Patrick J Hu et al. (2005) International Worm Meeting "EAK proteins function at the XXX cell membrane to potentiate AKT-1 dauer-inhibitory signals."

Patrick J Hu, Gary Ruvkun, Jinling Xu

[International Worm Meeting, 2005]

Akt/PKB proteins participate in evolutionarily conserved signal transduction pathways that are dysregulated in cancer and diabetes. In C. elegans, two Akt/PKB homologs, AKT-1 and AKT-2, prevent dauer formation under replete growth conditions by transducing insulin-like signals and inhibiting the FoxO transcription factor DAF-16 during early larval development. Genetic evidence in C. elegans supports the existence of undiscovered pathway components. In order to identify novel molecules that participate in C. elegans insulin-like signaling, we performed an Eak (Enhancer-of-akt-1) screen. A screen of ~20,000 genomes yielded 21 independent mutants that define 7 eak genes. Cloning and characterization of eak-3, eak-4, eak-5, and eak-6 reveal that they encode membrane-associated proteins that function in parallel to AKT-1 in the XXX cells to inhibit dauer formation. Consistent with a possible role in signal transduction, eak-5, which is allelic to the synthetic dauer formation gene sdf-9, and eak-6 encode proteins with homology to protein tyrosine phosphatases. These findings define a novel pathway that functions at the plasma membrane of the XXX cells to potentiate AKT-1 signals during larval development.

Patrick J Hu et al. (2003) International Worm Meeting "The enhancer-of-akt-1 gene eak-3 encodes a potential novel component of DAF-2 insulin/IGF-1-like signaling."

Gary B Ruvkun, Jinling Xu, Patrick J Hu

[International Worm Meeting, 2003]

The DAF-2 insulin/IGF-1-like signaling cascade regulates development, longevity, and metabolism. Structural and functional conservation of this pathway between C. elegans and humans suggests that novel components of DAF-2 signaling may be homologous to human proteins that play roles in the pathogenesis of diabetes and cancer.Genetic evidence is consistent with the existence of undiscovered DAF-2 pathway components. In order to identify such molecules, we performed an enhancer-of-akt-1 (Eak) screen. One gene identified in this screen, eak-3, has a weak Hid phenotype. eak-3 mutants enhance the dauer phenotype of two akt-1 loss-of-function alleles as well as that of age-1(hx546). The dauer phenotype of eak-3;akt-1 double mutants is suppressed by daf-16(mgDf47). Interestingly, although eak-3 mutations strongly enhance the dauer phenotype of akt-1 mutants, they do not affect lifespan. Therefore, eak-3 may encode a DAF-2 signaling component that specifically regulates dauer formation.

Patrick J Hu et al. (2002) East Coast Worm Meeting "The enhancer-of-akt-1 genes eak-1 through eak-5 may define novel components of DAF-2/insulin-like signaling"

Patrick J Hu, Gary B Ruvkun

[East Coast Worm Meeting, 2002]

DAF-2 insulin-like signaling regulates development, longevity, and metabolism in C. elegans. This conserved signal transduction pathway consists of the DAF-2/insulin receptor; AGE-1/PI 3-kinase; PDK-1, AKT-1, and AKT-2 kinases; the DAF-18/PTEN tumor suppressor homolog; and the DAF-16/Forkhead transcription factor. Although genetic screens have identified multiple alleles of all of these genes, epistasis experiments suggest the existence of undiscovered DAF-2/insulin receptor outputs that are independent of AGE-1/PI 3-kinase. To identify components of this parallel pathway, we screened for mutations that enhance the akt-1 null phenotype (Eak mutants). Since akt-1 null mutants are Daf-c at 270 C but not 250 C, we mutagenized akt-1 mutant animals and isolated F2 progeny that were now Daf-c at 250 C. We identified 30 independent mutants from a screen of approximately 21,000 haploid genomes. 26 mutants were suppressed by daf-16 RNAi, suggesting that they are components of DAF-2 signaling. Among these, 13 had a true Eak phenotype; they form dauers at 250 C only in a homozygous akt-1 null background. These 13 mutants define 5 complementation groups. Preliminary mapping analysis indicates that eak-1 through eak-5 do not correspond to known Daf or SynDaf genes. Therefore, the eak genes may define novel participants in DAF-2/insulin-like signaling. Fine mapping of these genes is ongoing.

Patrick J Hu et al. (2003) International Worm Meeting "The enhancer-of-akt-1 genes eak-1 through eak-6 may define novel components of DAF-2 insulin/IGF-1-like signaling."

Maurice Butler, Gary B Ruvkun, Patrick J Hu

[International Worm Meeting, 2003]

The DAF-2 insulin/IGF-1-like signaling cascade regulates development, longevity, and metabolism. Components of this pathway include AGE-1/PI 3-kinase, DAF-18/PTEN, PDK-1 and AKT-1/2 serine/threonine kinases, and the FoxO Forkhead transcription factor DAF-16. Structural and functional conservation of this pathway in humans suggests that novel components of DAF-2 signaling may be homologous to human proteins that play roles in the pathogenesis of diabetes and cancer.Genetic evidence is consistent with the existence of undiscovered DAF-2 pathway components. In order to identify such molecules, we performed an enhancer-of-akt-1 (eak) screen. akt-1 loss-of-function mutants form dauers at 270C but develop normally at 250C; therefore, we mutagenized akt-1 mutant animals and screened for F2 dauers at 250C. A screen of approximately 21,000 haploid genomes yielded 30 independent mutants. 22 of these define at least 6 eak genes. Single nucleotide polymorphism mapping analysis indicates that the eak genes are distinct from known Daf or SynDaf genes, suggesting that they encode novel components of DAF-2 signaling. One of these genes, eak-3, has been cloned; efforts to clone the other eak genes are ongoing.

Patrick J Hu et al. (2004) East Coast Worm Meeting "eak (enhancer-of-akt-1) genes encode membrane-associated proteins that potentiate AKT-1 signaling in the C. elegans XXX cells."

Patrick J Hu, Jinling Xu, Gary Ruvkun

[East Coast Worm Meeting, 2004]

Akt/PKB proteins transduce signals that regulate growth and apoptosis in mammals. Dysregulation of Akt/PKB signaling plays prominent roles in the pathogenesis of cancer and diabetes in humans. In C. elegans , AKT-1 prevents dauer formation by transducing insulin-like signals from the DAF-2 insulin/IGF-1 receptor to inhibit the FoxO transcription factor DAF-16. In order to identify novel components of DAF-2 signaling, we performed an Eak (enhancer-of- akt-1 ) screen. We have isolated 21 independent Eak mutants defining 7 complementation groups from a screen of approximately 20,000 haploid genomes. eak mutants have weak dauer-constitutive phenotypes and strongly enhance the weak dauer-constitutive phenotype of akt-1 loss-of-function mutants. eak phenotypes are suppressed by daf-16 null mutants, suggesting that EAK proteins function in DAF-2 signaling. Interestingly, all eak alleles assayed thus far have normal lifespans. 4 eak genes have been cloned. eak-3 and eak-4 encode novel proteins; eak-5 and eak-6 encode proteins with similarity to protein tyrosine phosphatases. Analysis of transgenic animals expressing transcriptional and translational fluorescent protein reporter constructs indicates that all 4 EAK proteins are predominantly expressed in and localize to the plasma membrane of the XXX cells. Expression of AKT-1 in the XXX cells fully rescues the dauer phenotype of eak;akt-1 double mutants, suggesting that the XXX cells are a major site of AKT-1 function in dauer regulation. Taken together, the data support a model whereby AKT-1 and EAK proteins function in parallel in the XXX cells to prevent dauer formation during larval development.

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.