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. 2020 Jan 20;21(2):671.
doi: 10.3390/ijms21020671.

DNA Methylation Is Correlated With Gene Expression During Diapause Termination of Early Embryonic Development in the Silkworm ( Bombyx mori)

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DNA Methylation Is Correlated With Gene Expression During Diapause Termination of Early Embryonic Development in the Silkworm ( Bombyx mori)

Bing Li et al. Int J Mol Sci. .
Free PMC article

Abstract

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause.

Keywords: Bombyx mori; DNA methylation; RNA-seq; diapause; embryonic development; whole-genome sequencing.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Enrichment analysis of differentially expressed genes (DEGs) following HCl treatment. (A) Annotation of DEGs with GO enrichment. Gene numbers and percentages are listed for each category. (B) Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment of DEGs. The red and green bars represent upregulated and downregulated genes, respectively, and the number represents the percentage of genes.
Figure 2
Figure 2
Analysis of methyltransferase expression. The expression fold change of BmDnmt1 and BmDnmt2 by qRT-PCR and RNA-seq, respectively, comparing diapause-terminated eggs with diapause-destined eggs.
Figure 3
Figure 3
Analysis of methylation levels and methylation sites of different samples. Percentage methylation in different samples (A) and the numbers of different methylation sites (B). Three biological replicates comprised each sample type. Significant differences are indicated by ** p < 0.01.
Figure 4
Figure 4
Validation of RNA-seq and whole-genome sequencing coupled with bisulfite DNA treatment (WGBS). Three genes that were significantly upregulated (101742044, 101739131 and 101739208) and three genes that were significantly downregulated (110385781, 101740063 and 101741941) from RNA-seq analyses were selected for mRNA quantification using RT-qPCR (A). DNA methylation levels of the hypermethylated gene, (101739208) and the hypomethylated gene, (110385781) from our WGBS were analyzed using methylation-sensitive McrBC-qPCR (B). Methylated DNA was digested by McrBC; thus, higher qPCR signals indicate lower methylation levels, and lower qPCR signals indicate higher methylation levels. Significant differences are indicated by ** p < 0.01 and * p < 0.05.
Figure 5
Figure 5
DNA methylation patterns in different genomic regions. Methylation percentages of different genetic elements, including CDS, downstream, exon, gene, intergenic, intron, inc-RNA, miRNA, mRNA, transcript, and upstream (A). Methylation percentages of transposon elements (TEs) and repeat elements (REs) (B). DNA, long interspersed nuclear elements (LINE), short interspersed nuclear elements (SINE), retrotransposons (RC) and long terminal repeats (LTR) are classified as TE, and Simple_repeat, Low_complexity, satellite and RNA are classified as RE.
Figure 6
Figure 6
Percentage methylation levels in gene bodies and 2 kb upstream and downstream regions. Different coloured lines represent different experimental repetitions.
Figure 7
Figure 7
KEGG pathway enrichment of different methylation genes (DMGs) and differentially methylated promoters (DMPs) following HCl treatment. KEGG pathway enrichment of DMGs located in differentially methylated regions (DMRs) (A). DMPs located in DMRs (B). The circle size represents gene numbers, and the colour represents the Q value.
Figure 7
Figure 7
KEGG pathway enrichment of different methylation genes (DMGs) and differentially methylated promoters (DMPs) following HCl treatment. KEGG pathway enrichment of DMGs located in differentially methylated regions (DMRs) (A). DMPs located in DMRs (B). The circle size represents gene numbers, and the colour represents the Q value.
Figure 8
Figure 8
Distribution and functional enrichment analysis of significant differences in methylation and expression levels in methylome- and transcriptome-associated genes (MTGs). The distribution of MTGs in four different quadrants; the number in each quadrant refers to the number of MTGs in that quadrant (A). MTGs were classified into cellular components, molecular function and biological processes by WEGO according to GO terms (B). KEGG pathway enrichment of MTGs. The circle size represents gene numbers, and the colour represents the Q value (C).
Figure 8
Figure 8
Distribution and functional enrichment analysis of significant differences in methylation and expression levels in methylome- and transcriptome-associated genes (MTGs). The distribution of MTGs in four different quadrants; the number in each quadrant refers to the number of MTGs in that quadrant (A). MTGs were classified into cellular components, molecular function and biological processes by WEGO according to GO terms (B). KEGG pathway enrichment of MTGs. The circle size represents gene numbers, and the colour represents the Q value (C).
Figure 9
Figure 9
Bisulfite sequencing validation of the hypermethylated gene, G2E3 (101739208).The number indicates the CG position of the target gene, and the percentage represents the statistical mean methylation level of the CG site or the whole CG site. Dark circles indicate CG site methylation, and open circles refer to unmethylated cytosines. Each row consists of a single sequenced clone.

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