The Oxidative Stress Response in Caenorhabditis elegans Requires the GATA Transcription Factor ELT-3 and SKN-1/Nrf2.
ABSTRACT: 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.
Project description: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.
Project description:Extracellular matrix barriers and inducible cytoprotective genes form successive lines of defense against chemical and microbial environmental stressors. The barrier in nematodes is a collagenous extracellular matrix called the cuticle. In Caenorhabditis elegans, disruption of some cuticle collagen genes activates osmolyte and antimicrobial response genes. Physical damage to the epidermis also activates antimicrobial responses. Here, we assayed the effect of knocking down genes required for cuticle and epidermal integrity on diverse cellular stress responses. We found that disruption of specific bands of collagen, called annular furrows, coactivates detoxification, hyperosmotic, and antimicrobial response genes, but not other stress responses. Disruption of other cuticle structures and epidermal integrity does not have the same effect. Several transcription factors act downstream of furrow loss. SKN-1/Nrf and ELT-3/GATA are required for detoxification, SKN-1/Nrf is partially required for the osmolyte response, and STA-2/Stat and ELT-3/GATA for antimicrobial gene expression. Our results are consistent with a cuticle-associated damage sensor that coordinates detoxification, hyperosmotic, and antimicrobial responses through overlapping, but distinct, downstream signaling.
Project description:GATA transcription factors play important roles in directing developmental genetic programs and cell differentiation, and are conserved in animals, plants and fungi. C. elegans has 11 GATA-type transcription factors that orchestrate development of the gut, epidermis and vulva. However, the expression of certain GATA proteins persists into adulthood, where their function is less understood. Accumulating evidence demonstrates contributions of 2 terminal differentiation GATA transcription factors, ELT-2 and ELT-3, to epithelial immune responses in the adult intestine and epidermis (hypodermis), respectively. Involvement in other stress responses has also been documented. We recently showed that ELT-2 acted as a tissue-specific master regulator, cooperating with 2 transcription factors activated by the p38 pathway, ATF-7 and SKN-1, to control immune responses in the adult C. elegans intestine. Here, we discuss the broader implications of these findings for understanding the involvement of GATA transcription factors in adult stress responses, and draw parallels between ELT-2 and ELT-3 to speculate that the latter may fulfill similar tissue-specific functions in the epidermis.
Project description: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.
Project description:GATA transcription factors play a crucial role in the regulation of immune functions across metazoans. In Caenorhabditis elegans, the GATA transcription factor ELT-2 is involved in the control of not only infections but also recovery after an infection. We identified RPT-6, part of the 19S proteasome subunit, as an ELT-2 binding partner that is required for the proper expression of genes required for both immunity against bacterial infections and recovery after infection. We found that the intact ATPase domain of RPT-6 is required for the interaction and that inhibition of rpt-6 affected the expression of ELT-2-controlled genes, preventing the appropriate immune response against Pseudomonas aeruginosa and recovery from infection by the pathogen. Further studies indicated that SKN-1, which is an Nrf transcription factor involved in the response to oxidative stress and infection, is activated by inhibition of rpt-6. Our results indicate that RPT-6 interacts with ELT-2 in vivo to control the expression of immune genes in a manner that is likely independent of the proteolytic activity of the proteasome.
Project description:The mechanisms involved in the recognition of microbial pathogens and activation of the immune system have been extensively studied. However, the mechanisms involved in the recovery phase of an infection are incompletely characterized at both the cellular and physiological levels. Here, we establish a Caenorhabditis elegans-Salmonella enterica model of acute infection and antibiotic treatment for studying biological changes during the resolution phase of an infection. Using whole genome expression profiles of acutely infected animals, we found that genes that are markers of innate immunity are down-regulated upon recovery, while genes involved in xenobiotic detoxification, redox regulation, and cellular homeostasis are up-regulated. In silico analyses demonstrated that genes altered during recovery from infection were transcriptionally regulated by conserved transcription factors, including GATA/ELT-2, FOXO/DAF-16, and Nrf/SKN-1. Finally, we found that recovery from an acute bacterial infection is dependent on ELT-2 activity.
Project description:GATA transcription factors play critical roles in cellular differentiation and development. However, their roles in mature tissues are less understood. In C. elegans larvae, the transcription factor ELT-2 regulates terminal differentiation of the intestine. It is also expressed in the adult intestine, where it was suggested to maintain intestinal structure and function, and where it was additionally shown to contribute to infection resistance. To study the function of elt-2 in adults we characterized elt-2-dependent gene expression following its knock-down specifically in adults. Microarray analysis identified two ELT-2-regulated gene subsets: one, enriched for hydrolytic enzymes, pointed at regulation of constitutive digestive functions as a dominant role of adult elt-2; the second was enriched for immune genes that are induced in response to Pseudomonas aeruginosa infection. Focusing on the latter, we used genetic analyses coupled to survival assays and quantitative RT-PCR to interrogate the mechanism(s) through which elt-2 contributes to immunity. We show that elt-2 controls p38-dependent gene induction, cooperating with two p38-activated transcription factors, ATF-7 and SKN-1. This demonstrates a mechanism through which the constitutively nuclear elt-2 can impact induced responses, and play a dominant role in C. elegans immunity.
Project description:Caenorhabditis elegans NRF (NF-E2-related factor)/CNC (Cap'n'collar) transcription factor, Skinhead-1 (SKN-1), is conservatively critical for promoting phase II detoxification gene expressions in response to oxidative stress. SKN-1 activity is controlled by well-known phosphorylation and recently-reported O-GlcNAcylation. Whether other kinds of posttanslational modifications of SKN-1 occur and influence its function remains elusive. Here, we found arginines 484 and 516 (R484/R516) of SKN-1 were asymmetrically dimethylated by PRMT-1. Oxidative stress enhanced the binding of PRMT-1 to SKN-1. Consequently, asymmetrical dimethylation of arginines on SKN-1 was elevated. Loss of prmt-1 or disruption of R484/R516 dimethylation decreased the enrichment of SKN-1 on the promoters of SKN-1-driven phase II detoxification genes, including gamma-glutamine cysteine synthetase gcs-1, glutathione S-transferases gst-7 and gst-4, which resulted in reduced ability of worms to defense against oxidative stress. These findings have important implications for investigating the physiological and pathological functions of arginine methylation on conserved NRF/CNC transcription factors in human diseases related to oxidative stress response.
Project description:Oxidative damage contributes to human diseases of aging including diabetes, cancer, and cardiovascular disorders. Reactive oxygen species resulting from xenobiotic and endogenous metabolites are sensed by a poorly understood process, triggering a cascade of regulatory factors and leading to the activation of the transcription factor Nrf2 (Nuclear factor-erythroid-related factor 2, SKN-1 in Caenorhabditis elegans). Nrf2/SKN-1 activation promotes the induction of the phase II detoxification system that serves to limit oxidative stress. We have extended a previous C. elegans genetic approach to explore the mechanisms by which a phase II enzyme is induced by endogenous and exogenous oxidants. The xrep (xenobiotics response pathway) mutants were isolated as defective in their ability to properly regulate the induction of a glutathione S-transferase (GST) reporter. The xrep-1 gene was previously identified as wdr-23, which encodes a C. elegans homolog of the mammalian ?-propeller repeat-containing protein WDR-23 Here, we identify and confirm the mutations in xrep-2, xrep-3, and xrep-4 The xrep-2 gene is alh-6, an ortholog of a human gene mutated in familial hyperprolinemia. The xrep-3 mutation is a gain-of-function allele of skn-1 The xrep-4 gene is F46F11.6, which encodes a F-box-containing protein. We demonstrate that xrep-4 alters the stability of WDR-23 (xrep-1), a key regulator of SKN-1 (xrep-3). Epistatic relationships among the xrep mutants and their interacting partners allow us to propose an ordered genetic pathway by which endogenous and exogenous stressors induce the phase II detoxification response.
Project description:The short, asymmetrical DNA sequence to which the vertebrate GATA family of transcription factors binds is present in some Caenorhabditis elegans gene regulatory regions: it is required for activation of the vitellogenin genes and is also found just 5' of the TATA boxes of tra-2 and the msp genes. In vertebrates GATA-1 is specific to erythroid lineages, whereas GATA-2 and GATA-3 are present in multiple tissues. In an effort to identify the trans-acting factors that may recognize this sequence element in C. elegans, we used a degenerate oligonucleotide to clone a C. elegans homolog to this gene. We call this gene elt-1 (erythrocytelike transcription factor). It is single copy and specifies a 1.75-kb mRNA that is present predominantly, if not exclusively, in embryos. The region of elt-1 encoding two zinc fingers is remarkably similar to the DNA-binding domain of the vertebrate GATA-binding proteins. However, outside of the DNA-binding domains the amino acid sequences are quite divergent. Nevertheless, introns are located at identical or nearly identical positions in elt-1 and the mouse GATA-1 gene. In addition, elt-1 mRNA is trans-spliced to the 22-base untranslated leader, SL1. The DNA upstream of the elt-1 TATA box contains eight copies of the GATA recognition sequence within the first 300 bp, suggesting that elt-1 may be autogenously regulated. Our results suggest that the specialized role of GATA-1 in erythroid gene expression was derived after separation of the nematodes and the line that led to the vertebrates, since C. elegans lacks an erythroid lineage.