DNA Methylation Patterns Provide Insights into the Epigenetic Regulation of Intersex Formation in the Chinese Mitten Crab (Eriocheir sinensis)

Abstract

DNA methylation is a form of epigenetic regulation that plays an important role in regulating gene expression of organisms. However, the DNA methylation pattern of intersex crabs has not yet been clarified. In order to reveal the DNA methylation in intersex Eriocheir sinensis, this study investigated the genome-wide DNA methylation profiles of female, male, and intersex individuals. The similar results across samples showed that the levels of cytosine methylation in the CG context were significantly higher than that in the CHG and CHH contexts. The methylation levels in the promoter region were higher than those in other functional element regions. We screened 149 differentially methylated genes (DMGs) in the promoter region between female and intersex crabs and 110 DMGs between male and intersex crabs. Three core gene networks were found in a comparison group of female and intersex crabs that involved heat shock proteins, ribosomes, and metabolism pathways; two core gene networks were found in the comparison group of male and intersex crabs that involved ribosomes and metabolism pathways. The six confirmed genes of Hsc70, Hsp90, Rpl18, Acsl1, Yip2, and Rpl7 had lower methylation levels in the promoter region of intersex crabs than that of female and male crabs. However, six genes showed higher expression in intersex crabs than in female and male crabs. Our results reveal that DNA methylation is involved in the formation and maintenance of life activities of intersex crabs through the regulation of gene expression, enriching the DNA methylation library of the whole genome of E. sinensis and providing new insights for a better understanding of the epigenetic regulation of the formation of intersex E. sinensis.

Keywords: DNA methylation; Eriocheir sinensis; intersex.

Figure 1

Overall methylation levels for fm, cm, and mm. The proportions of mCG, mCHG, and mCHH in mC. The red, blue, and green colors represent methylated (m) CG/mC, mCHH/mC, and mCHG/mC, respectively. mC represents cytosine methylation. H can be any of A, T, or C. fm, female crabs. mm, male crabs. cm, intersex crabs.

Figure 2

DNA methylation levels of three methylated forms of CG, CHH, and CHG among genomic functional elements. (a) CG, (b) CHH, and (c) CHG. H can be any of A, T, or C. The horizontal coordinates represent the different functional elements on the genome: A, B, and C are promoters. D, F, and I are exons. E, G, and H represent introns. J represents downstream. The left vertical coordinates indicate their methylation levels in their respective backgrounds. TSS is the transcription start site. TES is the transcription end site. fm, female crabs. mm, male crabs. cm, intersex crabs.

Figure 3

Kyoto encyclopedia of genes and genomes (KEGG) analysis of differentially methylated genes (DMGs) in the promoter region of cm vs. fm and cm vs. mm. (a) Scatter plot of KEGG pathways in the top 10 of cm vs. fm. (b) KEGG pathways for the top 10 of cm vs. mm. The ordinate represents the enriched pathway and the abscissa represents the Rich factor of the corresponding pathway. The size of the spots represents the number of DMGs enriched in each pathway, and the color of the spots represents the corrected p-value of each pathway. The Rich factor represents the ratio of the number of DMGs mapped to a pathway to the total number of genes mapped to that pathway. The larger the enrichment factor, the higher the degree of enrichment. fm, female crabs. mm, male crabs. cm, intersex crabs.

Figure 4

Protein–protein interaction (PPI) network and KEGG enrichment information for the cm vs. fm and cm vs. mm comparison groups. (a) The cm vs. fm PPI network of DMGs in the promoter region. (b) The core network of cm vs. fm identification using the MCODE plugin and enrichment analysis via the KEGG. (c) The cm vs. mm PPI network of DMGs in the promoter region. (d) The core network of cm vs. mm identification using the MCODE plugin and enrichment analysis via the KEGG. fm, female crabs. mm, male crabs. cm, intersex crabs.

Figure 5

Methylation levels of DMGs in the promoter region. (a) Methylation levels of fm and cm key functional DMGs. (b) Methylation levels of mm and cm key functional DMGs. Hsc70, heat shock cognate protein 71 kDa. Hsp90, heat shock protein 90. Rpl18, 60S ribosomal protein L18. Acsl1, long-chain acyl-CoA synthetase 1. Rpl7, 60S ribosomal protein L7. Yip2, Yippee-interacting protein 2. fm, female crabs. mm, male crabs. cm, intersex crabs.

Figure 6

Validation of enzymatic methyl sequencing (EM-seq) data by bisulfite sequencing PCR (BSP). (a) BSP results of Hsc70 gene between fm and cm. (b) BSP results of Hsp90 gene between fm and cm. (c) BSP results of Rpl7 gene between mm and cm. (d) BSP results of Yip2 gene between mm and cm. Black and white circles indicate methylated and unmethylated CG, respectively. Cross indicates mismatch or gap in alignment. fm, female crabs. mm, male crabs. cm, intersex crabs.

Figure 7

The relative expression of mRNA for the candidate gene in the muscle tissue of E. sinensis was detected using the qRT-PCR. (a) qRT-PCR of fm and cm key function DMGs (b) qRT-PCR of mm and cm key function DMGs. 18S was used as an internal control. The values are represented as the mean ± SEM of three replicates. Hsc70, heat shock cognate protein 71 kDa. Hsp90, heat shock protein 90. Rpl18, 60S ribosomal protein L18. Acsl1, long-chain acyl-CoA synthetase 1. Rpl7, 60S ribosomal protein L7. Yip2, Yippee-interacting protein 2. fm, female crabs. mm, male crabs. cm, intersex crabs.