE neuron terminal selector TFs (UNC-3,TTX-3/CEH-10,CHE-1) were compared to a control set of 1000 random genes or 93 collagen genes that we expect to exert their expression independently of these three TFs. Each criterion that CisOrtho uses to rank potential TF target genes was calculated for each orthologous region between 5 nematode species in upstream, intronic, exonic, and downstream regions. Known TF target genes ranked significantly better for all criteria in upstream promoter regions while intronic, exonic, downstream, and total gene ranking criteria were not always significantly different. For in vivo validation of CisOrtho, six highly ranked potential UNC-3 target genes were chosen. Four of these genes are completely uncharacterized while two have published expression patterns in the ventral nerve cord where UNC-3 exerts its regulation as a terminal selector of cholinergic motor neurons. GFP promoter fusions were made for four of the genes that are previously uncharacterized in the literature. Each reporter was injected into wild type C. elegans then crossed into an UNC-3 mutant, unc-3(e151). The four uncharacterized genes showed ventral nerve cord neuron expression and all 6 genes had significant loss of ventral nerve cord neuron expression in the UNC-3 mutant suggesting UNC-3 dependence. CisOrtho predicted target genes are also enriched in neurogenesis pathway genes for all three terminal selector genes providing ample candidates for further in vivo experimentation. These lines of evidence suggest that CisOrtho is able to successfully predict novel targets of TFs.

Passannante M et al. (2000) European Worm Meeting "A meiotic function for the Caenorhabditis elegans homolog of the human Blooms syndrome gene"

Rose AM, Mueller F, Wicky C, Passannante M

[European Worm Meeting, 2000]

The him-6 gene is required for meiotic recombination and consequently for the proper segregation of all chromosomes in Caenorhabditis elegans ( Hodgkin et al., Genetics 91, 67-94, 1979). him-6 encodes a putative recQ type of DNA helicase which shows strong homology to the human BLM (Bloom's Syndrome) gene product. It is also related to the E. coli RecQ helicase, the S. pombe rqh1+ gene and the S. cervisiae Sgs1 gene. Blooms syndrome (BS) is a rare autosomal recessive disorder characterized by genomic instability. This phenotype is not confined to mitotic cells. Men with BS are sterile and women are subfertile. Furthermore, the BLM protein has been shown to colocalize with RAD51/DMC1 during meiotic prophase (Moens et al., J. Cell Sci. 113, 663-672, 2000). Sequencing the mutant alleles of him-6 revealed that e1423 contains a T to A transition, which gives rise to a stop codon in the helicase domain. The mild allele e1104 shows a G to Q substitution at a conserved position in the helicase domain. Cytological observations of both him-6(e1423) and him-6(e1104) homozygotes revealed a high frequency of univalents in oocytes at diakinesis, suggesting that the chromosomes fail to undergo crossing-over or pairing. However, at pachytene stage, where they are fully paired in wild type animals, no visible defects were observed in him-6 mutants. Immunostaining with an antibody raised against the C-terminal part of the protein localized HIM-6 to the germ line nuclei of both hermaphrodite and male. The protein is present in mitotically dividing nuclei and in all subsequent stages of meiotic prophase. In addition, the protein was also detected in all nuclei of the early embryo. The strong meiotic phenotype suggests a role for the HIM-6 protein in the recombination pathway, but it might not be its sole function. We are currently investigating possible interactions of HIM-6 with other proteins.

Manoj Nanji et al. (2002) European Worm Meeting "Dissecting the complexities of C. elegans DAF-2 insulin/IGF like receptor signalling pathway"

David Gems, Manoj Nanji

[European Worm Meeting, 2002]

An insulin/IGF-like signalling pathway in C. elegans plays a role in early development, the dauer/non-dauer decision, fertility and life span (1). Components of this pathway identified by genetic analysis include DAF-2, a homologue of the vertebrate insulin and IGF-I receptors, AGE- 1, a subunit of phosphoinositide 3-kinase (PI3K), DAF-18 (PTEN) phosphatase, PDK- 1(phosphoinositide-dependent kinase), AKT-1 and AKT-2 (protein kinase Bs) and DAF-16, a forkhead transcription factor. Mutations in daf-2 or in age-1 cause dauer larva arrest, and can double adult life (2-4). Although this signal transduction pathway between daf-2 and daf-16 has been well defined, several findings suggest the existence of a second daf-2-daf-16 pathway. For example, phenotypic analysis of 15 daf-2 mutants, which fall into two distinct classes, 1 and 2, suggested that the gene has two functional components, daf-2A and daf-2B (4). These potentially correspond to different signalling pathways emanating from daf-2 into the cell. In addition, a gain-of-function allele of akt-1(5), and the hypomorphic allele daf-18(e1375), fully suppress the dauer constitutive (Daf-c) phenotype of several severe age-1 alleles yet only weakly suppress the relatively weak allele, daf-2(e1370). To investigate and characterise the putative second daf-2 pathway, we have carried out interaction studies between several class 1 and class 2 daf-2 alleles and various components of the PI3K and other signalling pathways. Results of these interaction studies together with a revised model of daf-2 signalling will be presented.

Manoj Nanji et al. (2001) International C. elegans Meeting "daf-2 signalling into the cell: looking for the second pathway"

Manoj Nanji, David Gems

[International C. elegans Meeting, 2001]

The daf-2/age-1/daf-16 pathway regulates numerous aspects of life history including early development, the dauer/non-dauer decision, fertility and life span (1). Mutations in daf-2 , a homologue of vertebrate insulin and IGF-I receptors, or in age-1 , a subunit of phosphoinositide 3-kinase (PI3K), cause dauer larva arrest, and can double adult life (2-4). Other components of the pathway include daf-18 (PTEN phosphatase), pdk-1 (phosphoinositide-dependent kinase), and akt-1 and akt-2 (protein kinase Bs). The latter inactivate DAF-16, a forkhead transcription factor, probably by phosphorylation. Although this signal transduction pathway between daf-2 and daf-16 has been well defined, several findings suggest the existence of a second daf-2 - daf-16 pathway. For example, phenotypic analysis of 15 daf-2 mutants, which fall into two distinct classes, suggested that the gene has two functional components, daf-2A and daf-2B (4). These potentially correspond to different signalling pathways emanating from daf-2 into the cell; daf-2B is likely to correspond to the PI3K pathway (4). In addition, a gain-of-function allele of akt-1 (5), and the hypomorphic allele daf-18(e1375) , fully suppress the dauer constitutive (Daf-c) phenotype of several severe age-1 alleles yet only weakly suppress the relatively weak allele, daf-2(e1370). To investigate and characterise the putative second daf-2 pathway, we are carrying out interaction studies between several class 1 and class 2 daf-2 alleles and various components of the PI3K and other signalling pathways. Preliminary results suggest a possible identity for the daf-2A pathway. We will present the results of these interactions studies. (1) Guarente, L., Kenyon, C. Nature 408: 255 (2000). (2) Kimura, K.D. et al. Science 277: 942 (1997). (3) Morris, J.Z. et al . Nature 382: 536 (1996). (4) Gems, D. et al ., Genetics 150: 129 (1998). (5) Paradis, S. & Ruvkun, G. Genes Dev . 12: 2488 (1998).

Heestand, Bree et al. (2021) International Worm Meeting "Adult longevity of late-generation Piwi/prg-1 mutants"

Lister-Shimauchi, Evan, Heestand, Bree, Ahmed, Shawn, Kennedy, Sophia, Simon, Matt

[International Worm Meeting, 2021]

The C. elegans Piwi Argonaute protein PRG-1 associated with piRNAs to preventing germ cells from expressing foreign genetic elements such as transposons. PRG-1 promotes germ cell immortality and lines of prg-1 mutants are initially fertile but become sterile if they are grown for many generations. prg-1 mutant sterility is a form of reproductive arrest (1), which may resemble reproductive arrest in response to environmental stresses such as starvation (2). We found that early-generation prg-1 mutants have normal lifespans and fertility, while later generations had reduced fertility yet longer lifespans. Mutation of the stress response transcription factor Daf-16/FOXO, caused the longevity phenotype of late-generation prg-1 mutants to disappear, leading to the conclusion that prg-1 mutant longevity may be a hormetic stress response. Phenotypes that arise from epigenetic defects can be inherited for at least 3 generations (3,4), and we found that longevity of late-generation prg-1 mutants is inherited by F1 but not F2 cross progeny. We are currently asking if the daf-12 germline signaling pathway that regulates aging is required of prg-1 mutant longevity. Overall, our results imply that the longevity of late-generation prg-1 mutant is distinct from the longevity that is transmitted for several generations by germ cells of mutants deficient for the ASH-2/SET-2/trithorax transcriptional activation complex (3,4). 1. Heestand, B., Simon, M., Frenk, S., Titov, D. & Ahmed, S. Transgenerational Sterility of Piwi Mutants Represents a Dynamic Form of Adult Reproductive Diapause. Cell Rep. 23, 156-171 (2018). 2. Angelo, G. & Van Gilst, M. R. Starvation Protects Germline Stem Cells and Extends Reproductive Longevity in C. elegans. Science 326, 954-958 (2009). 3. Greer, E. L. et al. Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans. Nature 479, 365-371 (2011). 4. Lee TW, David HS, Engstrom AK, Carpenter BS, Katz DJ. Repressive H3K9me2 protects lifespan against the transgenerational burden of COMPASS activity in C. elegans. Elife (2019). doi: 10.7554/eLife.48498

Chantal Wicky et al. (2001) International C. elegans Meeting "The C.elegans homolog of the human Bloom's syndrome gene is required for meiotic recombination and genome integrity"

Myriam Passannante, Fritz Mueller, Ann Rose, Chantal Wicky

[International C. elegans Meeting, 2001]

The him-6 gene is required for meiotic recombination and genome stability during proliferation of premeiotic cells in Caenorhabditis elegans . him-6 encodes a putative recQ type of DNA helicase which is homologous to the human BLM (Bloom's Syndrome) gene product. It is also related to the E. coli RecQ helicase, the S. pombe rqh1+ gene and the S. cervisiae Sgs1 gene. Bloom’s syndrome (BS) is a rare autosomal recessive disorder characterized by genomic instability. The BLM protein is thought to function as antirecombinase in induced recombination resulting, e.g., from blocked replication. The him-6 gene is predominantly expressed in the germ line. Mutations in him-6 result in a decrease in meiotic recombination. Oocyte chromosomes of both him-6(e1423) and him-6(e1104) homozygotes lack chiasmata, suggesting that the chromosomes fail to undergo crossing-over or pairing. However, at pachytene stage, where they are fully paired in wild type animals, no visible defects were observed in him-6 mutants. Unlike BLM - , however, him-6 mutations cause no apparent somatic phenotypes. Double mutants for him-6 and top-3 (the gene encoding topoiosomerase III a ) exhibits severe defects in the proliferation of the germ line nuclei, a phenotype which is not observed in top-3 single mutants. The number of nuclei in the gonad is greatly reduced and there are some evidences of chromosomal damage. These nuclei do not appear to progress through meiosis and the chromosomal defects are not suppressed by a mutation in the spo-11 gene, which is essential for the initiation of meiotic recombination. Altogether, this suggests a profound defect in the ability of him-6 top-3 double mutants to maintain genome integrity during the stage of germ line proliferation. We propose that HIM-6 has at least two distinct functions. First, it promotes normal levels of meiotic recombination, a novel function for eukaryotic BLM proteins, and second, together with topoisomerase III a , it has DNA repair activity during the proliferation of premeiotic cells. We are currently investigating the possibility that HIM-6 participates in a large repair complex, together with other factors such as, e.g., MRE-11.

Chin-Sang ID et al. (1998) West Coast Worm Meeting "The role of the VAB-2 Ephrin in C. elegans development"

Ding M, Chisholm AD, Chin-Sang ID

[West Coast Worm Meeting, 1998]

Mutations in the genes vab-1 and vab-2 cause similar defects in epidermal morphogenesis. Mutations in both genes cause variable defects in head epidermal morphogenesis (the Notched head phenotype) and in ventral closure during embryogenesis. vab-1 encodes an Eph receptor tyrosine kinase that is expressed in the developing nervous system, and which is required in neural cells for proper epidermal morphogenesis (George et al., Cell 92: 633, 1998). Eph receptors are the largest subfamily of vertebrate receptor protein-tyrosine kinases (RPTKs) and have been implicated in a variety of processes, including embryonic pattern formation and axonal guidance. Mutations affecting the kinase domain of VAB-1 cause a weak mutant phenotype, suggesting VAB-1 may have both kinase-dependent and kinase-independent signaling functions, possibly corresponding to the forward and reverse signaling functions described for some vertebrate Eph receptors. We have found that vab-2 encodes a C. elegans member of the Ephrin family of ligands for Eph receptors, previously only identified in vertebrates (1997 International Worm Meeting Abstracts, p. 386). Vertebrate Ephrins are either transmembrane proteins (Ephrin-Bs) or glycosylphosphatidylinositol (GPI)-linked proteins (Ephrin-As). The predicted VAB-2 protein contains a potential GPI attachment signal sequence, but is more similar to transmembrane Ephrins in its receptor binding domain. The molecular lesions of vab-2 alleles suggest they cause partial or complete loss of function. The VAB-2 Ephrin is thus a candidate ligand for the VAB-1 Eph receptor. We are taking three approaches to address whether VAB-2 is a ligand for VAB-1. First, we are using cell culture experiments to determine if VAB-1 and VAB-2 proteins bind in vitro. Second, we are determining whether the expression pattern of VAB-2 in worms is consistent with its function as a ligand for VAB-1. A VAB-2::GFP reporter construct is not expressed in epidermal cells during ventral enclosure but in many neuronal cells. VAB-2/VAB-1 signaling might therefore operate between neuronal cells. Finally, we are using genetic analysis to address whether vab-2 functions in a vab-1 pathway. Strong vab-2 alleles, which result in nonsense mutations in the protein, cause phenotypes much weaker than those of vab-1 null mutations, and cause some phenotypes not observed in vab-1 backgrounds. VAB-2 might therefore participate in a subset of the functions of VAB-1, or it might be redundant with additional ligands. We are examining the phenotypes of vab-1; vab-2 double mutant strains to address whether vab-1 and vab-2 function in the same pathway.