Brejning, Jeanette et al. (2009) International Worm Meeting "Characterization of hydroxyurea resistance and Aging in C. elegans."

Brejning, Jeanette, Olsen, Anders, Jakobsen, Helle, Scholer, Lone

[International Worm Meeting, 2009]

Checkpoints are evolutionary conserved surveillance mechanisms activated upon DNA damage to halt the progression of the cell cycle. Appropriate checkpoint function is required to preserve the genomic content of descendant cells and aberrant checkpoint function is connected to development of cancer. We have found that inactivation of certain checkpoint proteins, including p53, cause resistance to the chemotherapeutic drug hydroxyurea (HU) that stalls replication. HU is an inhibitor of ribonucleotide reductase (RNR) in many organisms, but the target in C. elegans is less clear. RNR is involved in synthesis of deoxyribonucleotide (dNTP) precursors for DNA replication and repair and consists of two homodimeric subunits RNR-1 and RNR-2. We report that in C. elegans, HU induces transcription of both RNR subunits, indicating that HU targets RNR in C. elegans as well. Inactivation of some S-M checkpoint proteins can increase stress resistance and lifespan of C. elegans1. Interestingly, several genes that influence HU resistance also influence lifespan and stress resistance. We find that at least one of these genes appears to function in the S-M checkpoint pathway. 1.A. Olsen, M. C. Vantipalli, G. J. Lithgow, Science 312, 1381 (2006).

Lawrence, Kate et al. (2013) International Worm Meeting "DNA damage response and spindle assembly checkpoint collaborate to elicit cell cycle arrest in response to replication defects in the C. elegans male germ line."

Engebrecht, JoAnne, Lawrence, Kate

[International Worm Meeting, 2013]

Persistent DNA damage in germline stem cells leads to embryonic lethality, progeny inviability or germline tumors. Consequently, cells closely monitor genomic integrity and delay progression through the cell cycle so repair precedes division. In C. elegans, genotoxic stress activates checkpoints that initiate cell cycle arrest in proliferating germ cells. Arrest in response to stalled replication forks induced by hydroxyurea (HU) results in enlarged nuclei and accumulation of S-phase markers. HU damage is sensed by the C. elegans homolog of ATR, a PI3-related protein kinase, which initiates a signaling cascade to induce arrest and repair. The signal transducers and downstream effectors of this DNA-damage-response (DDR) have been extensively studied in hermaphrodites, but not in males. While RNAi knockdown of several of these genes disrupts checkpoint output and thus prevents arrest in hermaphrodites, the same treatment only partially prevents HU-induced arrest in males. We took a candidate approach to identify compensatory pathways that contribute to HU-induced arrest in males. One well-characterized pathway that elicits cell cycle arrest is the spindle assembly checkpoint (SAC). The SAC is most often associated with monitoring kinetochore attachment to spindles during prometaphase/metaphase of mitosis and meiosis. We found that RNAi knockdown of several SAC components alone did not affect HU-induced cell-cycle arrest in males; however, knockdown of both ATR and SAC components resulted in a failure to arrest in the presence of HU, indicating that the DDR and SAC both function to elicit arrest in the presence of stalled forks. Consistent with this, SAC components become enriched at the nuclear periphery in an ATR-dependent manner in response to HU. Our data reveal that the SAC has a novel role in S-phase arrest, which is independent of fzy-1/cdc20, an APC activator and key regulator of SAC-induced metaphase arrest. Preliminary studies suggest that the other APC activator, fzr-1/cdh1 facilitates SAC-induced S-phase arrest. We hypothesize that FZR-1 forms an S-phase inhibiting complex with SAC subunits analogous to the MCC complex that mediates APC inhibition during SAC-induced M-phase arrest.

Olsen, Anders et al. (2009) International Worm Meeting "A whole genome screen for checkpoint functions that determine lifespan in C. elegans."

Zhang, Mike, J. Lithgow, Gordon, A. Caldwell, Guy, Hamamichi, Shusei, Olsen, Anders, Zeng, Xianmin, Mark, Karla, Benedetti, Michael, A. Caldwell, Kim, L. Knight, Adam, Bhaumik, Dipa, Vantipalli, Maithili

[International Worm Meeting, 2009]

Based on our previous observation that gene encoding cell cycle checkpoint functions determined lifespan in C. elegans, we aimed to define the genetic pathways at play. Three observations prompt this study. First we isolated a mutation in the gene checkpoint gene cid-1 which causes thermotolerance, increased lifespan and resistance to hydroxyurea (HU). Second we also found that mutations in other checkpoint genes and the tumor suppressor p53 confer resistance to HU. Third, since the nematode is post-mitotic, it is possible to study the effect of knocking down these genes in a whole organism without occurrence of lethal cancers. When wild-type eggs are placed on NGM plates spotted with HU the worms arrest development in a dose dependent manner. We screened for RNAi clones that suppressed this arrested development. All hits were re-tested at multiple doses of HU and resistance quantified as body size at three days of age. ~ 50 clones reproducibly result in HU resistance (hur genes) We are currently examining the hur genes for abnormal germline development, sterility and embryonic lethality. Many known cell cycle and checkpoint mutants show sensitivity to DNA damage. Therefore, to further test for cell cycle / checkpoint phenotypes we are exposing young worms to ionizing radiation and studying three different markers of DNA damage/checkpoint inefficiency, embryonic lethality, cell cycle arrest and apoptosis in the germline. We observed thermotolerance for 21 of the hits but not for all of them. Thus, HU resistance does not stem from a generalized increase in stress resistance but from a more specific mechanism. One of the clones increases thermotolerance of daf-2, daf-16 and eat-2 mutants and seems to function independently from insulin signaling and caloric restriction. Aging is one of the biggest risk factors for cancer. Our ongoing analysis suggests that the hur gene list is highly enriched for increased lifespan although some result in shortened lifespan. We are investigating these and similar genes in a number of neuronal cell death models including human cells.

Myers, Toshia et al. (2013) International Worm Meeting "Towards understanding the role of histone demethylation in replication-induced DNA damage repair."

Amendola, Pier Giorgio, Salcini, Anna Elisabetta, Myers, Toshia

[International Worm Meeting, 2013]

Post-translational modification (PTM) of histones is required to successfully repair DNA damage, which is caused by endogenous (e.g. replication stress) and exogenous (e.g. ionizing radiation) DNA stressors. Histone PTMs facilitate the DNA damage repair (DDR) response by regulating chromatin remodelling and recruitment of repair factors to sites of DNA damage. Although emerging evidence suggests that histone methylation promotes DDR, the role of pre-existing histone methylation and histone demethylation on cellular response to replication stress remains elusive. Therefore, we have taken steps to understand the role of the C. elegans Jumonji C-domain containing histone demethylase homologues in replication stress response. In a survival screen for sensitivity or resistance to hydroxyurea (HU), a chemical that inhibits ribonucleotide reductase leading to replication stress, we identified jmjd-1.1(hc184) and jmjd-1.2(tm3713) as mutations that confer HU resistance. JMJD-1.1 and JMJD-1.2 are histone demethylases specific for H3K9me2 and H3K27me2, marks associated with heterochromatin and transcriptional repression. These mutants have significantly less embryonic lethality than wild-type controls after treatment with 25mM HU (and other chemicals that cause replication stress induced DNA damage) but are not resistant to ionizing or ultraviolet radiation. Although these mutants have increased germline levels of both H3K9me2 and H3K27me2, they are otherwise phenotypically wild-type for germline morphology, brood size, and embryonic lethality under normal conditions. Similarly, mutants have normal cell cycle arrest, RAD-51 expression, and apoptosis, despite that H3K27me2 levels are diminished in the wild-type germlines but remain high in mutant germlines, after HU treatment. Currently, we are trying to understand the mechanism by which these proteins regulate replication stress response by testing if HU resistance is related to aberrant transcriptional regulation or to chromatin structure.

Lawrence, Kate (2011) International Worm Meeting "Spindle assembly checkpoint plays a role in DNA-damage-induced cell cycle arrest in C. elegans male germline."

Lawrence, Kate

[International Worm Meeting, 2011]

Persistent DNA damage in germline stem cells leads to embryonic lethality, progeny inviability or germline tumors. Consequently, cells closely monitor genomic integrity and can delay their progress through the cell cycle so that repair precedes division. In C. elegans, genotoxic perturbations to proliferative cells in the distal tip of the gonad activate checkpoints that initiate a cell cycle arrest. When this arrest is in response to stalled replication forks induced by hydroxyurea (HU), it is characterized by enlarged nuclei and can be visualized cytologically. HU damage is sensed by the C. elegans homolog of ATR, a PI3-related protein kinase, and launches a signaling cascade that results in a G1/S phase arrest. The signal transducers and downstream effectors of this DNA-damage-response (DDR) pathway have been studied extensively in hermaphrodites, but have not been investigated fully in males. We saw that while RNAi knockdown of several of these genes disrupts checkpoint output in hermaphrodites, the same treatment does not prevent HU-induced arrest in males. Our preliminary results strongly suggest that not all components of the DDR are essential for male cell-cycle arrest in response to stalled replication forks. We next investigated functional redundancy between the DDR and the spindle assembly checkpoint (SAC), which is most often associated with regulating kinetochore attachment to spindles during prometaphase/metaphase of mitosis and meiosis. We found that RNAi knockdown of several SAC components alone did not affect HU-induced cell-cycle arrest in males; however, knockdown of both ATR and SAC resulted in a failure to arrest in the presence of HU. This result suggests that, in males, the DDR and SAC work together to elicit arrest in the presence of stalled forks. To analyze this differentially regulated HU-induced arrest, we identified markers that characterize the stages of the cell cycle. Preliminary data suggests that the SAC, like the DDR, mediates an S phase arrest not predictive of its expected role as an inhibitor of cdc20 at metaphase. Future work aims to understand this novel role for SAC components and investigate the mechanisms used by the SAC to induce an S phase arrest.

Li SSL et al. (2000) East Coast Worm Meeting "Isolation of Mutants Defective for the Initiation of Postembryonic Development in Caenorhabditis elegans"

Pare C, Ambros VR, Li SSL, Roy RD

[East Coast Worm Meeting, 2000]

Normal postembryonic development of C. elegans depends on precise cellular proliferation and differentiation. Cells must respond to cues which govern proliferation, arrest and differentiation in synchrony with the organism's development. Environmental signals also play a major role in controlling these processes particularly during dauer formation and also at the transition from embryonic to larval development. Although the initiation of dauer development is well understood, little is known about the mechanism by which these environmental signals activate the onset of the postembryonic developmental program in C. elegans remains largely misunderstood. To understand the molecular basis that underlies the onset of larval development and the control of cell division at initiation of postembryonic development, screens were performed to identify temperature-sensitive mutants that are unable to initiate larval development and cell divisions typical of postembryonic development. Exposure of L1 larvae to a food source and hydroxyurea (HU), a potent inhibitor of DNA replication, causes worms to grow up to become uncoordinated and sterile adults. However, HU does not affect worms that harbor temperature sensitive mutations blocking them from initiating cell division.. We used this observation to screen for mutants that do not enter cell cycle at 25C (and hence are unaffected by HU) but activate postembryonic development and cell divisions normally at 15C. After a 48-hour culture in the presence of HU at 25C, worms were transferred to permissive temperature and allowed to grow to the adult stage. Healthy worms were isolated and maintained at 15C for further characterization. Two temperature sensitive mutants have been isolated which are normal at 15C but show complete developmental arrest as L1 larvae at 25C. Following this arrest, these animals can be shifted to 15C and development will proceed correctly. One mutant has been mapped to right arm of chromosome IV by STS mapping while mapping of the second mutant is presently under way.

Aristizabal, David et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The abnormal embryonic partitioning gene par-5 regulates replication stress-induced checkpoint response and cell proliferation in C. elegans germline"

Aristizabal, David, Schwartz, Simo, FONTRODONA, Laura, Porta-de-la-Riva, Montserrat, CERON, Julian

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

After DNA damage, cells respond through surveillance mechanisms in order to maintain the genomic stability by activating signal transduction cascades, so-called checkpoints. Such mechanisms promote mainly the inhibition of the cell cycle progression, repair of the damaged DNA and induction of apoptotic cell death if DNA is not repaired. In C. elegans germline, DNA damage and replicative stress (e.g. Hydroxyurea (HU) treatment) trigger a checkpoint response through conserved pathways. In our lab, by screening for human genes whose expression was induced after HU treatment (Guerra et al, unpublished results), we identified two genes coding for 14-3-3 proteins (Z and E) that are homologues of the C. elegans gene par-5. The partitioning defective PAR proteins regulate the asymmetric cell division that takes place at the first embryonic division. Interestingly, PAR-5 is also present in the adult germline, but its function there remains unknown. By using a HU-induced cell cycle arrest model in the germline we found that par-5 is required for a complete DNA damage checkpoint response. par-5 inactivation (by RNAi or mutation) severally impairs the cell cycle arrest induced by the DNA replication stress. Moreover, such inactivation also affects cell proliferation in the germline precursors as was evidenced by a significant decrease in the number of mitotic cells after RNAi knockdown. Supporting the function of par-5 in the checkpoint, we have observed a higher acc umulation of DNA repair foci (labeled with a HUS-1::GFP reporter) in germ cells after par-5 RNAi. This role in the DNA damage response is not general for par genes since we have observed that RNAi depletion of par-2 and par-3 do not affect the checkpoint response. The molecular mechanism ruling the function of PAR-5 in the HU-induced checkpoint will be discussed at the meeting.

Russell, L. et al. (2017) International Worm Meeting "SMRC-1 maintains genome integrity in C. elegans."

Xu, X., Maine, E., Sullenberger, M., Yanowitz, J., Russell, L., Yang, B.

[International Worm Meeting, 2017]

In order to maintain genomic integrity, DNA must be replicated without damaging the genetic code. This can be especially difficult under stressful conditions where additional barriers to replication may arise. Additionally, challenging repetitive sequences, such as those in telomeric DNA, provide endogenous barriers to replication. In humans, SMARCAL1 helps maintain genomic integrity by restarting stalled replication forks. This is a critical function, and defects in SMARCAL1 have been implicated in the rare disease Schimke Immuno-osseous Dysplasia. We used CRISPR to generate knockout alleles of the C. elegans SMARCAL1 ortholog, smrc-1. We found that these mutants show a decreased brood size and laid fewer eggs than wild type and also show increased germline apoptosis, phenotypes that are dependent on the activity of the DNA damage checkpoint. To further investigate the role of SMRC-1 in DNA damage repair, we subjected smrc-1 mutants to hydroxyurea (HU) and camptothecin (CPT), which can inhibit replication fork progression, as well as ionizing radiation (IR) which causes double stranded breaks. We found that smrc-1 mutants are sensitive to hydroxyurea (HU) and tagged SMRC-1 proteins relocalize from the nucleoplasm to chromatin-associated foci following HU exposure. Consistent with a role for SMRC-1 at stalled replication forks, smrc-1 mutants enhance the genomic instability phenotype of dog-1, showing increased mutations in a region that forms G-quadruplexes. smrc-1 mutants also exhibit a mortal germline (MRT) phenotype that we attribute to a buildup of mutations over generations. Interestingly, absence of SMRC-1 function enhances the MRT phenotype in the telomere mutant, trt-1, implicating SMRC-1 as a major determinant of telomere integrity. SMRC-1 protein co-immunoprecipiates with the H3K9 methyltransferase, MET-2 and double mutants show enhanced sterility. These studies introduce the exciting possibility that replication fork repair is coupled with the protection of repetitive sequences by methylation.

Michael Ailion et al. (2007) International Worm Meeting "Feeling unmotivated? Mapping and cloning suppressors of activated Gq."

Michael Ailion, Erik Jorgensen, Daniel Hu, Kim Schuske, Patrick Hullett, Andrea Pappas

[International Worm Meeting, 2007]

Gq/EGL-30 stimulates neurotransmission, in part by activating phospholipase C/EGL-8 and UNC-13. However, there is evidence that other pathways operate downstream of EGL-30 to regulate neurotransmission. To identify genes in such pathways, we screened for suppressors of the activated Gq mutant egl-30(tg26). We expected to find mutants in the canonical pathway (egl-30, egl-8, unc-13) as well as in other parallel pathways. We screened approximately 18,000 ENU-mutagenized genomes and isolated 43 mutants that suppress the hyperactive, small size and slow growth phenotypes of egl-30(tg26). These include recessive mutations in egl-30, egl-8, unc-13, ric-8 and unc-31/CAPS, all of which were expected from the screen. More interestingly, we isolated mutations in at least sixteen other genes not known previously to function in Gq signaling at the synapse. Many of the new mutants share similar phenotypes, suggesting that they function in a common pathway. When crossed out of the egl-30(tg26) background, the mutants fall into two distinct classes, unmotivated and fainter. Both classes are capable of coordinated movement when stimulated or in the absence of food, but show very little spontaneous movement on food. Thus, these genes appear to function in the regulation of movement rather than the execution of coordinated movements. Two of the new genes in the unmotivated class encode likely components of a small GTPase pathway. We identified mutations in the RhoGEF-2 specific isoforms of unc-73/Trio and in a novel protein with a small GTPase effector domain (the RUN domain). This suggests that the small GTPase Rho is a downstream effector of Gq. Other mutants in this class include hid-1, pkc-1 and a number of uncloned mutants that have been mapped to unique positions. In the fainter class, we identified alleles of unc-80. Other mutants with a fainter phenotype suppress activated Gq, and our screen also identified a novel fainter mutant. These genes may be downstream targets of Gq-regulated Rho activity. We are in the process of cloning the novel fainter mutant and several other mutants with an unmotivated phenotype similar to the unc-73 and RUN domain mutants. Finally, we identified an unusual allele of unc-104/kinesin that may regulate expression of the kinesin protein in specific cells relevant to the unmotivated phenotype. Together, these data provide preliminary evidence for a novel synaptic regulatory mechanism that affects behavior.

Gratien Dalpe et al. (2002) European Worm Meeting "plexin-1 is required for attraction and repulsion of cells in the epidermis of C.elegans"

Gratien Dalpe, Lijia W Zhang, Joseph G Culotti, Hong Zheng

[European Worm Meeting, 2002]

Plexins are semaphorin receptors conserved in vertebrates and invertebrates. However, their in vivo role in axon guidance, cell migration and embryonic development is not yet understood. Here we report the isolation of a mutation in the C.elegans plexin-1 (CEL-plx- 1(ev724)). Mutant animals have ventral enclosure defects and the first male ray is anteriorly positioned. Mutant ray 1 precursor cells are misplaced anteriorly during male tail development. semaphorin-1 mutants (smp-1/2(ev715, ev709)) have an identical male phenotype and we show that they function in the same genetic pathway as plx-1. CEL-plx- 1 shares high similarity with the C-terminal domain of human and D.melanogaster plexins, in which the presence of a RACGTP binding domain has been demonstrated (Hu (2001)). We find that known mutants for C.elegans rac genes (ced-10, mig-2) and a small GTPase activator (unc-73) have a ray 1 positioning defect similar to plx-1(ev724). mig-2 and ced-10 genetically act in parallel for this phenotype and these genes are likely activated by unc-73. Our genetic data suggest that plexin-1 and sema-1 are part of a pathway modulating the unc-73/mig-2/ced-10 pathway for ray 1 positioning. This model is consistent with known biochemical interactions between PLX-1 and RACGTP activated form (Vikis (2000); Driessens (2001); Hu (2001)), suggesting that upon SMP-1/2 binding the function of the PLEXIN-1/RAC complex is modulated.