Stress-responsive and metabolic gene regulation are altered in low S-adenosylmethionine

Abstract

S-adenosylmethionine (SAM) is a donor which provides the methyl groups for histone or nucleic acid modification and phosphatidylcholine production. SAM is hypothesized to link metabolism and chromatin modification, however, its role in acute gene regulation is poorly understood. We recently found that Caenorhabditis elegans with reduced SAM had deficiencies in H3K4 trimethylation (H3K4me3) at pathogen-response genes, decreasing their expression and limiting pathogen resistance. We hypothesized that SAM may be generally required for stress-responsive transcription. Here, using genetic assays, we show that transcriptional responses to bacterial or xenotoxic stress fail in C. elegans with low SAM, but that expression of heat shock genes are unaffected. We also found that two H3K4 methyltransferases, set-2/SET1 and set-16/MLL, had differential responses to survival during stress. set-2/SET1 is specifically required in bacterial responses, whereas set-16/MLL is universally required. These results define a role for SAM in the acute stress-responsive gene expression. Finally, we find that modification of metabolic gene expression correlates with enhanced survival during stress.

Fig 1. SAM plays an important role in the transcriptional response to stress.

A. Schematic showing the metabolic link between s-adenosylmethionine (SAM) production and histone methylation B. Diagram of the experimental rationale comparing knockdowns of the SAM synthase sams-1 and the H3K4 methyltransferases set-2/SET1 and set-16/MLL in the transcriptional response to three distinct stresses. Venn diagrams comparing stress responsive gene expression in control (C), sams-1 (D) set-2 (E) and set-16 RNAi upregulated genes (F). Upregulated genes were defined as increased by 2 or more fold with an FDR of less than 0.01 in each of the stresses.

Fig 2. Transcriptional response to P. aeruginosa requires sams-1.

(A) Heat map showing genes upregulated by more than 2-fold with an FDR of less than 0.01 in C. elegans exposed to P. aeruginosa. (B) Strip-plot comparing the top 20 genes upregulated in control vs. sams-1(RNAi) animals exposed to P. aeruginosa. The dotted line is placed at one on the Y-axis. Statistical significance calculated by KS value. (C) Venn Diagram comparing the overlap between genes upregulated more than 2-fold in control vs. sams-1(RNAi) animals exposed to P. aeruginosa.

Fig 3. Differential transcriptional responses to a xenotoxic stress and heat stress after sams-1(RNAi).

(A) Heat map showing genes upregulated by more than 2-fold with an FDR of less than 0.01 in C. elegans exposed to R24. (B) Strip plots showing that the top 20 genes upregulated in controls in response to R24 decreased are in sams-1(RNAi) animals. (C) Venn diagrams show that the majority of genes upregulated more than two-fold in sams-1 animals in response to R24 are also upregulated in controls. (D) Heat map showing genes upregulated by more than 2-fold with an FDR of less than 0.01 in C. elegans exposed to heat. (E) Strip-plot shows that the top 20 genes expressed after heat shock are upregulated similarly in control and sams-1(RNAi) animals. (F) Venn Diagram comparing gene sets regulated after heat stress shows many ectopic genes upregulated in sams-1(RNAi) animals.

Fig 4. Differential transcriptional responses to a bacterial, xenotoxic and heat stress after set-2(RNAi).

(A) Strip plots show that many of the top 20 genes upregulated in response to P. aeruginosa are reduced after set-2 RNAi. KS calculations were used to determine significance. (B) Venn diagrams show that the majority of genes upregulated after set-2(RNAi) in response to P. aeruginosa were also upregulated in controls. (C) Strip-plot shows that many of the top 20 genes induced by R24 in control animals are reduced after set-2 RNAi. (D) Venn diagram shows that set-2(RNAi) animals induce many genes outside the response to R24 seen in control animals. (E) Strip plots demonstrate that most of the top 20 genes induced in response to heat are expressed at similar levels in set-2 RNAi animals. (F) Venn diagrams show that many the majority of genes induced more than 2-fold in control animals are also upregulated after heat stress in set-2 RNAi animals and that expression of many additional genes also increases. RNA for sequencing was isolated from control, sams-1, set-2 and set-16 RNAi as a set for each stress. Therefore, control genes in A-F are the same as in Fig 1 for P. aeruginosa and Fig 2 for R24 and heat.

Fig 5. set-16 is important for gene expression after bacterial and xenotoxic stress genes.

(A) Strip plots show that many of the top 20 genes upregulated in response to P. aeruginosa are reduced after set-16 RNAi. KS calculations were used to determine significance. (B) Venn diagrams show that the majority of genes upregulated after set-16(RNAi) in response to P. aeruginosa were also upregulated in controls. (C) Strip-plot shows that the majority of the top 20 genes induced by R24 in control animals are reduced after set-16 RNAi. (D) Venn diagram shows that genes induced by more than two-fold set-16(RNAi) animals are also induced in controls. (E) Strip plots demonstrate that most of the top 20 genes induced in response to heat are expressed at similar levels in set-16 RNAi animals. (F) Venn diagrams show that many the majority of genes induced more than 2-fold in control animals are also upregulated after heat stress in set-16 RNAi animals, and that expression of many additional genes also increases. RNA for sequencing was isolated from control, sams-1, set-2 and set-16 RNAi as a set for each stress. Therefore, control genes in A-F are the same as in Fig 1 for P. aeruginosa and Fig 2 for R24 and heat.

Fig 6. Stress-responsive and metabolic gene regulation are linked to survival after bacterial or xenotoxic stress in low SAM or H3K4 MT knockdown.

(A) Representative Kaplan-Meier survival plot of set-2 or set-16 adults exposed to P. aeruginosa shows increased sensitivity to bacterial stress (data and additional statistics available in S5 Table). Statistical significance is shown by Log-rank test. (B) Bubble charts show broad category enrichment of upregulated genes determined by Worm-Cat in control, sams-1, set-2 or set-16 animals in genes changed more than 2-fold (FDR <0.01) after P. aeruginosa exposure. Specific category enrichment of stress (C) categories in sams-1, set-2 or set-16 animals in genes changed more than 4-fold (FDR <0.01) after P. aeruginosa exposure. Complete category enrichment data is available in S8 Table. (D) Representative Kaplan-Meier survival plot of sams-1, set-2 or set-16 RNAi adults exposed to R24 shows differential sensitivity to xenotoxic stress (data and additional statistics available in S9 Table). Statistical significance is shown by Log-rank test. (E) Bubble charts show broad category enrichment of upregulated genes determined by Worm-Cat in control, sams-1, set-2 or set-16 animals in genes changed more than 2-fold (FDR <0.01) after R24 exposure. Specific category enrichment of stress (F) or metabolic (G) categories in sams-1, set-2 or set-16 animals in genes changed more than 2-fold (FDR <0.01) after R24 exposure. Complete category enrichment data is available in S10 Table.

Fig 7. Down-regulation of metabolic gene regulation is linked to survival after heat stress in low SAM or H3K4 MT knockdown.

(A) Representative Kaplan-Meier survival plot of control, sams-1, set-2 or set-16 adults exposed to heat shows increased sensitivity to heat stress (data and additional statistics available in S11 Table). Statistical significance is shown by Log-rank test. For additional replicates. (B) Bubble charts show broad category enrichment of upregulated genes determined by Worm-Cat in control, sams-1, set-2 or set-16 animals in genes changed more than 4-fold (FDR <0.01) after heat exposure. Specific category enrichment of stress (C) or transcription (D) categories in control, sams-1, set-2 or set-16 animals in genes changed more than 4-fold (FDR <0.01) after heat exposure. (E) Bubble charts show broad category enrichment of downregulated genes in control, sams-1, set-2 or set-16 animals in genes changed more than 2-fold (FDR <0.01) after heat exposure. Specific category enrichment of metabolism (F), lipid metabolism (G) or transcription (H) categories in control, sams-1, set-2 or set-16 animals in genes changed more than 4-fold (FDR <0.01) after heat exposure. Complete category enrichment data is available in S12 Table.