High temperature influences DNA methylation and transcriptional profiles in sea urchins (Strongylocentrotus intermedius)

Abstract

Background: DNA methylation plays an important role in life processes by affecting gene expression, but it is still unclear how DNA methylation is controlled and how it regulates gene transcription under high temperature stress conditions in Strongylocentrotus intermedius. The potential link between DNA methylation variation and gene expression changes in response to heat stress in S. intermedius was investigated by MethylRAD-seq and RNA-seq analysis. We screened DNA methylation driver genes in order to comprehensively elucidate the regulatory mechanism of its high temperature adaptation at the DNA/RNA level.

Results: The results revealed that high temperature stress significantly affected not only the DNA methylation and transcriptome levels of S. intermedius (P < 0.05), but also growth. MethylRAD-seq analysis revealed 12,129 CG differential methylation sites and 966 CWG differential methylation sites, and identified a total of 189 differentially CG methylated genes and 148 differentially CWG methylated genes. Based on KEGG enrichment analysis, differentially expressed genes (DEGs) are mostly enriched in energy and cell division, immune, and neurological damage pathways. Further RNA-seq analysis identified a total of 1968 DEGs, of which 813 genes were upregulated and 1155 genes were downregulated. Based on the joint MethylRAD-seq and RNA-seq analysis, metabolic processes such as glycosaminoglycan degradation, oxidative phosphorylation, apoptosis, glutathione metabolism, thermogenesis, and lysosomes are regulated by DNA methylation.

Conclusions: High temperature affected the DNA methylation and expression levels of genes such as MOAP-1, GGT1 and RDH8, which in turn affects the metabolism of HPSE, Cox, glutathione, and retinol, thereby suppressing the immune, energy metabolism, and antioxidant functions of the organism and finally manifesting as stunted growth. In summary, the observations in the present study improve our understanding of the molecular mechanism of the response to high temperature stress in sea urchin.

Keywords: Epigenetic; High temperature; Methylation; Sea urchin (Strongylocentrotus intermedius); Transcriptome.

Fig. 1

Growth of Strongylocentrotus intermedius with different shell diameters at different temperatures. (A) The optimum and extreme temperature for growth of S. intermedius with different shell diameter. (B) Response surface plot of temperature, diameter and their interaction in growth of S. intermedius

Fig. 2

Statistical analysis of methylation sites in Strongylocentrotus intermedius under high temperature stress. (A) Distribution of CG methylation sites on different gene functional elements. (B) Distribution of CWG methylation sites on different gene functional elements. (C) Distribution of CG sites in TSSs. (D) Distribution of CG sites in TTSs. (E) Distribution of CG sites in TSSs, TTSs, and gene bodies. (F) Distribution of CWG sites in TSSs. (G) Distribution of CWG sites in TTSs. (H) Distribution of CWG sites in TSSs, TTSs, and gene bodies. (I) Distribution of CG differential methylation sites on different gene functional elements. (J) Distribution of CWG differential methylation sites on different gene functional elements. (K) Statistical analysis of differential methylation sites

Fig. 3

GO Enrichment analysis of differentially methylated genes at the site level of Strongylocentrotus intermedius under high temperature stress. (A) GO enrichment analysis of the top 30 of CWG differentially methylated genes at the site level. (B) GO enrichment analysis of the top 30 of CG differentially methylated genes at the site level

Fig. 4

KEGG Enrichment analysis of differentially methylated genes at the site level of Strongylocentrotus intermedius under high temperature stress. (A) KEGG enrichment analysis of the top 20 of CWG differentially methylated genes at the site level. (B) KEGG enrichment analysis of the top 20 of CG differentially methylated genes at the site level

Fig. 5

Analysis of methylation differences at the gene level in Strongylocentrotus intermedius under high temperature stress. (A) Volcano plot of differential expression of genes methylated at CG sites. (B) Volcano plot of differential expression of genes methylated at CWG sites. (C) Heatmap of differential methylation gene clustering among CG sites. (D) Heatmap of differential methylation gene clustering among CWG sites. (E) Bar chart of GO functional classification of differentially methylated genes. (F) KEGG pathway enrichment analysis of CG differentially methylated genes. (G) KEGG pathway enrichment analysis of CWG differentially methylated genes

Fig. 6

Transcriptome analysis of Strongylocentrotus intermedius under high temperature stress. (A) FPKM distribution. (B) MA plot of differential expression of genes. (C) GO enrichment analysis of the top 30 differentially expressed genes. (D) KEGG pathway enrichment analysis of the top 20 differentially expressed genes

Fig. 7

Combined DNA methylation and transcriptome analysis of Strongylocentrotus intermedius under high temperature stress. (A) Quadrant analysis of differentially expressed genes and CG differentially methylated genes. (B) Quadrant analysis of differentially expressed genes and CWG differentially methylated genes. Red indicates negatively associated loci of differential genes, and blue indicates positively associated loci of differential genes. (C) GO enrichment analysis of DMGs which were also DEGs. (D) Verification of 10 randomly selected DEGs by qRT-PCR.

Fig. 8

Diagram of the mechanism underlying the response of Strongylocentrotus intermedius to high temperature stress. Red represents upregulation/activation, and green represents downregulation/inhibition