s-Adenosylmethionine Levels Govern Innate Immunity through Distinct Methylation-Dependent Pathways

Abstract

s-adenosylmethionine (SAM) is the sole methyl donor modifying histones, nucleic acids, and phospholipids. Its fluctuation affects hepatic phosphatidylcholine (PC) synthesis or may be linked to variations in DNA or histone methylation. Physiologically, low SAM is associated with lipid accumulation, tissue injury, and immune responses in fatty liver disease. However, molecular connections among SAM limitation, methyltransferases, and disease-associated phenotypes are unclear. We find that low SAM can activate or attenuate Caenorhabditis elegans immune responses. Immune pathways are stimulated downstream of PC production on a non-pathogenic diet. In contrast, distinct SAM-dependent mechanisms limit survival on pathogenic Pseudomonas aeruginosa. C. elegans undertakes a broad transcriptional response to pathogens and we find that low SAM restricts H3K4me3 at Pseudomonas-responsive promoters, limiting their expression. Furthermore, this response depends on the H3K4 methyltransferase set-16/MLL. Thus, our studies provide molecular links between SAM and innate immune functions and suggest that SAM depletion may limit stress-induced gene expression.

Figure 1. Co-regulation of lipogenic and immune function genes with depletion of SAM

A. Schematic showing C. elegans pathways producing or utilizing SAM. MT is methyltransferase, SAM is s-adenosylmethionine, PC is phosphatidylcholine. Human names for orthologs are shown in parenthesis, see also Table S1. B. Bar graphs comparing p-values for GO categories of genes regulated more than 2.0 fold after sams-1(RNAi). Downregulated genes are shown in red bars as log p value. To distinguish upregulated genes, -(log p value) was used for blue bars. Immune categories are highlighted in yellow, lipogenic in green. Genes are identified in Table S2, tab: GO Categories. Genes that were upregulated (C-D), down regulated (E) or not changed in microarray studies were validated in qRT-PCR standardized to an exogenous “spike in” mRNA. G. Bar graphs comparing p-values for GO categories of genes regulated more than 2.0 fold after sbp-1(RNAi). Downregulated genes are shown in red bars as log p value. To distinguish upregulated genes, -(log p value) was used for blue bars. Immune categories are highlighted in yellow, lipogenic in green. Genes are identified in Table S3, tab: GO Categories.

Figure 2. Constitutive activation of innate immune pathway in after sams-1 depletion

A. Schematic showing p38/PMK-1 mitogen activated protein kinase signaling during response to bacterial infection in C. elegans (Kim, 2014). B. qRT-PCR comparing innate immune gene expression in sams-1(lof) and pmk-1(lof); sams-1(lof) mutants. C. qRT-PCR comparing expression of a lipogenic (fat-7) or other (arf-1.1) gene highly expressed gene in sams-1(lof) and pmk-1(lof); sams-1(lof) mutants. D. Immunoblot of phospho-PMK-1 in vehicle (Veh), phorbol acid treated (PMA) and Pseudomonas aeruginosa (PA) exposed wild type (WT), sams-1(lof) or tir-1(lof) mutants. Histone 3 shows loading. E. Wild type, pmk-1(lof) or tir-1(lof) animals were exposed to control or sams-1(RNAi) and immunoblotted with antibodies to phosph-PMK-1 or Histone 3. Error bars show standard deviation. Results from Student's T test shown by * <0.05, ** <0.01, *** <0.005.

Figure 3. Restoration of PC synthesis through dietary choline rescues innate immune phenotypes in sams-1(RNAi) animals

A. Heat map of genome wide expression changes in sams-1(RNAi), sams-1(RNAi) rescued by choline, or vector-only control (Vec) animals supplemented with choline. Heat map showing changes in expression level and choline rescue of lipogenic (B) and immune function (C) genes. Color values are shown in D. E. Control (Vec) or sams-1(RNAi) animals which were grown on normal media or media supplemented with dietary choline (CH) were immunoblotted with antibodies recognizing phospho-PMK-1, SAMS-1 or Histone 3.

Figure 4. Reduced resistance to Pseudomonas aeruginosa in sams-1(lof) animals

Representative Kaplan-Meir plot comparing survival of (WT), sams-1(lof), pmk-1(lof) and pmk-1(lof); sams-1(lof) mutants exposed to pathogenic Pseudomonas aeruginosa, PA14 (A), the attenuated Pseudomonas strain gacA (B) or E. coli OP50 (C). NS is not significant. Additional statistics are available in Table S4. All strains in panels A, B, and C were raised on cdc-25(RNAi) to prevent egg laying. D. Fluorescent micrograph showing Pseudomonas load (PA14 GFP) after 24 hour exposure in intestines of wild type, pmk-1(lof) and sams-1(lof) mutants. Red asterisks show pharynx position. Representative experiments showing quantitation of PA14 GFP and OP50 GFP after 24 (E) or 48 (F) hours exposure. Number of animals is shown in parentheses. “Partial” refers to light GFP in a section of the intestine, “full light” to light GFP along the length of the intestine and “full bright” to strong GFP in the entire intestinal tract.

Figure 5. sams-1(lof) animals lack a transcriptional response to pathogens

qRT-PCR comparing induction of infection response genes by Pseudomonas in wild type (WT) or sams-1(lof) mutants after 6 hours of exposure to PA14 compared to the value on E. coli (OP50). Infection response genes were selected from innate immune genes with moderate induction on E. coli (OP50) after sams-1(RNAi) (A, B) or were selected from Troemel et al. (2006) (C, D). E, F. Induction of infection response genes in sams-1(lof) and sams-1(lof) choline (CH) rescued animals were compared by qRT-PCR. Lipid droplet accumulation shown by Sudan Black staining in anterior intestine (G) and lipogenic gene expression shown by qRT-PCR (H) comparing C. elegans maintained on E. coli and those raised on E. coli and transferred at L4 to Pseudomonas gacA for 48 hours. Error bars show standard deviation. Results from Student's T test shown by * <0.05, ** <0.01, *** <0.005.

Figure 6. Infection response genes do not accumulate activating histone methylation marks in sams-1(lof) mutants exposed to Pseudomonas

A. H3K4me3 is diminished in nuclei of intestinal cells after sams-1(RNAi) and also in choline treated sams-1(RNAi). Yellow bar shows 2 microns. B. Quantitation of immuoflourescence showing an average of pixel intensity over area for 8-12 nuclei per sample. C. Immunostaining comparing markers of active phosphorylated RNA Polymerase II (Pol II PSer 5, PSer 2) with total Pol II (unP) See Figure S2 for quantitation. D. Other histone modifications associated with active transcription (H3K36me and H3K9ac) or with heterochromatin (H3K9me3) within intestinal nuclei in control or sams-1(RNAi) animals. See Figure S2 for quantitation. E-J. Chromatin immunoprecipitation comparing levels of H3K4me3 on infection response or control genes grown on E. coli (OP50) or Pseudomonas (PA14) in wild-type (WT) or sams-1(lof) mutants. Input levels were normalized to the WT E. coli value on the upstream primer pair. Numerical representation of primer location is based on translational start site. Legend in J refers to all panels. Error bars show standard deviation. Results from Student's T test shown by * <0.05, ** <0.01, *** <0.005.

Figure 7. set-16/MLL is important for expression of infection response genes upon Pseudomonas exposure

A. Immunostaining of intestinal nuclei with antibodies to H3K4me3 after RNAi of set-2 or set-16. Yellow bar shows 2 microns. B. Quantitation of immuoflourescence showing an average of pixel intensity over area for 8-12 nuclei per sample. Requirement for set-2/SET1 (B-D) or set-16/MLL (E-G) for induction of infection response genes upon a 6 hour exposure to PA14 compared to E. coli (HT115). Error bars show standard deviation. Results from Student's T test shown by * <0.05, ** <0.01, *** <0.005.