Cellular damage caused by reactive oxygen species is believed to be a major contributor to age-associated diseases. Previously, we characterized the 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 mutants display increased expression of SKN-1-dependent, phase II detoxification enzymes that is dependent on PMK-1 (a p38 MAPK ortholog). An RNA-interference screen was conducted using a transcription factor library to identify genes required for increased expression of the SKN-1 target in mutants. We identified ELT-3, a member of the GATA transcription factor family, as a positive regulator of:: expression. We found that ELT-3 interacts with SKN-1 to activate transcription and that is required for enhanced expression in the mutant 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 .
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 (Brca1 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.
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.
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