HIF-1 and SKN-1 coordinate the transcriptional response to hydrogen sulfide in Caenorhabditis elegans

Abstract

Hydrogen sulfide (H₂S) has dramatic physiological effects on animals that are associated with improved survival. C. elegans grown in H₂S are long-lived and thermotolerant. To identify mechanisms by which adaptation to H₂S effects physiological functions, we have measured transcriptional responses to H₂S exposure. Using microarray analysis we observe rapid changes in the abundance of specific mRNAs. The number and magnitude of transcriptional changes increased with the duration of H₂S exposure. Functional annotation suggests that genes associated with protein homeostasis are upregulated upon prolonged exposure to H₂S. Previous work has shown that the hypoxia-inducible transcription factor, HIF-1, is required for survival in H₂S. In fact, we show that hif-1 is required for most, if not all, early transcriptional changes in H₂S. Moreover, our data demonstrate that SKN-1, the C. elegans homologue of NRF2, also contributes to H₂S-dependent changes in transcription. We show that these results are functionally important, as skn-1 is essential to survive exposure to H₂S. Our results suggest a model in which HIF-1 and SKN-1 coordinate a broad transcriptional response to H₂S that culminates in a global reorganization of protein homeostasis networks.

Figure 1. Exposure to H₂S induces rapid and progressive changes in mRNA abundance.

A. Experimental design schematic. C. elegans were grown from synchronized first-stage larvae (L1) for 48 h to young adult before being collected for RNA extraction. Each bar represents 48 h from L1 to first-day adult for one experimental group. Time in room air is indicated in white and time in H₂S indicated in red. Exposure to H₂S (50 ppm in room air) was always immediately prior to isolating RNA. B. Changes in mRNA abundance measured by microarray. Plots show magnitude of change in transcript level (log₂ FC) as a function of adjusted p-value (log₁₀ p-value). Each point is data from one gene product. Significant changes (adj. p-value<0.05) are red. After 1 h exposure to H₂S (left), 16 genes were significantly up-regulated and one was down-regulated (Table 1). After 12 h exposure to H₂S (middle), 445 transcripts were significantly changed, with 259 up-regulated (Tables 1 and S1). After 48 h in H₂S (right), 5089 transcripts were significantly altered relative to untreated controls (Table S2).

Figure 2. HIF-1 is required for early transcriptional responses to H₂S.

A. H₂S-induced transcriptional changes require HIF-1. Changes in mRNA abundance after 1 h exposure to H₂S were measured by qRT-PCR in wild-type (N2, open bars) and hif-1(ia04) mutant animals (filled bars). Three biological replicates for each group were performed, and each PCR reaction was run in duplicate. Error bars represent the standard deviation of the biological replicates, as propagated through the ΔΔC_t and fold-change calculations. *Difference between induction in wild-type (N2) is statistically different than in hif-1(ia04) mutant animals, p<0.05. Red dashed line demarks where transcript levels in H₂S are the same as in room air. B. Transcriptional changes after 1 h exposure to H₂S overlap slightly with hif-1-dependent changes in response to hypoxia. 3 of 16 transcripts upregulated in response to 1 h exposure to H₂S were identified as hif-1-dependent targets in hypoxia (n = 68) . The probability of observing this overlap randomly is 0.001. C. There is minimal overlap between the transcriptional responses to hydrogen sulfide and hypoxia. Venn diagram shows overlap between genes induced by exposure to 12 h H₂S (n = 298) and all genes products that are altered by hypoxia (n = 654) . The probability of randomly observing an overlap of 8 genes between these datasets is 0.006.

Figure 3. SKN-1 is essential for appropriate response to H₂S.

A. Some H₂S-induced transcriptional changes require skn-1. Changes in mRNA abundance after 1 h exposure to H₂S were measured by qRT-PCR in N2 animals grown on control RNAi food L4440 (open bars) or on skn-1(RNAi) (filled bars). Three biological replicates for each group were performed, and each PCR reaction was run in duplicate. Error bars represent the standard deviation of the biological replicates, propagated through the ΔΔC_t and fold-change calculations. *Difference between induction in control is significantly different than skn-1(RNAi) p<0.05. Table shows the frequency that core skn-1 consensus sites (RTACT, [27]) are found within the upstream 2 kb flanking region of each transcript whose regulation in response to H₂S was altered by skn-1(RNAi). ^‡genes reported to have SKN-1 bound in the promoter in the ModENCODE database . B. There is little similarity between response to H₂S and other skn-1-dependent transcriptional responses. The overlap between the H₂S-regulated genes after 12 h (n = 445) was greater than chance when compared with skn-1-dependent gene products in unstressed conditions (n = 233, 16 common transcripts, hypergeometric probability 0.006) and for genes that require skn-1 for arsenic-induced upregulation (n = 118, 10 common transcripts, hypergeometric probability 0.01) . There was not significant overlap between transcripts altered by exposure to H₂S and skn-1 dependent transcripts that are downregulated in unstressed conditions (n = 63, hypergeometric probability 0.13), upregulated by tert-butyl hydroperoxide (n = 64, hypergeometric probability 0.06) or hyperoxia (n = 68, hypergeometric probability 0.15). C. skn-1 is required to survive exposure to H₂S. Unc animals (skn-1/nT1 heterozygotes) were compared to non-Unc, skn-1 homozygotes for sensitivity to H₂S (#animals alive/total after exposure to 50 ppm H₂S).