Santiago-Martinez, Dianaliz et al. (2011) International Worm Meeting "Exploring the Function of TRF-1 in Polycystin-Expressing Sensory Neurons."

Barr, Maureen M., Hodgkin, Jonathan, Santiago-Martinez, Dianaliz, Gravato-Nobre, Maria

[International Worm Meeting, 2011]

The Tumor Necrosis Factor (TNF) receptor-associated factors (TRAFs) are adaptor/scaffold molecules that interact with members of the interleukin-receptor (ILRs) or TNFR families and function mainly in the immune system. TNF-alpha signaling promotes autosomal dominant polycystic kidney disease (ADPKD). In mammals, there are six TRAFs (1-6) that share a conserved C-terminal TRAF domain. The C. elegans genome does not encode a TNF Receptor but does encode a sole TRAF gene, trf-1. In humans, mutations in the polycystin-1 (PC-1) or polycystin-2 (PC-2) ciliary mechanosensory complex cause ADPKD. In C. elegans, the polycystins LOV-1 and PKD-2 localize on the ciliary membrane and are required for male sensory behaviors. Hence, the connection between the polycystins and cilia seems to be an ancient one. We find that trf-1 is co-expressed with pkd-2 in the male specific CEM, RnB, and HOB neurons and is required for male mating behaviors. We are interested in exploring a potential connection between immune recognition and mate recognition. To this end, we examined the mating behavior of mutant males defective in the Toll pathway including tol-1 and ikb-1 (I kappa beta homolog). We performed four mating behavioral (leaving, retention, response, and vulva location). Like pkd-2 mutants, trf-1(nr2014) mutant males have response and Lov defects. In contrast, tol-1;him-5 and ikb-1;him-5 are not statistically different from the wild-type him-5. trf-1;him-5 and pkd-2;him-5 males are leaving assay (Las) defective, whereas tol-1;him-5 and ikb-1;him-5 are not Las. All mutant strains seem to be normal for the retention assay, indicating that these males can sense the presence of a mate. We conclude that trf-1 acts like pkd-2, suggesting the two function in a similar pathway. Genetic interactions between trf-1 and components of the C. elegans PKD pathway (lov-1, pkd-2, klp-6, and the cwp genes) are being examined. To ascertain the site of TRF-1 action, we generated transgenic animals expressing TRF-1::GFP. Although dimly expressed, TRF-1::GFP localizes to cell bodies of CEM and HOB neurons, and in rays 3, 8 and 9, TRF-1 localizes to cell bodies and ray dendrites. TRF-1::GFP is not detectable in sensory cilia. We are currently determining whether trf-1 is required for PKD-2 localization or abundance. We are also testing the hypothesis that TRF-1 physically associates with the intracellular domains of the membrane proteins LOV-1, PKD-2, and CWP-5, or with the kinesin-3 motor protein KLP-6.

Santiago-Martinez D et al. (2010) Biology of the C. elegans Male, Madison, WI "Exploring the Function of TRF-1 in Polycystin-Expressing Sensory Neurons"

Santiago-Martinez D, Barr M M, Hodgkin J, Gravato-Nobre M

[Biology of the C. elegans Male, Madison, WI, 2010]

The Tumor Necrosis Factor (TNF) receptor-associated factors (TRAFs) are adaptor/scaffold molecules that interact with members of the interleukin-receptor (ILRs) or TNFR families and function mainly in the immune system. TNF-alpha signaling promotes autosomal dominant polycystic kidney disease (ADPKD). In mammals, there are six TRAFs (1-6) that share a conserved C-terminal TRAF domain. The C. elegans genome does not encode a TNF Receptor but does encode a sole TRAF gene, trf-1. In humans, mutations in the polycystin-1 (PC-1) or polycystin-2 (PC-2) ciliary mechanosensory complex cause ADPKD. In C. elegans, the polycystins LOV-1 and PKD-2 localize on the ciliary membrane and are required for male sensory behaviors. Hence, the connection between the polycystins and cilia seems to be an ancient one. We find that trf-1 is co-expressed with pkd-2 in the male specific CEM, RnB, and HOB neurons and is required for male mating behaviors. We are interested in exploring a potential connection between immune recognition and mate recognition. To this end, we examined the mating behavior and mating efficiency of mutant males defective in the Toll pathway including tol-1 and ikb-1 (I kappa beta homolog). We performed four mating behavioral (leaving, retention, response, and vulva location) and mating efficiency assays. Like pkd-2 mutants, trf-1(nr2014) mutant males have response and Lov defects. In contrast, tol-1;him-5 and ikb-1;him-5 are not statistically different from the wild-type him-5. In the mating efficiency assay, trf-1;him-5 and pkd-2;him-5 scored low and are not statistically different, whereas tol-1 and ikb-1 appear wild type. trf-1;him-5 and pkd-2;him-5 males are leaving assay (Las) defective, whereas tol-1 and ikb-1 are not Las. All mutant strains seem to be normal for the retention assay, indicating that these males can sense the presence of a mate. We conclude that trf-1 acts like pkd-2, suggesting the two function in a similar pathway. Genetic interactions between trf-1 and components of the C. elegans PKD pathway (lov-1, pkd-2, klp-6, and the cwp genes) are being examined. To ascertain the site of TRF-1 action, we are generating transgenic animals expressing a functional TRF-1::GFP. We will determine whether TRF-1 is required for the localization, stability, or signaling properties of or physically associates with polycystin pathway components.

BeomSeok Seo et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Dissecting Alternative Lengthening of Telomeres in Caenorhabditis elegans"

BeomSeok Seo, Junho Lee

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Eukaryotes have linear chromosomes which causes ''the end-replication problem''. This is solved by telomerase which adds telomeric repeats to the end of the chromosomes. However, it was found that telomeres can be maintained without telomerase in yeast and cancer cells. This is called Alternative Lengthening of Telomere(ALT), which is presumed to be based on a process involving recombination. In Caenorhabditis elegans, it is known that trt-1 telomerase deficient mutant become sterile after several generations. We mutagenized trt-1 mutant worms and screened suppressor mutants which survived without telomerase. The Terminal Restriction Fragments (TRF) assay revealed that telomere length was lengthened and heterogeneous. Currently we are mapping suppressor mutants with single nucleotide polymorphism(SNP).

Crocker, Olivia et al. (2021) International Worm Meeting "Interrogating the role of paternally contributed tRNA fragments in C. elegans fertilization and development"

Conine, Colin, Crocker, Olivia

[International Worm Meeting, 2021]

Across organisms, males experiencing environmental stressors contribute non-genetic information to future generations. While sperm chromatin retains a small percentage of histones, the overwhelming exchange of histones for protamines in sperm paired with the erasure of DNA methylation after fertilization challenges the traditional epigenetic inheritance paradigms as mechanisms of intergenerational transmission. Prior work has demonstrated that modulating the diet of mammals has effects on the metabolism of offspring by altering the abundance of small RNAs in sperm, particularly the levels of highly abundant ~28-34 nt tRNA fragments (tRFs). After fertilization, the altered abundance of tRFs correlates with modified embryonic gene expression and development. While the potential role of tRFs as a carrier of epigenetic information in sperm is exciting, little is known about the molecular functions of tRFs. In mammals there are greater than 10 RnaseA and RnaseT2 enzymes with the potential to generate tRFs. Thus, redundancy of murine endonucleases makes it difficult to establish a tractable genetic model of tRF function. Recently, we have identified abundant tRFs in C. elegans males which are specifically enriched in sperm. Fortunately, the C. elegans genome encodes only one RnaseT2 (rnst-2) and no RnaseA homologs, allowing for simple genetic manipulation of tRF abundance. We have generated several loss-of-function and catalytically dead alleles of rnst-2 which all exhibit developmental and male fertility defects. Additionally, by endogenously tagging rnst-2 we have found that it is specifically expressed in sperm and in the developing embryo. Finally, rnst-2 regulates the levels of tRFs in male worms, establishing a model to determine if tRFs in sperm can transmit epigenetic information to progeny in C. elegans. To support these studies, we have developed a method for single embryo RNA-Sequencing in worms, permitting the quantitation of gene expression changes across the transcriptome from 2- and 8-cell embryos to determine the first molecular effects of modulating the levels of tRFs in sperm and early development.

Seo B et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Telomere lengthening in suppressor mutants of telomerase deficient Caenorhabditis elegans."

Kim C, Lee J, Seo B

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Eukaryotic cells have linear chromosomes, which cause 'the end-replication problem'. Telomerase , a reverse transcriptase, can solve this problem by adding telomeric repeats to the end of the chromosomes. However, it was also found that telomeres can be maintained without telomerase in yeast and some cancer cells by Alternative Lengthening of Telomere (ALT) mechanism, which is based on a process involving recombination or replication. In Caenorhabditis elegans, the telomerase deficient mutant strain becomes sterile (mortal germline phenotype) after several generations as a result of shortening of telomeres. We mutagenized telomerase mutant animals and isolated suppressor mutants which acquired immortality of germ cells. With Terminal Restriction Fragments(TRF) assay and Fluorescence In Situ Hybridization(FISH), we found that the telomeres of the suppressor mutants were lengthened and had distinct property from wild type telomeres. Currently, we are mapping suppressor mutants by Single Nucleotide Polymorphisms(SNPs) and whole genome sequencing technology.

Soon Baek Hwang et al. (2002) West Coast Worm Meeting "TEP-1, a C. elegans telomere binding protein, is required for chromosome stability"

Junho Lee, In Kwon Chung, Soon Baek Hwang, Seung Hyun Kim

[West Coast Worm Meeting, 2002]

We identified by yeast one-hybrid screening a Caenorhabditis elegans gene encoding a homeobox-containing protein that specifically binds double-stranded telomeres. We found that TEP-1 localizes to telomeres in vivo. Modeling of the TEP-1 homeodomain revealed that its three-dimensional structure has a strong similarity to that of hTRF1 and Taz1p, human and Schizosaccharomyces pombe telomere binding proteins, respectively, although TEP-1 does not share amino acid sequence similarity with these proteins. Like hTRF1, TEP-1 bends DNA by binding telomeric sequences. Distinct functions of the TEP-1 domains were identified: the N-terminal domain and the homeobox domain function in telomere binding, and the C terminal region functions in DNA bending and loop formation. Two dimensional gel electrophoresis and Southern analysis of telomeric DNA showed that, like TRF2, TEP-1 is required for loop formation at telomeric regions in vivo. Overexpression of TEP-1 enhances loop formation at the ends of telomeres, while a C terminal deletion mutant or a dominant negative mutant cannot form loops. We also show that tep-1 genetically interacts with mrt-2 and cep-1, which encode the telomere checkpoint protein and the C. elegans p53 homolog, respectively, indicating that tep-1 acts with these genes to maintain chromosome stability. We propose that TEP-1 is a functional homolog of the mammalian TRF proteins and that it acts as a telomere protecting protein in maintaining chromosome stability.

Kim, Chuna et al. (2011) International Worm Meeting "Telomere lengthening in suppressor mutants of telomerase deficient Caenorhabditis elegans."

Seo, Beomseok, Lee, Junho, Kim, Chuna

[International Worm Meeting, 2011]

Eukaryotic cells have linear chromosomes which cause 'the end-replication problem'. With telomeres, the chromosomal ends can be protected from degradation or fusion with another chromosome. Telomeres of somatic cells are gradually shortened with repeated replication. However, telomeres of cancer cells and germ cells are not shortened because they can maintain telomere length by the telomerase, which is a special kind of reverse transcriptase. However, it was also found that telomeres can be maintained without telomerase in yeast and some cancer cells by the Alternative Lengthening of Telomere (ALT) mechanism, which is based on a process involving recombination. But, until now, the mechanism that may suppress ALT in telomerase-negative cells have not been known. Therefore, we performed forward and reverse genetic screenings for finding ALT suppressor. In Caenorhabditis elegans it was known that the trt-1(ok410) mutant strain, which lacks the telomerase catalytic subunit, becomes sterile (mortal germline phenotype) after several generations as a result of shortening of telomeres. We mutagenized trt-1(ok410) mutant animals and isolated suppressor mutants that had re-acquired immortality of germ cells. With Terminal Restriction Fragments(TRF) assay, Single Telomere Length Analysis(STELA) and Fluorescence In Situ Hybridization(FISH), we found that the telomeres of the suppressor mutants were lengthened and had distinct property from normal telomeres. Currently, we are mapping these suppressor mutants by Single Nucleotide Polymorphisms(SNPs) and whole genome sequencing technology.

Iris Cheung et al. (2004) West Coast Worm Meeting "Telomere length measurement in C. elegans by Single Telomere Length Analysis"

Duncan M Baird, Mike Schertzer, Iris Cheung, Agnes Baross, Ann M Rose, Peter M Lansdorp

[West Coast Worm Meeting, 2004]

Telomeres are structural ends of eukaryotic chromosomes and are composed of telomeric repeats and associated proteins. One of the functions of telomeres is to prevent chromosome ends from being recognized as double-stranded breaks and to protect them from fusion and degradation. Telomere length affects telomere function because a minimal telomere length is required for the formation of a proper telomere structure, and telomere length may influence the equilibrium between the 'closed' and the 'open' states, which may be subjected to different enzymatic activities (eg telomerase). Terminal restriction fragment (TRF) analysis is the only method currently available for measuring telomere length in Caenorhabditis elegans . Its limitations include low sensitivity and interference by the presence of interstitial telomeric sequences in the C. elegans genome. Here we report the adaptation of single telomere length analysis (STELA) to measure the length of telomere repeats on the left arm of chromosome V in C. elegans. This highly sensitive PCR based method allows telomere length measurement from as few as a single worm. The application of STELA to eight wildtype C. elegans strains revealed considerable strain-specific differences in telomere length. Within individual strains, short outlying telomeres were observed that were clearly distinct from the bulk telomere length distributions, suggesting that processes other than end-replication losses and telomerase-mediated lengthening may generate telomere length heterogeneity in C. elegans. The utility of this method was further demonstrated by the characterization of telomere shortening in mrt-2 mutants. We are currently using STELA to study different mutants for telomere phenotypes.

Lu WX et al. (1996) East Coast Worm Meeting "MOLECULAR ANALYS1S OF THE CLASS B SYNTHETIC MULTIVULVA GENE lin-37"

Lu WX, Horvitz HR

[East Coast Worm Meeting, 1996]

The receptor tyrosine kinase/ras/kinase cascade pathway for vulval induction is negatively regulated by two functionally redundant pathways The activity of one pathway is disrupted by Class A mutations, while the activity of the second pathway is disrupted by Class B mutations. Animals are multivulva (Muv) only when they are doubly mutant in both Class A and B genes, and have a wildtype vulval phenotype when carrying two mutations of the same class. To understand how these pathways function on a molecular level, we have cloned the Class B synthetic multivulva gene lin-37 and are further characterizing this gene. Iin-37 maps on LGIII between sma-3 and mec-14. We rescued the Muv phenotype of the strain lin-8(n111); lin-37(n758) with both cosmic C48B6 and cosmic F31H1. This result indicates that lin-37 activity is located within the overlapping region of the two cosmids. The sequence of this region has been determined by the Genome project. There are multiple open reading frames (ORFs) within this 14 kb genomic region as predicted by Genefinder. Our attempts to further narrow the rescuing activity have failed, suggesting that lin-37 could be part of an operon. We therefore disrupted each ORF individually and identified one ORF required for rescue. The predicted polypeptide product of 27.3 kD is novel. We are in the process of isolating cDNAs. lin-37 acts cell nonautonomously (2), presumably in hyp7, similarly to lin-15 (3), which encodes both class A and class B activities. On the other hand, the class B gene lin-36 has been shown to act cell autonomously in the Pn.p cells (Jeff Thomas, personal communication). Further characterization of lin-37 and study of the interactions among synthetic multivulva genes should allow us to gain insights into the roles of these genes in regulating the Ras signaling pathway. Ref. 1. Ferguson and Horvitz, Genetics 123:109121, 1989 2. Hedgecock and Herman, Genetics 141:9891006, 1995 3. Herman and Hedgecock, Nature 348: 169171, 1990

EM Link et al. (1999) International C. elegans Meeting "C. elegans immunity? A reverse genetic analysis of components of the Toll signaling pathway"

G Hardiman, LX Liu, J Hodgkin, EM Link, L Nelson

[International C. elegans Meeting, 1999]

The Toll signaling pathway is involved in innate immunity in both flies and mammals. The Drosophila pathway, including the Toll receptor, Pelle kinase, and Dorsal transcription factor, regulates both dorsal-ventral polarity in the embryo and antimicrobial peptide production in adult flies. The human inflammatory cytokine IL-1 signals through the IL-1 receptor (whose cytoplasmic domain is homologous to that of Toll) and the Pelle homolog IRAK, ultimately activating NF k B, a rel protein homologous to Dorsal. Human Toll homologs also signal to NF k B(1). To investigate this pathway in C. elegans , we identified orthologs of four genes and generated deletion mutations for each: tol-1 = Tol l receptor, pik-1 = P elle/ I RAK k inase, trf-1 = TR A F , and ikb-1 = I k B . Worms with a deletion affecting all of tol-1 except the N-terminal leucine-rich repeats arrest as small, deformed larvae, a phenotype rescued by the tol-1 cosmid. However, a second tol-1 deletion in the intracellular domain seems wild-type. Deletion mutants of the other three genes show no obvious phenotype. To test whether these deletion alleles affect putative C. elegans host defenses, we exposed worms to an Aureobacterium strain that causes anal infection and local tissue swelling (2). The swelling phenotype was not altered in the four viable mutants. We also examined the expression of abf-1 and abf-2 , which encode homologs of Ascaris antibacterial peptides (3). abf-1 and abf-2 expression was not altered in the four mutants compared to wild type, following either usual culture or Aureobacterium infection. Orthologs for other downstream elements of the Toll pathway, including NIK, IKK and NF k B (1), are conspicuously absent from the complete worm genome, suggesting that C. elegans has evolved an alternative transcriptional output for tol-1 . Although our preliminary assays were negative, we cannot rule out a possible role for tol-1 in worm immunity, given the dearth of known microbial pathogens of C. elegans . (1) Kopp ER & Medzhitov R, Curr Opin Immunol 1999; 11: 13. (2) Hodgkin J & Corneliussen B, WBG 1997; 15 (1):73. (3) Kato Y, WBG 1996; 14 (4):37.