SKN-1-dependent oxidative stress response in C. elegans
ABSTRACT: Oxidative stress may play a role in normal aging. SKN-1 is a transcription factor necessary for intestine development in Caenorhabditis elegans, which also regulates the response to oxidative stress post-embryonically. Using DNA microarrays, we found that oxidative stress induces the antioxidant response, the heat shock response, and detoxification genes, while the expression of genes involved in homeostasis, development, and reproduction were decreased. Both up-regulated and down-regulated genes can be wholly, partially, or not at all dependent on SKN-1 action. However, induction of the heat shock response by oxidative stress was not affected by SKN-1 removal. Keywords: stress response Overall design: TJ1060 was grown at 16°C. Eggs were collected by hypochlorite, hatched overnight at 20°C (Emmons, 1979 #52) and first-stage larvae (L1) were placed onto 2 % peptone plates (1.7 % agar, 20 g/l peptone, 25 mM NaCl, 50 mM KH2PO4 pH 6.0, 5 ug/ml cholesterol, 1 mM CaCl2, 1 mM MgSO4) at 25°C. Sterile 3 day old young adult animals on peptone plates were treated with 99 % O2 at 40 PSI for 6 hours at 20°C in a hyperbaric O2 chamber. Half of the worms were untreated and served as a control population. A small plate of strain CL2166 [dvIs19 pAF15(gst-4::GFP::NLS)] was used as an indicator strain and checked for GFP induction in both control and experimental conditions. Worms were harvested by washing off plates with S-basal and quick frozen in liquid nitrogen. Total RNA was prepared using TRIZOL reagent and standard protocols and cleaned up on Qiagen RNeasy columns. The Affymetrix labeling kit was used to prepare labeled cRNA as a target for the Affymetrix GeneChip C.elegans Genome Array. Gene chip hybridization and scanning were done at Genome Explorations (Memphis, MN) and the University of Colorado at Boulder Gene Chip Core Facility (Boulder, CO).
INSTRUMENT(S): [Celegans] Affymetrix C. elegans Genome Array
Project description:Oxidative stress may play a role in normal aging. SKN-1 is a transcription factor necessary for intestine development in Caenorhabditis elegans, which also regulates the response to oxidative stress post-embryonically. Using DNA microarrays, we found that oxidative stress induces the antioxidant response, the heat shock response, and detoxification genes, while the expression of genes involved in homeostasis, development, and reproduction were decreased. Both up-regulated and down-regulated genes can be wholly, partially, or not at all dependent on SKN-1 action. However, induction of the heat shock response by oxidative stress was not affected by SKN-1 removal. Experiment Overall Design: TJ1060 was grown at 16 C. Eggs were collected by hypochlorite, hatched overnight at 20 C (Emmons, 1979 #52) and first-stage larvae (L1’s) were placed onto 2 % peptone plates (1.7 % agar, 20 g/l peptone, 25 mM NaCl, 50 mM KH2PO4 pH 6.0, 5 µg/ml cholesterol, 1 mM CaCl2, 1 mM MgSO4) at 25 C. Sterile 3 day old young adult animals on peptone plates were treated with 99 % O2 at 40 PSI for 6 hours at 20 C in a hyperbaric O2 chamber. Half of the worms were untreated and served as a control population. A small plate of strain CL2166 [dvIs19 pAF15(gst-4::GFP::NLS)] was used as an indicator strain and checked for GFP induction in both control and experimental conditions. Worms were harvested by washing off plates with S-basal and quick frozen in liquid nitrogen. Total RNA was prepared using TRIZOL reagent and standard protocols and cleaned up on Qiagen RNeasy columns. The Affymetrix labeling kit was used to prepare labeled cRNA as a target for the Affymetrix GeneChip® C.elegans Genome Array. Gene chip hybridization and scanning were done at Genome Explorations (Memphis, MN) and the University of Colorado at Boulder Gene Chip Core Facility (Boulder, CO).
Project description:Reactive Oxygen Species increase gradually with aging and Steadily diminish the cell's ability to maintain homeostasis. Nuclear Factor-like 2 and its C elegans ortholog, SKN-1 are transcription factors that play a pivotal role in oxidative stress response, cellular homeostasis and lifespan. But like other defence systems, Nrf2-mediated stress response is compromised in aging and neurodegenerative diseases. In this study we provide evidence that this FDA-approved drug is a bona fide activator of Nrf2/SKN-1 pathway.
Project description:Hormesis occurs when a low level stress elicits adaptive beneficial responses that protect against subsequent exposure to severe stress. Recent findings suggest that mild oxidative and thermal stress can extend lifespan by hormetic mechanisms. Here we show that the botanical pesticide plumbagin, while toxic to C. elegans nematodes at high doses, extends lifespan at low doses. Because plumbagin is a naphthoquinone that generates free radicals in vivo, we investigated whether it extends lifespan by activating an adaptive cellular stress response pathway. Mammalian NF-E2-related factor 2 (Nrf2) and its C. elegans ortholog SKN-1, mediate protective responses to oxidative stress by promoting target gene expression via antioxidant response elements (ARE). Genetic analyses showed that skn-1 mediates plumbagin’s lifespan-extending effect in C. elegans. Further screening of a series of plumbagin analogs identified three additional naphthoquinones that could induce SKN-1 targets in C. elegans. Naphthazarin showed skn-1-dependent lifespan extension, over an extended dose range compared to plumbagin, while the other naphthoquinones, oxoline and menadione, had differing effects on C. elegans survival and failed to activate ARE reporter expression in cultured mammalian cells. Our findings reveal the potential for low doses of naturally occurring naphthoquinones to extend lifespan by engaging a specific adaptive cellular stress response pathway. Overall design: Approximately 1,000 age-synchronized young adults were treated for two days with either 100 µM plumbagin or DMSO as a vehicle control and then washed three times in M9 and flash frozen with dry ice. RNA was isolated with the Absolutely RNA Miniprep Kit (Agilent Technologies, USA). RNA concentration and quality was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, USA). Microarray hybridizations were performed with the C. elegans 4x44K Oligo Microarray (Agilent Technologies, USA). Raw microarray hybridization intensity data from four separate experiments were log-transformed and normalized to generate z-scores and subsequent z-ratios (Cheadle et al. 2003).
Project description:Gene BbMBF1 playes an essential role in fungal response to oxidative stress, a determinant to the biocontrol potential of entomopathogenic fungi. The genome-wide exprssion analysis involved in stress response was analyzed by using high throughput sequencing (RNA-Seq). Our transcriptional profiles revealed that numerous differentially expressed genes (DEGs), of which involved in metabolism, cell transport and cell rescue, were significantly controlled by BbMBF1 during response to oxidative stress. Overall design: Total RNA obtained from BbMBF1 disruption mutant were compared to that of wild type strain (WT) grown in SDB mediua (Glucose 4%, Peptone 1%, and Yeast extract 1%).
Project description:The Caenorhabditis elegans oxidative stress response transcription factor, SKN-1, is essential for the maintenance of redox homeostasis and is a functional ortholog of the Nrf family of transcription factors. The numerous levels of regulation that govern these transcription factors underscore their importance. Here, we add a thioredoxin, encoded by trx-1, to the expansive list of SKN-1 regulators. We report that loss of trx-1 promotes nuclear localization of intestinal SKN-1 in a redox-independent, cell non-autonomous fashion from the ASJ neurons. Furthermore, this regulation is not general to the thioredoxin family, as two other C. elegans thioredoxins TRX-2 and TRX-3 do not play a role in this process. Moreover, TRX-1-dependent regulation requires signaling from the p38 MAPK signaling pathway. However, while TRX-1 regulates SKN-1 nuclear localization, SKN-1 transcriptional activity remains largely unaffected. Interestingly, RNA-Seq revealed that loss of trx-1 elicits a general, organism-wide down-regulation of several classes of genes; those encoding for collagens and lipid transport and localization being most prevalent. However, one prominent lipase-related gene, lips-6, is highly up regulated upon loss of trx-1 in a skn-1-dependent manner. Together, these results uncover a novel role for a thioredoxin in regulating intestinal SKN-1 nuclear localization in a cell non-autonomous manner, thereby contributing to the understanding of the processes involved in maintaining redox homeostasis throughout an organism. Four samples were analyzed: Two nematode strains were analyzed, each under non-stressed and stressed (10mM NaAs) conditions
Project description:In pregnancies involving preeclampsia (PE), there is evidence that the fetal-placental unit is under oxidative stress. Here we examined primary cell lines generated from umbilical cords (UC) delivered by mothers who had either a normal pregnancy or experienced early onset PE to determine whether the two had distinguishable phenotypes. While all UC provided outgrowths when established in 4 % O2, success was less assured for PE cords under ambient (20 % O2) conditions (P < 0.05). Moreover, proliferation rates of established PE lines, although similar to controls in 4 % O2, were significantly lower in 20 % O2. PE lines grown in 4 % O2 were also more susceptible to the pro-oxidant diethylmaleate than control lines, and unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few differentially regulated genes between PE lines and controls in 4 % O2 conditions, but confirmed the more severely stressed phenotype of the PE lines under 20 % O2. We conclude that the primary UC cell lines generated from PE births maintain a susceptibility to oxidative stress that is stable over many cell divisions, but whether the basis of this vulnerability is genetic or epigenetic remains unclear. RNA was isolated from early passages (< p5) of UC fibroblast lines (15 PE and 9 CTL lines, grown in T25 flasks) when they reached ~90% confluency in 4 % O2 conditions. In order to collect RNA from cells under 20 % O2, cell lines at either p 4, 5, or 6 were switched from 4 % O2 conditions to 20 % O2 conditions when they were approximately 20 % confluent. When they reached ~90% confluency (generally 2 days in 4 % O2 and 3-4 days in 20 % O2 conditions), medium was removed and RNA STAT60 (I ml; Tel-Test, Friendswood, TX) was immediately added to each T25 flask and RNA extracted by following the manufacturer’s instructions. The samples of RNA were submitted to University Texas Southwestern Medical Center Microarray Core Facility (https://microarray.swmed.edu/) and microarray analysis performed with Illumina HumanHT-12 v4 expression BeadChips. Raw intensity data were background subtracted by using BeadStudio software and analyzed further by GeneSpring 12.6 software (Agilent Technologies Inc., Santa Clara CA), according to the advanced workflow protocol: percentile shift and filter by flags (detected).
Project description:Vitamin D is a secosteroid that has multiple regulatory roles including the regulation of bone and calcium homeostasis. Deficiency of 25-hydroxyvitamin D, the major circulating form of vitamin D, is associated with an increased risk of age-related chronic diseases including Alzheimer’s disease, Parkinson’s disease, cognitive impairment, and cancer. In this study, we utilized Caenorhabditis elegans to examine the mechanism by which vitamin D influences aging. We found that Vitamin D3-induced lifespan extension requires the stress response pathway genes SKN-1, IRE-1, and XBP-1. Vitamin D3 induced expression of SKN-1 target genes, but not canonical targets of IRE-1/XBP-1. Vitamin D3 suppressed an important molecular pathology of aging, that of widespread protein insolubility, and prevented the toxicity caused by human β-amyloid. Our observation that vitamin D3 improves protein homeostasis and slows aging highlights the importance of maintaining appropriate vitamin D serum levels, and may explain why such a wide variety of human age-related diseases are associated with a vitamin D deficiency. Overall design: The experimental design consisted of contrasting gene expression data derived from RNA extracted from pools of synchronized aged worms. L4 worms were placed on either vehicle (DMSO) or Vitamin D (100uM) for 44 hours prior to extraction. A pool of 50 worms was considered a single biological replicate. For the Vitamin D treated group, there were 6 independent biological replicates, and were compared with a group of untreated (vehicle) wild-type N2 animals, also using 6 biological replicates.
Project description:Analysis of constitutive changes in gene expression patterns in populations of the nematode Caenorhabditis remanei experimentally evolved to either acute heat stress, acute oxidative stress or the lab environment. Results of this study demonstrate that acute heat stress and acute oxidative stress are complex traits with a unique genetic basis. Overall design: Pooled population RNA sequencing libraries were obtained from the ancestor population and three evolved populations: one biological replicate of the control selected population, one heat stress selected population and one oxidative stress selected population per replicate. Tissue was collected from populations of L1 larval worms. Overall design: mRNA profiles of ancestral and three experimentally evolved populations of C. remanei at 20°C, 6- 8 replicates/evolved line for each population
Project description:Corynebacterium glutamicum was adapted in a chemostat for 1,900 h with gradually increasing H2O2 stress to understand the oxidative stress response of an industrial host. After 411 generations of adaptation, C. glutamicum developed the ability to grow under stress of 10 mM H2O2, whereas the wild-type did not. The adapted strain also showed increased stress resistance to diamide and menadione, SDS, Tween 20, HCl, NaOH, and ampicillin. A total of 1,180 genes in the RNA-seq transcriptome analysis of the adapted strain were up-regulated twice or higher (corresponding to 38.6 % of the genome), and 126 genes were down-regulated half or less (4.1 % of genome) under 10 mM H2O2-stress conditions compared with those of the wild-type under a no stress condition. Especially the aromatic compound-degrading gene clusters (vanRABK, pcaJIRFLO, and benABCDRKE) were more than threefold up-regulated. Plausible reasons for the H2O2-stress tolerance of the adapted strain are discussed as well as the potential strategy for development of oxidative stress-tolerant strain. Comparison of global gene expression profiling using RNA-seq data of wild-type strain under normal condition (WT_MCGC) vs. 10 mM H2O2-adapted strain under oxidative stress condition Global gene expression profiling_WT vs. H2O2 adapted C. glutamicum strains