Research ArticleORGANISMAL BIOLOGY

DNA methylation regulates transcriptional homeostasis of algal endosymbiosis in the coral model Aiptasia

See allHide authors and affiliations

Science Advances  15 Aug 2018:
Vol. 4, no. 8, eaat2142
DOI: 10.1126/sciadv.aat2142
  • Fig. 1 DNA methylation landscape.

    (A) Relative distribution of methylated CpGs across intergenic (18%), genic (82%), intronic (42%), and exonic (40%) regions in the Aiptasia genome. (B) Normalized frequencies of methylated CpGs in different regions. Chi-square test shows significant differences between intergenic and genic regions and between exons and introns (****P < 0.0001). (C) Relative frequencies of methylated positions across a normalized gene model. All frequencies were normalized to the lengths of the corresponding regions.

  • Fig. 2 DNA methylation is associated with higher expression.

    (A) Gene expression is positively correlated with median methylation levels. t test P values are 7.65 × 10−21, 3.75 × 10−14, and 1.75 × 10−13 for Q1 (first quartile) and Q2 of methylation (meth) levels, Q2 and Q3, and Q3 and Q4, respectively. FPKM, fragments per kilobase per million mapped fragments. ****P < 0.0001. (B) ChIP-seq analysis of H3K36me3 signals shows significant enrichment in methylated genes. t test P values are 2.48 × 10−20 for highly methylated genes (HM) and all methylated genes (allM) and 1.06 × 10−72 for unmethylated genes (UM) and all methylated genes. Highly methylated genes show the strongest enrichment of H3K36me3, followed by all methylated genes. In contrast, unmethylated genes show only weak enrichment of H3K36me3 over input controls. ****P < 0.0001. (C) Distribution of H3K36me3 enrichment and DNA methylation levels across two exemplary gene models. H3K36me3 and DNA methylation show coinciding distribution patterns over genes.

  • Fig. 3 DNA methylation regulates transcriptional homeostasis.

    (A) Spurious transcription in gene bodies is significantly lower in methylated and highly methylated genes. The y axis shows the natural logarithm of the coverage fold change (FC) of exons 1 to 6 versus exon 1, with lower values meaning less spurious transcription. *P < 0.05, ***P < 0.001, ****P < 0.0001. (B) There is a linear relationship between the inverse of transcriptional noise and log expression level [log10(mean FPKM)]. Given the same expression level, methylated genes always show lower levels of transcriptional noise. Methylated genes, n = 8561, r2 = 0.46, P < 2.2 × 10−16; unmethylated genes, n = 2491, r2 = 0.52, P < 2.2 × 10−16.

  • Fig. 4 PCA and KEGG pathway enrichment analysis.

    (A and B) PCA of gene expression and median methylation levels of Aiptasia genes. Both gene expression and DNA methylation separate samples by symbiosis state. PC1, first principal component; PC2, second principal component. (C) KEGG pathway enrichment analysis of previously identified symbiosis-related pathways. The y axis shows the ranks of these pathways in the pathway enrichment analyses of DEGs, DEMGs, and DMGs, respectively. The combined set of DEMG provides significant lower P values (front ranks) for symbiosis-related pathways. NF-κB, nuclear factor κB.

  • Fig. 5 Schematic diagram of symbiosis establishment– and breakdown-associated genes.

    Every node represents a category of genes and generally has multiple corresponding genes. The inside colors of nodes represent the expression changes of corresponding genes, including non-DEGs (cyan), up-regulated (red), down-regulated (blue), and up- and down-regulated DEGs (light red). The colors of node edges represent the methylation level changes of corresponding genes, including non-DMGs (light blue), hypermethylated (red), hypomethylated (blue), and hypermethylated and hypomethylated DMGs (light red). Numbers in circles denote genes/proteins: 1, complement receptor; 2, scavenger receptor; 3, C-type lectin; 4, integrin; 5, Toll-like receptor; 6, Ras-related C3 botulinum toxin substrate 1 [RAC1; Ras homolog (Rho) family]; 7, collagen; 8, vesicle-associated membrane protein; 9, autophagy-related protein 16 (ATG16); 10, Ras-related protein 5 (Rab5); 11, Rab7; 12, reduced form of nicotinamide adenine dinucleotide oxidase; 13, syntaxin 12; 14, ATG5; 15, ATG10; 16, programmed cell death 6–interacting protein; 17, sorting nexin (SNX); 18, cytoplasmic dynein; 19, tubulin alpha chain; 20, tubulin beta chain; 21, nitric oxide synthase (NOS); 22, lysosome-associated membrane glycoprotein/cluster of differentiation; 23, cathepsin L; 24, kinesin; 25, phosphatidylinositol 4,5-bisphosphate 3-kinase; 26, RAC serine/threonine-protein kinase; 27, B cell lymphoma 2 (Bcl-2) antagonist of cell death; 28, tumor necrosis factor receptor–associated factor; 29, NF-κB; 30, caspase-8 (CASP8); 31, CASP7; 32, apoptosis regulator/Bcl-2 (BCL2); 33, Rho; 34, Rho-associated protein kinase; 35, phosphatidylinositol 4-phosphate 5-kinase/phosphatidylinositol 5-phosphate 4-kinase/phosphatidylinositol 3-phosphate 5-kinase; 36, vinculin; 37, radixin; 38, profilin; 39, actin; 40, CD63; 41, lysosomal-associated transmembrane protein; 42, V-type proton adenosine triphosphatase (ATPase); PI3P, phosphatidylinositol-3-phosphate; PIP3, phosphatidylinositol (3,4,5)-trisphosphate.

  • Fig. 6 Schematic diagram of symbiosis maintenance–associated genes.

    Every node represents a category of genes and generally has multiple corresponding genes. The inside colors of nodes represent the expression changes of corresponding genes, including non-DEGs (cyan), up-regulated (red), down-regulated (blue), and up-regulated and down-regulated DEGs (light red). The colors of node edges represent the methylation level changes of corresponding genes, including non-DMGs (light blue), hypermethylated (red), hypomethylated (blue), and hypermethylated and hypomethylated DMGs (light red). Numbers in circles denote genes/proteins: 1, carbonic anhydrase (CA); 2, ammonium transporter; 3, glucose transporter; 4, amino acid transporter; 5, glutamine synthetase; 6, glutamate synthase; 7, glutamate dehydrogenase; 8, phosphate transporter; 9, V-type proton ATPase; 10, sugar transporter; 11, lipid transfer protein; 12, aquaporin 3 (glycerol transporter); 13, monocarboxylate transporter; 14, alcohol dehydrogenase; 15, aldehyde dehydrogenase; 16, NOS; 17, arginase; 18, carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase; 19, carbamoyl-phosphate synthase (ammonia); 20, ABC transporter.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/8/eaat2142/DC1

    Fig. S1. Circos visualization of different data at the genome-wide level.

    Fig. S2. Methylated genes in Aiptasia have lower CpG O/E.

    Fig. S3. Methylation patterns.

    Fig. S4. Sequence conservation of histone H3 homologs.

    Fig. S5. Western blot.

    Fig. S6. Correlation matrices of replicates.

    Fig. S7. Validation of methylation level.

    Fig. S8. qPCR validation of gene expression levels.

    Fig. S9. Correlation between DNA methylation changes and gene expression changes.

    Table S1. Aiptasia DMGs.

    Table S2. Aiptasia DEGs.

    Table S3. GO enrichment analysis of DMGs—biological process.

    Table S4. GO enrichment analysis of DMGs—molecular function.

    Table S5. GO enrichment analysis of DMGs—cellular component.

    Table S6. GO enrichment analysis of DEGs—biological process.

    Table S7. GO enrichment analysis of DEGs—molecular function.

    Table S8. GO enrichment analysis of DEGs—cellular component.

    Table S9. KEGG pathway enrichment analysis of DEGs.

    Table S10. KEGG pathway enrichment analysis of DMGs.

    Table S11. KEGG pathway enrichment analysis of DEMGs and statistics of symbiosis related pathways’ ranks.

    Table S12. Cnidarian-dinoflagellate symbiosis–related Aiptasia genes.

    Table S13. qPCR primers for gene expression validation.

    Table S14. qPCR raw Ct (cycle threshold) values for gene expression validation.

    Table S15. qPCR: ΔΔCt approach for fold-change calculations.

    Table S16. Primers for bisulfite PCR-based methylation validation.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Circos visualization of different data at the genome-wide level.
    • Fig. S2. Methylated genes in Aiptasia have lower CpG O/E.
    • Fig. S3. Methylation patterns.
    • Fig. S4. Sequence conservation of histone H3 homologs.
    • Fig. S5. Western blot.
    • Fig. S6. Correlation matrices of replicates.
    • Fig. S7. Validation of methylation level.
    • Fig. S8. qPCR validation of gene expression levels.
    • Fig. S9. Correlation between DNA methylation changes and gene expression changes.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Aiptasia DMGs.
    • Table S2 (Microsoft Excel format). Aiptasia DEGs.
    • Table S3 (Microsoft Excel format). GO enrichment analysis of DMGs—biological process.
    • Table S4 (Microsoft Excel format). GO enrichment analysis of DMGs—molecular function.
    • Table S5 (Microsoft Excel format). GO enrichment analysis of DMGs—cellular component.
    • Table S6 (Microsoft Excel format). GO enrichment analysis of DEGs—biological process.
    • Table S7 (Microsoft Excel format). GO enrichment analysis of DEGs—molecular function.
    • Table S8 (Microsoft Excel format). GO enrichment analysis of DEGs—cellular component.
    • Table S9 (Microsoft Excel format). KEGG pathway enrichment analysis of DEGs.
    • Table S10 (Microsoft Excel format). KEGG pathway enrichment analysis of DMGs.
    • Table S11 (Microsoft Excel format). KEGG pathway enrichment analysis of DEMGs and statistics of symbiosis related pathways’ ranks.
    • Table S12 (Microsoft Excel format). Cnidarian-dinoflagellate symbiosis–related Aiptasia genes.
    • Table S13 (Microsoft Excel format). qPCR primers for gene expression validation.
    • Table S14 (Microsoft Excel format). qPCR raw Ct (cycle threshold) values for gene expression validation.
    • Table S15 (Microsoft Excel format). qPCR: ΔΔCt approach for fold-change calculations.
    • Table S16 (Microsoft Excel format). Primers for bisulfite PCR-based methylation validation.

    Download Tables S1 to S16

    Files in this Data Supplement:

Navigate This Article