Xiaohui Qiu et al. (2004) West Coast Worm Meeting "ARI-1, a ubiquitin ligase involved in C. elegans pharyngeal development"

Xiaohui Qiu, Iqbal Ahmed, David S Fay, Bethany L Johanson

[West Coast Worm Meeting, 2004]

The Class B SynMuv gene lin-35 encodes the C. elegans homolog of the human Retinoblastoma tumor suppressor protein (Rb). Unlike most other systems, loss of lin-35/Rb activity in C. elegans is not highly deleterious. In addition to a well-documented role in cell cycle control, genetic screens carried out in our laboratory have discovered additional functions for lin-35 including a role in pharyngeal development (Fay et al., 2003, 2004). In double mutants of lin-35; ubc-18 and lin-35; pha-1, an early stage of pharyngeal morphogenesis is defective, resulting in an abnormal cellular architecture and a failure of the pharynx to elongate and attach to the buccal cavity. UBC-18 is a conserved ubiquitin-conjugating enzyme while the molecular function of PHA-1 is not clear. However, multiple lines of evidence indicate that PHA-1 and UBC-18 are likely to act together with LIN-35 to control the activity or abundance of a common target. This includes the observation that the synthetic phenotypes of lin-35; ubc-18 and lin-35; pha-1 are nearly identical as is their spectrum of genetic interactions with other SynMuv genes. In addition, both double mutants are suppressable by the same suppressor mutations (also see abstract for B. Johanson et al.) and pha-1 and ubc-18 are themselves synthetically lethal (Fay et al., 2003, 2004). Our interest is to find the common target and to elucidate the underlying biology of the observed defects. Since UBC-18 is a ubiquitin-conjugating enzyme and presumably interacts with at least one cognate ubiquitin ligase, we carried out a yeast two hybrid screening using UBC-18 as the bait molecule. Our screen identified three ubiquitin ligases, one of which when inactivated by RNAi, produced or enhanced pharyngeal defects in a manner identical to that of ubc-18. We term this gene ari-1 (C27A12.8), based on its homology with the fly ariadne protein. Given the fact that UBC-18 physically interacts with ARI-1 and that RNAi feeding generates an identical synthetic defect, we believe that ARI-1 functions downstream UBC-18 to coordinately regulate an early step of pharyngeal morphogenesis with LIN-35 and PHA-1. To further search for the relevant protein substrate of ARI-1, we have conducted two-hybrid screens using ARI-1 as bait. Using several different bait fusion constructs, we are currently carrying out a saturating screen and have identified one or more very good candidate ARI-1 substrates to date. We have also constructed a variety of full-length ari-1::GFP fusion constructs to determine the expression pattern of ARI-1, and have been screening for deletion mutations in the ari-1 locus. 1. Lu, X., and H.R. Horvitz. Cell 95: 981-91, 1998 2. Fay et al. Genes & Dev. 16: 503-17, 2002 3. Fay et al. Development 130: 3319-3330, 2003 4. Fay et al. Dev. Biol. 271:11-25, 2004 5. Aguilera et al. Genetics 155: 1231-44, 2000

Frezal, Lise et al. (2017) International Worm Meeting "Some C. elegans wild isolates display a heat-triggered mortal germ line phenotype."

Braendle, Christian, Frezal, Lise, Demoinet, Emilie, Felix, Marie-Anne, Miska, Eric, Zhang, Gaotian

[International Worm Meeting, 2017]

The mortal germ line phenotype was defined by Ahmed and Hodgkin as a multigenerational phenotype, whereby a selfing C. elegans line becomes sterile after several generations (1). The N2 reference strain does not display this phenotype, but Ahmed and Hodgkin could isolate mutants that are mortal. Some of these mutations are temperature-sensitive and affect small RNA and chromatin modification pathways, which may explain the multigenerational nature of the phenotype (e.g. 2,3). We discovered serendipitously that many C. elegans wild isolates display a strong mortal Germ Line (Mrt) phenotype: chronic exposure to high temperature, e.g. 25 deg C, progressively leads to sterility within several generations. Here we discuss this surprising finding. We first assayed a reference panel of 97 C. elegans wild isolates (4) for their Mrt phenotype, scoring the number of generations at 25 deg C after which full sterility occurred. Wild isolates showed strong, reproducible differences. We found no association between the severity of the Mrt phenotype and the climatic origin of C. elegans isolates. In the generations before full sterility, we observed a progressive brood size decrease, germ line differentiation defects, chromosomal aberrations at diakinesis and DNA damage. The mortal germline phenotype was however fully reversed by switching back from 25 deg C to 15 deg C before the last generation. Most surprisingly, germline immortality of some isolates was rescued by artificial infection with Nematocida microsporidia, natural intracellular C. elegans pathogens infecting intestinal cells, which suggests signaling from soma to germline. We suggest that the Mrt phenotype observed under laboratory conditions is likely not commonly displayed under natural conditions, yet provides an exciting model to test whether and how epigenetic inheritance systems are modulated by natural genetic variation. In order to pinpoint the molecular inheritance system underlying this mortal phenotype, we focused on determining the genetic basis of the quantitative variation among the C. elegans wild isolates, using both association mapping and laboratory crosses. The two approaches yielded three candidate regions, which we confirmed through introgression experiments. Thus, although the multigenerational nature of the Mrt phenotype may stem from inheritance of small RNAs and chromatin marks that are temperature-sensitive, phenotypic variation in the Mrt phenotype among C. elegans wild isolates is due to genetic variation. (1) Ahmed & Hodgkin. 2000. Nature; (2) Ashe et al. 2012 Cell; (3) Buckley et al. 2012 Nature; (4) Andersen et al. 2012 Nature Genetics.

Ahmed S et al. (2000) European Worm Meeting "A mutator phenotype found in C. elegans checkpoint mutants"

Hodgkin JA, Ahmed S

[European Worm Meeting, 2000]

DNA damage checkpoint proteins are caretakers of the genome. These checkpoint proteins function by sensing DNA damage and inducing either CELL CYCLE ARREST, which allows for the DNA damage to be repaired, or APOPTOSIS, which eliminates the cell that has been damaged. Alteration of the p53 checkpoint protein is frequently observed in cancer cells. Since mutations in multiple genes occur in developing cancers, it seems reasonable to surmise that a DNA damage checkpoint defect might result in an elevated mutation rate that leads to cancer. However, controversial results have been obtained regarding mutation rates in mammalian cells with checkpoint defects, and no mutator phenotype has been reported for yeast checkpoint mutants. Several different C. elegans mutants with DNA damage checkpoint defects have been recently identified (1,2) - the germlines of these checkpoint mutants are hypersensitive to X-rays and to other agents that damage DNA. We observed an elevated rate of spontaneous mutation in one of these C. elegans checkpoint mutants, and another checkpoint mutant was previously shown to be a mutator (3). This increase in mutation frequency is much higher for some genes than for others. These results may explain the controversy surrounding mutation rates in human cells with checkpoint defects. 1. Ahmed S, Hodgkin J (2000). MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403, 159-164. 2. Gartner A, Milstein S, Ahmed S, Hodgkin J, Hengartner MO (2000). A conserved checkpoint pathway mediates DNA damage-induced apoptosis and cell cycle arrest in C. elegans. Molecular Cell, in press. 3. Hartman PS, Herman RK (1982). Radiation-sensitive mutants of Caenorhabditis elegans. Genetics 102, 159-178.

Shawn Ahmed et al. (2001) International C. elegans Meeting "C. elegans DNA damage checkpoint mutants mutate"

Shawn Ahmed, James Thomas, Monika Tzoneva, Jonathan Hodgkin

[International C. elegans Meeting, 2001]

The DNA damage checkpoint is composed of a group of proteins that sense DNA damage and induce either cell cycle arrest and DNA repair (which mends the damage) or programmed cell death (which removes the damaged cell). Although many cancer cells are checkpoint-defective, efforts to show that mammalian cells with defects in the DNA damage checkpoint are mutating spontaneously have produced controversial results. In addition, it is unclear which DNA damage checkpoint protein physically binds to damaged DNA. We have been addressing both of these issues using the nematode C. elegans . Three C. elegans DNA damage checkpoint mutants have been identified to date: mrt-2 (e2663) , op241 , and rad-5 (mn159) (1,2). The rates and nature of spontaneous mutation in these mutants have been examined using a variety of tests. We find that all of these DNA damage checkpoint mutants display a spontanaeous mutator phenotype, and that most of these spontaneous mutations are deletions. Although mrt-2 (e2663) and op241 mutate at all loci tested, rad-5 (mn159) is mutating primarily at a single locus. This surprising result agrees with the observation that rad-5 (mn159) is Temperature-sensitive and is therefore unlikely to be a null allele (3). Since rad-5 (mn159) is mutating mostly at a single locus, rad-5 (mn159) may be defective in sensing a particular kind of spontaneous DNA damage that occurs at this locus. Thus, the RAD-5 checkpoint protein may be a primary 'sensor' of DNA damage, a protein whose identity has eluded the DNA damage checkpoint community for many years. 1. Gartner, A, Milstein, S, Ahmed, S, Hodgkin, J, and Hengartner, MO (2000). A conserved checkpoint pathway mediates DNA damage-induced apoptosis and cell cycle arrest in C. elegans . Molecular Cell 5: 435-443. 2. Ahmed, S, and Hodgkin, J (2000). MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans . Nature 403: 159-164. 3. Hartman, PS and Herman RK (1982). Radiation-sensitive mutants of C. elegans . Genetics 102: 159-178.

Aisa Nakashima et al. (2008) European Worm Meeting "Two new germ cell immortality mutants"

Jan Laroque, Theresa M Zucchero, Julie Hall, Aisa Nakashima, Shawn Ahmed, Lauren Garwood

[European Worm Meeting, 2008]

Germ cells represent canonical stem cells that possess the remarkable. quality of being able to proliferate from one generation to the next,. indefinitely, free of replicative damage. mortal germline mutants initially. display normal levels of fertility, but become progressively sterile when. grown for multiple generations. Of 16 original mortal germline mutants (1),. most were temperature-sensitive and only became sterile when grown at 25oC.. Of the ts mutants, two were resistant to RNAi feeding constructs targeting. embryonic lethal genes at all temperatures: 1c and 10i. The 10i mutation. mapped to the right arm of chromosome IV, between unc-22 and dpy-4, and. fails to complement rsd-2 for its RNAi resistance phenotype. rsd-2 has been. reported to be is deficient for spreading of dsRNA-mediated RNAi from. somatic cells to germ cells (2), suggesting that this function may be. relevant to maintaining germ cells over multiple generations during growth. at high temperatures. The mechanism by which this occurs is being studied.. (1) Ahmed S, Hodgkin J (2000). MRT-2 checkpoint protein is required for. germline immortality and telomere replication in C. elegans. Nature.. (2) Tijsterman M, May RC, Simmer F, Okihara KL, Plasterk RH (2004). Genes. required for systemic RNA interference in Caenorhabditis elegans. Current. Biology.

Bettina Meier et al. (2002) European Worm Meeting "Studying telomere replication in C. elegans"

Jonathan Hodgkin, Bettina Meier, Shawn C Ahmed, Yan Liu

[European Worm Meeting, 2002]

The stable length of telomeres, the native ends of linear chromosomes, is maintained by a specialized reverse transcriptase, telomerase. Whereas the telomerase catalytic subunit and its RNA component have been shown to be sufficient to confer the addition of repetitive sequences to chromosome ends in vitro, studies in yeast suggest that a larger protein complex is required to ensure access of telomerase in vivo. mrt-2 was the first C. elegans mutant that exhibits progressive telomere shortening and eventually sterile mutant worms (Ahmed and Hodgkin, 2000), phenotypes that are similar to yeast and mouse telomerase mutants. Strikingly, mutating the S. pombe homologue of the mrt-2 checkpoint gene does not abolish telomerase action and instead results in short but stable telomeres (Dahlen et al., 1998). Thus, we are interested in studying telomere replication in higher organisms like the nematode C. elegans. We are currently characterizing two novel mortal germline mutants that are defective for telomere replication, trt-1 and mrt-1. One mutant, trt-1, carries a mutation in DY3.4, a C. elegans reverse transcriptase that is similar to other telomerase catalytic subunits, although DY3.4 appears to lack the T-motif that is found in most other telomerases. A second mutant, mrt-1, maps closely to DY3.4, and we are trying to determine which gene is mutated in mrt-1. Furthermore, we have initiated a largescale screen to identify most genes that are involved in telomerase function in C. elegans.

Nakashima, Aisa et al. (2009) International Worm Meeting "RNAi Spreading Mutants rsd-2 and rsd-6 are Deficient for Germ Cell Immortality."

Ahmed, Shawn, Garwood, Lauren, Nakashima, Aisa, Alvares, Stacy, Hall, Julie, LaRoque, Jan, Zucchero, Theresa

[International Worm Meeting, 2009]

Germ cells represent canonical stem cells that possess the remarkable quality of being able to proliferate from one generation to the next, indefinitely, free of replicative damage. mortal germline (mrt) mutants initially display normal levels of fertility, but become progressively sterile when grown for multiple generations. Of 16 mrt mutants identified in a pilot EMS screen (Ahmed and Hodgkin, 2000), most were temperature-sensitive and only became sterile when grown at 25 deg C. Of the ts mutants, two were resistant to RNAi feeding constructs targeting embryonic lethal genes: 1c and 10i. Genetic mapping experiments, complementation tests for RNAi resistance, and DNA sequencing revealed that 1c and 10i contain mutations in rsd-6 and rsd-2, respectively. Independently isolated alleles of rsd-6 and rsd-2 confer progressive sterility when propagated at 25 deg C, and complementation tests confirmed that these genes are required for germ cell immortality. rsd-6 and rsd-2 have been reported to be deficient for spreading of dsRNA-mediated RNAi from somatic cells to germ cells (Tijsterman et al., 2004). However, our results indicate that RSD-6 may be required in germ cells to promote germ cell immortality, whereas it functions in somatic but not germ cells to promote RNAi spreading. In addition, other RNAi-spreading defective mutants do not exhibit progressive sterility. These results indicate that RSD-6 and RSD-2 are likely to promote germ cell immortality via a mechanism that is distinct from their role in RNAi spreading.

Frezal, Lise et al. (2015) International Worm Meeting "Evolutionary variation in heat-triggered multigenerational onset of sterility in C. elegans."

Miska, Eric, Felix, Marie-Anne, Frezal, Lise

[International Worm Meeting, 2015]

We serendipitously discovered that many C. elegans wild isolates display a strong Mortal Germ Line (Mrt) phenotype (1) at high temperature, whereby upon chronic exposure to temperatures such as 25degC, they progressively become sterile after several generations. Here we will report on our systematic studies of this multigenerational onset of sterility in wild isolates of C. elegans. We assayed a reference panel of 97 genetically distinct isogenic C. elegans wild isolates (2) for the number of generations at 25degC after which full sterility occurred. Reproducible variation was found among these wild isolates showing that C. elegans displays natural variation in the Mrt phenotype, likely of genetic nature. The N2 strain is among the most resistant ones. In order to genetically map this natural variation in the multigenerational onset of sterility, we generated and investigated 120 recombinant inbred lines derived from parental isolate pairs with contrasted Mrt phenotypes. Previous studies (e.g. 3, 4) strongly suggest that the observed temperature-sensitive Mrt phenotype could be regulated through small RNAs and/or chromatin state modifications that are transmitted across generations. Therefore, we are investigating the evolution of small RNA contents over several generations in naturally Mrt and non-Mrt C. elegans isolates. The aim of this work is to test whether and how epigenetic inheritance systems are modulated by natural genetic variation. We are particularly intrigued by the evolutionary and ecological contexts that allow the evolution of conditional sterility defects.(1) Ahmed & Hodgkin. 2000. Nature. (2) Andersen et al. 2012 Nature Genetics (3) Ashe et al. 2012 Cell (4) Buckley et al. 2012 Nature..

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

Gartner A et al. (2000) East Coast Worm Meeting "Genetic analysis of DNA damage induced germline apoptosis"

Hengartner MO, Schumacher B, Gartner A, Milstein S, Hofmann ER

[East Coast Worm Meeting, 2000]

DNA damage induces cell cycle arrest that allows for repair of the lesion. However, in metazoans, when damage to the genome is excessive, cells undergo apoptosis. The importance of DNA damage in tumorigenesis has directed extensive research on DNA repair and cell cycle arrest in yeast and bacteria, yet not much is known about the molecular and genetic pathways of checkpoint mediated apoptosis. In C. elegans, gamma irradiation induces apoptotic cell death of meiotic germ cells as well as proliferation arrest of mitotic germ cells. The DNA damage-mediated apoptosis is dependent on ced-3, ced-4 and is negatively regulated by ced-9. The positive death regulator, egl-1, participates in, but is not essential for radiation-induced apoptosis (1). Recently, three mutants - dam-1(op241), rad-5(mn159), and mrt-2(e2663) - have been identified in C. elegans that block DNA damage induced apoptosis and cell cycle arrest (1). The MRT-2 protein is homologous to S. pombe RAD1, demonstrating for the first time that RAD1 homologs participate in apoptosis. We believe that many other genes are important for this DNA damage response. C. elegans provides an excellent genetic model to study DNA damage-induced apoptosis. In order to understand the mechanisms of DNA damage-induced germ cell cycle arrest and apoptosis, we plan to use C. elegans for a genetic and molecular analysis of these pathways. We will isolate and characterize three candidate checkpoint deletion mutants using reverse genetics. These deletion mutants will be characterized genetically for their involvement in DNA damage induced apoptosis and cell cycle arrest. We will use forward genetics to identify new, possibly metazoan specific, dam mutants that are defective in their apoptotic response to DNA damage. We expect that these studies will lead to a greater understanding of the genetic and molecular pathway(s) that regulate DNA damage-induced cell cycle arrest and apoptosis 1) Gartner, A., Milstein, S., Ahmed, S., Hodgkin, J., Hengartner, M.O. 2000. Mol. Cell 5:435-443.