Terrance J. Kubiseski

In this paper, the novel effect of electric field (EF) on adult C. elegans egg-laying in a microchannel is discovered and correlated with neural and muscular activities. The quantitative effects of worm aging and EF strength, direction, and exposure duration on egg-laying is studied phenotypically using egg-count, body length, head movement, and transient neuronal activity readouts. Electric egg-laying rate increases significantly when worms face the anode and the response is EF-dependent, i.e. stronger (6V/cm) and longer EF (40s) exposure result in a shorter egg laying response duration. Worm aging significantly deteriorates the electric egg-laying behaviour with 88% decrease in the egg-count from Day-1 to Day-4 post young-adult stage. Fluorescent imaging of intracellular calcium dynamics in the main parts of the egg-laying neural circuit demonstrate the involvement and sensitivity of the serotonergic hermaphrodite specific neurons (HSNs), vulva muscles, and ventral cord neurons to the EF. HSN mutation also results in a reduced rate of electric egg-laying allowing the use of this technique for cellular screening and mapping of the neural basis of electrosensation in C. elegans . This novel assay can be parallelized and performed in a high-throughput manner for drug and gene screening applications.

Oxidative stress causes damage to cells by creating reactive oxygen species (ROS) and the overproduction of ROS have been linked to the onset of premature aging. We previously found that a brap-2 (BRCA1 associated protein 2) mutant significantly increases the expression of phase II detoxification enzymes in C. elegans. An RNAi suppression screen to identify transcription factors involved in the production of gst-4 mRNA in brap-2 worms identified SEM-4 as a potential candidate. Here, we show that knockdown of sem-4 suppresses the activation of gst-4 caused by the mutation in brap-2. We also demonstrate that sem-4 is required for survival upon exposure to oxidative stress and that SEM-4 is required for expression of the transcription factor SKN-1C. These findings identify a novel role for SEM-4 in ROS detoxification by regulating expression of SKN-1C and the phase II detoxification genes.

Oxidative stress causes damage to cells by creating reactive oxygen species (ROS) and the overproduction of ROS have been linked to the onset of premature ageing. We previously found that a brap-2 (BRCA1 associated protein 2) mutant significantly increases the expression of phase II detoxification enzymes in C. elegans. An RNAi suppression screen to identify transcription factors involved in the production of gst-4 mRNA in brap-2 worms identified SEM-4 as a potential candidate. Here, we show that knockdown of sem-4 suppresses the activation of gst-4 caused by the mutation in brap-2. We also demonstrate that sem-4 is required for survival upon exposure to oxidative stress and that SEM-4 is required for expression of the transcription factor SKN-1C. These findings identify a novel role for SEM-4 in ROS detoxification by regulating expression of SKN-1C and the phase II detoxification genes. Article Summary: Reactive oxygen species have been implicated as a harmful agent in many age-related diseases as well as an important signaling molecule. The transcription factor SKN-1 in C. elegans is an important regulator of reactive oxygen species levels. Here we show that the transcription factor SEM-4 is required to activate the expression of skn-1c and promote phase II detoxification gene expression. These findings identify a novel role for SEM-4 in regulating reactive oxygen species levels.

The overproduction of reactive oxygen species (ROS) in cells can lead to the development of diseases associated with aging. We have previously shown that C. elegans BRAP-2 (Brca-1 associated binding protein 2) regulates phase II detoxification genes such as gst-4, by increasing SKN-1 activity. Previously, a transcription factor (TF) RNAi screen was conducted to identify potential activators that are required to induce gst-4 expression in brap-2(ok1492) mutants. The lipid metabolism regulator NHR-49/HNF4 was among 18 TFs identified. Here, we show that knockdown of nhr-49 suppresses the activation of gst-4 caused by brap-2 inactivation and that gain-of-function alleles of nhr-49 promote gst-4 expression. We also demonstrate that nhr-49 and its cofactor mdt-15 are required to express phase II detoxification enzymes upon exposure to chemicals that induce oxidative stress. Furthermore, we show that NHR-49 and MDT-15 enhance expression of skn-1a/c. These findings identify a novel role for NHR-49 in ROS detoxification by regulating expression of SKN-1C and phase II detoxification genes.

Background. Mutations in C. elegans can produce visible and quantifiable defects in morphology, lifespan, and development. BRAP2/IMP (BRCA1-associated binding protein 2) has been characterized as an E3 ubiquitin ligase, a general cytoplasmic retention factor, a potential scaffold protein, and is found to be widely expressed throughout various mammalian tissues, most highly in testes. However, its role in the development or health of these tissues has not been addressed. Results. The focus of this study is to determine the role of BRAP-2 in C. elegans germline development. We determined that brap-2 mutants display defects in germline morphology and a reduction in brood size. We also found that chromosomal abnormalities and embryonic lethality are elevated in brap-2 mutants following DNA damage, suggesting a potential role for BRAP-2 in facilitating DNA repair. Conclusions. Our findings indicate that BRAP-2 is required for C. elegans germline health and identifies a novel role for BRAP-2 in germline development.

Background. Mutations in C. elegans can produce visible and quantifiable defects in morphology, lifespan, and development. BRAP2/IMP (BRCA1-associated binding protein 2) has been characterized as an E3 ubiquitin ligase, a general cytoplasmic retention factor, a potential scaffold protein, and is found to be widely expressed throughout various mammalian tissues, most highly in testes. However, its role in the development or health of these tissues has not been addressed. Results. The focus of this study is to determine the role of BRAP-2 in C. elegans germline development. We determined that brap-2 mutants display defects in germline morphology and a reduction in brood size. We also found that chromosomal abnormalities and embryonic lethality are elevated in brap-2 mutants following DNA damage, suggesting a potential role for BRAP-2 in facilitating DNA repair. Conclusions. Our findings indicate that BRAP-2 is required for C. elegans germline health and identifies a novel role for BRAP-2 in germline development.

As part of the DNA damage response (DDR) network, the tumour suppressor Breast cancer susceptibility gene 1 (BRCA1) is activated to facilitate DNA repair, transcription and cell cycle control. BRC-1, the Caenorhabditis elegans ortholog of BRCA1, has conserved function in DNA double strand break repair, wherein a loss of brc-1 results in high levels of germline apoptosis. BRAP2/IMP was initially identified as a BRCA1 associated binding protein and previously we have shown that the C. elegans brap-2 deletion mutant experiences BRC-1 dependent larval arrest when exposed to low concentrations of paraquat. Since BRC-1 function in the germline is conserved, we wanted to determine the role of BRAP-2 in DNA damage induced germline apoptosis in C. elegans. We examined levels of germ cell death following DNA damage and found that brap-2(ok1492) mutants display reduced levels of germline apoptosis when compared to the wild type, and the loss of brap-2 significantly reduced germ cell death in brc-1 mutant animals. We also found increased mRNA levels of skn-1 following DNA damage in brap-2 mutants and that skn-1 RNAi knockdown in brap-2;brc-1 double mutants and a loss of pmk-1 mutation in brap-2 mutants increased apoptosis to wild type levels, indicating that brap-2 promotion of cell survival requires PMK-1 and SKN-1. Since mammalian BRAP2 has been shown to bind the AKT phosphatase PHLPP1/2, it suggests that BRAP2 could be involved in the Insulin/Insulin-like growth factor Signaling (IIS) pathway. We found that this interaction is conserved between the C. elegans homologs and that a loss of akt-1 in brap-2 mutants increased germline apoptosis. Thus in response to DNA damage, our findings suggest that BRAP-2 is required to attenuate the pro-cell survival signals of AKT-1 and PMK-1/SKN-1 to promote DNA damage induced germline apoptosis.

Cellular damage caused by reactive oxygen species is believed to be a major contributor to age-associated diseases. Previously, we characterized the Caenorhabditis elegans Brap2 ortholog (BRAP-2) and found that it is required to prevent larval arrest in response to elevated levels of oxidative stress. Here, we report that C. elegans brap-2 mutants display increased expression of SKN-1-dependent, phase II detoxification enzymes that is dependent on PMK-1 (a p38 MAPK C. elegans ortholog). An RNA-interference screen was conducted using a transcription factor library to identify genes required for increased expression of the SKN-1 target gst-4 in brap-2 mutants. We identified ELT-3, a member of the GATA transcription factor family, as a positive regulator of gst-4p::gfp expression. We found that ELT-3 interacts with SKN-1 to activate gst-4 transcription in vitro and that elt-3 is required for enhanced gst-4 expression in the brap-2(ok1492) mutant in vivo. Furthermore, nematodes overexpressing SKN-1 required ELT-3 for life-span extension. Taken together, these results suggest a model where BRAP-2 acts as negative regulator of SKN-1 through inhibition of p38 MAPK activity, and that the GATA transcription factor ELT-3 is required along with SKN-1 for the phase II detoxification response in C. elegans.