S-adenosylmethionine synthases specify distinct H3K4me3 populations and gene expression patterns during heat stress
- Program in Molecular Medicine, UMASS Chan Medical School, United States
- Cancer Center, UMASS Chan Medical School, United States
- Department of Molecular, Cell, and Cancer Biology, UMASS Chan Medical School, United States
Figures
Figure 1 with 3 supplements
acquisition of H3K4me3 in heat-shocked animals.
(A) Methionine intake through diet enters the 1 carbon cycle and is used by SAM synthases for the synthesis of SAM which is used by methyltransferases to add methyl moieties to proteins, nucleic acids and lipids. (B) Representative confocal images of animals co-expressing RFP::SAMS-1and GFP::SAMS-4 in the germline and intestine. Scale bar represents 50 microns. Kaplan-Meier survival plots of sams-1(lof) (C) or sams-4(ok3315) (D) following heat shock. Statistical significance is shown by Log-rank test. Each graph represents the compiled data from three biologically independent repeats; data is compiled in Supplementary file 2. Representative immunofluorescence images of intestinal nuclei stained with H3K4me3-specific antibody and quantification in sams-1(lof) animals (E, H), sams-4(RNAi) (F, I) or in sams-1(lof); sams-4(RNAi) animals (G, J). sams-3 may also be targeted; see also (Figure 3E). Scale bar represents 25 microns. Error bars show average and standard deviation. Statistical significance was calculated using unpaired Student’s t-test. ns = not significant, ****=p < 0.0001, ***=p < 0.001. Graph represents compiled data from three biologically independent repeats per condition with each point representing a single animal.
Figure 1—figure supplement 1
Expression patterns of SAM synthases in adult C. elegans.
(A) Comparison of polyA +RNA levels of SAM synthases with selected other metabolic genes in adult animals from the modEncode data set (Gerstein et al., 2010). (B) Representative confocal images of animals expressing RFP::SAMS-1or GFP::SAMS-4. hypodermis is (h), intestine (i) and germline (gl). Scale bars represent 50 microns. (C) Confocal projections of GFP::SAMS-4 and SAMS-3::mKate subjected to sams-3 or sams-4(RNAi). Scale bars represent 25 microns. (D) Absolute quantification of the SAM level in animals fed on control RNAi or sams-4(RNAi). The levels are expressed as mM/mg tissue. Mean and standard deviation are shown representing 4 biological replicates. Statistical significance is measured by Students T Test.
Figure 1—figure supplement 2
Distinct patterns of gene expression after sams-1 or sams-4 RNAi in basal conditions.
(A) Principal component analysis showing overlapping components between genes regulated in sams-3 and sams-4(RNAi) animals. (B) Venn diagram showing the overlap in upregulated genes in animals fed sams-1 or sams-4(RNAi). (C) Bubble charts show broad category enrichment of upregulated genes in animals fed sams-1 or sams-4(RNAi). (D) Bubble charts show broad category enrichment of downregulated genes in animals fed sams-1 or sams-4(RNAi). (E) Venn diagram showing the overlap in upregulated genes involved in lipid metabolism in animals fed sams-1 or sams-4(RNAi). (F) Venn diagram showing the overlap in upregulated genes involved in pathogen stress response in animals fed sams-1 or sams-4(RNAi).
Figure 1—figure supplement 3
sams-4 is important for survival and H3K4me3 in sams-1 animals after heat shock.
(A) Schematic for the heat stress assay. (B) Survival assays comparing response to heat in SAM synthase mutants. (C) Lifespan assay with sams-4(RNAi) animals where sams-3 may also be targeted. (D) Heat shock survival assays showing that genetic loss of sams-4 limits survival in sams-1(RNAi) animals after heat shock. For B-D, statistical significance is shown by Log-rank test. Each graph represents compiled data from three3 biologically independent repeats. Data for each replicate is compiled in Supplementary file 2. (E) Quantification of immunofluorescence imaging of intestinal nuclei stained with HK4me3 antibody after heat shock from sams-4(ok3315); sams-1(RNAi) animals. Statistical significance was calculated using unpaired Student’s t-test. ns = not significant, ****=p < 0.0001, ***=p < 0.001. Graph represents compiled data from three biologically independent repeats per condition. Black bars show mean and standard deviation. LC/MS relative quantitation of SAM (F), Methionine (G) and S-adenosylhomocysteine (SAH) (H). Graphs represent four independent biological replicates (1–4: red, blue, orange and green) that were normalized for protein levels before quantitating relative levels of metabolites. Black bars show means.
Figure 2 with 1 supplement
H3K4me3 demethylases modulate SAM synthase phenotypes.
Kaplan-Meier plots of survival assays comparing basal and heat shocked wild type (N2) or sams-1(lof) animals grown on RNAi for the histone methyltransferases set-2 (A) and set-16 (B), or demethylases rbr-2 (C) and spr-5 (D).Scale bar is 25 microns. Heat shock survival assays for sams-4(ok3315) animals exposed to set-2 or set-16 RNAi are shown in (E, F). Statistical significance is shown by Log-rank test. Each graph represents compiled data from 3 biologically independent repeats. Data for each replicate is compiled in Supplementary file 2. Black bars show mean and standard deviation. Statistical significance is determined by Student T test.
Figure 2—figure supplement 1
H3K4me3 demethylases modulate SAM synthase phenotypes.
Representative immunofluorescence images and quantitation of intestinal nuclei stained with H3K4me3 specific antibody for set-2 (A, D), set-16 (B, E) and rbr-2 (C, F). Statistical significance was calculated using unpaired Student’s t-test. ns = not significant, ****=p < 0.0001, ***=p < 0.001. Graph represents compiled data from three biologically independent repeats per condition. Heat shock survival assays examining the impact of demethylase knockdown on sams-1(lof) animals for amx-1 (G) and lsd-1 (H). Survival was determined by plotting Kaplan-Meier survival plots. Statistical significance is shown by Log-rank test. Each assay represents compiled data from three biologically independent repeats (Supplementary file 2).
Figure 3 with 3 supplements
H3K4me3 modifying enzymes modulate SAM synthase phenotypes.
(A) Bar graph showing the distribution of the enrichment of H3K4me3 over different genomic loci in animals fed control RNAi, sams-1(RNAi) or sams-4(RNAi) at 15°C and 37°C. (B) Aggregation plots showing TSS enrichment in the H3K4me3 peaks identified in animals fed control RNAi at 15°C and 37°C. The Y axis on TSS plots shows Peaks per base pair of gene. (C) Venn diagram comparing the overlap in the H3K4me3 peaks identified in animals fed control RNAi at 15°C and 37°C. (D) Aggregation plots showing TSS enrichment in the H3K4me3 peaks identified in animals fed control RNAi or sams-1(RNAi) or sams-4(RNAi) at 15 °C and Venn diagram comparing the overlap in the H3K4me3 peaks identified in animals fed control RNAi or sams-1(RNAi) or sams-4(RNAi) at 15 °C. (E) Aggregation plots showing TSS enrichment in the H3K4me3 peaks identified in animals fed control RNAi or sams-1(RNAi) or sams-4(RNAi) at 15 °C and Venn diagram comparing the overlap in the H3K4me3 peaks identified in animals fed control RNAi or sams-1(RNAi) or sams-4(RNAi) at 37 °C. (F) Bubble chart showing enriched gene categories in differential peaks as determined by WormCat in animals fed control RNAi at 15 °C only, 37 °C only and common between 15°C and 37°C (G) or sams-1(RNAi) and sams-4(RNAi) at 37 °C. Aggregation plots showing TSS enrichment of Control peaks that did not change after sams-1(RNAi) and sams-4(RNAi) (independent) (H) 15 °C or (I) 37 °C. Shaded areas in the Venn diagrams indicate the population of genes used for plotting the TSS enrichment plots. Aggregation plots showing TSS enrichment of Control peaks that were dependent on sams-1(RNAi) or sams-4(RNAi) (J) 15 °C or (K) 37 °C. Shaded areas in the Venn diagrams indicate the population of genes used for plotting the TSS enrichment plots.
Figure 3—figure supplement 1
H3K4me3 C&T correlation with published H3K4me3 ChipSeq data.
(A) Correlation plots showing r values for C&T replicates. (B) Comparison of H3K4me3ChIP seq from modEncode (L3) Ho et al., 2014, Pu et al (Adult glp-1(e2141)) Pu et al., 2018, Wan et al., 2022 (adult) and our C&T data. (C) IGV browser tracks showing no antibody controls around the pcaf-1 gene, which has been used as positive control for H3K4me3 5 prime peaks in C. elegans (Ding et al., 2015; Xiao et al., 2011).
Figure 3—figure supplement 2
Distinct H3K4me3 patterns after heat shock in sams-1 and sams-4 RNAi animals.
Sunburst diagram showing the enriched gene categories in animals fed control RNAi at (A) 15 °C or (B) 37 °C. Sunburst diagram showing the overall enriched gene categories (C) and genes involved in metabolism (D) in animals fed sams-1(RNAi) at 37 °C. Sunburst diagram showing the overall enriched gene categories (E) and genes involved in metabolism (F) in animals fed sams-4(RNAi) at 37 °C. Aggregation plots showing average enrichment of reads around the transcription start site (TSS) for genes which are sams-1 dependent only dependent on either sams-1 or sams-4 or sams-4 dependent only at (G) 15 °C or (H) 37 °C. The Y axis on TSS plots shows Peaks per base pair of gene.
Figure 3—figure supplement 3
SAM synthase-specific patterns H3K4me3 in germline nuclei.
(A) Representative immunofluorescence images of H3K4me3 staining in the germline in animals fed on control, sams-1 or sams-4(RNAi). Scale bar is 5 microns. (B) Venn diagrams showing the overlap in H3K4me3 peaks identified on ubiquitously expressed genes in control animals at 15 °C or 37 °C. (C) Venn diagrams showing the overlap in H3K4me3 peaks identified on germline-specific genes in control animals at 15 °C or 37 °C. Aggregation plots showing average enrichment of reads around the transcription start site (TSS) of (D) ubiquitously or (E) germline-specific or (F) intestine-specific genes in animals fed control, sams-1 or sams-4(RNAi) at 15 °C. The Y axis on TSS plots shows Peaks per base pair of gene. Aggregation plots showing average enrichment of reads around the transcription start site (TSS) of (G) ubiquitously or (H) germline-specific or (I) intestine-specific genes in animals fed control, sams-1 or sams-4(RNAi) at 37 °C.
Figure 4 with 1 supplement
Distinct gene expression and H3K4me3 patterns after heat shock in sams-1 and sams-4 RNAi animals.
(A) Venn diagram showing overlap of genes upregulated by heat shock in control, sams-1 or sams-4 RNAi animals. sams-1 data is from Ding et al., 2018. (B) Bubble charts show broad category enrichment of up genes determined by Worm-Cat in control (RNAi) or sams-1 or sams-4 animals in genes changed (FDR <0.01) after heat shock. (C) Heat map for heat shock response genes upregulated following heat shock in animals fed control RNAi, sams-1 or sams-4(RNAi). TSS plots showing aggregation of H3K4me3 in genes upregulated in control, sams-1 or sams-4 RNAi at (D) 15 °C or (E) 37 °C. TSS plots showing aggregation of H3K4me3 in all genes upregulated in control or sams-1 dependent or sams-4 RNAi dependent at (F) 15 °C or (G) 37 °C. The Y axis on TSS plots shows Peaks per base pair of gene. Genome browser tracks for (H) fbxa-59 and (I) T27F6.8 to visualize changes in H3K4me3 enrichment in animals fed control, sams-1 or sams-4(RNAi) at 15 °C or 37 °C.
Figure 4—figure supplement 1
sams-1 and sams-4 have distinct gene expression patterns after heat shock.
(A) PCA plot showing groupings of up and downregulated genes from Control, sams-1, or sams-4(RNAi) animals. Survival curves examining heat shock responses after RNAi of T27F6.8 or fbxa-59. Survival was determined by plotting Kaplan-Meier survival plots. Statistical significance is shown by Log-rank test. Each assay represents compiled data from three biologically independent repeats (Supplementary file 2).
Figure 5
Genes that depend on sams-1 or sams-4 for expression have reduced H3K4me3.
(A) Venn diagram showing overlap in downregulated genes in animals fed control, sams-1, or sams-4(RNAi) at 37 °C. (B) Bubble charts show broad category enrichment of metabolism genes determined by Worm-Cat in sams-1 or sams-4 animals in genes changed (FDR <0.01) after heat shock. (C) Bubble charts show broad category enrichment of transcription factor and metabolism genes determined by Worm-Cat in sams-1 or sams-4 animals in genes changed (FDR <0.01) after heat shock. Aggregation plots showing average enrichment of reads around the transcription start site (TSS) in animals fed (D) control, (E) sams-1 or (F) sams-4(RNAi) at 15 °C or 37 °C. The Y axis on TSS plots shows Peaks per base pair of gene.
Figure 6 with 1 supplement
nhr and lipid beta oxidation genes lose H3K4me3 after sams-1 RNAi but expression after heat shock.
(A) Venn diagram showing the overlap between H3K4me3 peaks identified in animals fed control or sams-1(RNAi) at 15 °C and downregulated genes identified in heat shocked animals fed sams-1(RNAi). Heat map for (B) nuclear hormone response genes and (C) lipid β-oxidation genes downregulated following heat shock in animals fed control RNAi, sams-1 or sams-4(RNAi). Genes linked to nhr-68 feedback loop (Bulcha et al., 2019) are marked in red. (D) Genome browser tracks for nhr-68 to visualize changes in H3K4me3 enrichment in animals fed control, sams-1, or sams-4(RNAi) at 15 °C or 37 °C.
Figure 6—figure supplement 1
Schematic of potential nhr-68 module regulation in sams-1 animals.
(A) Genome browser tracks for ges-1 showing H3K4me3 enrichment in animals fed control, sams-1 or sams-4(RNAi) at 15 °C or 37 °C. (B) Schematic showing the dynamic changes in the transcription and H3K4me3 landscape in low SAM animals following heat shock.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (C. elegans) | sams-1 | Wormbase | WBGene00008205 | |
| Gene (C. elegans) | sams-4 | Wormbase | WBRNAi00010322 | |
| Gene (C. elegans) | set-2 | Wormbase | WBGene00004782 | |
| Gene (C. elegans) | set-16 | Wormbase | WBGene00011729 | |
| Gene (C. elegans) | rbr-2 | Wormbase | WBGene00004319 | |
| Gene (C. elegans) | spr-5 | Wormbase | WBRNAi00004611 | |
| Gene (C. elegans) | lsd-1 | Wormbase | WBGene00011615 | |
| Genetic reagent (C. elegans) | N2 | Caenorhabditis Genetics Centre | Wild type | |
| Genetic reagent (C. elegans) | sams-1(ok3033) | CGC | HA1975 | sams-1(ok3033) X |
| Genetic reagent (C. elegans) | sams-4(ok3315) | CGC | RB2420 | C06E7.3(ok3315) IV |
| Genetic reagent (C. elegans) | rfp::sams-1(ker5); sams-1(ok3033) | This paper | WAL500 | WAL500. See ‘Materials and methods, section C. elegans strains’ |
| Genetic reagent (C. elegans) | gfp::sams-4(ker6); sams-4(ok3315) | This paper | WAL501 | WAL501; See ‘Materials and methods, section C. elegans strains’ |
| Genetic reagent (C. elegans) | Pges-1::NHR-68::GFP | Bulcha, J.T Cell Reports 26, 460–468.e4. 10.1016 /j.celrep.2018.12.064. | VL1296 | See ‘Materials and methods, section C. elegans strains’ |
| Genetic reagent (C. elegans) | sams-3::mKate(nu3139) | This paper, InVivo Biosystems | WAL503 | WAL503; See ‘Materials and methods, section C. elegans strains’ |
| Genetic reagent (C. elegans) | rfp::sams-1;gfp(ker5)::sams-4(ker6) | This paper | WAL502 | See ‘Materials and methods, section C. elegans strains’ |
| Genetic reagent (C. elegans) | Escherichia coli OP50 | CGC | N/A | Used as food source for C. elegans |
| Strain, strain background (E. coli) | sams-1 RNAi | Source Bioscience | X-5P21 | Used to knock down target mRNA |
| Strain, strain background (E. coli) | sams-4 RNAi | Source Bioscience | IV-3C01 | Used to knock down target mRNA |
| Strain, strain background (E. coli) | set-2 RNAi | ORFeome RNAi library | mv_C26E6.9 | Used to knock down target mRNA |
| Strain, strain background (E. coli) | set-16 RNAi | Source Bioscience | III-6D12 | Used to knock down target mRNA |
| Strain, strain background (E. coli) | rbr-2 RNAi | Source Bioscience | IV-5D22 | Used to knock down target mRNA |
| Strain, strain background (E. coli) | spr-5 RNAi | Source Bioscience | I-6H02 | Used to knock down target mRNA |
| Strain, strain background (E. coli) | lsd-1 RNAi | Source Bioscience | X-5P17 | Used to knock down target mRNA |
| Antibody | Tri-Methyl-Histone H3 Lys4 (Rabbit monoclonal) | Cell Signaling Technology | C42D8 | Used for IF (1:200) and CUT&Tag (1:50) |
| Software, algorithm | GraphPad Prism v8 | https://www.graphpad.com | N/A | Used for statistical analysis of data and generate graphs |
| Software, algorithm | HOMER | https://homer.ucsd.edu/homer/, Heinz et al., 2010 | Bioinformatic data analysis software | |
| Software, algorithm | Dolphin | https://www.umassmed.edu/biocore/introducing-dolphin/, Yukselen et al., 2020 | Bioinformatic data analysis software | |
| Software, algorithm | DeBrowser | https://debrowser.umassmed.edu, Kucukural et al., 2019 | Bioinformatic data analysis software | |
| Software, algorithm | ChipPeakAnno | https://bioconductor.org/packages/release/bioc/html/ChIPpeakAnno.html, Zhu et al., 2010 | Bioinformatic data analysis software | |
| Software, algorithm | BioVenn | https://biovenn.nl, Hulsen et al., 2008 | Used to generate Venn diagrams | |
| Software, algorithm | WormCat | https://www.wormcat.com/, Higgins et al., 2022 | Used to generate WormCat data | |
| Software, algorithm | BowTie2 | https://bowtie-bio.sourceforge.net/bowtie2/index.shtml, Langmead et al., 2009 | Bioinformatic data analysis software | |
| Commercial assay or kit | KAPA Library Quantification Kits | https://sequencing.roche.com/global/en/products/group/kapa-library-quantification-kits.html#productInfo | N/A | Used to quantify DNA for CUT&Tag |
| Commercial assay or kit | NEBNext High-Fidelity 2 X PCR Master Mix | https://www.neb.com/products/m0541-nebnext-high-fidelity-2x-pcr-master-mix#Product%20Information | N/A | Used to amplify libraries in CUT&Tag |
| Chemical compound, drug | ConA beads | https://www.bangslabs.com/sites/default/files/imce/docs/PDS%20720%20Web.pdf | N/A | Used in CUT&Tag ‘See Materials and methods section; CUT&Tag’ |
| Other | Potter-Elvehjem Tissue Grinder With PTFE Pestle | https://www.thomassci.com/Equipment/Grinders/_/POTTER-ELVEHJEM-TISSUE-GRINDER-WITH-PTFE-PESTLE?q=Kontes%20Glass | 3432 N59; Mfr. No. 886000–0020 | Used for homogenizing worms in CUT&Tag ‘Materials and methods section CUT&Tag’ |
Additional files
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Supplementary file 1
RNA seq for SAM synthase knockdown in basal conditions.
Tabs A-C show sams-3, sams-4, sams-5 (RNAi) RNA seq data then Tabs D-F show WormCat gene enrichment. sams-1 data is from Ding et al., 2018. Enriched categories from WormCat. Red color denoted categories with a p value of less than 0.01. NS is not significant, NV is no value, RGS is regulated gene set.
- https://cdn.elifesciences.org/articles/79511/elife-79511-supp1-v3.xlsx
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Supplementary file 2
Statistics for survival curves.
Each tab contains data for replicate experiments (R1, R2, R3). Statistical information from GraphPad Prism is also included.
- https://cdn.elifesciences.org/articles/79511/elife-79511-supp2-v3.xlsx
-
Supplementary file 3
CUT&TAG peaks for H3K4me3 from sams-1 and sams-4 animals in basal and heat shocked samples.
Tabs A-F: Cut and Tag peaks from Control, sams-1 and sams-4 RNAi animals at 15 and 37 degrees determined by HOMER. Tabs G-I: Enriched categories from WormCat. Color denoted categories with a p value of less than 0.01 NS is not significant, NV is no value, RGS is regulated gene set.
- https://cdn.elifesciences.org/articles/79511/elife-79511-supp3-v3.xlsx
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Supplementary file 4
Limited activation of heat shock response in sams-4 RNAi animals.
Tabs show RNA seq from control (A), sams-1 (B) or sams-4 (C) animals subjected to heat shock that was used for comparison with C&T data. Differential genes were identified using Deseq2 in DolphinNext. Data for control and sams-1 RNAi animals is from Ding et al., 2018. WormCat batch output of two-fold regulated genes for Categories 1, 2, and 3 are in tabs (E-G). Highlighting denotes genes with significantly p values. NS is not significant, NV is no value, RGS is regulated gene set.
- https://cdn.elifesciences.org/articles/79511/elife-79511-supp4-v3.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/79511/elife-79511-mdarchecklist1-v3.docx
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S-adenosylmethionine synthases specify distinct H3K4me3 populations and gene expression patterns during heat stress
eLife 12:e79511.
https://doi.org/10.7554/eLife.79511
