Genome-wide methylation analysis identified sexually dimorphic methylated regions in hybrid tilapia

Abstract

Sexual dimorphism is an interesting biological phenomenon. Previous studies showed that DNA methylation might play a role in sexual dimorphism. However, the overall picture of the genome-wide methylation landscape in sexually dimorphic species remains unclear. We analyzed the DNA methylation landscape and transcriptome in hybrid tilapia (Oreochromis spp.) using whole genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). We found 4,757 sexually dimorphic differentially methylated regions (DMRs), with significant clusters of DMRs located on chromosomal regions associated with sex determination. CpG methylation in promoter regions was negatively correlated with the gene expression level. MAPK/ERK pathway was upregulated in male tilapia. We also inferred active cis-regulatory regions (ACRs) in skeletal muscle tissues from WGBS datasets, revealing sexually dimorphic cis-regulatory regions. These results suggest that DNA methylation contribute to sex-specific phenotypes and serve as resources for further investigation to analyze the functions of these regions and their contributions towards sexual dimorphisms.

Figure 1. Overall methylation levels in 13 species of eukaryotes, including Oreochromis niloticus.

Tree topology is generated from NCBI Taxonomy. The DNA methylation levels are from the main chromosomes of each organism, whereby chloroplast and mitochondria genomes are not included. Cytosine methylation data for T. nigroviridis, C. intestinalis, T. castaneum, B. mori, A. mellifera and O. sativa are taken from Zemach et al.. Cytosine methylation data for D. rerio, M. musculus, P. trichocarpa, A. thaliana, C. reinhardtii are taken from Feng et al.. Cytosine methylation data for C. semilaevis are taken from Shao et al..

Figure 2. Correlation matrix showing the Pearson correlation of base resolution CpG methylation genome-wide between the skeletal muscle tissue samples of hybrid tilapia.

Histograms showed CpG methylation level of each sample from 0% to 100% distributed across 20 bins of 5% intervals. The red line and green line represent linear regression and loess fit, respectively, to model the relationship of differential CpG methylation sites between compared individual pairs. Comparing the genome-wide CpG methylation profile between the sexes showed similar CpG methylation profiles in skeletal muscle tissue, with obvious differences between the sexes. Also, majority of CpG sites are highly methylated, with most of the CpG sites at more than 75% methylation level.

Figure 3. CpG Methylation level in relation to gene body, 5 Kb region upstream of TSS and 5 Kb downstream of TTS in hybrid tilapia.

CpG methylation level dropped gradually to 25% approaching the TSS before rising sharply to 75% in the gene body. CpG methylation level then dipped rapidly to 50% approaching the TTS and subsequently returned to earlier intragenic CpG methylation level.

Figure 4. Distribution of CpG methylation level across various genomic elements such as promoters, introns, exons, DNA transposons, SINEs, LINEs and LTRs in male and female tilapia.

The percentage of repeat elements is summarized according to chromosome locations.

Figure 5. CIRCOS chart summarizing the differentially methylated regions in two sexes of hybrid tilapia.

Track 1 shows an ideogram representing the 22 chromosomes in hybrid tilapia. Track 2 represents differentially methylated regions between the male and female individuals. Histograms in the blue regions show regions hypermethylated in males while orange regions show regions hypermethylated in females. Histograms were expanded for illustration purposes and histogram width is not a direct representation of DMR size. Track 3 displays the locations of all CpG islands in hybrid tilapia.

Figure 6. Numbers of hypermethylated DMRs in male (blue) and female (red) tilapia skeletal muscle tissue.

Dotted line graph represents the Z-score of number of DMRs in each chromosome. In this plot, chromosome LG1 shows very high number of DMRs as compared to the other chromosomes (Z-score > 3).

Figure 7. Low CpG methylation at promoter regions are linked with high gene expressions in skeletal muscle of hybrid tilapia.

(A) Log₂ RPM vs CpG methylation levels (%) at gene promoter regions. Gene promoter regions are arbitrary defined as 1000 bp upstream of TSSs. (B) Averaged log₂ (RPM) profile of male (Y-axis) and female (X-axis). Differentially expressed genes (DEGs) are highlighted in green with multiple testing corrections. (C) WEGO output of sexually dimorphic DEGs.

Figure 8. Gene network analysis of DEGs.

Genes upregulated in males are designated as red nodes while genes upregulated in females are designated as blue nodes. Relationship confidence level is depicted in terms of green colour tones. The gene networks involved are listed in the table with the corresponding biological process associated with the networks shown. P-values shown are FDR corrected.

Figure 9. Enrichments of sexually dimorphic active cis-regulatory motifs in hybrid tilapia skeletal muscles.

Only the top 50 genes in each sex with the highest enrichments of LMRs in the promoter regions were selected for analyses. Transcription factor motif binding sites were drawn from the database JASPAR. (A) Male coordinates of LMRs (red triangle) and UMRs (blue square) in chromosome LG18. (B) Consensus sequence motifs of promoters enriched with LMRs in males. (C) Consensus sequence motifs of promoters enriched with LMRs in females.