41 Results
for author "Yue-wen Wang"
Figure 1Mutations to the xenobiotics response pathway-4 (xrep-4) gene inhibit activation of gst-4. (A) Schematic of the xrep-4 locus marked with the F-box domain and mutations identified in this study. (B) Summary of six xrep-4 EMS alleles identified. (C) Activation of the Skinhead family member-1 (SKN-1)-dependent gst-4p::GFP reporter in wild-type or xrep-4(zj26) mutant worms when exposed to prooxidants juglone or acrylamide. gst-4p::GFP scoring was as described in Materials and Methods, ***P < 0.001, Chi-square tests from 103 to 105 worms scored per condition for one trial. Representative images are shown. Bar, 100 μM.
Figure 2xrep-4 is required for induction of detoxification genes upstream of wdr-23 and skn-1. (A) Knockdown of xrep-4 by RNAi phenocopies xrep-4(zj26). ***P < 0.001 Chi-square tests from 50 to 70 worms screened per condition for one trial. Representative images are shown. Bar, 100 μM. (B) Knockdown of xrep-4 does not reduce constitutive activation of gst-4p::GFP in wdr-23(tm1817) loss-of-function or skn-1(k1023) gain-of-function mutants. A total of 35–56 worms were screened per condition for one trial. (C) Fold change in mRNA levels of SKN-1 downstream target genes in N2 control worms fed xrep-4 dsRNA, xrep-4(zj26) mutants fed control dsRNA under basal conditions, or after exposure to juglone. **P < 0.01, ***P < 0.001 compared to N2;control(RNAi) as determined by two-way ANOVA with Bonferroni post hoc tests. (D) Expression of SKN-1 target genes can be rescued in the xrep-4(zj26) mutant by an extrachromosomal array carrying xrep-4 genomic DNA (zjEx121). ***P < 0.001 compared to N2;control(RNAi) as determined by two-way ANOVA with Bonferroni post hoc tests. N = 4 replicates of 200–300 worms. dsRNA, double-stranded RNA; mRNA, messenger RNA; RNAi, RNA interference; skn-1, Skinhead family member-1; xrep-4, xenobiotics response pathway-4.
Figure 3Loss of xrep-4 reduces C. elegans resistance to multiple stressors, but does not shorten normal life span. (A) Longevity curve for wild-type and xrep-4(zj26) mutant worms. Results for trial #1 are shown (full results and statistics are in Table S1 in File S1). (B) Survival curve of wild-type and xrep-4(zj26) mutant worms exposed to 175 µM juglone. ***P < 0.001 Log-rank test compared to N2. Results for trial #1 are shown (full results and statistics in Table S1 in File S1). (C) Survival curve of wild-type and xrep-4(zj26) mutant worms exposed to 7 mM acrylamide. ***P < 0.001 Log-rank test compared to N2. Results for trial #5 are shown (full results and statistics are in Table S2 in File S1). (D) Survival curve of wild-type and skn-1(k1023) worms fed control or xrep-4 dsRNA and exposed to 7 mM acrylamide; all conditions were P < 0.001 Log-rank test compared to N2; control(RNAi), results for trial #1 are shown (full results and statistics in Table S2 in File S1). (E) Representative images of normal and coiler worm phenotypes induced by acrylamide. Bar, 100 μM. (F) Percentage of worms displaying a coiler phenotype at days 3, 4, and 5 of adulthood after initiating exposure to 7 mM acrylamide on day 1. ***P < 0.001 two-way ANOVA with Bonferroni post hoc tests. Shown are combined results from three to six trials for each condition, with >30 worms per trial. RNAi, RNA interference; xrep-4, xenobiotics response pathway-4.
Figure 4Overexpression of xrep-4 can activate SKN-1 downstream genes and increase oxidative stress resistance. ***P < 0.001 compared to N2 as determined by Student's t-test. (A) Levels of xrep-4 mRNA in a strain carrying an extrachromosomal array expressing the full length xrep-4 genomic DNA. (B) Overexpression of xrep-4 in a wild-type background increases basal levels of gst-4p::GFP. myo-2p::tdTomato was used as an injection comarker; representative images are shown. Bar, 100 μM. (C) Fold change in mRNA levels of SKN-1 downstream target genes under basal and juglone conditions in wild-type and xrep-4 overexpression strains. *P < 0.05 and ***P < 0.001 compared to N2 as determined by two-way ANOVA with Bonferroni post hoc tests. N = 4 replicates of 200–300 worms. (D) Survival curve of wild-type and xrep-4 OE worms exposed to 175 μM of juglone. Results from trial #1 are shown, ***P < 0.001 Log-rank test compared to N2. (E) Longevity curve of wild-type and xrep-4 OE worms. Results for trial #3 are shown. (D and E) Results for all trials are in Table S3 in File S1. Two other independent lines were generated for the xrep-4 OE strain that showed similar results (Figure S1 in File S1). mRNA, messenger RNA; OE, overexpressing; RNAi, RNA interference; SKN-1, Skinhead family member-1; xrep-4, xenobiotics response pathway-4.
Figure 5Epistatic analysis of xrep-4 with known regulators of SKN-1. (A) Activation of gst-4p::GFP upon knockdown of SKN-1 upstream regulators in wild-type or xrep-4(zj26) mutant backgrounds. Shown are representative images and categorical scoring of each RNAi. Bar, 100 μM. *P < 0.05 and ***P < 0.001 as determined by the Chi-square test. Shown are results from one trial with a total of 91–226 worms scored for each condition. (B) Relative levels of gst-4 mRNA in N2 and daf-2(e1370) mutants fed with control or xrep-4 dsRNA. ***P < 0.001 as determined by one-way ANOVA and Tukey’s post hoc test. N = 4 replicates of 200–300 worms per condition. (C) Longevity curve of wild-type and daf-2(e1370) worms fed with either control or xrep-4 dsRNA. Knockdown of xrep-4 had no effect on life span in either the daf-2(e1037) or N2 wild-type strain. Trial #3 is shown for both strains, with statistics for all trials shown in Table S4 in File S1. dsRNA, double-stranded RNA; mRNA, messenger RNA; ns, not significant; RNAi, RNA interference; SKN-1, Skinhead family member-1; xrep-4, xenobiotics response pathway-4.
Figure 6XREP-4 interacts with SKR-1 and WDR-23. (A) XREP-4 interacts with WDR-23 and SKR-1 when coexpressed in HEK293 cells. (B) Knockdown of skr-1 by RNAi reduces expression of SKN-1 downstream genes in response to juglone in N2 wild-type worms. All conditions are significantly different from N2; control(RNAi) with ***P < 0.001 as determined by ANOVA Bonferroni test; xrep-4(zj26);control(RNAi) and xrep-4(zj26);skr-1(RNAi) are not significantly different. N = 4 replicates of 200–300 worms. ns, not significant; RNAi, RNA interference; SKN-1, Skinhead family member-1; SKR, Skp1-related; xrep-4, xenobiotics response pathway-4.
Figure 7A mutation that disrupts XREP-4 and SKR-1 interaction can inhibit the oxidative stress response. (A) Schematic and DNA chromatogram of the xrep-4(zj28) mutation that causes a predicted proline to leucine single amino acid substitution at position 20 in XREP-4. (B) XREP-4 carrying the P20L mutation has normal interactions with WDR-23, but reduced binding to SKR-1 when coexpressed in HEK293 cells. (C) The xrep-4(zj28) mutation inhibits activation of gst-4p::GFP upon exposure to juglone or acrylamide. ***P < 0.001 Chi-square test with a total of 99–109 worms scored per condition for one trial. Representative images are shown. Bar, 100 μM. (D) Fold change in SKN-1 downstream target mRNA levels in the xrep-4(zj28) mutant compared to N2 wild-type under basal and juglone exposure, P < 0.05 and ***P < 0.001 compared to N2 as determined by two-way ANOVA with Bonferroni post hoc tests. N = 4 replicates of 200–300 worms. (E) SKR-1, XREP-4, and WDR-23 form a protein complex when coexpressed in HEK293 cells. Coexpression of XREP-4 enhances the interaction between SKR-1 and WDR-23, and this interaction is reduced by substituting the wild-type XREP-4 protein with the P20L mutated variant. mRNA, messenger RNA; SKN-1, Skinhead family member-1; SKR, Skp1-related; xrep-4, xenobiotics response pathway-4.
Figure 8xrep-4 negatively regulates the expression level and nuclear localization of the WDR-23::GFP fusion protein. (A and B) Knockdown of xrep-4 by RNAi increases the overall GFP fluorescence of worms carrying the integrated WDR-23::GFP translational fusion protein; ***P < 0.001 compared to control as determined by Student’s t-test. N = 7 slides containing a total of 69–70 worms; representative images are shown. Bar, 100 μM. (C and D) Knockdown of xrep-4 increased the total number of worms with visible nuclear accumulation of the WDR-23::GFP protein compared to worms fed with control dsRNA. Arrowheads indicates WDR-23::GFP in the hypodermal nuclei. A total of 69–70 worms were scored for each condition from two trials, ***P < 0.001 Chi-square test. Representative images are shown. Bar, 25 μM. (E) Proposed model of XREP-4 regulation of the SKN-1 oxidative stress response. XREP-4 serves as an F-box protein that together with ubiquitin ligase adapter SKR-1 regulates WDR-23, which is a principal suppressor of SKN-1. Loss of either SKR-1 (Wu et al. 2016) or XREP-4 attenuates the SKN-1-dependent response to oxidative stress. XREP-4 appears to regulate SKN-1 downstream of DAF-2, TORC1, GSK-3, and CCT-1, but could function upstream or independent of the proteasome genes pas-5 and pas-6. CCT-1, chaperonin-containing TCP-1; TORC1, target of rapamycin complex 1; dsRNA, double-stranded RNA; GSK-3, glycogen synthase kinase-3; RNAi, RNA interference; Skinhead family member-1; SKR, Skp1-related; xrep-4, xenobiotics response pathway-4.
Figure 1Protein interactions between PPE and PCP core proteins. (A and C) Yeast-two-hybrid plates for interaction between Fy and Dsh (A), Frtz and Dsh (C). The plates are 4 aa (Ade/-His/-Leu/-Trp) drop out plates containing x-α-gal. Only the colonies with interacting protein pairs grow and turn blue. (B and D) Co-immunoprecipitation results for Fy and Dsh (B) Frtz and Dsh (D), respectively. (B) The co-immunoprecipitation of Dsh and Fy. This experiment used UAS-fy-GFP driven by ptc-Gal4 and dsh-myc transgenes (Bloomington Drosophila Stock Center stock number 25385). Wing disc samples were immunoprecipitated using rabbit GFP antibody then detected with mouse GFP and Myc antibody for western blotting. Arrows point to Fy-GFP and dsh-myc on the Western blots. Part D shows the co-immunoprecipitation of Dsh and Frtz. This experiment used UAS-frtz driven by ptc-Gal4 and dsh-myc transgenes. Wing disc samples were immunoprecipitated using rabbit Myc antibody then detected with rat Frtz and mouse Myc antibody for western blotting. Arrows point to dsh-myc and Frtz on the Western blots. (E and F) Yeast-two-hybrid plates for interactions between Dvl2 and Intu (E) and WDPCP and Intu (F).