Comparative analysis of the molecular and physiological consequences of constitutive SKN-1 activation

Abstract

Molecular homeostats play essential roles across all levels of biological organization to ensure a return to normal function after responding to abnormal internal and environmental events. SKN-1 is an evolutionarily conserved cytoprotective transcription factor that is integral for the maintenance of cellular homeostasis upon exposure to a variety of stress conditions. Despite the essentiality of turning on SKN-1/NRF2 in response to exogenous and endogenous stress, animals with chronic activation of SKN-1 display premature loss of health with age, and ultimately, diminished lifespan. Previous genetic models of constitutive SKN-1 activation include gain-of-function alleles of skn-1 and loss-of-function alleles of wdr-23 that impede the turnover of SKN-1 by the ubiquitin proteasome. Here, we define a novel gain-of-function mutation in the xrep-4 locus that results in constitutive activation of SKN-1 in the absence of stress. Although each of these genetic mutations results in continuously unregulated transcriptional output from SKN-1, the physiological consequences of each model on development, stress resistance, reproduction, lipid homeostasis, and lifespan are distinct. Here, we provide a comprehensive assessment of the differential healthspan impacts across multiple models of constitutive SKN-1 activation. Although our results reveal the universal need to reign in the uncontrolled activity of cytoprotective transcription factors, we also define the unique signatures of each model of constitutive SKN-1 activation, which provides innovative solutions for the design of molecular "off-switches" of unregulated transcriptional homeostats.

Keywords: Aging; C. elegans; Cytoprotection; Proteostasis; Transcription; skn-1.

Fig. 1

Identification of xrep-4gf mutation that induces gst-4 expression. A Cartoon model of EMS screen for activators of the gst-4p::GFP reporter. B Whole-genome sequencing and InDel mapping identifies genetic linkage of lax137 to the middle of chromosome I, as marked by dashed lines. Amino acid substitution in xrep-4gf(lax137) marked by the arrow. C–F RNAi of xrep-4 suppresses the observed gst-4p::GFP activation in lax137;gst-4p::GFP animals. F. Ectopic expression of xrep-4gf phenocopies the increased expression of gst-4p::GFP (G) as compared to non-transgenic siblings (NTS) (H). I Phyre2 predicted structure of XREP-4 WT (cyan), with amino acid substitution in xrep-4gf (purple)

Fig. 2

xrep-4gf mutants activate SKN-1 transcription. A Volcano plot of differentially expressed genes in xrep-4gf mutants compared to wild type (WT) animals, all genes have a p-value < 0.01. GO-term enrichment of xrep-4gf genes are associated with activated SKN-1. B 1796 genes are upregulated in both xrep-4gf and skn-1gf mutants that are associated with expression in foraging behaviors, lipid metabolism, and the sensory nervous systems. SKN-1wt-GFP expression is restricted to ASI neurons (arrows) in WT (C) and xrep-4gf mutants (D). As compared to control RNAi (E), RNAi of skn-1 reduces xrep-4gf(lax137) activation of the gst-4p::gfp reporter (F)

Fig. 3

Models of constitutive SKN-1 activation. A Genetic pathway for xrep-4 regulation of skn-1 survival responses. B skn-1gf and xrep-4gf mutants at day 1 adulthood display enhanced resistance to acute hydrogen peroxide exposure. Statistical comparisons by one-way ANOVA test. **p < 0.005. n=150–200; N=3 per condition. C skn-1gf and xrep-4gf animals display shortened maximal lifespan (4th quartile) relative to WT, while xrep-4gf animals have a median lifespan more similar to WT controls

Fig. 4

xrep-4gf mutants display age-dependent somatic depletion of fat (Asdf). A–F Representative images of Nile Red fixed-staining of age-matched L4-stage animals and total stored lipids quantified G Statistical comparisons by one-way ANOVA test. ****p < 0.0001. n=50; N=3 per condition. Intracellular lipid distribution in day 3 constitutive SKN-1 activation mutants via Oil Red O (ORO) staining (H–J) and quantification of the percentage of the population displaying non-Asdf, intermediate, and Asdf phenotypes at day 3 (K) and day 5 (L) of adulthood. n=50–100; N=3 per condition. CTCF: corrected total cell fluorescence

Fig. 5

Constitutive activation of SKN-1 drives premature loss of muscle function. A–D Movement is progressively impaired in skn-1gf and xrep-4gf mutants at L4 and day 3 of adulthood. Statistical comparisons by one-way ANOVA test. *p<0.05, *** p<0.0005, **** p<0.0001. n=30–50; N=3 per condition