All strains are available upon request. Supplemental Figures S1-S5 and Supplementary Tables 741
S1-S5 can be found in ‘Supp Figures and Tables.docx’. Supplementary Table S4 contains 742
genotypes for each strain used in this study. Supplementary Table S5 contains nucleotide 743
sequences for each primer used in qRT analysis. RNA Seq gene expression data are available 744
in excel format in file ‘Supplementary Excel Data Set S6.xlsm’ or at GEO with the accession 745
number: xxxxxxx (**submitted, to be assigned) 746
ACKNOWLEDGMENTS: 748
We thank T. K. Blackwell and the Caenorhabditis Genetics Center, which is funded by the NIH 749
National Center for Research Resources (NCRR), for the C. elegans strains. T. K. Blackwell is 750
also acknowledged for sharing an aliquot of the FC4 antibody. We also thank V. Garcia and J. 751
Hourihan for helpful advice regarding the use of phospho-specific antibodies for Western 752
Blotting. The research reported in this publication was supported by the National Institute of 753
Allergy and Infectious Diseases and the National Institute of General Medical Sciences of the 754
National Institutes of Health under award numbers R01AI076406 to DAG and R01GM98200 to 755
SA. SA was also supported by ACS RSG014-044-DDC. A.M.-V. is a member of the EU-ROS 756
(BM1203) and GENiE (BM1408) COST Actions and his work was financed by grants from the 757
Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629). P.S. was supported by the 758
Swedish Research Council, the Torsten Söderberg and Åhlén Foundations, and by the 759
Karolinska Institute Strategic Neurosciences Program. The content is solely the responsibility of 760
the authors and does not necessarily represent the official views of the granting agencies. 761 762 763 764 765 766 767 768 769
770 771 772 773 774 REFERENCES 775
An, J. H. and T. K. Blackwell (2003). "SKN-1 links C. elegans mesendodermal specification to a 776
conserved oxidative stress response." Genes Dev 17(15): 1882-1893. 777
An, J. H., K. Vranas, et al. (2005). "Regulation of the Caenorhabditis elegans oxidative stress 778
defense protein SKN-1 by glycogen synthase kinase-3." Proc Natl Acad Sci U S A 779
102(45): 16275-16280. 780
Anderson, M. E. (1998). "Glutathione: an overview of biosynthesis and modulation." Chem Biol 781
Interact 111-112: 1-14. 782
Arner, E. S. and A. Holmgren (2000). "Physiological functions of thioredoxin and thioredoxin 783
reductase." Eur J Biochem 267(20): 6102-6109. 784
Berndt, C., C. H. Lillig, et al. (2008). "Thioredoxins and glutaredoxins as facilitators of protein 785
folding." Biochim Biophys Acta 1783(4): 641-650. 786
Bowerman, B., B. W. Draper, et al. (1993). "The maternal gene skn-1 encodes a protein that is 787
distributed unequally in early C. elegans embryos." Cell 74(3): 443-452. 788
Buchanan, B. B., A. Holmgren, et al. (2012). "Fifty years in the thioredoxin field and a bountiful 789
harvest." Biochim Biophys Acta 1820(11): 1822-1829. 790
Cacho-Valadez, B., F. Munoz-Lobato, et al. (2012). "The characterization of the Caenorhabditis 791
elegans mitochondrial thioredoxin system uncovers an unexpected protective role of 792
thioredoxin reductase 2 in beta-amyloid peptide toxicity." Antioxid Redox Signal 16(12): 793
1384-1400. 794
Carroll, B. T., G. R. Dubyak, et al. (2006). "Sulfated signal from ASJ sensory neurons modulates 795
stomatin-dependent coordination in Caenorhabditis elegans." J Biol Chem 281(47): 796
35989-35996. 797
Chavez, V., A. Mohri-Shiomi, et al. (2009). "Ce-Duox1/BLI-3 generates reactive oxygen species 798
as a protective innate immune mechanism in Caenorhabditis elegans." Infect Immun 799
77(11): 4983-4989. 800
Choe, K. P., A. J. Przybysz, et al. (2009). "The WD40 repeat protein WDR-23 functions with the 801
CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in 802
Caenorhabditis elegans." Mol Cell Biol 29(10): 2704-2715. 803
Du, H., S. Kim, et al. (2015). "A cytosolic thioredoxin acts as a molecular chaperone for 804
peroxisome matrix proteins as well as antioxidant in peroxisome." Mol Cells 38(2): 187- 805
194. 806
Edgar, L. G. and J. D. McGhee (1986). "Embryonic expression of a gut-specific esterase in 807
Caenorhabditis elegans." Dev Biol 114(1): 109-118. 808
Ewald, C. Y., J. N. Landis, et al. (2014). "Dauer-independent insulin/IGF-1-signalling implicates 809
collagen remodelling in longevity." Nature. 810
Fierro-Gonzalez, J. C., A. Cornils, et al. (2011). "The thioredoxin TRX-1 modulates the function 811
of the insulin-like neuropeptide DAF-28 during dauer formation in Caenorhabditis 812
elegans." PLoS One 6(1): e16561. 813
Fierro-Gonzalez, J. C., M. Gonzalez-Barrios, et al. (2011). "The thioredoxin TRX-1 regulates 814
adult lifespan extension induced by dietary restriction in Caenorhabditis elegans." 815
Biochem Biophys Res Commun 406(3): 478-482. 816
Folick, A., H. D. Oakley, et al. (2015). "Aging. Lysosomal signaling molecules regulate longevity 817
in Caenorhabditis elegans." Science 347(6217): 83-86. 818
Fujino, G., T. Noguchi, et al. (2007). "Thioredoxin and TRAF family proteins regulate reactive 819
oxygen species-dependent activation of ASK1 through reciprocal modulation of the N- 820
terminal homophilic interaction of ASK1." Mol Cell Biol 27(23): 8152-8163. 821
Fujino, G., T. Noguchi, et al. (2006). "Thioredoxin and protein kinases in redox signaling." Semin 822
Cancer Biol 16(6): 427-435. 823
Garsin, D. A., C. D. Sifri, et al. (2001). "A simple model host for identifying Gram-positive 824
virulence factors." Proc Natl Acad Sci U S A 98(19): 10892-10897. 825
Glover-Cutter, K. M., S. Lin, et al. (2013). "Integration of the unfolded protein and oxidative 826
stress responses through SKN-1/Nrf." PLoS Genet 9(9): e1003701. 827
Gonzalez-Barrios, M., J. C. Fierro-Gonzalez, et al. (2015). "Cis- and trans-regulatory 828
mechanisms of gene expression in the ASJ sensory neuron of Caenorhabditis elegans." 829
Genetics 200(1): 123-134. 830
Grant, B. and D. Hirsh (1999). "Receptor-mediated endocytosis in the Caenorhabditis elegans 831
oocyte." Mol Biol Cell 10(12): 4311-4326. 832
Hoeven, R., K. C. McCallum, et al. (2011). "Ce-Duox1/BLI-3 generated reactive oxygen species 833
trigger protective SKN-1 activity via p38 MAPK signaling during infection in C. elegans." 834
PLoS Pathog 7(12): e1002453. 835
Holmgren, A. (1985). "Thioredoxin." Annu Rev Biochem 54: 237-271. 836
Holmgren, A. and J. Lu (2010). "Thioredoxin and thioredoxin reductase: current research with 837
special reference to human disease." Biochem Biophys Res Commun 396(1): 120-124. 838
Hope, I. A. (1999). C. elegans A Practical Approach. The Practical Approach Series. Oxford, 839
Oxford University Press. 840
Huang da, W., B. T. Sherman, et al. (2009). "Bioinformatics enrichment tools: paths toward the 841
comprehensive functional analysis of large gene lists." Nucleic Acids Res 37(1): 1-13. 842
Huang da, W., B. T. Sherman, et al. (2009). "Systematic and integrative analysis of large gene 843
lists using DAVID bioinformatics resources." Nat Protoc 4(1): 44-57. 844
Hybertson, B. M., B. Gao, et al. (2011). "Oxidative stress in health and disease: the therapeutic 845
potential of Nrf2 activation." Mol Aspects Med 32(4-6): 234-246. 846
Inoue, H., N. Hisamoto, et al. (2005). "The C. elegans p38 MAPK pathway regulates nuclear 847
localization of the transcription factor SKN-1 in oxidative stress response." Genes Dev 848
19(19): 2278-2283. 849
Jee, C., L. Vanoaica, et al. (2005). "Thioredoxin is related to life span regulation and oxidative 850
stress response in Caenorhabditis elegans." Genes Cells 10(12): 1203-1210. 851
Jimenez-Hidalgo, M., C. L. Kurz, et al. (2014). "Functional characterization of thioredoxin 3 852
(TRX-3), a Caenorhabditis elegans intestine-specific thioredoxin." Free Radic Biol Med 853
68: 205-219. 854
Kahn, N. W., S. L. Rea, et al. (2008). "Proteasomal dysfunction activates the transcription factor 855
SKN-1 and produces a selective oxidative-stress response in Caenorhabditis elegans." 856
Biochem J 409(1): 205-213. 857
Langmead, B., C. Trapnell, et al. (2009). "Ultrafast and memory-efficient alignment of short DNA 858
sequences to the human genome." Genome Biol 10(3): R25. 859
Leung, C. K., K. Hasegawa, et al. (2014). "Direct interaction between the WD40 repeat protein 860
WDR-23 and SKN-1/Nrf inhibits binding to target DNA." Mol Cell Biol 34(16): 3156-3167. 861
Liu, Y. and W. Min (2002). "Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit 862
ASK1-mediated apoptosis in a redox activity-independent manner." Circ Res 90(12): 863
1259-1266. 864
Loria, P. M., J. Hodgkin, et al. (2004). "A conserved postsynaptic transmembrane protein 865
affecting neuromuscular signaling in Caenorhabditis elegans." J Neurosci 24(9): 2191- 866
2201. 867
Lushchak, V. I. (2011). "Adaptive response to oxidative stress: Bacteria, fungi, plants and 868
animals." Comp Biochem Physiol C Toxicol Pharmacol 153(2): 175-190. 869
Lynn, D. A., H. M. Dalton, et al. (2015). "Omega-3 and -6 fatty acids allocate somatic and 870
germline lipids to ensure fitness during nutrient and oxidative stress in Caenorhabditis 871
elegans." Proc Natl Acad Sci U S A 112(50): 15378-15383. 872
Mahajan-Miklos, S., M. W. Tan, et al. (1999). "Molecular mechanisms of bacterial virulence 873
elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis 874
model." Cell 96(1): 47-56. 875
Marinho, H. S., C. Real, et al. (2014). "Hydrogen peroxide sensing, signaling and regulation of 876
transcription factors." Redox Biol 2: 535-562. 877
Maruyama, T., T. Araki, et al. (2014). "Roquin-2 promotes ubiquitin-mediated degradation of 878
ASK1 to regulate stress responses." Sci Signal 7(309): ra8. 879
McCord, J. M. and I. Fridovich (1969). "Superoxide dismutase. An enzymic function for 880
erythrocuprein (hemocuprein)." J Biol Chem 244(22): 6049-6055. 881
Miranda-Vizuete, A., J. C. Fierro Gonzalez, et al. (2006). "Lifespan decrease in a 882
Caenorhabditis elegans mutant lacking TRX-1, a thioredoxin expressed in ASJ sensory 883
neurons." FEBS Lett 580(2): 484-490. 884
Oliveira, R. P., J. Porter Abate, et al. (2009). "Condition-adapted stress and longevity gene 885
regulation by Caenorhabditis elegans SKN-1/Nrf." Aging Cell 8(5): 524-541. 886
Page, A. P. and I. L. Johnstone (2007). "The cuticle." WormBook: 1-15. 887
Park, S. K., P. M. Tedesco, et al. (2009). "Oxidative stress and longevity in Caenorhabditis 888
elegans as mediated by SKN-1." Aging Cell 8(3): 258-269. 889
Powis, G. and W. R. Montfort (2001). "Properties and biological activities of thioredoxins." Annu 890
Rev Biophys Biomol Struct 30: 421-455. 891
Prahlad, V., T. Cornelius, et al. (2008). "Regulation of the cellular heat shock response in 892
Caenorhabditis elegans by thermosensory neurons." Science 320(5877): 811-814. 893
Ray, P. D., B. W. Huang, et al. (2012). "Reactive oxygen species (ROS) homeostasis and redox 894
regulation in cellular signaling." Cell Signal 24(5): 981-990. 895
Robida-Stubbs, S., K. Glover-Cutter, et al. (2012). "TOR signaling and rapamycin influence 896
longevity by regulating SKN-1/Nrf and DAF-16/FoxO." Cell Metab 15(5): 713-724. 897
Salazar, M., A. I. Rojo, et al. (2006). "Glycogen synthase kinase-3beta inhibits the xenobiotic 898
and antioxidant cell response by direct phosphorylation and nuclear exclusion of the 899
transcription factor Nrf2." J Biol Chem 281(21): 14841-14851. 900
Schackwitz, W. S., T. Inoue, et al. (1996). "Chemosensory neurons function in parallel to 901
mediate a pheromone response in C. elegans." Neuron 17(4): 719-728. 902
Schmeisser, S., S. Priebe, et al. (2013). "Neuronal ROS signaling rather than AMPK/sirtuin- 903
mediated energy sensing links dietary restriction to lifespan extension." Mol Metab 2(2): 904
92-102. 905
Steinbaugh, M. J., S. D. Narasimhan, et al. (2015). "Lipid-mediated regulation of SKN-1/Nrf in 906
response to germ cell absence." Elife 4. 907
Sun, J., V. Singh, et al. (2011). "Neuronal GPCR controls innate immunity by regulating 908
noncanonical unfolded protein response genes." Science 332(6030): 729-732. 909
Taylor, R. C. and A. Dillin (2013). "XBP-1 is a cell-nonautonomous regulator of stress resistance 910
and longevity." Cell 153(7): 1435-1447. 911
Thanan, R., S. Oikawa, et al. (2014). "Oxidative Stress and Its Significant Roles in 912
Neurodegenerative Diseases and Cancer." Int J Mol Sci 16(1): 193-217. 913
Tobiume, K., M. Saitoh, et al. (2002). "Activation of apoptosis signal-regulating kinase 1 by the 914
stress-induced activating phosphorylation of pre-formed oligomer." J Cell Physiol 191(1): 915
95-104. 916
Trapnell, C., D. G. Hendrickson, et al. (2013). "Differential analysis of gene regulation at 917
transcript resolution with RNA-seq." Nat Biotechnol 31(1): 46-53. 918
Trapnell, C., A. Roberts, et al. (2012). "Differential gene and transcript expression analysis of 919
RNA-seq experiments with TopHat and Cufflinks." Nat Protoc 7(3): 562-578. 920
Tullet, J. M., M. Hertweck, et al. (2008). "Direct inhibition of the longevity-promoting factor SKN- 921
1 by insulin-like signaling in C. elegans." Cell 132(6): 1025-1038. 922
van Oosten-Hawle, P. and R. I. Morimoto (2014). "Organismal proteostasis: role of cell- 923
nonautonomous regulation and transcellular chaperone signaling." Genes Dev 28(14): 924
1533-1543. 925
van Oosten-Hawle, P. and R. I. Morimoto (2014). "Transcellular chaperone signaling: an 926
organismal strategy for integrated cell stress responses." J Exp Biol 217(Pt 1): 129-136. 927
Walker, A. K., R. See, et al. (2000). "A conserved transcription motif suggesting functional 928
parallels between Caenorhabditis elegans SKN-1 and Cap'n'Collar-related basic leucine 929
zipper proteins." J Biol Chem 275(29): 22166-22171. 930
Wang, T. S., C. F. Kuo, et al. (1996). "Arsenite induces apoptosis in Chinese hamster ovary 931
cells by generation of reactive oxygen species." J Cell Physiol 169(2): 256-268. 932
Yoshioka, J., E. R. Schreiter, et al. (2006). "Role of thioredoxin in cell growth through 933
interactions with signaling molecules." Antioxid Redox Signal 8(11-12): 2143-2151. 934
Zhang, P., M. Judy, et al. (2013). "Direct and indirect gene regulation by a life-extending FOXO 935
protein in C. elegans: roles for GATA factors and lipid gene regulators." Cell Metab 936 17(1): 85-100. 937 938 939 940 941 942 943 944
945 946 947 948 949 950 951 ARTICLE SUMMARY 952
The accumulation of free radical damage is an underlying component of numerous human 953
pathologies, ranging from diabetes to aging. This accumulation can be counteracted, however, 954
by an oxidative stress response, which is characterized by the increased production of 955
antioxidants controlled by stress response transcription factors like those of the Nrf family found 956
in mammals. In C. elegans, SKN-1 is a functional ortholog of the Nrf proteins, and this model 957
organism has proven useful to understanding the regulation and the impact of various stress 958
responses. Herein, we demonstrate that intestinal SKN-1 subcellular localization is controlled in 959
part by a thioredoxin, TRX-1, expressed from the distant ASJ neurons, in a manner independent 960
of its redox activity. Interestingly, while the subcellular localization of SKN-1 is affected, 961
activation of this transcription factor, as measured by the expression of SKN-1-dependent genes 962
encoding antioxidants, remains unchanged. This work highlights a novel mechanism of SKN-1 963
regulation in which intestinal localization is impacted in a cell non-autonomous fashion. 964
Moreover, TRX-1-dependent regulation of SKN-1 demonstrates an example in which subcellular 965
localization and transcriptional activation are uncoupled. In conclusion, these findings elucidate 966
another mechanism by which SKN-1 is regulated, adding to the complexity, but exemplifying the 967
importance of tightly regulating stress responses. 968